CN1174099C - Nucleic acid vaccines encoding G protein of respiratory syncytial virus - Google Patents
Nucleic acid vaccines encoding G protein of respiratory syncytial virus Download PDFInfo
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- CN1174099C CN1174099C CNB988093014A CN98809301A CN1174099C CN 1174099 C CN1174099 C CN 1174099C CN B988093014 A CNB988093014 A CN B988093014A CN 98809301 A CN98809301 A CN 98809301A CN 1174099 C CN1174099 C CN 1174099C
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Abstract
Non-replicating vectors, such as plasmid vectors, containing a nucleotide sequence coding for a G protein of respiratory syncytial virus (RSV) and a promoter for such sequence, preferably a cytomegalovirus promoter, are described. Such vectors also may contain a further nucleotide sequence located adjacent to the RSV G protein encoding sequence to enhance the immunoprotective ability of the RSV G protein when expressed in vivo. Such non-replicating vectors may be used to immunize a host, including a human host, against RSV infection by administration thereto. Such non-replicating vectors also may be used to produce antibodies for detection of RSV infection in a sample.
Description
Invention field
The present invention relates to the field of respiratory syncytial virus (RSV) vaccine, relate in particular to the vaccine of the proteic nucleotide sequence of absorption (G) that comprises the RSV that encodes.
Background of invention
The respiratory syncytial virus (RSV) that belongs to the Paramyxoviridae of virus is a kind of minus-stranded rna virus, it is that (reference 1-each reference in this application marks in bracket, and these reference are done more fully explanation to the state of the art in field under the present invention for the main viral pathogens of decision infant's bronchitis and pneumonia.The complete reference documentation ﹠ info of relevant each part reference document this specification sheets at last, and then provide before claims.The content of these reference is used as reference and takes in this specification sheets).In the U.S., the annual acute respiratory infection that is caused by RSV can cause about 90,000 people's hospital cares, and 4,500 people's death (reference 2) are arranged.Only be used for the treatment of the required expense of rsv infection every year and just surpass 3.4 hundred million dollars (reference 3) in the U.S..Also do not have now licensed-in, at the vaccine of RSV.The main path of exploitation RSV vaccine comprises virus, attenuated live virus and the subunit vaccine of deactivation.
Protective immune response at RSV is considered to induce the neutralizing antibody (reference 4) that merges (F) and absorption (G) glycoprotein at the surface.In addition, cytotoxic T lymphocyte (CTL) is replied and has been participated in virus sweep.F albumen is guarded between RSV A and B hypotype.
The G albumen (33kDa) of RSV is that height O-is glycosylated, and producing apparent molecular weight is the glycoprotein (reference 5) of 90kDa.Two big hypotype: the A and the B (reference 6) of RSV virus had been determined already.Major antigen gender gap between these two kinds of hypotypes is present in (reference 3,7) on the G glycoprotein.
Rsv protein may be had any problem as vaccine.Up to now, to be proved the immunogenicity of inducing neutralizing antibody in the seronegativity chimpanzee very weak for the vaccine candidate object taken of parenteral.Exist under the condition of passive acquired antibody, can further weaken, described passive acquired antibody such as most of infant had passes through the maternal antibody that placenta obtains by the serum antibody response of described antigen induction.The subunit vaccine material standed for of RSV comprises fusion (F) glycoprotein of the purifying of the cell culture that comes from rsv infection, and described glycoprotein is affine or ion exchange chromatography purifying (reference 8) by immunity.With said preparation seronegativity or seropositivity chimpanzee are carried out the parenteral immunity, in the seronegativity animal, tire, need the dosage of 3 50 μ g in order to induce about 1:50RSV serum neutralization.When inoculating described animal, do not detect effect or the clinical disease that immunity is got rid of virus subsequently at the upper respiratory tract with wild-type RSV.There is not the research effect that this vaccine immunity is got rid of virus in lower respiratory tract, although this position is the position that may have its maximum efficiency by the serum antibody of parenteral immune induction.Security and immunogenicity research in minority seropositivity individuality, had been carried out already.Described vaccine has confirmed its security in seropositivity children and 3 seronegativity children (all children's age all surpasses 2.4 years old).Owing to accept the children's of immunity comparatively small amt, still can not determine the influence of immunity to lower respiratory illness.An immunizing dose can induce the intravital virucidin of vaccination person of 40%-60% to tire in seropositivity children body increases by 4 times.Therefore, studying on a small scale more than to provide enough data that the effectiveness of the anti-RSV-inductive of described vaccine disease is estimated.Another problem that subunit's RSV vaccine is run into is might cause increasing the weight of of disease with immunogen preparation inoculation seronegativity acceptor.In the sixties in 20th century, with the immune baby of RSV preparation (FI-RSV) of formalin deactivation, can cause contacting once more subsequently the enhancing of pulmonary disorder when viral alive, also be known as immunostimulant (reference 9,10).Described vaccine inoculation person can form strong serum and reply, but can not preventing infection, and some inoculator can produce serious even fatal sometimes respiratory tract disease when natural infection.Although accurate mechanism it be unclear that, the immunologic facilitation that has shown this form already might reflect the antigenic structural changes of RSV (reference 11), remaining serum and/or cell contamination thing (reference 12), virus absorption (G) proteic special characteristic (reference 13,14) or unbalanced cell-mediated immune responses (reference 13,15).Confirmed already that the FI-RSV vaccine can be induced TH2-type immunne response in mouse, and can not cause immunostimulant, replied (reference 15) but bring out a kind of TH1 with the RSV immunity of living.
In some research, carry out the enhancing that immune immunne response can cause the disease of rodent with synthetic RSV G fusion rotein, be similar to vaccine-induced result by the RSV of formalin deactivation.With expressing the proteic recombinant vaccine virus immune mouse of RSV G, can cause in lung, occurring the G-specific T-cells and reply, described cell can only be collected from the inferior pedigree of CD4+T cell, and obviously is partial to Th2.The G-specific T-cells can be induced pulmonary apoplexy, lung neutrophil collection (shock lung), and a large amount of lung eosinophils, and in the mouse acceptor of the transfer of subculture, cause death (reference 14) sometimes.Non-ly duplicate antigenic some vaccine preparation and carry out the dependency that immunity increases the weight of with disease with comprising, show that it is used as vaccine should act cautiously in the seronegativity human body.
Attenuated vaccine at the work of the disease that is caused by RSV is hopeful because of two kinds of major causes.At first, the vaccine virus infection of being lived can be induced the equilibrated immunne response, comprises mucous membrane and serum antibody and cytotoxic t-lymphocyte.Secondly, infect the baby, can not cause the enhanced disease during natural infection once more subsequently with the wild-type virus of attenuated vaccine material standed for of living or natural acquisition.Producing following attenuated vaccine alive is a kind of challenge, and this vaccine has immunogenicity to the infant with parent virucidin, and has been attenuated and can be used for more than or equal to seronegativity baby in age in June.The live-virus vaccine of attenuation also has residual virulence and the instable danger of genetics.
In several studies, confirmed to inject the plasmid DNA of the sequence that comprises a kind of foreign protein of encoding already, can cause the expression of described foreign protein, and induce at described antigenic antibody and cytotoxic t-lymphocyte (CTL) and reply (for example, reference 16,17,18).Plasmid DNA inoculation is used to express the method that viral protein concludes above has some advantages.At first, can import virus inside itself, can't render a service owing to the comprehensive virus of described antibody makes its forfeiture at the DNA that the virus antigen of will encoding under the condition of antibody is arranged.Secondly, the antigen of Biao Daing should have natural conformation and suitable glycosylation in vivo.Therefore, described antigen should be induced the antibody response that is similar to by the antibody response of the antigen induction that is present in the wild-type virus infection.On the contrary, some method that is used for purifying protein can be induced the change of conformation, therefore causes protective epitope's immunogenic forfeiture, and might cause immunostimulant.The 3rd, in live body, can detect by the time of the protein expression that carries out of plasmid DNA of injection and obviously be longer than time in the cell of virus infection, and the theoretic advantage that it had is, has prolonged cytotoxic T-cell induction and enhanced antibody response.The 4th, antigenic expression in vivo might provide provide protection, and does not need external adjuvant.
Before the present invention, still do not understand by taking the proteic dna molecular of coding RSV G carries out immunity to the disease that is caused by RSV ability.Specifically, still do not understand the effectiveness of carrying out immunity with the proteic gene pairs RSV of the RSV G inductive disease of coding secreted form.The infection of RSV can cause serious disease.It is useful with favourable that the coding proteic isolating gene of RSV G and nonreplication vector are provided, described carrier comprises plasmid vector, uses it in the body and takes, and be used to comprise the immunological reagent of vaccine, so that the disease that prevention is caused by RSV, and be used to prepare diagnostic preparation and test kit.Specifically, need provide the immunogen and the protectant vaccine that can be used as mankind's (comprising the seronegativity baby), this vaccine can not cause the enhancing (immunostimulant) of disease.
Summary of the invention
The present invention relates to immune host and make it resist the method for the disease that causes by respiratory syncytial virus, relate to the nonreplication vector that contains the nucleic acid molecule in the immune composition that is useful on described purpose, and relate to the diagnostic method that uses described carrier and nucleic acid molecule.Specifically, the present invention relates to provide the proteic nucleic acid vaccine of G of coding respiratory syncytial virus.
According to an aspect of the present invention; providing a kind of is used for to the immune composition of taking in the host; so that in described host, produce at the proteic protection antibody of respiratory syncytial virus (RSV) G, comprise a kind of nonreplication vector and pharmaceutically useful supporting agent, this carrier comprises:
First nucleotide sequence of encode a RSV G albumen or a RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction,
One is operably connected to the promoter sequence that described first nucleotides sequence lists, be used for described host express RSV G albumen and
Second nucleotide sequence between described first nucleotide sequence and described promoter sequence is used for strengthening described carrier and carries out the proteic expression in vivo of RSV G described host.
Described first nucleotide sequence can be the proteic nucleotide sequence of coding complete length RSV G.This first nucleotide sequence can comprise that nucleotide sequence shown in Figure 2 (sequence 1) or coding have the RSV G albumen (sequence 2) of the complete length of aminoacid sequence shown in the sequence 2.
In addition, described first nucleotide sequence can be that coding has lacked the proteic sequence of RSV G of striding film encoding sequence and its upstream sequence.Proteic first nucleotide sequence of RSV G of described brachymemma of encoding can comprise nucleotide sequence shown in Figure 3 (sequence 3) or can comprise that coding has the proteic nucleotide sequence of RSV G of the brachymemma of aminoacid sequence shown in Figure 3 (sequence 4).The shortage of striding the expression of film district can cause the proteic generation of RSV G of secreted form.
Described nonreplication vector can also comprise the allos signal peptide coding nucleotide sequence of 5 ' the terminal upstream that is positioned at described first nucleotide sequence.Can the encode signal peptide of human tissue plasmin activator of described signal coding sequence.
Described promoter sequence can be early stage immediately cytomegalovirus (CMV) promotor.Described second nucleotide sequence can comprise the human cytomegalic inclusion disease virus intron A.
Described nonreplication vector is plasmid vector normally.Be specially pXL5 or pXL6 by the proteic plasmid vector of coding G that immune composition comprised that this aspect of the present invention provided, its make up and the peculiar element that had shown in Fig. 4 or 5.
According to a further aspect in the invention, provide a kind of immune host to make it resist the method that is infected the disease that causes by respiratory syncytial virus (RSV), this method comprises the nonreplication vector of taking significant quantity to described host, and this carrier comprises:
First nucleotide sequence of encode a RSV G albumen or a RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction,
One is operably connected to the promoter sequence that described first nucleotides sequence lists, so as in described host, to express RSV G albumen and
Second nucleotide sequence between described first nucleotide sequence and described promoter sequence is used for strengthening described carrier and carries out the proteic expression in vivo of described RSV G described host.
Can implement described immunization method, so that induce equilibrated Th1/Th2 immunne response.
The present invention comprises that also the novel method of the disease that caused by respiratory syncytial virus infection takes place the gene prevention host who uses coding respiratory syncytial virus (RSV) G albumen or RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction, and this method comprises:
Separate described gene;
Described gene is operatively coupled at least one control sequence, so that produce a kind of nonreplication vector, described control sequence can instruct the proteic expression of RSVG after described carrier is imported into the host, so as to produce at the proteic immunne response of RSV G and
Described carrier is imported described host.
Can also may further comprise the steps by the method that this aspect of the present invention provided:
Described gene is operably connected on the immunoprotection enhancement sequences; so that in described host, produce the enhanced immanoprotection action by RSV G albumen; preferably, be included in and import immunostimulation CpG sequence in the described carrier by the immunoprotection enhancement sequences is imported between described control sequence and the described gene.
In addition, the present invention includes a kind of production and be used to prevent the host that the method for the vaccine of the disease that causes takes place to be infected by respiratory syncytial virus (RSV), this method comprises:
First nucleotide sequence that separates coding RSV G albumen or RSV G protein fragments, this nucleotide sequence can produce the antibody with RSV G albumen specific reaction,
Described first nucleotide sequence is operably connected at least one control sequence, so that produce a kind of nonreplication vector, described control sequence can instruct the proteic expression of described RSV G after importing the host, when in described host, carrying out expression in vivo by described carrier, generation is at the proteic immunne response of RSV G
Described first nucleotide sequence is operably connected to second nucleotides sequence lists, so as to strengthen described carrier in described host, carry out the proteic expression in vivo of described RSV G and
The vaccine that described preparing carriers is become to be used for taking in the body.
Described carrier can be the plasmid vector that is selected from pXL5 and pXL6.The present invention also comprises the vaccine of being taken to the host who comprises human host by this method being used for of producing.
As indicated above, carrier provided by the present invention can be used for diagnostic purpose.Therefore, in another aspect of this invention, provide a kind of mensuration respiratory syncytial virus (RSV) G the method that albumen exists in sample, may further comprise the steps:
A) with a kind of nonreplication vector immunity host, so that produce to the special antibody of described RSV G albumen, described nonreplication vector comprises first nucleotide sequence of coding RSV G albumen or RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction, one is operably connected to the promoter sequence that described first nucleotides sequence lists, so that in described host, express described RSV G albumen, and second nucleotide sequence between described first nucleotide sequence and described promoter sequence, so that in described host, strengthen the proteic expression in vivo of RSV G that is undertaken by described carrier;
B) separate described RSV G protein specific antibody;
C) allow described sample contact, comprise any RSV G albumen that is present in the described sample and the complex body of RSV G protein specific antibody so that produce with isolated antibody; With
D) generation of the described complex body of mensuration.
Being used to induce the nonreplication vector of described antibody can be plasmid vector pXL5 and pXL6.
The present invention comprises that also a kind of being used for detect the diagnostic kit that respiratory syncytial virus (RSV) G albumen exists at sample, and this test kit comprises:
A) a kind of nonreplication vector, this carrier can produce after taking to the host the special antibody of described RSV G albumen, described nonreplication vector comprises first nucleotide sequence of coding RSV G albumen or RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction, one is operably connected to the promoter sequence that described first nucleotides sequence lists, so that in the host, express described RSV G albumen, and second nucleotide sequence between described first nucleotide sequence and described promoter sequence, so that in described host, strengthen the proteic expression in vivo of RSV G that is undertaken by described carrier;
B) tripping device of the described RSV G protein specific antibody of separation;
C) device that allows described isolating RSV G albumen specific antibody contact with described sample comprises any RSV G albumen that is present in the described sample and the complex body of RSV G protein specific antibody so that produce; With
D) measure the identification apparatus that described complex body produces.
The invention still further relates to the method for a kind of production, comprising the special antibody of respiratory syncytial virus (RSV) G albumen:
A) nonreplication vector with significant quantity carries out immunity to the host, so that produce RSV G protein specific antibody, described nonreplication vector comprises:
First nucleotide sequence of encode a RSV G albumen or a RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction,
One is operably connected to the promotor that described first nucleotides sequence lists, be used for the host express described RSV G albumen and
Second nucleotide sequence between described first nucleotide sequence and described promoter sequence is so that strengthen the proteic expression in vivo of described RSV G that is undertaken by described carrier in described host;
B) from described host, separate RSV G protein specific antibody;
The invention still further relates to and a kind ofly be used for producing, may further comprise the steps the special monoclonal antibody method of respiratory syncytial virus (RSV) G albumen:
A) make up a kind of carrier, this carrier comprises first nucleotide sequence of coding RSV G albumen or RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction, one is operably connected to the promoter sequence that described first nucleotides sequence lists, so that in described host, express described RSV G albumen, and second nucleotide sequence between described first nucleotide sequence and described promoter sequence, so that in the host, strengthen the proteic expression in vivo of RSV G that is undertaken by described carrier;
B) take described carrier at least one mouse, so that produce at least one mice immunized;
C) in described at least one immune mouse body, take out the B-lymphocyte;
D) will merge from the B-lymphocyte and the myeloma cell of described at least one immune mouse, so that produce hybridoma;
E) clone described hybridoma;
F) screening can produce the clone of anti--RSV G protein antibodies;
G) cultivate described generation anti--clone of RSV G protein antibodies;
H) separate anti--RSV G protein monoclonal antibody.
Described monoclonal antibody can be used for albumen from virus purifying RSV G.
In this application, term " RSV G albumen " is used to limit the RSV G albumen of complete length, described albumen has variation in its aminoacid sequence, comprise the natural G albumen that is present in various RSV strains, the RSV G albumen that lacks the secreted form of striding the film district, and the proteic functional analogue of RSV G.In this application, if first kind of albumen is relevant with second kind of albumen and/or have and second kind of albumen identical function on immunology, described first kind of albumen is second kind proteic " functional analogue ".For example, described functional analogue can be described proteic immunologic competence fragment or its immunologic competence replacement, interpolation or deletion mutantion type.
The accompanying drawing summary
By following general explanation and embodiment, and can further understand the present invention with reference to the accompanying drawings, wherein:
The restriction figure of the proteic gene of Fig. 1 presentation code respiratory syncytial virus (RSV) G.
The nucleotide sequence (sequence 1) of the proteic gene of respiratory syncytial virus G of Fig. 2 presentation code film combining form, and by its coded proteic aminoacid sequence of RSV G (sequence 2);
Fig. 3 presentation code lacks the nucleotide sequence (sequence 3) of the proteic gene of secreted form RSV G of described membrane-spanning domain, and by the proteic aminoacid sequence of RSVG (sequence 4) of its coded brachymemma that lacks described membrane-spanning domain.
Fig. 4 represents the structure of plasmid pXL5, and this plasmid comprises the proteic gene of RSV G of the film combining form of the complete length of encoding, and comprises CMV intron A sequence;
Fig. 5 represents the structure of plasmid pXL6, and this plasmid contains the proteic gene of RSV G that coding lacks the secreted form of described membrane-spanning domain, and comprises CMV intron A sequence, and the nucleotide sequence of the signal peptide of coding human tissue plasmin activator (TPA).
Fig. 6 represents the nucleotide sequence (sequence 5) of plasmid VR-1012;
Fig. 7 represent 5 ' non-translational region and human tissue plasmin activator (TPA) signal peptide nucleotide sequence (sequence 6) and
Fig. 8 is illustrated in RSV stimulates the intravital pneumonocyte factor expression of dna immunization mouse feature afterwards.
The general remark of invention
As indicated above, present invention relates in general to polynucleotides, comprise DNA, carry out immunity, in order to obtain Respiratory Syncytial Virus(RSV) (RSV) is infected in order to obtain protection, and relate to the diagnostic method that uses special nonreplication vector. In the present invention, some recombinant plasmid vectors have been made up, in order to comprise the nucleotide sequence of coding RSV G albumen.
The nucleotides of complete length RSV G gene is (sequence 1) as shown in Figure 2, some structure provided by the present invention comprises the nucleotide sequence of coding complete length RSV G (sequence 2) albumen, and other comprise by disappearance and stride RSV G gene that the nucleotides of film coded sequence and its upstream modifies (referring to Fig. 3, sequence 3), in order to produce RSV G albumen secretion or brachymemma (sequence 4) that lacks described membrane-spanning domain.
The nucleotide sequence of coding RSV G albumen is operably connected on the promoter sequence, so that the coded RSV G albumen of expression in vivo. Described promoter sequence can be immediately early stage cytomegalovirus (CMV) promoters of the mankind, and this promoter is disclosed in the list of references 19. Can use any other common promoter, comprise constitutive promoter, such as sarcoma virus LTRs, and inducible promoter, such as metallothionein promoter, and tissue-specific promoter.
Nonreplication vector provided by the present invention when taking to animal with the immune composition form of pharmaceutically useful supporting agent assembly, can be realized RSV G protein expression in the body, and this point is confirmed by the antibody response in the animal body of having taken described composition. Described antibody can be used for the rsv protein in the test sample in the present invention, as hereinafter further disclosing. Take described nonreplication vector, particularly plasmid pXL5 and pXL6 can produce anti--G antibody, virucidin, after virus stimulates, equilibratory Th1/Th2 replys in lung, and generation can illustrate this point to the prevention effect of rsv infection alive by following examples in Mice Body.
Described recombinant vector can also comprise second nucleotide sequence that is positioned near RSV G encoding histone nucleotide sequence, in order to strengthen the immune protection of RSV G albumen when carrying out expression in vivo in the host. Described humidification can provide by strengthening expression in vivo, for example, transcribes and/or translates by improving mRNA stability, strengthening. This additional sequences is usually located between described promoter sequence and the RSV G albumen coded sequence. This enhancing sequence can comprise immediately early stage cytomegalovirus intron A sequence.
Nonreplication vector provided by the present invention can also comprise an extra nucleotide sequence, and this sequential coding comes from the another kind of antigen of RSV, comes from antigen or at least a immunomodulator of at least a other pathogen, such as cell factor. Described carrier can also be included as the extra nucleotide sequence of chimeric or bicistronic mRNA structure. In addition, can make up respectively the carrier that contains described extra nucleotide sequence, and take to the host simultaneously with nonreplication vector provided by the present invention.
Described nonreplication vector can also comprise the nucleotide sequence of a coding allos virus or eucaryon signal peptide, such as human tissue plasmin activator (TPA) signal peptide, so that the endogenous signal peptide on the RSV G albumen of replacement brachymemma. Described nucleotide sequence can be arranged in the upstream that described carrier is close to RSV G coded sequence.
Can strengthen the immunogenicity of described non-replicating dna vector by in described carrier, inserting immunostimulation CpG sequence.
Those skilled in the art are very clear, and various embodiments of the present invention have a lot of purposes in rsv infection immunity, diagnosis and treatment field. Further non-limiting explanation to described purposes will be provided below.
1. vaccine prepares and purposes.
Can prepare the immune composition that is suitable for use as vaccine by the present invention disclosed RSV G gene and carrier. Described vaccine can cause immune response in animal body, and this replys the generation that comprises anti--RSV G antibody. The immune composition that comprises vaccine that contains described nucleic acid can be prepared into injection with acceptable liquid solution or emulsion on the physiology, is used for taking polynucleotides. Described nucleic acid can be combined with liposome, as (for example being combined into the nucleic acid liposome with lecithin liposome or other liposome well known in the art, disclosed in the WO93/24640, list of references 20) or described nucleic acid can be combined with a kind of adjuvant, to further disclose such as following institute. The lipid physical efficiency that comprises cationic lipid independently and promptly interacts with polyanion such as DNA and RNA, produces liposome/nucleic acid compositions, and said composition can be caught 100% polynucleotides. In addition, described polycation type complex and cell membrane merge, and cause interior conveying of cell of polynucleotides, avoid thus the digestive ferment of described liposome compartment. Disclosed PCT application WO94/27435 has disclosed the science of heredity immune composition that comprises cationic lipid and polynucleotides. It is favourable that use can help the preparation of cellular uptake nucleic acid, such as calcium ion, virus protein and other transfection promoter.
The polynucleotides immune formulation can also be prepared into microcapsules, comprises biodegradable long-acting granular. Therefore, US5,151,264 have disclosed the pelleted substrate of a kind of phosphatide/glycolipid/polysaccharide properties, and this carrier is named as Bio Vecteurs Supra Moleculaires (BVSM). Described granulating carrier is used in one deck in its some layer and carries various molecules with BA.
US5,075,109 has disclosed the encapsulated of antigen trinitrophenyl keyhole chirp hemocyanin in 50: 50 poly-(the DL-lactide is glycolide altogether) and SEB. Also propose other and be used for encapsulated polymer, such as poly-(glycolide), poly-(the DL-lactide is glycolide altogether), copolymerized oxalate, PCL, poly-(lactide is the caproic acid lactone altogether), poly-(carboxylic acid amide esters), poe and poly-(8-hydroxybutyric acid) and polyanhydride.
Disclosed PCT application WO91/06282 has disclosed a kind of delivery vehicles that contains some biological attachment microspheres and antigen. Described microsphere is starch, gel, glucan, collagen or albumin. This delivery vehicles is specially adapted to by the Nasal Mucosa Absorption vaccine. Described delivery vehicles can additionally contain absorption enhancer.
Contain nonreplication vector RSV G gene can with compatible mixed with excipients that can be medicinal. Described excipient can comprise water, salt solution, glucose, glycerine, ethanol and combination thereof. Described immune composition and vaccine can also contain auxiliary substance, such as humidification or emulsifying agent, and the pH buffer, or strengthen the adjuvant of its effect. Immune composition and vaccine can by in subcutaneous, intravenous, the corium or intramuscular injection take by parenteral route, and can the injection before carry out the local anaesthesia preliminary treatment to the injection site. In addition, immune composition produced according to the invention can cause that the mode of immune response prepares and carries at mucomembranous surface. Therefore, can be by such as nasal cavity or oral cavity (in the stomach and intestine) approach described immune composition being used for mucomembranous surface. In addition, other mode of administration comprises that suppository and oral formulations may be desirable. For suppository, binding agent and carrier can comprise, for example PAG or triglycerides. Oral formulations can comprise normally used material, such as pharmaceutical grade asccharin, cellulose and magnesium carbonate.
Described immune formulation and vaccine are taken in the mode of coincideing with formulation, and its consumption can play treatment, prevention and immunization. Dose depends on the object of receiving treatment, and for example, comprises the ability of the synthetic RSV G albumen of individual immune system and antibody thereof, if essential, also depends on to produce cell-mediated immune response. The accurate dosage of the active component that need to take depends on doctor's judgement. But, those skilled in the art can determine easily that suitable dose scope, this scope are about 1 μ g contains RSV G gene to about 2mg carrier. The suitable scheme of taking at first with booster also is variable, but can be included in take for the first time after follow-up taking and then. Described dosage can also depend on route of administration, and changes according to host's size. The vaccine that can only prevent a kind of pathogen is univalent vaccine. The vaccine that contains the antigen-like material of some pathogen is combination-vaccine, and belongs to the present invention. For example, described combination-vaccine contains the various strains that come from various pathogen or same pathogen or the material that comes from the combination of various pathogen.
If take simultaneously described carrier with adjuvant, can obviously improve its immunogenicity, usually use with the solution form of the 0.05-0.1% in the salting liquid that is dissolved in phosphoric acid buffer. Adjuvant can improve the immunogenicity of antigen, but itself immunogene not necessarily. Adjuvant can also work by described antigen being remained near the place of using the position, in order to produce storage effect, promotes antigen slowly to discharge enduringly in described immune cell. Adjuvant can also arrive the antigen storing position with described immune cytotaxis, and stimulates described cell to bring out immune response.
For many years immunostimulant or adjuvant be used to strengthen the host already to the immune response such as vaccine. Therefore, already identified the adjuvant that can strengthen the immune response of antigen. But, some are poisonous for certain in the described adjuvant, and can cause undesirable side effect, make it not be suitable for human and a lot of animals. In fact, only there are aluminium hydroxide and aluminum phosphate (usually being commonly referred to as alum) to be often used as the adjuvant of human and live vaccine.
Multiple exogenous adjuvant and other immunological regulation material can cause the effective immune response for antigen. Comprising with saponin(e and Membrane protein antigen combination producing immunostimulating complex (ISCOMS), pluronic polymer with the mineral oil combination, be present in the deactivation mycobacterium in the mineral oil, Freund's complete adjuvant, bacterial preparation, such as muramyl dipeptide (MDP) and lipopolysaccharides (LPS), and Monophosphoryl lipid A, QS21 and polyphosphazene.
In particular of the present invention, the nonreplication vector of first nucleotide sequence of the described G albumen that comprises the RSV that encodes can use with oriented molecule, in order to make described carrier be oriented to specific cell, comprises described immune cell.
The immunogenicity of described nonreplication vector can be by taking with the DNA carrier of the express cell factor or chemotactic factor (CF) or by being strengthened with bicistronic mRNA or the described molecule of fusion structure form coexpression simultaneously.
Can described nonreplication vector be flowed to the host by several different methods, for example, Tang etc. (list of references 21) have disclosed the DNA gold particulate that will scribble coding BGH (BGH) and have imported mouse skin, cause in this Mice Body, producing anti-BGH antibody, and Feurth etc. (list of references 22) confirm, a kind of jet injector can be used for skin, muscle, fat and the breast tissue of transfection animal alive.
2. immunoassays
RSV G gene of the present invention and carrier can be used as immunogene, so as for the preparation of immunoassays anti--G antibody, comprise enzyme linked immunosorbent assay (ELISA) (ELISA), RIAs and other non-enzyme len antibody are in conjunction with measuring or means known in the art. In ELISA measures, at first take described nonreplication vector to the host, in order to produce the special antibody of RSV G albumen. Described RSV G-specific antibody is fixed on the surface of selection, for example, can be in conjunction with the surface of described antibody, such as the hole of polystyrene microtiter plates. After the antibody of removing not absorption by washing, the nonspecific proteins such as bovine serum albumin(BSA) (BSA) solution can be attached on the surface of described selection, known described non-specific albumen can play the antigen neutralization effect to described specimen. Therefore can seal so the non-specific adsorption site on the described fixed surface, and reduce because antiserum non-specific binding caused background to the described surface.
Then described fixed surface is contacted with sample such as clinical or biologic material, measure in the mode that can induction of immunity complex (antigen/antibody) forms. The method can comprise that the diluent of the salting liquid (PBS) used such as BSA solution, ox gamma Globulin (BGG) and/or phosphoric acid buffer/Tween dilutes described sample. With described sample incubation about 2-4 hour, heated culture temperature was approximately 20-37 ℃ then. After incubation, the surface that washing sample is contacted is in order to remove not immune compound material. Described washing methods can comprise the solution washing of using such as PBS/Tween or borate buffer. Specific immune complex between the RSV G specific antibody that forms specimen and combination, and through after the washing, can measure formation or even the quantity of immunocomplex.
Biologic material
Some plasmid of the gene that contains coding RSV G albumen that this paper is related was transferred to American type culture collection (ATCC) preservation according to budapest treaty already before the application's the applying date, the address at preservation center is 12301 Parklawn Drive, Rockville, Maryland, 20852, USA.
Take this U.S. Patent application after the basis is patented, the public can obtain the sample of preservation plasmid, restrictedly all will remove the institute that obtains the preservation thing till that time. If described preservation mechanism can not provide activated sample, can change the sample of described preservation plasmid. This paper disclosed and claimed invention be not limited to plasmid limited range by institute's preservation because the embodiment of preservation only is used as explanation of the present invention. Any same or similar plasmid of the antigen that coding is similar or identical with the disclosed antigen of the application all belongs to scope of the present invention.
| Plasmid | The ATCC preserving number | Preservation date |
| PXL5 | 209143 | On July 16th, 1997 |
| PXL6 | 209144 | On July 16th, 1997 |
Embodiment
More than explanation has disclosed the present invention on the whole.Can more fully understand the present invention by following specific embodiment.Disclosing these embodiment only is for purpose of explanation, rather than will limit scope of the present invention.The change of form and the replacement of equivalent are regarded as that the present invention hints or conspicuous scheme.Although used concrete term herein, these terms are used for illustration purpose, rather than are used to limit purpose.
Employed molecular genetics, protein biochemistry and immunological method clearly do not disclose in this manual, and this class example is reported in the scientific literature in a large number, and belongs to those skilled in the art's general technical ability.
Example 1
Present embodiment has disclosed the structure of the carrier that contains RSV G gene.
The restriction figure of the proteic gene of G of Fig. 1 presentation code respiratory syncytial virus, and the aminoacid sequence (sequence 2) of the nucleotide sequence (sequence 1) of the proteic gene of Fig. 2 presentation code complete length RSV G and supposition.Proteic gene of Fig. 3 presentation code excretory RSV G (sequence 3) and the aminoacid sequence of inferring (sequence 4).
RSV G albumen for The expressed length prepares plasmid pXL5 (Fig. 4) as follows:
The reorganization Bluescript plasmid (RSV G12) that will contain the proteic cDNA of G of the coding clinical RSV isolate of complete length (A subgroup) is used to make up RSV DNA-G immune carrier.With AflIII and EcoRI digestion RSV G12, and mend flat with the Klenow fragment of archaeal dna polymerase.The resulting 1.23kb fragment that contains the proteic encoding sequence of described complete length G is carried out gel-purified, and be connected in use in advance the linearizing VR-1012 of EcoRV (Vical) (Fig. 6) on.This process places the downstream of the intron A sequence of early stage immediately cytomegalovirus (CMV) promotor and human cytomegalic inclusion disease virus (CMV) with RSV G cDNA, and is positioned at upstream, Trobest (BGH) poly-A site.Confirm that by sequencing analysis described cDNA fragment is bonded in the described plasmid structure.Resulting plasmid is named as pXL5.
For expression-secretion RSV G albumen, prepare plasmid pXL6 (Fig. 5) as follows:
With EcoRI digestion RSV G12, put down with the Klenow benefit, and digest once more with BamHI.Described BamHI cracking meeting causes the segmental generation of the proteic cDNA of coding RSV G of the terminal brachymemma of N-.This dna fragmentation is carried out gel-purified, and be connected under the condition that has a pair of 11 aggressiveness oligodeoxynucleotide (5 ' GATCCAATCAG3 ') (sequence 7) (3 ' GTGAGTCCTAG5 ') (sequence 8) on the VR-1020 (Vical) that digested with BglII in advance, mend flat with Klenow, digest once more with BamHI, and carry out gel-purified.This process places immediately the downstream of the intron A sequence of CMV promotor and people CMV and the upstream in BGH polyA site in early days with the RSV GcDNA (coding region that lacks terminal 91 amino-acid residues of N-that comprise membrane-spanning domain) of brachymemma.In addition, imported the 5 ' non-translational region of about 100bp and the encoding sequence of the proteic signal peptide of the former activator of human plasmin (Fig. 7), this sequence frame endomixis is at the N-end of the RSV G albumen coded sequence that is positioned at CMV promotor/intron A sequence downstream.Confirmed that by sequencing analysis described cDNA fragment is combined on the described plasmid structure.Resulting plasmid is named as pXL6.
Example 2
This embodiment has disclosed the immunity of mouse.Disclosed in reference 24, mouse susceptible RSV.
By twice cesium chloride centrifugation plasmid DNA purification.In order to carry out intramuscular (i.m.) immunity, (male from both sides to BALB/c mouse with pXL5, pXL6 or the V-1012 of 2X50 μ g (1 μ g/ μ l is dissolved among the PBS), age in 6-8 week) (Jackson Lab., BarHarbor, ME, shin bone front side muscle USA) is injected.Handled described muscle with the digoxigenin (Latoxan, France) of 2 * 50 μ l (10 μ M are dissolved among the PBS) in 5 days before injection DNA, DNA absorbs and enhancing immunity is replied so that strengthen, as disclosed (reference 23) such as Davis.After 6 weeks and 13 weeks, described animal is carried out reinforced immunological respectively with the plasmid DNA of same dose.In order to carry out intradermal (i.d.) immunity, the plasmid DNA (2 μ g/ μ l are dissolved among the PBS) of 100 μ g is expelled to its tail root place, and respectively at 6 weeks and 13 weeks reinforced immunologicals afterwards.With the clinical RSV strain of the A2 blood subgroup that is grown in 106 plaque-forming units (pfu) in the Hep2 cell mouse in the positive controls is carried out immunity by mode in the nose (i.n.), described bacterial strain is so kind as to give (reference 24) by doctor B.Eraham.
After immune 4 weeks for the third time, by the RSV A2 bacterial strain stimulation mouse of approach in the nose with 106pfu.After 4 days, under aseptic condition, take out lung, weigh, and homogenate in the perfect medium of 2ml (reference 25).By the quantity of the method (reference 26) that disclosed in the past with the pfu in the vaccine quality Vero cell replication lung homogenate thing.
Example 3
This embodiment has disclosed the immunogenicity and the provide protection of polynucleotide immunity.
With specific enzymes linked immunosorbent assay (ELISA) analysis sero-fast anti--RSV G IgG antibody titer, and analyze the special plaque reduction of RSV-and tire available from immune mouse.ELISAs uses 96 hole flat boards of the immune serum that scribbles immunoaffinity purification RSV G albumen (50ng/mL) and 2 times of serial dilutions to carry out.To be used as second antibody with the goat anti-mouse IgG antibody that alkaline phosphatase (JacksonImmunoRes., Mississauga, Ontario, Canada) puts together.Reduce with vaccine quality Vero raji cell assay Raji plaque according to the method (reference 26) of Prince etc. and to tire.The RSV Long strain (ATCC) with 50pfu of 4 times of serial dilutions of immune serum is placed in the substratum, under the condition that 5% carbonic acid gas is arranged, cultivated 1 hour down, and this mixture is used for vero cells infection at 37 ℃.Methyl alcohol with 80% is plaque fixedly, and uses the mouse anti RSV F mono-clonal IgG1 antibody and the anti-mouse IgG antibody colour developing of donkey of puting together with peroxidase (JacksonImmunoRes., Mississauga, Ontario, Canada) after 5 days.The special plaque reduction of described RSV-is tired and is defined as causing plaque quantity to reduce by 60% serum samples diluted degree.ELISA and plaque reduce mensuration and all repeat, and given data are the mean value of twice mensuration.
Shown in the result who the is obtained Table I and II below:
Table I .DNA-G is in the intravital immunogenicity of BALB/c mouse
| Immunogene is anti--RSV G IgG tire RSV specificity plaque tire (Log2 (tire/100) reduce 10 (Log2 tires) 17 weeks in all 17 weeks of 6 weeks |
| VR-1012(i.m.) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 pXL5(i.m.) 3.10±2.77 9.70±1.06 8.60±1.17 5.40±1.65 pXL6(i.m.) 5.78±1.20 9.30±0.82 8.89±1.54 7.26±0.82 pXL5(i.d.) 1.50±1.27 8.60±1.43 8.30±1.25 7.92±0.59 pXL6(i.d.) 3.70±1.25 10.30±1.06 9.44±1.24 6.92±0.94 RSV(i.n.) 6.83±0.41 9.67±0.52 9.83±0.4 1 11.80±0.08 |
The immune protection of Table II .DNA-G in BALB/c mouse
The average viral lung of the immunogen number of mice * that tires adequately protects
The number of mice # of (pfu/g lung)
(Log10±SD)
VR-1012(i.m.) 6 4.81±0.01 0
pXL5(i.m.) 6 0.29±0.90 5
pXL6(i.m.) 6 0.40±1.20 5
pXL5(i.d.) 6 0.30±1.10 5
pXL6(i.d.) 6 0.29±0.90 5
RSV(i.n.) 6 0.00±0.00 6
* the sensitivity of Ce Dinging: 10
1.96The pfu/g lung
The mouse that the # term adequately protects is meant after virus stimulates 4 days, animal that can not detected RSV in lung.
As shown in Table I, after by i.m. or i.d. immunity, plasmid pXL5 and pXL6 are the immunogens that produces anti--G antibody and virucidin.In addition, as shown in Table II, plasmid pXL5 and pXL6 can protect the lower respiratory tract of the immune mouse that crosses not to be subjected to initial rsv infection.Control vector can not produce immunne response, and provide protection can not be provided.
Example 4
This embodiment has disclosed determining with local pulmonary cytokine-expressing feature in pXL5 and the immune mouse that crosses of pXL6 after stimulating with RSV.
In the 0th week and the pXL5 and the immune BALB/c mouse of pXL6 that prepared according to the method for example 1 in the 6th week, and stimulate with RSV by i.n. in the 10th week with 100 μ g.Control animal, and stimulates with RSV with the blank PI-RSV and the RSV immunity of living with identical method.In addition, according to the applying date be the pending U.S. Patent Application 08/476,397 in June 7 nineteen ninety-five, the method that discloses in (WO96/40945) is used the pXL2 immune animal, and stimulates with RSV by the same way.After virus stimulates 4 days, lung is taken out in the mouse body of immunity, and at once freezing in liquid nitrogen.Homogenate lung in the TRIzol/ beta-mercaptoethanol prepares total RNA by chloroform extraction and isopropanol precipitating.Use IL-4, IL-5 or IFN-γ special primer that described RNA sample is carried out reversed transcriptive enzyme polymerase chain reaction (RT-PCR) then available from CloneTech.Then with the product of amplification with available from the cytokine specificity of CloneTech
32The probe that the P-mark is crossed carries out liquid hybridization, separates on 5% polyacrylamide gel, and is undertaken quantitatively by the radiated signal that scans in the described gel.From each treatment group, take out 3 mouse lungs, and analyze the pneumonocyte factor expression, carry out at least 2 times.The result is shown in Figure 8, and, show the mean value and the standard error of described mensuration in the drawings.
The data that provide from Fig. 8 are as can be seen:
1. carry out immunity meeting equilibratory cytokine curve (IFN-γ, IL-4 and IL-5) with the RSV that lives by nasal cavity (i.n.) method, can produce Th2 advantage (higher IL-4 and IL-5) and carry out immunity by intramuscular (i.m.) method with FI-RSA.Above result is similar to the result who reports in relevant document.
2. can produce the equilibrated cytokine curve of the RSV immunity of similar work with pXL5 or pXL6 immunity by i.m. or intradermal (i.d.) method.
3. carry out the intensity of pXL6 (RSV G) and the immune cytokine response that is produced of pXL2 (RSV F) by i.m. apparently higher than immune intensity with the structure of energy expression-secretion type albumen (SEC) with the RSV immunity generation of living.
With can The expressed the film of length carry out the intensity of the cytokine response that pXL5 immunity and i.d.pXL6 produced a little more than immune intensity in conjunction with the structure of RSV G albumen (MA) with the RSV immunity generation of living.
5. with the result's different (reference 13) who is reported by Openshaw etc., observed the equilibrated local cells factor with the DNA-G immunity and replied.Described investigator uses the proteic recombinant vaccine virus of expression G to report local T h2 by the bronchoalveolar lavage analysis and replys.Result of the present invention obtains by the single-gene method, shows that Th2 replys the not necessarily proteic internal characteristics of G.
Specification is summed up
As the summary of this specification, the invention provides the novel nonreplication vector of the gene that contains coding RSV G albumen, with the method for described carrier immunity, and the method for diagnosing with described carrier. It is possible improving within the scope of the present invention.
Reference
1.McIntosh K., Canock, R.M.In:Fields B.N, Knipe, DM writes. virusology. New York: Raven press: 1990:1045-1072
2.Heilman, C.A., transmissible disease magazine .1990,161:402 to 406.
3.Wertz GW, Sullender WM., biotechnology 1992; 20:151-176
4.Murphy, B.R. etc., 1994, virus research .32:13-36.
5.Levine, S., Kleiber-France, R., and Paradiso, the hereditary Journal of Virology .69 of P.R. (1987), 2521-2524.
6.Anderson, L.J., Hierholzer, J.C., Tsou, C., Hendry, R.M., Fernie, B.F., Stone, Y. and McIntoah, K. (1985) transmissible disease magazine .151,626-633.
7.Johnson etc., Journal of Virology 1987,61:3163-3166
8.Crowe, J.E., vaccine 1995,13:415-421
9.Kapikian A.Z. etc. 1969, U.S. epidemiology magazine .89:422-434.
10.Kim H.W. waits 1969 U.S. epidemiology magazine .89:422-434.
11.Murphy, 1986 clinical microbiology magazine .24:197-202. such as B.R.
12.Vaux-Peretz, 1992 vaccine 10:113-118. such as F.
13.Openshaw, P.J.1995 Springer-Semine immunopathology .17:187-201.
14.Alwan Deng 1994 The Journal of Experimental Medicine .179:81-89.
15.Graham, B.S.1995 U.S. respiratory tract clinical care medical journal 152:563-6
16.WO90/11092
17.WO94/21797
18.Ulmer，Current Opinion，Invest Drugs，1993，2：983-989
19.Chapman, B.S., Thayer, R.M., Vincent, K.A. and Haigwood, N.L., nucleic acids research .1991,19:3979-3986.
20.Nabel, G.J.1993, the periodical 90:11307-11311. of institute of NAS
21.Tang etc., nature 1992,356:152-154
22.Furth etc., analytical biochemistry, 1992,205:365-368
23.Davis etc., vaccine 1994,12:1503-1509
24.Graham, B.S.; Perkins M.D.; Wright, P.F. and Karzon, the modern Journal of Virology .1988 of D.T. 26:153-162.
25.Du R.P etc. 1994., biotechnology 12:813-818.
26.Prince, G.A. etc., 1978. American Journal of Pathology 93:771-790.
Claims (23)
1. one kind is used for using so that generation is at the immune composition of the proteic protection antibody of respiratory syncytial virus G in this host to host is for oral administration, and the equilibratory in vivo Th1/Th2 cytokine of described composition curve, it is characterized in that:
A kind of after importing the host who will protect the not carrier and the pharmaceutically useful supporting agent of reproducible, this carrier comprises:
First nucleotide sequence of encode a RSV G albumen or a RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction,
One is operably connected to the promoter sequence that described first nucleotides sequence lists, so as in described host, to express described RSV G albumen and
Second nucleotide sequence between described first nucleotide sequence and described promoter sequence is so that strengthen the proteic expression in vivo of RSV G that described carrier carries out in described host.
2. composition as claimed in claim 1 is characterized in that the RSV G albumen of the described first nucleotide sequence coded complete length or coding have lacked the RSVG albumen of striding film coding region and its upstream sequence.
3. composition as claimed in claim 2 is characterized in that described first nucleotide sequence comprises that nucleotide sequence shown in Figure 2 or coding have the proteic nucleotide sequence of complete length RSV G of aminoacid sequence shown in Figure 2.
4. composition as claimed in claim 2 is characterized in that described first nucleotide sequence comprises that nucleotide sequence shown in Figure 3 or coding have the RSV G albumen of the brachymemma of aminoacid sequence shown in Figure 3.
5. as each composition among the claim 1-4, it is characterized in that described nonreplication vector also comprises an allos signal peptide coding nucleotide sequence near described first nucleotide sequence 5 ' terminal upstream.
6. composition as claimed in claim 5 is characterized in that the signal peptide of described signal coding sequence coding human tissue plasmin activator.
7. as each composition among the claim 1-4, it is characterized in that described promoter sequence is early stage immediately cytomegalovirus promoter.
8. as each composition among the claim 1-4, it is characterized in that described second nucleotide sequence is the human cytomegalic inclusion disease virus intron A.
9. as each composition among the claim 1-4, it is characterized in that described nonreplication vector is a plasmid vector.
10. composition as claimed in claim 9 is characterized in that described plasmid vector is pXL5 or pXL6.
11. a carrier, reproducible not after importing the host that will protect as the medicine that the disease that is caused by respiratory syncytial virus infection is carried out immunity, the equilibratory in vivo Th1/Th2 cytokine of described medicine curve is characterized in that:
First nucleotide sequence of encode a RSV G albumen or a RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction,
One is operably connected to the promoter sequence that described first nucleotides sequence lists, so that express described RSV G albumen in described host.
12., it is characterized in that the RSV G albumen of the described first nucleotide sequence coded complete length or coding have lacked the RSVG albumen of striding film coding region and its upstream sequence as the carrier of claim 11.
13., it is characterized in that described nucleotide sequence comprises nucleotide sequence shown in Figure 2 or encodes the proteic nucleotide sequence of complete length RSV G shown in Figure 2 as the carrier of claim 12.
14., it is characterized in that described first nucleotide sequence comprises that nucleotide sequence shown in Figure 3 or coding have the proteic nucleotide sequence of RSV G of the brachymemma of aminoacid sequence shown in Figure 3 as the carrier of claim 12.
15., it is characterized in that described nonreplication vector also comprises an allos signal peptide coding nucleotide sequence near 5 ' terminal upstream of described first nucleotide sequence as each carrier among the claim 11-14.
16., it is characterized in that the signal peptide of described signal coding sequence coding human tissue plasmin activator as the carrier of claim 15.
17., it is characterized in that described promoter sequence is early stage immediately cytomegalovirus promoter as each carrier among the claim 11-14.
18. as each carrier among the claim 11-14, also comprise second nucleotide sequence between described first nucleotide sequence and described promoter sequence, so that in described host, strengthen the proteic expression in vivo of RSV G that is undertaken by described carrier.
19., it is characterized in that described second nucleotide sequence is the human cytomegalic inclusion disease virus intron A as the carrier of claim 18.
20., it is characterized in that described carrier is a plasmid vector as each carrier among the claim 11-14.
21., it is characterized in that described plasmid vector is pXL5 or pXL6 as the carrier of claim 20.
22. a production is used to protect the host to avoid because the method for the vaccine of the disease that respiratory syncytial virus infection causes is characterized in that:
First nucleotide sequence that separates coding RSV G albumen or RSV G protein fragments, described protein fragments can produce the antibody with RSV G albumen specific reaction,
Described first nucleotide sequence is operably connected at least one control sequence; so that produce the carrier that does not duplicate after the host that a kind of importing will protect; described control sequence can instruct the proteic expression of described RSV G after importing the host; so that produce at the proteic immunne response of RSV G
Described first nucleotide sequence is operably connected to second nucleotides sequence lists, so as in described host, to strengthen the proteic expression of described RSV G undertaken by described carrier and
Described preparing carriers is become to be used for to the vaccine of taking in the host.
23., it is characterized in that described carrier is selected from following one group: pXL5 and pXL6 as the method for claim 22.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US89644297A | 1997-07-18 | 1997-07-18 | |
| US08/896,442 | 1997-07-18 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1271389A CN1271389A (en) | 2000-10-25 |
| CN1174099C true CN1174099C (en) | 2004-11-03 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB988093014A Expired - Fee Related CN1174099C (en) | 1997-07-18 | 1998-07-16 | Nucleic acid vaccines encoding G protein of respiratory syncytial virus |
Country Status (8)
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| EP (1) | EP0996730A1 (en) |
| JP (1) | JP2001512662A (en) |
| CN (1) | CN1174099C (en) |
| AU (1) | AU756222B2 (en) |
| BR (1) | BR9815496A (en) |
| CA (1) | CA2296089A1 (en) |
| NZ (1) | NZ502626A (en) |
| WO (1) | WO1999004010A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6083925A (en) * | 1995-06-07 | 2000-07-04 | Connaught Laboratories Limited | Nucleic acid respiratory syncytial virus vaccines |
| FR2790959B1 (en) * | 1999-03-15 | 2003-06-27 | Pf Medicament | USE OF BACTERIAL MEMBRANARY FRACTIONS WITH ADJUVANT EFFECT, THEIR PREPARATION METHODS AND PHARMACEUTICAL COMPOSITION CONTAINING THEM |
| FR2798857B1 (en) * | 1999-09-23 | 2003-06-06 | Pf Medicament | USE OF AN OMPA MEMBRANE PROTEIN OF ENTEROBACTERIA ASSOCIATED WITH AN RSV IMMUNOGENIC PEPTIDE FOR THE PREPARATION OF NASAL ADMINISTRATIVE VACCINES |
| ATE413190T1 (en) * | 1999-12-01 | 2008-11-15 | Novartis Vaccines & Diagnostic | PRODUCING ANTIBODIES SPECIFIC TO HEPATITIS C VIRUS (HCV) |
| GB0700133D0 (en) | 2007-01-04 | 2007-02-14 | Humabs Llc | Human cytomegalovirus neutralising antibodies and use thereof |
| US7947274B2 (en) | 2007-01-04 | 2011-05-24 | Humabs, LLC. | Human cytomegalovirus neutralising antibodies and use thereof |
| KR100938105B1 (en) * | 2007-09-21 | 2010-01-21 | 이화여자대학교 산학협력단 | Respiratory Syncytial Virus Vaccine |
| MX343490B (en) | 2008-07-16 | 2016-11-08 | Inst For Res In Biomedicine | Human cytomegalovirus neutralizing antibodies and use thereof. |
| WO2012053856A2 (en) | 2010-10-21 | 2012-04-26 | 이화여자대학교 산학협력단 | Respiratory syncytial virus vaccine composition and method for preparing same |
| CN111303278B (en) * | 2013-04-15 | 2022-09-06 | 扬森疫苗与预防公司 | Human antibodies binding to RSV G protein |
| AU2020329654A1 (en) | 2019-08-12 | 2022-03-10 | Advaccine (Suzhou) Biopharmaceuticals Co. Ltd. | Immune composition comprising respiratory syncytial virus (RSV) G polypeptide |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5620896A (en) * | 1992-03-23 | 1997-04-15 | University Of Massachusetts Medical Center | DNA vaccines against rotavirus infections |
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1998
- 1998-07-16 CA CA002296089A patent/CA2296089A1/en not_active Abandoned
- 1998-07-16 AU AU84278/98A patent/AU756222B2/en not_active Ceased
- 1998-07-16 EP EP98934710A patent/EP0996730A1/en not_active Withdrawn
- 1998-07-16 WO PCT/CA1998/000697 patent/WO1999004010A1/en not_active Application Discontinuation
- 1998-07-16 JP JP2000503216A patent/JP2001512662A/en not_active Ceased
- 1998-07-16 CN CNB988093014A patent/CN1174099C/en not_active Expired - Fee Related
- 1998-07-16 NZ NZ502626A patent/NZ502626A/en not_active IP Right Cessation
- 1998-07-16 BR BR9815496-6A patent/BR9815496A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0996730A1 (en) | 2000-05-03 |
| NZ502626A (en) | 2002-09-27 |
| WO1999004010A1 (en) | 1999-01-28 |
| AU756222B2 (en) | 2003-01-09 |
| CN1271389A (en) | 2000-10-25 |
| BR9815496A (en) | 2001-12-18 |
| CA2296089A1 (en) | 1999-01-28 |
| JP2001512662A (en) | 2001-08-28 |
| AU8427898A (en) | 1999-02-10 |
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