CN101495139A - Differentiating therapeutic composition - Google Patents

Differentiating therapeutic composition Download PDF

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Publication number
CN101495139A
CN101495139A CN 200680050074 CN200680050074A CN101495139A CN 101495139 A CN101495139 A CN 101495139A CN 200680050074 CN200680050074 CN 200680050074 CN 200680050074 A CN200680050074 A CN 200680050074A CN 101495139 A CN101495139 A CN 101495139A
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experimenter
antigen
infect
labelling
virus
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T·M·埃利斯
S·G·芬威克
C·M·詹姆斯
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DIVA SOLUTIONS Pty Ltd
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DIVA SOLUTIONS Pty Ltd
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Priority claimed from AU2005906282A external-priority patent/AU2005906282A0/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

The present invention provides a method of tagging a subject to which at least a first antigen has been administered or will be administered, wherein the antigen is derived from an agent capable of infecting the subject and is able to either generate or assist in generating a therapeutic effect in the subject, the method comprising the step of: administering to the subject a marker or reporter that generates a detectable signal in the subject and is capable of identifying the subject as having received the first antigen.

Description

Differentiate therapeutic combination
Technical field
The present invention relates to a kind of discriminating therapeutic combination (differentiating therapeuticcomposition) and be used to detect that immunity is crossed and the experimenter's that crosses of immunity purposes.More specifically, the invention provides a kind of vaccine that is used for immune experimenter, described vaccine provides a kind of labelling that immunity had taken place that is used to indicate.The invention still further relates to a kind of with the vaccine immunity experimenter so that can detect the immunity method.
Background technology
The program of using vaccination to prevent and treat virus outbreak and infection must have a kind of system that can effectively monitor the lasting viral infection that exists in the population.Yet vaccination makes monitors the complexity that becomes by serological method on a large scale to transmission of infection, because vaccinated experimenter all can produce specificity at this viral antibody with the experimenter who contacts virus.The infectious street strain of virus and the antigen similarity between the viral vaccine when particularly using inactivation of viruses as vaccine (if) have hindered distinguishing experimenter who infects and vaccinated experimenter, because the vaccination meeting causes antibody to produce and continue existing, described antibody can't be distinguished in experimenter who infects and vaccinated experimenter.
(for example there is multiple viral disease, foot and mouth disease, bird flu (AI), Newcastle, west Nile virus and feline immunodeficiency virus (FIV)), wherein owing to the experimenter that can't distinguish infection and vaccinated experimenter have hindered monitoring to virus outbreak and propagation.At present, in many countries and regions (for example China, Indonesia, Vietnam and Hong Kong), utilize the H5 avian influenza vaccine to realize the control that bird flu is propagated gradually according to government's program of preventing and treating.
The whole world increases gradually to the concern of DIVA (can differentiate infection with vaccinated animal) vaccination strategy.For example, recommended to use DIVA to implement all vaccination, can monitor the propagation of infection like this about the WHO/FAO/OIE joint conference of bird flu strain H5N1HPAI.Yet as mentioned below, present DIVA method still is difficult to the expansion scale, and is difficult to differentiate the infection of vaccination and other epidemic virus strains usually.
Present monitoring method comprises to be carried out body marker, uses the sentry animal, virusology is tested and use the recombinant heterologous vaccine vaccinated animal.Yet these existing methods have many limitation.
Vaccinated animal is carried out body marker comprise use physical method (for example ear tag, lower limb hoop or wing mark) the vaccinated experimenter's individuality of identification.Yet, because logistics and these methods of economic cause are difficult to large-scale application.Because logistics and economic cause, not vaccinated sentry animal also is difficult to be used in many affected areas, small-scale high-risk rural domestic animal is arranged, for example group of birds or single domestic animal in the described area.In addition, also the risk of Cun Zaiing is to give human risk if the sentry by described viral infection, has then increased to propagate.
By screening and the virusology test or the RT-PCR supervision test that detect the individuality that live virus carries out be the very expensive and method very high of a kind of price to equipment requirements, this method also is not suitable for many areas, particularly, all very common at these regional numerous diseases (for example bird flu and foot and mouth disease) than the poverty-stricken area.This method also is difficult to the expansion scale.In addition, be used to detect viral virusology test and RT-PCR the information that only can provide about the current infection conditions of individual subjects is provided, and can not analyze this experimenter's infection history and/or vaccination history.
Recently, developed many being called as the recombinant heterologous vaccine of " can differentiate infection with vaccinated animal " vaccine or DIVA vaccine.After carrying out vaccination with this class recombinant vaccine, vaccinated birds can produce the N antibody response different with the birds of natural infection.Use to differentiate then antibody test measure described experimenter by wild-type virus infect still be by described recombinant virus infect (for example, the H5N2 vaccine can be used in the area that the H5N1 infection occurs, test the N1 antibody of birds then, described N1 antibody can be used as the indicator of contact virus; Perhaps can use heterologous vaccine H7N3 to inoculate with antagonism H7N1 disease, so just can by whether existing N3 antibody to discern vaccinated birds).
Yet, being faced with in the infectious area of general H5N1 many, other low pathogenicity bird flu viruss (for example H9N2, H6N1 etc.) can be popular in aquatic bird and poultry.The bird vaccine inoculation (for example H5N2 vaccine) of anti-H5N1 can not make this birds exempt from the infection of low pathogenicity bird flu virus (for example H6N1) subsequently.Therefore, if test, will provide false positive results based on the DIVA antibody test of N hypotype antibody at N1.In addition, some areas are just being used H5N1 vaccine of the same race and xenogenesis H5N2 vaccine simultaneously in their vaccination program, and described vaccination program can make the application of DIVA serology test become complicated.In addition, there are 16 kinds of H types and 9 kinds of N types can merge in the avian influenza virus subtype, therefore need many new tests and vaccine hypotype that described method is expanded to every kind of new virus subtype.
The present invention has satisfied in this area the demand for the new virus vaccine that can differentiate vaccinated and the experimenter that infects, and described new virus vaccine can improve one or more problems that exist in the prior art at least.
General explanation
Those skilled in the art will recognize that the present invention that the application describes can be easy to change and revise, and be not limited to the specifically described embodiment of this paper.The present invention includes all such changes and modifications.The present invention also comprise individually or fully in the description related or illustrated institute in steps, feature, preparation and chemical compound, and wantonly two or more multinomial any and whole combination of described step and feature.
The full text of every piece of file, list of references, patent application or patent that this paper quoted all by reference mode is included this paper clearly in, and this is meant that the reader should read above-mentioned quoted passage and consider as the part of this description.Why the file that this paper quoted, list of references, patent application or patent do not repeat in description only is for simplicity.
The manufacturer's operation instruction of being mentioned in arbitrary file that this paper or this paper quoted for any product, description, the description of product and product Dan Jun mode are by reference included this paper in, and may be used to implement the present invention.
The present invention is not limited to the scope of any specific embodiments as herein described.These embodiments only have been intended to exemplary effect.Obviously, the product of functional equivalent, preparation and method all belong to scope of the present invention as herein described.
The present invention as herein described may comprise one or more numerical rangies (for example size, concentration etc.).Numerical range should be understood to include all numerical value in this scope, the numerical value that comprises the numerical value that limits this scope and contiguous this scope, the numerical value of described this scope of vicinity can produce with the identical or essentially identical result of its next-door neighbour's the numerical value that defines this range limit.
This description in the whole text in, unless in addition requirement in the context, term " comprises " or its synonym " comprises " or " containing " all should be understood to mean and comprise illustrated whole or whole group, but does not get rid of any other whole or whole group.Should also be noted that, in content disclosed herein, particularly in claim and/or each section, term for example " comprises ", " comprising ", " containing " etc. should have united states patent law and give its implication, for example, they can represent " comprising ", " comprising ", " comprising " etc.; Term for example " basically by ... constitute " and " basically by ... composition " have united states patent law and give its implication, for example they can comprise the element of clearly not describing, but get rid of the element that exists in the prior art or can influence basic feature of the present invention or new feature.
Other definition of the term that this paper is selected can and can be used in the whole text referring to specific embodiment part.Except as otherwise noted, every other Science and Technology term used herein all has the implication of persons of ordinary skill in the technical field of the present invention institute common sense.
Summary of the invention
On the one hand, the invention provides a kind of labelling and be given the method that maybe will be given at least a first antigenic experimenter, wherein said antigen derives from the infectious agent that can infect described experimenter, and described antigen can produce in described subject or help to produce therapeutical effect, described method comprises the following steps: to give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the described first antigenic experimenter.
Aspect preferred, the invention provides a kind of labelling and accepted a kind of first antigenic experimenter's method, described first antigen derives from the infectious agent that can infect described experimenter, wherein said antigen can produce or have at least in described subject and help produce therapeutic response, described method comprises the following steps: to give described experimenter a kind of labelled antigen, described labelled antigen is detectable and can differentiating and processing crosses and untreated experimenter that the expression of described labelled antigen is ectogenic to described first antigen in described subject.
On the other hand, the invention provides the method that a kind of labelling has been given a kind of first antigenic experimenter, described first antigen derives from the virus that can infect described experimenter, and described method comprises the following steps:
(a) give described experimenter a kind of labelled antigen, described labelled antigen is not to derive from the biology that can infect described experimenter natively; And
(b) wherein said labelled antigen can cause detectable replying in described subject.
Aspect producing detectable signal for example positive antibody is replied, selected first antigen and labelled antigen should not interfere with each other.
The present invention also provides a kind of and at a kind of infectious agent the experimenter has been carried out immune method, and described method comprises the following steps:
(a) give described experimenter a kind of first antigen, wherein said first antigen can in described subject, produce or help to produce therapeutical effect and
(b) give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the first antigenic experimenter described in the step (a).
More preferably, described at a kind of virus and to the experimenter carry out the immunity method comprise the following steps:
(a) give described experimenter at least a first antigen, described first antigen derives from the virus that can infect described experimenter and can send and pass or produce curative effect;
(b) give described experimenter a kind of labelled antigen simultaneously, described labelled antigen is not to derive from the biology that can infect described experimenter natively; With
(c) wherein said labelled antigen can cause detectable immunne response in described subject.
The present invention also provides a kind of and at a kind of virus the experimenter has been carried out immune method, described method comprises the following steps: to give described experimenter a kind of single dosage form, described single dosage form comprises that (a) derives from first antigen of the virus that can infect described experimenter and (b) be not to derive from the labelled antigen that can infect described experimenter's biology natively, and wherein said labelled antigen can cause detectable immunne response in described subject.
The present invention also provides a kind of pharmaceutically acceptable preparation:
(a) a kind of can in described subject, produce or help to produce therapeutical effect first antigen and
(b) a kind ofly can in described subject, produce detectable signal and can identify labelling or the reporter of having accepted the first antigenic experimenter described in the step (a).
More preferably, described pharmaceutically acceptable preparation comprises:
(a) a kind of first antigen that derives from the virus that can infect described experimenter;
(b) a kind of is not to derive from the labelled antigen that can infect described experimenter's biology natively, and wherein said labelled antigen can cause detectable immunne response in described subject.
The present invention also provides the method for a kind of vaccine of a kind of labelling, described vaccine can resist can infected subjects virus, described method comprises the following steps: a kind of first antigen and a kind of labelling or reporter combination, described first antigen can produce in described subject or help to produce therapeutical effect, and described labelling or reporter can produce detectable signal and can identify in described subject has accepted the described first antigenic experimenter.
More preferably, the method for a kind of vaccine of described labelling comprises the following steps:
(a) described vaccine is contacted with a kind of labelled antigen, described labelled antigen is not to derive from the biology that can infect described experimenter natively; With
(b) wherein said labelled antigen can cause detectable immunne response in described subject.
The method that the present invention also provides a kind of discriminating to use the experimenter that preparation as herein described inoculated, described method comprise the following steps: to measure sample from described experimenter to detect described labelling.
More preferably, described discrimination method comprises the following steps:
(a) measure sample from the experimenter to detect the antibody of anti-described labelled antigen; With
(b) wherein the existence of the antibody of anti-described labelled antigen shows that they are at least by described labelled antigen immunity mistake.
The present invention also provides a kind of test kit that is used for the experimenter is carried out immunity, comprising:
(a) a kind of first antigen that derives from the virus that can infect described experimenter;
(b) a kind of is not to derive from the labelled antigen that can infect described experimenter's biology natively.
The present invention also provides a kind of test kit, comprising: a kind of instrument that is used to detect the antibody of the anti-labelled antigen that is produced, described labelled antigen is not to come from the biology that can infect described experimenter.
With reference to following description, other aspects of the present invention and advantage will be conspicuous for a person skilled in the art, and present invention is described with reference to following illustrative embodiments.
Description of drawings
Fig. 1 shows Equivac TMThe chicken of T inoculation is tetanus toxoid (TT) antibody positive.
The chicken that Fig. 2 shows the TT+Alum inoculation shows the TT antibody positive after vaccination.
The duck that Fig. 3 shows the TT+Alum inoculation shows the TT antibody positive after the increased dosage amount inoculation.
Fig. 4 shows with the positive control of the chicken of taking from inoculation and compares, the TT antibody horizontal from broiler (35 age in days) serum that collect in the slaughterhouse.
Fig. 5 shows with the positive control of the chicken of taking from inoculation and compares, the TT antibody horizontal from meat kind chicken (1.5-2 age) serum that collect in the slaughterhouse.
Fig. 6 shows with the positive control of the chicken of taking from inoculation and compares, the TT antibody horizontal from laying hen (1.5-2 age) serum that collect in the slaughterhouse.
Fig. 7 shows with the positive control of the chicken of taking from inoculation and compares, the TT antibody horizontal in Australian northwestward sentry chicken (surpassing for the 1 age) serum.
Fig. 8 shows the intravital TT level of Hong Kong/China's Mainland chicken (90 age in days).
Fig. 9 shows the TT antibody horizontal of Ke Wunula (Kununurra) duck.
Figure 10 shows the TT antibody horizontal of Jian Dakete (Jandakot) duck.
Figure 11 shows the level that gives chicken TT antibody behind bird flu and the TT vaccine jointly.
The intravital HA of chicken that Figure 12 shows co-inoculation tires.
Intravital TT of duck and AI that Figure 13 shows co-inoculation tire.
Intravital TT of duck and AI that Figure 14 shows co-inoculation tire.
Figure 15 shows the intravital TT level of vaccinated chicken.
After Figure 16 showed co-inoculation TT and AI vaccine, the intravital HI of chicken tired.
The specific embodiment
According to the present invention, the inventor is verified can partly to solve problems of the prior art at least by following method: with the labelling or the reporter marking animals that can be detected in subject, wherein this labelling can produce its signal in the mode that does not rely on described first antigen presentation.By producing signal, can demonstrate the described first antigenic curative effect that is used for the treatment of the experimenter at an easy rate, and not need to change detection architecture basically with in the methods of the invention in the mode that does not rely on described first antigen presentation.In addition, separate, can carry out higher levels of control the effect and the described first antigenic effect of described labelling by the generation of detectable signal that the described first antigenic expression and described labelling are caused.
Labelling experimenter's method
According to the present invention, provide a kind of labelling to be given the method that maybe will be given at least a first antigenic experimenter, wherein said antigen derives from the infectious agent that can infect described experimenter, and described antigen can produce in described subject or help to produce therapeutical effect, described method comprises the following steps: to give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the described first antigenic experimenter.
In a preferred embodiment, the invention provides a kind of labelling and accepted a kind of first antigenic experimenter's method, described first antigen derives from the infectious agent that can infect described experimenter, described antigen can produce or have at least in described subject and help produce therapeutic response, described method comprises the following steps: to give described experimenter a kind of labelled antigen, described labelled antigen is detectable and can differentiating and processing crosses and untreated experimenter that the expression of described labelled antigen is ectogenic to described first antigen in described subject.
In another embodiment, the invention provides the method that a kind of labelling has been given a kind of first antigenic experimenter, described first antigen derives from the virus that can infect described experimenter, and described method comprises the following steps:
(a) give described experimenter a kind of labelled antigen, described labelled antigen is not to derive from the biology that can infect described experimenter natively; And
(b) wherein said labelled antigen can cause detectable replying in described subject.
Described first antigen to its medicable virus can be any can infected subjects and cause the virus of disease.Preferably, described virus is for can infect the virus that includes but not limited to following animal species: human and/or other higher mammals, fish and birds.Described virus may be to be selected from following infective virus: the member of pico+ribonucleic acid+virus section is Aphthovirus (virus that for example causes foot and mouth disease) for example, the member of orthomyxoviridae family is bird flu virus for example, the member of Retroviridae is FIV for example, the member of Paramyxoviridae for example causes the virus of Newcastle, the member of Rhabdoviridae for example causes rabic rabies virus, the virus that the member of Parvoviridae is for example relevant with gastroenteritis and the member of papovaviridae for example cause the human papillomavirus of tumor and wart.In a specific embodiments of the present invention, described virus is bird flu virus.
When selecting described labelled antigen, to test to guarantee that described antigen can not disturb first antigen to cause detectable signal (for example positive antibody is replied) at least.Yet described labelled antigen can promote described first antigen to reply.
Phrase used herein " being not to derive from the antigen that can infect described experimenter's biology natively " typically refers to the antigen that derives from a kind of like this biology, described biology be not extensively observed or discovery meeting infected subjects under the biology of group member.According to this understanding, described antigenic detectability can not be sheltered by the intravital natural infection situation of described experimenter.More specifically, the signal that is produced by described labelled antigen must surpass the intravital any noise of the described group member of experimenter, and described noise is that the congenital infection by described biology is produced.Therefore, described labelled antigen must be able to be detected in the animal body of immunity.Preferably, in the animal population of being treated, because the accidental noise that exists described labelled antigen to cause should be very low.In addition, described labelled antigen should not induced harmful immunne response that will endanger described experimenter.
When selecting described first antigen and described labelled antigen, need act with caution to guarantee that these two kinds of antigens can not cause the ability that positive antibody is replied by interfering with each other its.
Described experimenter is variable, but preferably is selected from mammal, birds and fish.More preferably, described experimenter is a mammal, and for example farm-animals comprises sheep, goat, pig, milch cow, horse, vigone, and house pet is Canis familiaris L. and cat for example, and primates; Human; Birds, for example chicken, goose and duck; And fish.
For example, as the experimenter that used a kind of first antigen inoculation from infective virus and when protecting its further infection that exempts from this virus, can use method of the present invention.Then, can be by coming " labelling " if described experimenter detects the anti-described first antigenic antibody with proof in this subject with the inoculation of described labelled antigen, then these antibody sources are in vaccination but not infect.
Though those of ordinary skill can be recognized described labelled antigen and can be the antigen of the endogenous expression consistent with described first antigen, but in an embodiment preferred of the present invention, with respect to described first antigen, described labelled antigen is exogenous expression's a antigen.
The mode that phrase used herein " expression of described labelled antigen is ectogenic to described first antigen " typically refers to first antigen presentation does not relate to or does not rely on the method that produces labelled antigen.Preferably, described labelled antigen is not expressed to rely on the first antigenic mode.More preferably, use another kind of expression vector to express described labelled antigen.
The present invention also provides a kind of labelling to be given a kind of first antigenic experimenter's method, described first antigen derives from the infectious agent that can infect described experimenter, described antigen can produce therapeutic response in described subject, described method comprises the following steps: to give described experimenter a kind of labelled antigen, described labelled antigen is detectable and can differentiating and processing crosses and untreated experimenter that the expression of described labelled antigen is ectogenic to described first antigen in described subject.
The method according to this invention, described first antigen can produce curative effect to described experimenter usually.Preferably, described first antigen is used to produce protectiveness or neutrality is replied, and this replying may obtain by vaccine, yet can obtain the curative effect of any kind by described first antigen.According to described first antigenic this effect, those of ordinary skill should be understood that described first antigen can comprise and a plurality ofly can produce the antigen part that panimmunity is replied.Described first antigen also can be single antigen.It should be understood, however, that the experimenter can be given many antigens usually, for example two or more antigenic mixture.Consider this point, some vaccines are designed to comprise multiple antigen so that cause the most effective antibody response in subject.
In a highly preferred embodiment of the present invention, described first antigen can derive from and be selected from following source: complete inactivation virus or deactivation virus or a kind of virus former (viral agent), the former modified recombinant that passed through of described virus has lacked its virulence, but is still keeping its natural immune characteristic.Preferably, described first antigen derives from naturally occurring virus.Yet it can also derive from the genetic modification form or the recombinant form of naturally occurring virus.
Preferably, described first antigen can bring out protection antibody in subject, and therefore protects described experimenter to exempt from the infection of this virus.When described virus is bird flu virus, hemagglutinin and neuraminidase and/or can constitute described first antigen from the capsid protein of west Nile virus.Perhaps, described first antigen is selected from inner conservative virus antigen, for example substrate or nucleoprotein.When described virus was bird flu virus, the first antigenic example was the combination of H5 and N2.
The labelling system
Just as described herein, the requirement of described labelling system is that described labelling can be distinguished the animal that crosses that immunity is crossed and not immune.The labelling that can be used in the method for the present invention should be that those of ordinary skills are known.They comprise any can labelling or report animal that existing immunity is crossed and immunity the method for animal.Desirable labelling is harmless and does not have the labelling of overt toxicity that described labelling can easily be detected and make it possible to distinguish fast the population of subjects that immunity is crossed.For example, described labelling can be genetic marker or expressed proteins labelling or immune labeled.
Based on the genetic marker this respect, the feature of this class labelling can obtain by following technology from described labelling system: for example simple repeated sequence (SSR) of restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), arbitrarily primed polymerase chain reaction (AP-PCR), DNA cloning fingerprint (DAF), sequence signature amplification region (SCAR), amplified fragment length polymorphism (AFLP), the microsatellite that is otherwise known as and single nucleotide polymorphism (SNP).For example, referring to Berry, Don, et al., " Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles:Applications to Maize Hybrids and Inbreds ", Genetics, 2002,161:813824 and Berry, Don, et al., " Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles:Applications to Maize InbredLines and Soybean Varieties ", Genetics, 2003,165:331342, the two all by reference mode include this paper in.
Based on protein labeling system this respect, described labelling can be not to be the protein of expressing in described population of subjects usually, still can carry out test biology in described labelling system.For example, described protein can detect by the monoclonal antibody of labelling.Perhaps, described labelling can be the sequence of green, yellow or blue fluorescent protein (being respectively GFP, YFP and BFP) gene.
In a highly preferred embodiment of the present invention, described Mk system is based on immune labeled.Preferably, described immune labeled for deriving from the antigen of tape label of the biology that can not infect described experimenter.For example, the labelled antigen that is used for foot-and-mouth disease vaccine can comprise and derives from the antigen that can not infect the biology of domestic animal natively.Perhaps, described antigen can be synthetical antigen.
Preferably, described labelled antigen derives from tetanus pathogen, for example tetanus toxoid.In this respect, birds is to tetanus toxoid insensitive (toxic dose of birds is 350,000 times of toxic dose of horse).In addition, birds can not infected by Clostridium tetani (Clostridium tetani) or be infected by it, but they can produce antibody response to this antigen really.Perhaps, described labelled antigen can be selected from the another kind of biology that can not infect or infect described species, for example from the diphtheria toxoid of diphtheria corynebacterium (Corynebacterium diphtheriae).
Preferably, described labelled antigen is a kind of known antigens, and described antigen is in large-scale production and be registered and be used to give the mankind and/or animal.Even more preferably, described labelled antigen is applicable to and carries out large-scale production at lower cost.
Preferably, described labelled antigen can not infect that given experimenter belongs to or any member of species, and therefore can be used in the multiple vaccine or be used in can immune multiple experimenter's vaccine in.
Give antigen
The present invention also provides a kind of and at a kind of infectious agent the experimenter has been carried out immune method, and described method comprises the following steps:
(a) give described experimenter a kind of first antigen, wherein said first antigen can in described subject, produce or help to produce therapeutical effect and
(b) give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the first antigenic experimenter described in the step (a).
When being delivered to animal, described first antigen and described labelling or reporter be give jointly or give simultaneously or give continuously with any order.
Preferably, described first antigen and labelled antigen be give jointly or give simultaneously.Therefore, the present invention also provides a kind of and at a kind of virus the experimenter has been carried out immune method, and described method comprises the following steps:
(a) give described experimenter a kind of first antigen that derives from the virus that can infect this experimenter;
(b) give or give simultaneously described experimenter a kind of labelled antigen jointly, described labelled antigen is not to derive from the biology that can infect this experimenter natively, and wherein said labelled antigen can cause detectable immunne response in described subject.
For the present invention, be meant simultaneously basically and give at one time or within 24 hours.
When described antigen is administration simultaneously, preferably give described antigen with single dosage form.
The present invention also provides a kind of and at a kind of infectious agent the experimenter has been carried out immune method, and described method comprises the following steps:
(a) give described experimenter a kind of first antigen, wherein said first antigen can produce in described subject or help to produce therapeutical effect; With
(b) give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the first antigenic experimenter described in the step (a).
More preferably, described at a kind of virus and to the experimenter carry out the immunity method comprise the following steps:
(a) give described experimenter at least a first antigen, described first antigen derives from the virus that can infect described experimenter and can send or produce curative effect;
(b) give described experimenter a kind of labelled antigen simultaneously, described labelled antigen is not to derive from the biology that can infect this experimenter natively, and wherein said labelled antigen can cause detectable immunne response in described subject.
The present invention also provides a kind of and at a kind of virus the experimenter has been carried out immune method, described method comprises the following steps: to give described experimenter a kind of single dosage form, described single dosage form comprise (a) a kind of derive from first antigen of the virus that can infect described experimenter and (b) a kind of be not to derive from the labelled antigen that can infect described experimenter's biology natively, wherein said labelled antigen can cause detectable immunne response in described subject.
Described single dosage form can comprise a kind of described first antigenic preparation and a kind of mixture or other compositionss that contains the preparation of described labelled antigen of containing.Perhaps, described single dosage form can comprise a kind of preparation that contains described first antigen and described labelled antigen.
According to the present invention, the recombinant precursor that comprises described first antigen or described labelled antigen can following form give the experimenter: for example deactivation is viral or the avirulence virus relevant with described infective virus.This class deactivation virus or avirulence virus are bred in described vaccinated subject and can be provided lasting antigenic stimulus by expressing described first antigen and labelled antigen in one period.Expressing the virus of the recombinant precursor of described first antigen and labelled antigen can also be handled further so that the whole virus vaccine of deactivation to be provided.
Described first antigen and/or labelled antigen can also be the dna vaccination form.Therefore, the form that described first antigen and/or described labelled antigen can naked DNAs is sent and is passed to carry out vaccination.Preferably, described dna vaccination comprises the recombinant precursor that contains described first antigen or described labelled antigen.Described dna vaccination construct also can comprise plasmid DNA.
Perhaps, the form that described first antigen and labelled antigen can two or more separate dosage forms gives simultaneously.In this embodiment of the present invention, described experimenter accepts a plurality of dosage forms simultaneously.
The selection of first antigen and labelled antigen and their administering mode are very important, because these antigens should not disturb each other effect when administration.Preferably, described first antigen and labelled antigen combined effect or synergism, the another kind of antigen that promptly wherein at least a antigenic effect can be existed strengthens.Most preferably, described labelled antigen can strengthen vaccinated experimenter to described first antigenic the replying, and also can cause the detectable immunne response at himself simultaneously.
Preferably, described antigen is sent with single dose, but can send this antigen by multiple dose in one period.When needs are kept immune state at this infective virus, can give multidose.Usually, when the needs multiple dosing, the vaccination scheme can comprise the vaccine of initial dose, and the interval with 2-4 week gives 1-4 booster shot more then.After first vaccination, the experimenter can accept 1 booster shot in about 4 weeks.
Preparation/route of administration/dosage
It is a kind of pharmaceutically acceptable that the present invention also provides SystemAgent:
(a) a kind of can in described subject, produce or help to produce therapeutical effect first antigen and
(b) a kind of labelling or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the first antigenic experimenter described in the step (a).
More preferably, described pharmaceutically acceptable preparation comprises:
(a) a kind of first antigen that derives from the virus that can infect described experimenter;
(b) a kind of is not to derive from the labelled antigen that can infect described experimenter's biology natively, and wherein said labelled antigen can cause detectable immunne response in described subject.
Preferably, described preparation is for giving the vaccine of described experimenter's protective immunity.Even more preferably, described preparation has curative properties, and promptly it can be given to by the experimenter of described viral infection and described experimenter's health is improved.
Described first antigen and/or labelled antigen can be to put together or be connected on another kind of peptide or the polysaccharide.For example, can use immunogenic protein well known in the art.Useful immunogenic protein comprises keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, human serum albumin (HAS), people's gamma Globulin, chicken immune Lysozyme and cattle gamma Globulin.Useful immunogenicity polysaccharide comprises A family streptococcus bacterium wall polysaccharide (streptococcal polysaccharide), the streptococcic C polysaccharide of B family or streptococcus pneumoniae (Streptococcus pneumoniae) or the streptococcic capsular polysaccharide of B family.
Preparation of the present invention can any suitable way give, and concrete approach depends primarily on the antigenic manner of formulation that is used to send.Therefore, described antigen can be with parenteral mode administration in appropriate carriers, is generally hypodermically (SC), (IM), intravenous ground (IV), intraperitoneal ground (IP) or Intradermal ground (ID) intramuscularly.Yet other administration model also is an acceptable, for example oral, nasal delivery there or send through suppository.Perhaps, the application's preparation can be delivered in the embryonated egg to be used to resist the vaccine of poultry disease by (in ovo) in the direct ovum.
The preparation that is used for parenteral comprises sterile aqueous or non-aqueous solution, suspension or emulsion.The example of non-aqueous solvent is a for example olive oil of propylene glycol, Polyethylene Glycol, vegetable oil, and injectable organic ester ethyl oleate for example.Aqueous carrier comprises water, alcohol/aqueous solution, emulsion or suspension, contains salt or buffer medium.The parenteral carrier comprises sodium chloride solution, Lin Geshi glucose, glucose sodium chloride, Lactated Ringer'S Solution or fixed oil.Intravenous vehicles comprises liquid nutritional supplement, electrolyte replenisher (for example those are based on the supplement of woods Ge Shi glucose) etc.Can also there be antiseptic and other additives, for example antimicrobial, antioxidant, chelating agen and noble gas etc.
For suppository, traditional binding agent and carrier can comprise for example ployalkylene glycol or triglyceride; This class suppository can be made of the active component that described mixture contains 0.5%-10% following mixture, preferably contains the active component of 1%-2%.
The intranasal administration preparation can comprise the carrier that neither can stimulate nasal mucosa also can obviously not upset fibre function.The present invention can use diluent, for example water, saline or other known substances.Described nose preparation also can comprise antiseptic, such as but not limited to chlorobutanol and benzalkonium chloride.Can exist surfactant to strengthen the absorption of nasal mucosa to destination protein.
In addition, described vaccine can also the oral administration administration.Peroral dosage form comprises the excipient of common employing, for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.These compositionss adopt solution, suspension, tablet, pill, capsule, extended release preparation or form of powder, and contain the active component of 10%-95%, preferred 25%-70%.Be used for capsule, tablet and pill that oral administration gives the patient and can have enteric coating, described enteric coating comprises for example acrylic resin " S ", acrylic resin " L ", cellulose acetate, cellulose acetate phthalate or hydroxypropyl emthylcellulose.
Oral dosage form as herein described is at Martin, Remington ' s PharmaceuticalSciences, in 18th Ed. (1990 Mack Publishing Co.Easton PA 18042) the 89th chapter general description is arranged, mode is by reference included it in this paper.Solid dosage forms comprises tablet, capsule, pill, lozenge (troche) or lozenge (lozenge), cachet or sublimed preparation.Can also use liposome or albuminoid to seal to prepare compositions of the present invention (for example U.S. Patent No. 4,925, the proteinoid microsphere of being reported in 673).Can use liposomal encapsulated and come liposome is carried out derivatization (for example U.S. Patent No. 5,013,556) with various polymer.Marshall, in ModernPharmaceutics, Chapter 10, and Banker and Rhodes ed. has provided the description to possible solid therapeutic dosage forms in (1979), and described document mode is by reference included this paper in.Usually, described preparation can comprise inert fraction, and described inert fraction makes can be protected described preparation opposing gastric environment and described bioactive substance is discharged into enteral.
Oral liquid can be for example aqueous or butyrous suspension, solution, gel, emulsion, syrup or elixir form, perhaps the product of preparing again for water before use or other suitable carriers.This class dosage form can comprise conventional additive, for example suspending agent, emulsifying agent, non-aqueous carrier (can comprise edible oil) or antiseptic.Perhaps, described vaccine can be a kind of form of food carriers material component, for example pudding or Yoghurt.Particularly, live vaccine may need to be packaged into a kind of like this form, described form makes and this vaccine can be delivered in people's the digestive tract as a complete virion that described virion can be absorbed by gastral first (being the oral cavity) or other sites (for example intestinal) at least.Described preparation can comprise gelatin, cellulose or various other excipient composition, and perhaps described preparation can or similarly comprise the preparation of the described virus of component form for gel or food carriers (for example pudding).As another embodiment, can comprise flavouring agent, emulsifying agent or other additives in other components of described product formulation, delivery vector or described packaging material.Vaccine of the present invention can be packaged into solution, single dose, paste or gel or be contained in food in the plastic containers or nutrient substance, pillow type packaging (pillow-pack), tearable packing (tear-pack), suction pipe packing or other are applicable to the packing of liquid, gel or food carriers dosage form.
Dosage or effective dose should be enough to prevent, improve or reduce infective virus to experimenter's infection and/or be enough to produce antibody response at described labelled antigen.Those skilled in the art can determine described effective dose at an easy rate.Active component can account for about 95% (w/w) of about 0.01%-of described compositions usually, if perhaps be fit to even can be higher or lower.Preferably, the live vol of described first antigen and described labelled antigen be lower than about 10% (w/w) of described compositions, more preferably less than about 1% (w/w) of described compositions, most preferably be lower than about 0.05% (w/w) of described compositions.
Antigen amount in selected every kind of dosage is for can bring out in typical vaccination subject at the described first antigenic protective immune response with at the immunne response of described labelled antigen, the amount that can not bring out tangible harmful side effect again simultaneously.This amount can change according to employed concrete immunogen.Usually, estimate that every kind of dosage can comprise 0.1-1000 μ g albumen, preferred 0.2-200 μ g albumen.The optimal dose of concrete vaccine can utilize conventional study to determine, described conventional study comprises that observing the intravital antibody titer of experimenter replys with other.
The amount that gives depends on multiple factor, for example considers to carry out the experimenter's of vaccination age, body weight and health.This amount also depends on the ability of experimenter's immuning system synthesising antibody, needed degree of protection and the needed degree of replying to labelled antigen.Those of ordinary skills set up the method for dose response curve and can determine effective dose at an easy rate by carrying out normal experiment.
Formulation additives
Usually, with vaccine SystemInjectable liquid solution or suspension are made in agent (for example in the inventive method used those).Yet, can also be prepared into and be suitable for before injection, being dissolved in or being suspended in the solid form that is mixed with solution or suspension in the liquid.Described preparation can also be emulsive, or is encapsulated in the albumen in the liposome.
At Pharmaceutical Biotechnology, Vol.61 Vaccine Design-thesubunit and adjuvant approach, Powell and Newman write, Plenum Press, 1995 and New Trends and Developments in Vaccines, people such as Voller write, University Park Press, Baltimore, Md. has carried out general description to bacterin preparation among the U.S.A.1978.The technology that is encapsulated in the liposome is described in the U.S. Patent No. 4,235,877 of for example Fullerton to some extent.
Usually with active immne ultimate constituent and pharmaceutically acceptable mixed with excipients that can be compatible with described active component.Suitable excipient is, for example water, saline, glucose, glycerol, ethanol etc., and their combination.
In addition, if necessary, described vaccine can contain a spot of auxiliary agent, for example wetting agent or emulsifying agent, pH buffer agent and/or can strengthen the adjuvant of vaccine potency.
Suitable adjuvant includes but not limited to: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor--muramyl-L-alanyl-D-isoglutamine (CGP11637, nor-MDP is otherwise known as), N-acetyl muramyl-L-alanyl-D-isoglutamine base-L-alanine-2 (1 '-2 '-two palmityls-sn-glycerol-3-hydroxyl phosphorus acyloxy)-ethamine (CGP 19835A, MTP-PE is otherwise known as) and RIBI (contain the composition of three kinds of extractions: monophosphoryl lipid A from antibacterial, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) are dissolved in zamene/Tween 80 emulsion of 2%); Surfactant, for example hexadecylamine, octadecylamine, LYSOLECITHIN SUNLECITHIN A, DDA, N, the two octadecyl-N ' of N--N-two (2-ethoxy-propanediamine), methoxyl group cetyl-glycerol and pluronic polyhydric alcohol; Polyanion (polanion), for example DIVEMA (pyran), dextran sulfate, poly-IC, polyacrylic acid, carbopol; Peptide, for example muramyldipeptide, MPL, dimethylglycine (aimethylglycine), tuftsin, oil emulsion, aluminium potassium sulfate (alum) and their mixture.Other possible adjuvants comprise the B peptide subunit of escherichia coli (E.coli) heat-labile toxin or the B peptide subunit of cholera toxin.McGhee，J.R.，et al.，“On vaccinedevelopment，”Sem.Hematol.，30：3-15(1993)。
Other examples of adjuvant and other reagent comprise aluminium hydroxide, aluminum phosphate, aluminium potassium sulfate (alum), beryllium sulfate, silicon dioxide, Kaolin, carbon, water in oil emulsion, oil in water emulsion, muramyldipeptide, bacterial endotoxin, lipid X, coryne bacterium parvum (Corynebacterium parvum) (propionibacterium acnes (Propionibacterium Acnes), bordetella pertussis (Bordetella pertussis), polyribonucleotide, sodium alginate, lanoline, LYSOLECITHIN SUNLECITHIN A, vitamin A, saponin, liposome, levamisole, the DEAE-glucosan, block copolymer or other synthetic adjuvants.This class adjuvant can be available from various sources, for example the Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or incomplete Freund and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan).
Usually, can use the mixture of for example Amphigen (oil in water emulsion), Alhydrogel (aluminium hydroxide) or Amphigen and Alhydrogel.Only ratify aluminium hydroxide and be used for the mankind.
The ratio of immunogen and adjuvant can change in a bigger scope, as long as the two all exists with effective dose.For example, the amount of aluminium hydroxide is that vaccine mixture is (based on Al 2O 3) 0.5%.Easily, described vaccine is mixed with the immunogenic preparation that to contain final concentration be 0.2-200 μ g/ml, be preferably 5-50 μ g/ml.
After preparation is finished, described vaccine can be mixed in the sterile chamber, closed container and preserve under low temperature (for example 4 ℃) then is perhaps with its lyophilizing.Freeze-drying makes can the stable form Long-term Storage.
Preferably, also can contain carrier in the vaccine combination of the present invention.Described carrier can be oil in water emulsion or aluminum salt, for example aluminum phosphate or aluminium hydroxide.
One particularly preferred aspect, the antigen in the vaccine combination of the present invention can go with adjuvant 3--O-acidylate monophosphoryl lipid A (3D-MPL) and aluminum salt alum combination.When being generally used for human administration, adjuvant QS21 and the 3D-MPL content in vaccine is 1 μ g-200 μ g, 10 μ g-100 μ g for example, preferred every kind of dosage 10 μ g-50 μ g.
If vaccine is given with nontoxic oil in water emulsion form, then described oil in water emulsion preferably contains a kind of nontoxic oil, for example zamene, zamene and/or alpha tocopherol, and emulsifying agent is Tween 80 for example, and aqueous carrier.Described aqueous carrier for example can be phosphate buffered saline.In addition, described oil in water emulsion can contain sorbester p37 and/or lecithin and/or tricaprylin.Usually, described oil in water emulsion can comprise the zamene of 2-10%, the alpha tocopherol of 2-10% and the Tween 80 of 0.3-3%.Preferably, zamene: the ratio of alpha tocopherol is equal to or less than 1, because this can provide a kind of more stable Emulsion.The content of sorbester p37 also can be 1%.In some cases, vaccine of the present invention also advantageously contains stabilizing agent.Describe a kind of especially effectively adjuvant formulation among the WO95/17210, comprised QS21, the 3D-MPL and the tocopherol that are dissolved in the oil in water emulsion.
Therapeutic scheme of the present invention and pharmaceutical preparation can give jointly with other immune response-enhancing agents or biological response modifier, and described immune response-enhancing agents or biological response modifier include but not limited to the immunocyte that cytokine IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF or other cytokines infect.According to this aspect of the invention, preparation of the present invention is by being given with the above-mentioned cytokine of one or more therapeutic activity amounts therapy linked together.Term " cytokine " used herein " be meant any secrete polypeptide that can influence other cell-mediated immune response functions.Therefore, should consider that preparation of the present invention can give so that enhance immunity is replied jointly with cytokine.Preferred cytokine includes but not limited to interleukin-1-α (IL-1-α), interleukin-1-β (IL-1-β), interleukin II (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin-6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interferon-' alpha ' (IFN-α), interferon-beta (IFN-β), interferon-(IFN-γ), tumor necrosis factor (TNF variant), tumor necrosis factor (TNF-β), granulocyte colony-stimulating factor (G-CSF), granulocyte/M-CSF (GM-CSF) and transforming growth factor (TGF-β).
Described first antigen and/labelled antigen can neutral form or salt form make vaccine.Officinal salt comprises acid-addition salts (forming with the free amine group group of proteantigen) and the acid-addition salts that forms with mineral acid (for example hydrochloric acid or phosphoric acid) or organic acid (for example acetic acid, oxalic acid, mandelic acid, tartaric acid and maleic acid).Can also use inorganic base (for example sodium hydroxide, potassium hydroxide, aluminium hydroxide, calcium hydroxide or hydrated ferric oxide .) or organic base (for example 2-aminopropane., trimethylamine, 2-ethylaminoethanol, histamine and procaine) and free carboxy group to form salt.
The method of marker vaccine
As indicated above, by being mixed or make up with a kind of labelled antigen of the present invention, a kind of existing vaccine forms preparation of the present invention.Therefore, the present invention also provides the method for a kind of vaccine of a kind of labelling, described vaccine can resist can infected subjects virus, described method comprise the following steps: with described vaccine with a kind of be not to derive from the labelled antigen that can infect described experimenter's biology natively to contact, wherein said labelled antigen can cause detectable immunne response in described subject.
Preferably, simply described labelled antigen is joined in the vaccine to form the vaccine of tape label.
Carrier, host cell etc.
The present invention also provides a kind of carrier that contains a kind of polynucleotide, described labelled antigen of described polynucleotide encoding or described first antigen.Described carrier can be used to duplicate described nucleic acid in compatible host cell.Therefore, in another embodiment, the invention provides a kind of method for preparing the described labelled antigen of coding or the first antigenic polynucleotide through the following steps: polynucleotide of the present invention are introduced in the replicating vector, described carrier is introduced in the compatible host cell, and under the condition that described carrier is duplicated, made described host cell growth.From host cell, reclaim described carrier.Proper host cell comprises mammal cell line and other eukaryotic cell lines, for example insecticide Sf9 cell.
Preferably, described carrier also comprises control sequence, and described control sequence effectively is connected with described coded sequence and can makes the described coded sequence of host cell expression, and promptly described carrier is an expression vector.Term " effectively connects " relation between the described assembly that is meant can make them work in its expection mode.The regulating and controlling sequence that " effectively is connected " with coded sequence connects in such a way, and described mode is to realize the expression of described coded sequence under the condition compatible with described control sequence.
Can modify described control sequence, for example other transcriptional regulatory elements are modified so that responsive more to transcription regulatory factor by the transcriptional level of control sequence guidance by adding.
As mentioned below, carrier of the present invention can be transformed or transfection in the proper host cell to express albumen of the present invention.This method can comprise: under the condition of the vector expression that can make the coded sequence that contains encoding said proteins, cultivate and use above described expression vector transformed host cells, and randomly reclaim expressed proteins.
Described carrier can be for example plasmid or viral vector, and described plasmid or viral vector contain origin of replication, randomly contain and be useful on the promoter of expressing described polynucleotide, and randomly contain the regulator of promoter.Described carrier can contain one or more optionally marker gene, for example, for bacterial plasmid, contains ampicillin resistance gene, perhaps for the mammal carrier, contains neomycin or kalamycin resistance gene.For example can use carrier to come transfection or transformed host cell.
Comprise promoter/enhancer and other expression conditioning signals with the control sequence that coding proteinic sequence of the present invention effectively is connected.Can select the above-mentioned control sequence compatible, use the expression vector that designs for it in the described host cell with host cell.Term " promoter " is as known in the art and contains nucleic acid region, the size of described nucleic acid region and the scope of complexity between the promoter of minimum and contain upstream element and the promoter of enhancer between.
Described promoter is selected from the promoter that works usually in mammalian cell, but the promoter that also can use prokaryotic promoter and in other eukaryotic cells, work.Described promoter derives from the promoter sequence of virus or eukaryotic gene usually.For example, it can be the genomic promoter that derives from the cell that will express therein.
Preferably, described promoter is derivable, can regulate and control the expression of heterologous genes level like this in the total life cycle of cell." can induce " expression that is meant the described promoter acquisition of use to regulate.
In addition, any above-mentioned promoter all can be modified by adding other regulating and controlling sequences (for example enhancer sequence).Also can use chimeric promoters, described chimeric promoters comprises the sequential element from the promoter mentioned above of different two or more.
Carrier of the present invention can be introduced in the host cell, so that duplicate described carrier/polynucleotide and/or express antigen of the present invention by polynucleotide encoding of the present invention.Although can use prokaryotic cell to produce antigen of the present invention, preferably use eukaryotic cell, for example yeast, insecticide or mammalian cell, particularly mammalian cell as host cell.
Preferably, described recombinant precursor be can host cells infected the virion form.Perhaps, described recombinant precursor is not reproducible plasmid construction body or naked DNA vaccine form.
Discriminating is through the experimenter's of labelling method
After can or giving in described processing, experimenter that the inventive method is handled or the experimenter that is given a kind of preparation as herein described differentiated out.Therefore, the present invention also provides a kind of discriminating to use the experimenter's that a kind of preparation that contains first antigen and labelled antigen inoculated method, described first antigen derives from the virus that can infect described experimenter, described labelled antigen is not to derive from the labelled antigen that can infect described experimenter's biology natively, and described method comprises the following steps: to measure sample to detect described labelled antigen.The method that is used to detect described labelled antigen will depend on the character of described labelling.If for example described antigen only is used to produce antibody, then described method will comprise the following steps: to measure sample from described experimenter to detect the antibody of anti-described labelled antigen, and wherein the existence of the antibody of anti-described labelled antigen shows that the experimenter is at least by described labelled antigen immunity mistake.
Described method also comprises measures described sample to detect described first antigen.
The existence of the anti-described first antigenic antibody shows that described experimenter is by infection or contacted with the antigen that derives from described infective virus by vaccination, the antibody of the intravital anti-described labelled antigen of experimenter shows that then they are at least by described labelled antigen immunity mistake, because this antigen is not to derive from the biology that can infect described experimenter.
Preferably, use enzyme-linked immunosorbent assay (ELISA) to detect the antibody of anti-described first antigen and/or labelled antigen, wherein make antibody combine and use enzyme-antigen conjugate to detect antibody in the sample and/or quantitative with solid phase it.Perhaps, can use Western blotting, dissolved and isolating one or more antigens are combined with nitrocellulose filter.Then, utilize enzyme or hatch filter membrane under the precipitable situation that maybe can detect substrate and detect described antibody by existing through the anti-immunoglobulin of puting together (Ig) (for example horseradish peroxidase-Ig conjugate) of labelling.The advantage that Western blotting is measured do not need to be one or more required antigenic purity to surpass 50%.At Ausubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY can find the description about ELISA and Western blotting technology in the 10th and 11 chapters of John Wiley and Sons (1988).Perhaps, can utilize Immunofluorescent Antibody Test (for example those known tests of technical staff) to detect described antibody.
Test kit
Can use method and ingredient thereof of the experimenter being carried out immunity of the present invention with the form of test kit easily.Therefore, the present invention also provides a kind of test kit that is used for the experimenter is carried out immunity, comprising:
(a) a kind of first antigen that derives from the virus that can infect described experimenter;
(b) a kind of is not to derive from the labelled antigen that can infect described experimenter's biology natively.
Also can utilize suitable test kit to carry out the method for discriminating of the present invention through the experimenter of labelling.Therefore, the present invention also provides a kind of test kit, comprising: a kind of instrument that is used to detect the antibody of the anti-labelled antigen that is produced, described labelled antigen is not to come from the biology that can infect described experimenter.
Preferably, described test kit also comprises a kind of instrument that is used to detect the anti-first antigenic antibody that is produced, and described first antigen comes from the virus that can infect described experimenter.
Embodiment
The following example is used for describing the mode of using foregoing invention more all sidedly, and is used to illustrate the best mode of implementing each side of the present invention.It should be understood that the true scope that these methods do not limit the present invention in any way, and only play illustration.
Embodiment 1: with tetanus toxoid immunity chicken
Material and method
Animal and drylot feeding condition: chicken: obtain age in 6-7 week the little laying hen of the brown kind of female Hy-Line (layerpullet) (Altona Hatchery Pty.Ltd., Forrestfield, WA) and with its random packet, 5 chickens in every hurdle.Ji Lan makes away from ground and by steel wire.All chickens are all raised in big or small identical chicken hurdle adjacent one another are.
Abide by strict animal ethics guide (AEC numbering: 6-05-55 and 4-06-37), all animals are (the Department ofAgriculture Research Station) drylot feeding all at the agricultural research center that is positioned at Western Australia Medina.
Vaccination:, use the syringe of being furnished with the 21G syringe needle birds to be carried out vaccination by subcutaneous injection (s.c.) in the 0th week and the 4th week.As shown in Figure 1, with tetanus toxoid 1mL or 0.5mL purification, that be equipped with the alum adjuvant (TT) vaccine Equivac T (CSL Ltd., Australia) inoculation birds.As shown in Figure 2, (described mixture contains alum adjuvant (the 1mg aluminium hydroxide can combine with 50-200 μ g antigen) for Pfizer, WA) mixture inoculation birds with rough, unpurified TT.Use a series of TT dosage to determine optimal dose.
Collect blood: utilize venipuncture to collect blood sample in the 0th, 2,4,6 weeks after vaccination first from the wing vein.
With the blood sample of birds from syringe be injected into the glass blood taking tube or the plastics vacuum test tube handled with the serum coagulant (Greiner Bio-One, NC, USA) in, and spend the night at 18-22 ℃ of following standing demix.At room temperature test tube is collected serum with centrifugal 10 minutes of 1000g and by drawing supernatant.Before being used for measuring serum is preserved under 4 ℃ then, Long-term Storage can be preserved under (1 month) under-20 ℃ and-80 ℃ (above 6 months).
Measure the TT antibody titer: use collected serum to measure the intravital TT antibody titer of chicken by indirect ELISA (embodiment 5).In 4 ℃ moist chamber, use TT antigen formalin-inactivated, purification [0.0125 μ g/100 μ L] (List Biological Laboratories, Inc., CA, USA) or with the unpurified TT antigen that is dissolved in 0.05M carbonate buffer solution (pH 9.6) [14.58 μ g/100 μ L] [Pfizer, Western Australia] spend the night the bag by the immunoadsorption elisa plate (GreinerBioOne, Germany).
Statistical analysis: suppose heterogeneity of variance between the whole bag of tricks, use Students t-check that all data are carried out statistical analysis.Think that P value＜0.01 o'clock is significant.
The result
Pullet (10 every group) directly be obtained from business-like hatching and with the Equivac of 1.0mL or 0.5mL TMThe T vaccine carries out subcutaneous vaccination.Measure the anti-TT antibody (referring to Fig. 1) in the serum.Before immunity, there is not the tetanus toxoid antibody of preformation in the chicken body.In two weeks after the vaccination for the first time, two groups of vaccinated chickens have all successfully produced compared with the control and have surpassed 100% anti-TT antibody.After 4 weeks of vaccination first, the level of tiring of TT antibody descends, if but carry out enhance immunity after one month one time in the vaccination first time, then this level can be recovered during the 6th week.
Comprise that the subsequent experimental of 50 chickens is so that assess the optimal dose (referring to Fig. 2) of the TT that gives as vaccine.In this experiment, TT is obtained from the pharmaceutical companies that a family supplies raw materials for the commercialization vaccine.In laboratory, this TT preparation is mixed with aluminium hydroxide (alum).This vaccine is similar to employed commercialization vaccine in the above-mentioned research.Use a series of TT dosage to assess the optimal dose that can cause the highest TT antibody horizontal.
After Fig. 2 showed 2 vaccination, anti-TT antibody horizontal was up to 70%.Adopt there was no significant difference (p＞0.01) between the TT antibody horizontal that all proof loads obtain.Aspect causing and keeping the TT antibody horizontal, the effect of the TT dosage of 0.3mg and the TT dosage of 3mg is suitable.
Embodiment 2: use the tetanus toxoid immune duck
Material and method
Animal and drylot feeding condition: the young Muscovy duck (muscovy duckling) in age in duck: 6-8 week is obtained from the family-raise person (backyard breeder) of locality, Western Australia.Duck is raised in the duck hurdle, on concrete floor, be paved with Caulis et Folium Oryzae simultaneously.
Abide by strict animal ethics guide (AEC numbering: 6-05-55 and 4-06-37), all animals all are being positioned at the agricultural research center drylot feeding of Western Australia Medina.
Vaccination:, use the syringe of being furnished with the 21G syringe needle birds to be carried out vaccination by subcutaneous injection (s.c.) in the 0th week and the 4th week.As shown in Figure 3, (described mixture contains alum adjuvant (the 1mg aluminium hydroxide can combine with 50-200 μ g antigen) for Pfizer, WA) mixture inoculation birds with rough unpurified TT.Use a series of TT dosage to determine optimal dose.
Collect blood: utilize venipuncture to collect blood sample in the 0th, 2,4,6 weeks after vaccination first from the wing vein.
With the blood sample of birds from syringe be injected into the glass blood taking tube or the plastics vacuum test tube handled with the serum coagulant (Greiner Bio-One, NC, USA) in, and spend the night at 18-22 ℃ of following standing demix.At room temperature test tube is collected serum with centrifugal 10 minutes of 1000g and by drawing supernatant.Before being used for measuring serum is preserved under 4 ℃ then, Long-term Storage is under (after 1 month) under-20 ℃ and-80 ℃ (above 6 months) afterwards.
Measure the TT antibody titer: use collected serum to measure the intravital TT antibody titer of duck by indirect ELISA (embodiment 5).In 4 ℃ moist chamber, use TT antigen formalin-inactivated, purification [0.0125 μ g/100 μ L] (List Biological Laboratories, Inc., CA, USA) or with the unpurified TT antigen that is dissolved in 0.05M carbonate buffer solution (pH 9.6) [14.58 μ g/100 μ L] [Pfizer, Western Australia] spend the night the bag by the immunoadsorption elisa plate (GreinerBioOne, Germany).
Statistical analysis: suppose heterogeneity of variance between the whole bag of tricks, use Students t-check that all data are carried out statistical analysis.Think that P value＜0.01 o'clock is significant.
The result
The ability that duck after the vaccination produces anti-TT antibody is studied in the experiment that comprises 40 ducks.According to chicken shown in Figure 2 experiment in the identical method of method therefor, add the vaccination duck of alum with the TT of various dosage.The serum sample of collecting shows that (referring to Fig. 3) duck after strengthening inoculation has successfully produced anti-TT antibody.For the experimenter is not the mensuration of chicken, uses competitive ELISA, thinks that the inhibition of antagonism TT antibody horizontal was significant above 50% o'clock this moment.
Embodiment 3: have the anatoxic natural antibody of tetanus in the birds body
Material and method
Animal and drylot feeding condition: the little laying hen of the brown kind of female Hy-Line in age in chicken: 6-7 week is obtained from AltonaHatchery Pty.Ltd., Forrestfield, WA.
The young Muscovy duck in age in duck: 6-8 week is obtained from the family-raise person of locality, Western Australia.
Various birdss are collected the blood of meat kind chicken, broiler and laying hen from the two tame commercialization slaughterhouses that are positioned at the Western Australia.Always the sentry family chicken in place is on one's body and whether have a tetanus antibody of preformation in the wild sharp wing tree duck (plumed whistling duck) that Ke Wunula catches is taken a blood sample with the serum that is determined at them on one's body from different places from the northwestward, Western Australia.
Collect blood: utilize venipuncture to collect blood sample from the wing vein.The blood of the poultry that butcher in the slaughterhouse is collected from jugular vein.Cheryl doctor's Johansen of Univ Western Australia research group has been collected the blood sample that obtains from the wing vein of area, the northwestward, Western Australia birds.
With the blood sample of birds from syringe be injected into the glass blood taking tube or the plastics vacuum test tube handled with the serum coagulant (Greiner Bio-One, NC, USA) in, and spend the night at 18-22 ℃ of following standing demix.At room temperature test tube is collected serum with centrifugal 10 minutes of 1000g and by drawing supernatant.Before being used for measuring serum is preserved under 4 ℃ then, Long-term Storage can be preserved under (1 month) under-20 ℃ and-80 ℃ (above 6 months).
Measure the TT antibody titer: use collected serum to measure the TT antibody titer by indirect ELISA.In 4 ℃ moist chamber, use TT antigen formalin-inactivated, purification [0.0125 μ g/100 μ L] (List Biological Laboratories, Inc., CA, USA) or with the unpurified TT antigen that is dissolved in 0.05M carbonate buffer solution (pH 9.6) [14.58 μ g/100 μ L] [Pfizer, Western Australia] spend the night the bag by the immunoadsorption elisa plate (Greiner BioOne, Germany).
Statistical analysis: suppose heterogeneity of variance between the whole bag of tricks, use Students t-check that all data are carried out statistical analysis.Think that P value＜0.01 o'clock is significant.
The result
Poultry from diverse geographic location and source is determined under the situation of not carrying out the tetanus vaccine inoculation whether have TT antibody (referring to Fig. 4-10).General description to the various poultry of being screened has been shown in the table 1.
Table 1: the male quantity of poultry of being screened and tetanus antibody
Poultry Tested number TT antibody positive quantity
Broiler 294 0
Meat kind chicken 360 0
Laying hen 262 0
The garden sentry of the Australia northwestward 339 0
Hong Kong/China's Mainland chicken 230 0
Experiment is with hatching the institute birds 280 0
Wild sharp wing tree duck (Ke Wunula) 236 0
Muscovy duck (Mus covy duck) (Jandakot farm) 33 0
Amount to 2034 0
Fig. 4-6 shows with the positive control from vaccinated chicken and compares, the TT antibody horizontal (referring to material and method) that exists from the chicken serum that collect in the slaughterhouse.The background level of finding poultry reaches as high as 20% of positive control tetanus antibody, and old meat kind chicken and the background level of laying hen are lower than 40%.The background antibody horizontal that has also confirmed Australian northwestward sentry (Fig. 7) reaches as high as 40% of positive control level.In a word, described background level is lower than viewed positive TT antibody horizontal among Fig. 1 and Fig. 2, and thinks that 40% background level is a TT antibody response feminine gender.
The background TT antibody horizontal of also having tested another geographic chicken is used as contrasting the result who uses the geographic birds in Western Australia to obtain with us and compares.Use method and the reagent identical with reagent with the described method of method part with material is measured in Hong Kong quarantine laboratory.Chicken serum sample (230 samples) is obtained from Hong Kong and farm, China's Mainland.In these samples, have 75 chickens be from the import of farm, China's Mainland and inoculated with H5N2; Have 45 chickens be obtain from farm, Hong Kong and inoculated with H5N2, remaining chicken is the sentry chicken that did not inoculate from farm, Hong Kong.All birdss are about 90 ages in days and demonstrate the tetanus antibody level negative.
Also studied the situation that exists of the intravital natural TT antibody of duck.Geographic wild duck of Ke Wunula (Fig. 9) and local Muscovy duck (Figure 10) of raising have been carried out test and found that their TT antibody is negative.
Embodiment 4: the tetanus toxoid labelling and the influenza vaccines of combination
Material and method
Animal and drylot feeding condition: chicken: obtained the 6-7 little laying hen of the brown kind of female Hy-Line in age in week (Altona Hatchery Pty.Ltd., Forrestfield, WA) and with its random packet, every hurdle 5 chickens.Ji Lan makes away from ground and by steel wire.All chickens are all raised in big or small identical chicken hurdle adjacent one another are.
The young Muscovy duck in age in duck: 6-8 week is obtained from the family-raise person of locality, Western Australia.Duck is raised in the duck hurdle, on concrete floor, be paved with Caulis et Folium Oryzae simultaneously.
Abide by strict animal ethics guide (AEC numbering: 6-05-55 and 4-06-37), all animals all are being positioned at the agricultural research center drylot feeding of Western Australia Medina.
The preparation of H2N6 bird flu (AI) virus: cultivate H6N2AI virus original seed according to the standard method that OIE (http://www.oie.int/fr/normes/mmanual/A 00037.htm, visit on March 22nd, 2006) provides.In brief, under aseptic condition, H6N2 infectiousness AF is inoculated in the allantois of SPF Embryo Gallus domesticus ovum (100 μ L/ ovum).After hatching 4 days under 37 ℃, the ovum that infects is cooled to 4 ℃.The shell of each ovum is removed, under aseptic condition, collect AF and place under 4 ℃.Before merging collected AF, the HA activity (vide infra) of random test ovum.Came deactivation AF in 65 minutes by stirring at 37 ℃ of following formaldehyde (formaldehyde of operational analysis research grade) with 0.1% (v/v).Under 4 ℃, inoculate the inactivation of testing HA with Embryo Gallus domesticus with described suspension standing over night and by above-mentioned standard method.
Vaccination:, use the syringe of being furnished with the 21G syringe needle birds to be carried out vaccination by subcutaneous injection (s.c.) in the 0th week and the 4th week.
Figure 11-14 shows the screening to TT dosage, and with TT and oil adjuvant MontanideISA 70 VG (Seppic, France) mixing.Form of medication with the inactivated whole virus H6N2 vaccine of oil preparation is independently 1mL subcutaneous injection agent.Suggested design (Seppic, France) usefulness glass syringe and 21G syringe needle mixed vaccine goods according to manufacturer.
Data represented shown in Figure 15-16 is used to optimize used TT dosage and TT and H6N2 vaccine product is merged into the experiment of single injection.By mixing described bacterin preparation until obtaining uniform water in oil emulsion.
Collect blood: utilize venipuncture to collect blood sample in the 0th, 2,4,6 weeks after vaccination first from the wing vein.
With the blood sample of birds from syringe be injected into the glass blood taking tube or the plastics vacuum test tube handled with the serum coagulant (Greiner Bio-One, NC, USA) in, and spend the night at 18-22 ℃ of following standing demix.At room temperature test tube is collected serum with centrifugal 10 minutes of 1000g and by drawing supernatant.Before being used for measuring serum is preserved under 4 ℃ then, Long-term Storage is under (after 1 month) under-20 ℃ and-80 ℃ (above 6 months) afterwards.
Measure the TT antibody titer: use collected serum to measure the intravital TT antibody titer of chicken by indirect ELISA (embodiment 5).In 4 ℃ moist chamber, use TT antigen formalin-inactivated, purification [0.0125 μ g/100 μ L] (List Biological Laboratories, Inc., CA, USA) or with the unpurified TT antigen that is dissolved in 0.05M carbonate buffer solution (pH 9.6) [14.58 μ g/100 μ L] [Pfizer, Western Australia] spend the night bag by the immunoadsorption elisa plate.
Statistical analysis: use Students t-check that all data are carried out statistical analysis, suppose heterogeneity of variance between the whole bag of tricks.Think that P value＜0.01 o'clock is significant.
Hemagglutination (HA) test: according to standard scheme (terrestrial animal diagnostic test and vaccine handbook (Manual Diagnostic Tests and Vaccines for Terrestrial Animals), http://www.oie.int/fr/normes/mmanual/A 00037.htm, on June 7th, 2006 visit) the H6N2 bird flu virus of titration inactivation.In brief, with 0.1M PBS described viral suspension is diluted to 25 μ L continuously.In the hole, add 25 μ L PBS again so that final volume is 50 μ L.Then the chicken red blood cell (RBC) of 25 μ L 0.5% (v/v) is joined in each hole and with plate and under 4 ℃, hatched 60 minutes.Incubation period,, the RBC contrast should be a tangible speckle (button) in the bottom sedimentation of described hole when finishing.
By whether existing the RBC stream of tear shape to determine that HA tires 45 ° of plate inclinations and observation.The high dilution that provides complete HA (do not have flow) is that end points is tired, and represents 1 HA unit (HAU).
Measuring hemagglutination inhibition (HI) tires: use HI to measure to determine chicken and the intravital HA antibody horizontal of duck.Handle the influenza virus antiserum of duck with the deactivation nonspecific inhibitor with receptor destroying enzyme (RDE).Chicken antiserum without RDE handle and can use (diagnosis of WHO animal influenza and monitoring (WHO manualon Animal Influenza Diagnosis and Surveillance), WHO/CDS/CSR/NCS/2002.5, p.31).According to the directions for use of manufacturer, (Denka Seiken Co.Ltd. Japan) heavily is dissolved in (0.85%NaCl) in the physiological saline solution with lyophilized RDE.Then, the RDE of 3 times of volumes is joined in the serum of 1 times of volume and overnight incubation in 37 ℃ water-bath.Made described enzyme deactivation in 30 minutes by heating down then at 56 ℃.After the serum cooling that makes described processing, it is 1: 10 that the adding normal saline makes final dilution factor.
Carry out HI mensuration by continuously serum being diluted to 25 μ L with PBS onboard.(25uL 4HAU) adds in each hole and hatched under 4 ℃ 60 minutes, adds the chicken RBC of 25uL 0.5% afterwards and mixes gently with influenza antigen then.After under 4 ℃, hatching 60 minutes, assess HI by definite highest serum dilution factor that can suppress the virus antigen of 4HAU fully and tire.The serum contrast (virus-free antigen) parallel with serum sample is set to deduct non-specific HI activity.Except that the HI of duck tired, all data all were expressed as average antibody and tire+SEM (standard error of meansigma methods), the HI of described duck the expression separately of tiring.
The result
Next, studied use tetanus toxoid as biomarker to distinguish the combination of inoculate with the birds that did not inoculate.Two groups of experiments have been carried out.First group relates to the H6N2 avian influenza vaccine that gives described tetanus vaccine and inactivation with the form of injection independent of each other jointly, and second group of experimentation two kinds of vaccines are merged into the feasibility of single injection.Two kinds of vaccines all relate to use Montanide oil as adjuvant, and Montanide oil is used in the business-like poultry inoculation usually.
In co-administered experiment, use 30 chickens and 30 ducks to study the effectiveness of the vaccine of subcutaneous administration.Select the dosage (0.1mg, 0.3mg, 1.0mg) of tetanus toxoid according to result shown in Figure 2.It is 27 that the H6HA of used bird flu virus tires.The antibody horizontal of tetanus toxoid and HI tire shown in Figure 11-14.
Figure 11 shows the TT antibody horizontal that is caused by vaccination.Obtaining positive TT antibody horizontal after the inoculation for the second time, antibody titer reaches as high as 100%.Described powerful antibody is tired and can be lasted till postvaccinal first the 20th week always.There was no significant difference between the various dose, but compare with 1mg TT dosage, and 0.1mg TT dosage always provides high slightly TT and tires.
The intravital HI of chicken that Figure 12 shows co-inoculation tires.Successfully caused HA and tire after vaccination, tiring of all dosage groups all reached 29 after the vaccination second time simultaneously.It should be noted that 2 inoculation back tiring of (26) of Al contrast are lower than the co-inoculation group, show to have following two kinds of probabilities: exist extra adjuvant or some synergistic activity to cause the higher HI of co-inoculation group to tire when sending these two kinds of vaccines.Do not observe when giving specific antibody that TT and Al vaccine induce anti-TT or Al jointly and have interference effect.
Figure 13-14 shows the intravital TT of duck and Al tires.Aspect antibody response, the immune sensitivity of duck is usually not as chicken.In all TT dosage groups, positive anti-TT antibody titer (inhibition more than 50%) only just can obtain after the inoculation for the second time.0.1mg TT dosage has provided the strongest anti-TT antibody response once more, following closely be 1mg TT dosage.
The HA that is caused in the duck body tires and is lower than the HA that is caused in the chicken body and tires.Be not that all ducks all can produce immunne response to described vaccine, but 0.3mg TT dosage (containing Al) has caused the highest HA antibody response really.
Next studied the strategy of TT and Al vaccine being merged into single administration of vaccines.Clear and definite cause positive antibody reply aspect these two kinds of vaccines can be not interfering with each other after, with oil with these two kinds of vaccines mixing and be mixed with a kind of subcutaneous preparation that gives chicken (10 every group) that is used for.
Selected the best TT dosage of 0.3mg based on the result shown in Figure 11.This dosage reduced by half (0.15mg) and double (0.6mg) with the difference between the TT antibody response that is relatively caused.Comprised that also the matched group of only accepting the Al vaccine and only accepting 0.3mg TT is as the background reference.The intravital TT level of the chicken that inoculated is shown in Figure 15.
Detect the TT antibody titer in all TT inoculation group.The post-vaccine antibody level significantly increases for the second time, and 0.3mg TT and 0.6mg TT group show suitable antibody horizontal, and this antibody horizontal is similar to the contrast of only accepting TT.It should be noted that the control group A l inoculation chicken of not using the TT vaccine immunity in ELISA as not having detectable anti-TT antibody with expecting.Compare with the contrast of only accepting TT, do not have significant difference (P＞0.01) between the intravital TT antibody horizontal of the birds of co-inoculation.
Figure 16 shows HA after the vaccination and tires and be similar to the HA that obtains among Figure 12 and tire.It should be noted that the matched group of only accepting 0.3mg TT vaccine and not accepting the Al vaccine do not tire as not having the HI of detectable specificity at the H6N2 influenza virus with expecting.The group of all co-inoculation and only accept that there is not significant difference (P＞0.01) in HI between tiring in the matched group of Al.
Embodiment 5:ELISA diagnosis
In this TT labelling system, utilize the indirect elisa method can be quantitative to the intravital anti-TT antibody response of chicken at an easy rate, described indirect elisa method comprises that a kind of enzyme puts together antibody, described antibody recognition and in conjunction with the chicken immune globulin to detect the anti-TT antibody (the anti-chicken IgG of rabbit that for example, has puted together horseradish peroxidase) in the Sanguis Gallus domesticus.Yet this method can not be used to detect the antibody of other avian species (for example duck) except that chicken, because the described antibody of puting together is species specificity and limited in one's ability with the cross reaction of heterologous antibody.Therefore, indirect ELISA only limits to detect the intravital antibody of chicken.Because other avian species can (for example, be used to monitor poultry so need another kind of method to be used for detecting and to monitor TT antibody by the HPAI strain infection in our labelling vaccination strategies; Duck, turkey, goose, Carnis Coturnicis japonicae or leisure Wildlife; Stork, flamingo).
A kind of alternative method of indirect ELISA is to use a kind of competitive ELISA, and described competitive ELISA antagonist step is revised.In described competitive ELISA, the commercially available lowlenthal serum for preparing (containing the anti-TT antibody of specificity) is preceding adding, and described birds serum is combined by the TT antigen of described plate with bag.These two types of antibody can be competed in conjunction with general TT antigen.If the specificity T T antibody (positive serum) in the described birds serum is in conjunction with TT antigen, the combination of then described goat TT antibody can be blocked and compete.Otherwise, if described birds serum does not contain TT specific antibody (negative serum), the combination of described goat TT specific antibody then takes place, and does not compete.For the general mensuration of this class, the serum source that derives from avian species is not limited.
The TT specific antibody is represented with the percent that the average OD of TT antibody in the test sera (two repetitions) accounts for the average OD of positive control (taking a blood sample from the chicken of TT inoculation when the 6th week) on one's body in the chicken serum that will record by indirect ELISA, revises by the average OD of the negative control that deducts each sample (blood sampling before the inoculation when being equivalent to for the 0th week) respectively.
The TT specific antibody is represented with the inhibition percent that the average OD of TT antibody in the test sera (two repetitions) accounts for the average OD of negative control (taking a blood sample in advance when the 0th week) in the birds serum (being not limited to the chicken class) that will record by competitive ELISA.
Suppress %=100-(the average OD of the average OD/ negative control of 100 * test)
Think that the serum sample that suppresses above 50% is the TT antibody positive.
Material and method
Indirect TT ELISA:
In 4 ℃ moist chamber, use TT antigen formalin-inactivated, purification [0.0125 μ g/100 μ L] (List Biological Laboratories, Inc., CA, USA) or with the unpurified TT antigen that is dissolved in 0.05M carbonate buffer solution (pH 9.6) [14.58 μ g/100 μ L] [Pfizer, Western Australia] spend the night the bag by the immunoadsorption elisa plate (Greiner BioOne, Germany).(Nunc International Australia), cleans plate 6 times with PBS pH7.6/0.05% polysorbas20 (PBST) to use 12 hole Immuno cleaning equipments.Be 1/200 with the chicken serum dilution and in each hole, add 100 μ L with the PBST/4% skimmed milk, establish two repetitions.Then plate was hatched under 37 ℃ 1 hour, clean 6 times with PBST afterwards.Use anti-chicken IgG (the Chemicon InternationalInc. of the rabbit of having puted together horseradish peroxidase (HRP) with 1/4000 dilution of PBST/4% skimmed milk, Australia) and with its volume join in every hole, under 37 ℃, hatched 1 hour then with 100 μ L.All then PBST clean plates, add afterwards 100 μ L substrate solution (TMB Onesolution, Promega Corp., USA).Come cessation reaction by adding 50 μ L 2M sulphuric acid after 5 minutes.After 15 minutes, (Bio-Rad Model 680, CA USA) read plate at the 450nm place, and use 630nm as the reference wavelength to use microplate reader.
Competitive TT ELISA:
Similar described in indirect ELISA spent the night plate, and cleaned 6 times with PBST with the TT bag.Add first antibody (chicken or Sanguis Anas domestica are clear, 1/10 dilution, 100 μ L/ holes), establish two repetitions, and under 37 ℃, hatched 1 hour.With PBST plate is cleaned 6 times, add the anti-TT IgG of goat antibody (1/3200 dilution) (Accurate Chemical﹠amp then; Scientific Corp., NY USA), was hatched under 37 ℃ 1 hour.After with PBST the hole being cleaned 6 times, the anti-chicken IgG of rabbit that will put together HRP joins in every hole and hatched under 37 ℃ 1 hour again.And then use the PBST clean plate, add TMB OneSolution afterwards.Come cessation reaction by adding 2M sulphuric acid after 5 minutes, and read plate, use 630nm as the reference wavelength at the 450nm place.

Claims (13)

1. a labelling has been given the method that maybe will be given at least a first antigenic experimenter, wherein said antigen derives from the infectious agent that can infect described experimenter and can produce in described subject or help to produce therapeutical effect, described method comprises the following steps: to give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the described first antigenic experimenter.
2. a labelling has been accepted a kind of first antigenic experimenter's method, described first antigen derives from the infectious agent that can infect described experimenter, described first antigen can produce or have at least in described subject and help produce therapeutic response, described method comprises the following steps: to give described experimenter a kind of labelled antigen, described labelled antigen is detectable and can differentiating and processing crosses and untreated experimenter that the expression of described labelled antigen is ectogenic for described first antigen in described subject.
3. a labelling has been given a kind of first antigenic experimenter's method, and described first antigen derives from the virus that can infect described experimenter, and described method comprises the following steps:
(a) give described experimenter a kind of labelled antigen, described labelled antigen is not to derive from the biology that can infect described experimenter natively; And
(b) wherein said labelled antigen can cause detectable replying in described subject.
One kind at a kind of infectious agent and to the experimenter carry out the immunity method, described method comprises the following steps:
(a) give described experimenter a kind of first antigen, wherein said first antigen can in described subject, produce or help to produce therapeutical effect and
(b) give a kind of labelling of described experimenter or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the first antigenic experimenter described in the step (a).
One kind at a kind of virus and to the experimenter carry out the immunity method, comprise the following steps:
(a) give described experimenter at least a first antigen, described first antigen derives from the virus that can infect described experimenter and can send or produce curative effect;
(b) give described experimenter a kind of labelled antigen simultaneously, described labelled antigen is not to derive from the biology that can infect this experimenter natively, and wherein said labelled antigen can cause detectable immunne response in described subject.
One kind at a kind of virus and to the experimenter carry out the immunity method, described method comprises the following steps: to give described experimenter a kind of single dosage form, described single dosage form comprise (a) a kind of derive from first antigen of the virus that can infect described experimenter and (b) a kind of be not to derive from the labelled antigen that can infect described experimenter's biology natively, wherein said labelled antigen can cause detectable immunne response in described subject.
7. pharmaceutically acceptable preparation comprises:
(a) a kind of can in described subject, produce or help to produce therapeutical effect first antigen and
(b) a kind of labelling or reporter, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the first antigenic experimenter described in the step (a).
8. pharmaceutically acceptable preparation comprises:
A kind of first antigen that derives from the virus that can infect described experimenter; With a kind of be not to derive from the labelled antigen that can infect described experimenter's biology natively, wherein said labelled antigen can cause detectable immunne response in described subject.
9. the method for a marker vaccine, described vaccine can resist can infected subjects virus, described method comprises the following steps: a kind of first antigen and a kind of labelling or reporter combination, described first antigen can produce in described subject or help to produce therapeutical effect, described labelling or reporter can produce detectable signal in described subject, and can identify and accepted the described first antigenic experimenter.
10. the method for a marker vaccine comprises the following steps:
(a) described vaccine is contacted with a kind of labelled antigen, described labelled antigen is not to derive from the biology that can infect described experimenter natively; With
(b) wherein said labelled antigen can cause detectable immunne response in described subject.
11. a discriminating has used the described preparation of the application to carry out the experimenter's of vaccination method, described method comprises the following steps: to measure sample from described experimenter to detect described labelling.
12. a test kit that is used for the experimenter is carried out immunity comprises: (a) a kind of first antigen that derives from the virus that can infect described experimenter; (b) a kind of is not to derive from the labelled antigen that can infect described experimenter's biology natively.
13. a test kit comprises: a kind of instrument that is used to detect the antibody of the anti-a kind of labelled antigen that is produced, described labelled antigen is not to come from the biology that can infect described experimenter.
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