CN1281277C

CN1281277C - DNA vaccine for preventing influenza virus infection

Info

Publication number: CN1281277C
Application number: CN 200310110549
Authority: CN
Inventors: 陈则
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2003-11-21
Filing date: 2003-11-21
Publication date: 2006-10-25
Anticipated expiration: 2023-11-21
Also published as: CN1544089A

Abstract

The present invention relates to a DNA vaccine for preventing influenza viruses. Because the present invention adopts CD40L and influenza virus DNA to jointly immunize animals, the immune responses generated by the inducement of the present invention can favorably protect the mice against the attacks of a lethal dose of influenza viruses, and the anti-influenza effects are better than the effects of DNA vaccines without the CD40L. The DNA vaccine of the present invention used for immunizing animals has the advantages of simple preparation method, low price, convenient use and favorable immune effect, overcomes the defects of the known influenza prevention preparations, and provides a relatively safe and effective influenza prevention preparation.

Description

A kind of flu-prevention virus DNA vaccine

Technical field:

The invention belongs to a kind of biological product, be specifically related to a kind of flu-prevention viral vaccine.

Background technology:

Existing influenza biological preventing goods and preparation method have following kind: (one), inactivated influenza virus vaccine.The totivirus inactivated vaccine is on the basis of intact virus, the method for cause a disease virion Applied Physics or chemistry is made it complete inactivation (losing infectivity) and the viral vaccine made.Vaccinum influenzae inactivatum is compared other vaccines has production method fairly simple, advantages such as the easy control of production process, but its significant disadvantages is also arranged.Because the influenza virus that the preparation vaccine is used needs incubation growth in the allantoic cavity of Embryo Gallus domesticus, manufacturing cycle is long, and immunogenicity can be lowered or change.The inactivated vaccine of influenza not tool is long-lasting.By its inductive immunne response even also very of short duration, therefore need annual or twice inoculation in a year to the effect of congenerous disease strain.(2), influenza subunit vaccine.The influenza subunit vaccine is meant the effective viral surface antigen composition that is obtained by wild strains of influenza viruses behind cultivation, cracking, purification, mainly be HA and NA vaccine.But the traditional preparation process method production technology of subunit vaccine is loaded down with trivial details relatively, is difficult to obtain in a large number antigen, therefore causes subunit vaccine to cost an arm and a leg, and is difficult to apply.(3), Gripovax.Verified in mice, Gripovax has more broad-spectrum immunne response.As an effective vaccine, the cold adaptation strain of gene resortment must have the coding HA that derives from wild strain and 2 RNA sections of NA, and other 6 segments then must derive from the strain of cold adaptation donor.Prepare about 5 time-of-weeks that need of attenuated live vaccine from obtaining being cloned into of wild virus strain.Before the inoculation crowd, also vaccine strain must be cultivated in special aseptic egg allantoic cavity, further detect its toxicity.Attenuation treatment to Gripovax is experimental, and still there is the probability of back mutation in its virulence.Especially for the cold adaptation vaccine strain, chronic owing to need repeatedly going down to posterity, when viral prevalence, epidemic isolates is carried out cold adaptation go down to posterity also unrealistic.(4), influenza synthetic peptide vaccine.Synthetic and the identical peptide section of aminoacid sequence influenza virus protective antigen (HA, NA etc.) determinant; after being prepared into immunogen, animal or human's body is inoculated; can impel body to produce protection antibody, this peptide species is exactly the synthetic peptide vaccine of influenza.Synthetic peptide vaccine exists some theoretical and actual difficulties: the one, how to find and make up the polypeptide of best antigenic determinant; The 2nd, the less immunogenic of synthetic peptide must the adapted adjuvant during use.Because Freund adjuvant can not use at human body, thus the clinical evaluation of synthetic peptide be badly in need of development a kind of effectively, can be at the adjuvant of human body use.(5), influenza genetic engineering subunit vaccine.Induce influenza antigens determinant (HA, the NA etc.) gene of protective immune response to be inserted in the expression vector dna coding; then carrier is imported yeast, insecticide or mammalian cell; make it to express virus antigen albumen, promptly get the genetic engineering subunit vaccine of influenza behind the product purification.The weak point of genetic engineering subunit vaccine is that the workload of positive expression colony screening is big.Expressed proteins can not correctly fold or modify sometimes, thereby influences immunogenicity.The technology of recombiant protein separation and purification is relative complex sometimes.(6), influenza virus dna vaccination.Dna vaccination (DNA Vaccine) be with the coding certain antigen protein exogenous gene directly import in the animal somatic cell, and the expression system synthetic antigen albumen by host cell, induce the host to produce immunne response, to reach the purpose of prevention and treatment disease to this antigen protein.Dna vaccination is made up of the protective antigen gene and the vector plasmid two parts of pathogen (comprising virus and antibacterial etc.).Plasmid DNA must possess following condition: 1. have antibacterial replicon (Ori), guarantee that plasmid can duplicate in escherichia coli; 2. have the resistant gene that is used to screen, screening-gene can be selected resistant genes such as kanamycin, ampicillin or neomycin for use; 3. Eukaryotic promoter (have contain enhancer) is as CMV, SV40 promoter; 4. have Pol y A sequence, can guarantee mRNA stability in vivo, this stability is different different because of Poly A source.Think that at present Poly A is from bovine growth hormone gene (BGH) preferably.The lead-in mode of dna vaccination is various, and particle gun immunity that intramuscular injection, intradermal injection, intranasal instillation or nasal spray, liposome method and new development get up and the immunity of live body electric shock etc. are arranged.Influenza virus is still so far and causes human dead main diseases therefore, because influenza virus easily suddenlys change, so the mankind still can't conquer influenza so far.Vaccination is a kind of effective ways of flu-prevention, and dna vaccination (DNA vaccine) provides a kind of new selection for China.1993, Ulmer JB etc. inject the plasmid of the NP gene of encoding influenza virus A/PR/8/34 in the quadriceps femoris of BALB/c mouse, having produced strong and special CTL replys, cross-protection can be provided, start research [the Ulmer JB et al Science.1993 Mar 19 that nucleic acid vaccine is used for flu-prevention virus; 259 (5102): 1745-9.]; Chen Ze etc. are cloned into the HA encoding gene in the β actin expression vector of chicken, with 3 weekly intervals inoculation 2 times, adopt the mode of particle gun or live body electric shock immunity inoculation, dosage of inoculation is every mice of particle gun 1 μ g DNA, every mice of live body electric shock immunization method 30 μ g DNA.Behind the booster immunization the 7th day attacked with the congenerous disease strain.The result shows that the mice of inoculation HA dna vaccination is highly resistant to viral infection [Chen Z et al Vaccine.1998Oct; 16 (16): 1544-9; Chen Z et al Vaccine.1999 Feb 26; 17 (7-8): 653-9.], so the HA gene should be as the important component part of influenza dna vaccine.Dna vaccination can induce body to produce humoral immunization and cellular immunization in immunne response, dna vaccination is applied to animals such as mice, sheep, chicken, cat, cattle, pig, horse, monkey and chimpanzee and all succeeds on one's body, but its efficient in induce immune response is relatively low, influenced its practical application, therefore seeking other dna molecular then is the problem that needs solution with the immune efficient that improves nucleic acid vaccine.

Summary of the invention:

The present invention is intended to develop a kind of flu-prevention virus efficient DNA vaccine, to improve the immune efficient of dna vaccination.

The foregoing invention purpose is achieved through the following technical solutions.Vaccine of the present invention is a dna vaccination of making the flu-prevention virus of adjuvant with CD40L, and HA dna vaccination and CD40L are immune jointly with 1: 1 mass ratio.

Be described in further detail the present invention below.

Description of drawings:

Fig. 1 is for virus attack mice weight loss (g) and infect back restoration result figure.

Fig. 2 is the serum IgG titration results figure of virus-specific.

Fig. 3 is the level figure as a result of IgG antibody subtype in the mice of common immune HA and CD40L (behind the booster immunization) back.

CD40L is one of member of the super family of tumor necrosis factor, contains 261 aminoacid, for II type transmembrane glycoprotein, is positioned on the Xq24, and its relative molecular weight (Mr) is 39000.[Eur J Immunol.1992Dec；22(12)：3191-4]

Flu-prevention of the present invention prepares as follows with the efficient DNA vaccine: the acquisition of influenza antigen HA and CD40L gene, the structure of HA and CD40L dna vaccination, the evaluation of HA and CD40L dna vaccination.

The acquisition of influenza antigen HA and CD40L gene

Viral RNA is to extract from breeding in the virus that obtains the instar chicken embryo on 10th.RNA obtains strand cDNA through reverse transcription reaction.With cDNA is template, and the HA gene is carried out pcr amplification.Forward primer is 5 ' AAC CTC GAGAAT GAA GGC AAA CCT ACT GGT CC-3 ', reverse primer is 5 ' AAC CCC GGGTCT CAG ATG CATATT CTG CAC TGC A-3 '.Forward primer contains Xho I restriction enzyme site, and reverse primer contains Sma I restriction enzyme site.

CDNA library (Clontech company) with the mouse spleen cell is a template, clone the CD40L gene with PCR method, forward primer is 5 ' TTC CTC GAG CAT GAT AGA AAC ATA CAG-3 ', and reverse primer is 5 ' TGA CCC GGG GTA TAG GGA AGA CTG CCA-3 '.Forward primer contains Xho I restriction enzyme site, and reverse primer contains Sma I restriction enzyme site.

The structure of HA and CD40L dna vaccination

The low melting-point agarose gel reclaims the PCR product, is connected into the pGEM-T carrier, and transformed into escherichia coli identifies to determine by plasmid extraction and enzyme action whether HA, CD40L gene have been connected into the T carrier.The recon that a large amount of then amplifications are identified, purification reclaims.The T carrier that has HA, CD40L gene digests with Xho I-Sma I, low melting point reclaims HA, the CD40L fragment that scales off from the T carrier, be cloned in the expression vector pCAGGSP7 that same enzyme action is handled, obtain recombiant plasmid pCAGGSP7/HA, pCAGGSP7/CD40L.HA, CD40L gene nucleotide series confirm through 377 dna sequencing instrument (Applied Biosystem.U.S.A.) order-checking.

Carrier for expression of eukaryon pCAGGSP7 derives from the pCAGGSP7[Niwa H et al.Gene 1991 that is made up by Niwa et al, 108:103-200.], be with multiple clone site Kpnl, Xhol, Clal, EcoRV, Smal, the EcoRl site that Notl and Sacl insert pCAGGS obtains pCAGGSP7.Plasmid pCAGGS contains the beta-actin promoter composition of chicken, the ori site of SV40 (Ori), ampicillin resistance gene, the instantaneous enhancer of CMV, bovine growth hormone gene (BGH) poly A.

The evaluation of HA and CD40L dna vaccination

The plasmid of coding HA and CD40L increases in Escherichia coli XL1-blue respectively, with QIAGEN purification kit (QIAGEN, Tip500) purification.With the concentration and the purity of determined by ultraviolet spectrophotometry plasmid, the concentration of DNA and purity are determined by OD260, OD280, are chosen OD260/OD280 ratio and remove immune mouse in 1.8～2.0 plasmid DNA.

HA and CD40L plasmid are frozen respectively at-20 ℃ of low temperature, thaw before the immune mouse and according to consumption HA are mixed with the CD40L plasmid.

Immunization experiment step and experimental result

The HA that immunization experiment uses is the strains of influenza viruses (A/PR/8/34) that comes from the H1N1 hypotype, and its full name is hemagglutinin.

The plysiochemical characteristic of A/PR/8/34 HA (hemagglutinin):

The main surface glycoprotein of this Strain is hemagglutinin (HA).It has the character of the multiple animal erythrocyte of coagulation.

HA is typical type I glycoproteins by Influenza Virus RNA fragment 4 codings, and it contains 4 domains: signal peptide (targeting sequencing), cytosolic domain, membrane-spanning domain and extracellular domain.HA approximately is made up of 562-566 aminoacid, at the aminoterminal of HA by a signal peptide of forming by 16 hydrophobic amino acids.Be right after the HA1 part of being made up of 328 amino acid residues of signal peptide, c-terminus constitutes HA2 by 221 amino acid residues.One arginine residues is arranged between HA1 and HA2.HA2 c-terminus (185-211 amino acid region) mainly is made up of hydrophobic amino acid residue.10 amino acid residues of least significant end are hydrophilic mostly.

The three dimensional structure of HA: the HA albumen of influenza virus is present on the BLM with the form of trimer (trimer), and promptly fine the dashing forward of HA is made of monomer total length 13.4nm three HA monomer molecules.It can be divided into two parts, and a part is to be globular head, is made up of HA1, contains receptor binding site and antigen decision family; Another part is a handle, is made up of HA2 and section H A1, links to each other with cyst membrane, is about 7.6nm.HA2 amino terminal and HA1 carboxyl terminal are at a distance of 2.1nm.The HA2 amino terminal is positioned at the trimer interface of molecule, with viromembrane at a distance of 3nm, it has been rich in glycine residue, forms a unusual helical structure and extends trimerical interface.

Laboratory animal choose 8 the week age BALB/c female mice.

Intraperitoneal injection of anesthesia medicine (ketalar, lobeline hydrochloride mixture) makes the mice general anesthesia, with alcohol wipe mice right rear leg quadriceps femoris, draw with disposable syringe then and be dissolved in TE (1M Tris, 0.5M dna solution EDTA), pin is vertically inserted quadriceps femoris, slowly inject DNA.One group of BALB/c mouse mixed immunity 30 μ gHA (1 μ g/ μ l) and 30 μ g CD40L (1 μ g/ μ l), cumulative volume 30 μ l; One group of BALB/c mouse immunity 30 μ gHA (1 μ g/ μ l), cumulative volume 30 μ l; With the empty carrier immune mouse that does not insert exogenous gene as negative control.Insert two electrodes of electric shock instrument in the both sides of intramuscular injection pin hole, at a distance of 0.5cm, electric shock (voltage 100v, electric shock time 50ms shock by electricity positive and negative each three times, at interval 1s) is finished once immunity then.The 3rd week of immunity back is used the DNA booster immunization of same dose and composition.

Use the chloroformization mice, take a blood sample from heart.The mice back of the body is crouched in the experiment desktop, and little taking back sentenced 15～20 degree angles insertion syringe needle below xiphoid, extracts painstaking effort slowly and stops until blood flow.The blood of collecting places room temperature about 1 hour.Separate out serum after solidifying.4000 rev/mins centrifugal 10 minutes.Sucking-off serum under aseptic condition ,-20 degree refrigerators are preserved.

Survey the HA specific IgG antibodies with enzyme-linked immunosorbent assay (ELISA).With the inactivated vaccine bag of 10 μ g/mL by 96 hole ELISA Plate, 37 ℃ 2 hours; PBS-T (contains NaCl 8.0g in 1 liter of solution, KCl 0.2g, Na ₂HPO ₄1.15g, KH ₂PO ₄0.2g, Tween-20 0.454ml) and the back of giving a baby a bath on the third day after its birth time adds confining liquid and (contains NaCl8.0g in 1 liter of solution, KCl 0.2g, Na ₂HPO ₄1.15g, KH ₂PO ₄0.2g, the 10g bovine serum albumin), 4 ℃ are spent the night.With confining liquid with 2 multiple dilution antiserum after, add ELISA Plate, 37 ℃ of incubations 1 hour.After PBS-T gives a baby a bath on the third day after its birth time, add with the anti-Mus IgG of biotin (Biotin) labelled goat two anti-ly, what be used for antibody typing is the mountain sheep anti-mouse igg ₁, IgG _2aTwo anti-(γ-chain specific, Southern Biotechnology Associates, Inc.USA), 37 ℃ of incubations 1 hour.After PBS-T gives a baby a bath on the third day after its birth time, add alkali phosphatase enzyme mark the chain mycoprotein (SouthernBiotechnology Associates, Inc.USA), 37 ℃ of incubations 1 hour.At last, after PBS-T gives a baby a bath on the third day after its birth time, add 10mg/ml PNPP (Southern Biotechnology Associates, Inc.USA) colour developing.In 30 minutes, utilize dual wavelength (414nm-405nm) to measure the OD value with microplate reader (Labsystems Multiskan Ascent Finland product), finally determine the high dilution of IgG antibody, determine the height of antibody amount with this.

Take out trachea and the lung of mice, PBS (containing 0.1%BSA) notes are given a baby a bath on the third day after its birth inferior.The centrifugal cell debris that goes of rinsing liquid is used to survey virus titer.With 10 times of serial dilutions of lung washing liquid work, get each dilution factor 0.1ml and cultivated altogether 1 hour with the mdck cell that is adsorbed on 6 orifice plates, allow cell and virus fully adsorb, cover 6 holes with 2ml agar powder culture medium, at CO ₂Cultivated in the incubator two days, plaque forms, and calculates the number of plaque, represents what of virus quantity with plaque forming unit in every ml virus liquid.Each experimental group virus titer is that the mean ± SD with every milliliter of virus titer of all mices of each experimental group represents.

1 week was used lethal dose influenza virus (40LD behind the mice booster immunization ₅₀) attack mice by nasal drip (17 μ l viral suspension).In three weeks, judge the protection effect of Novel DNA vaccine with the survival rate of mice.

Mixed immunity HA and CD40L can protect mouse anti lethal dose influenza viruse attack

Mice is used the lethal dose influenza viruse attack, attacks 3 weeks of back and adds up dead mice and survival mice, determines the survival rate of mice with this.As shown in table 1, the result shows that the survival rate of mixing HA and CD40L immune mouse reaches 100%, and the survival rate of only immune HA dna vaccination mice also can reach 100%.

The survival rate of mice after the virus attack of table 1. lethal dose

The immunity plasmid	The protection effect
	The protection effect	Survival number/attack sum
	HA+CD40L HA empty carrier	Survival number/attack sum	5/5 ^* 5/5 ^* 0/5

^*Significant difference (P＜0.05)

CD40L can improve the attack of mouse anti lethal dose influenza virus

After mice was used the lethal dose influenza viruse attack, the protective rate of mice was as broad as long, yet body weight change had difference after mice was attacked, as shown in Figure 1.For the immunity twice altogether of examination mice, three weeks at interval.In a week behind the booster immunization, use the lethal dose influenza viruse attack.Monitored body weight change and the death of mice at 0～14 day.Weight loss is the average weight of mice among the figure.Wherein control group mice is all dead at the 7th day, and immune HA group and HA+CD40L group mice all survive.The result shows with independent immune HA and compares, common immune HA and CD40L group mice weight loss obviously reduces and weight recovery quickens.Though obtained proof in the experiment before us; 30 μ gHA can provide protection fully; but after the virus attack; the weight loss of mice is more; and behind common immune HA and the CD40L; losing weight seldom of mice, and weight recovery is very fast, and weight loss is very tangible clinical symptoms behind the A type influenza infection mice.This illustrates common immune HA and the intravital virus quantity of the CD40L dna vaccination group mice experiment mice of HA dna vaccination that only will be lower than immunity.

Common immune HA and CD40L dna vaccination have improved the titre of IgG antibody

With the IgG antibody in the ELISA method survey serum.We find to compare with independent immune HA, no matter be just exempt from or booster immunization after, the IgG antibody that common immune HA and CD40L have significantly improved anti-HA produces (Fig. 2), and the antibody horizontal behind the booster immunization is higher than the antibody horizontal after just exempting from.

(Fig. 2 A) mice is just exempted from the back and three weeks got blood by the docking method from afterbody, surveys the IgG antibody of the anti-HA in the serum, and serum was with dilution in 1: 200;

By heart extracting blood, survey the IgG antibody of the anti-HA in the serum behind (Fig. 2 B) booster immunization, serum was with dilution in 1: 4096.Strengthened the specific antibody of mouse anti HA behind common immune HA and the CD40L.Vertical bar and vertical line submeter are represented the meansigma methods and the SD of isotypes absorbance value.

* Student t test is used in significant difference p＜0.05.

The variation of the common immunity of CD40L and HA back IgG antibody subtype

Whether influence the variation of IgG antibody subtype in order to detect the CD40L plasmid, we have detected IgG among the specific serum IgG of anti-HA behind the booster immunization ₁And IgG _2aRelative populations, as shown in Figure 3,10 of every group of mices, immunity twice, at interval three weeks, every group of dosage is 30 μ g HA, 30 μ g HA+30 μ g CD40L.A week is used the lethal dose influenza viruse attack behind the booster immunization, and after three days, every group of mice got five from heart extracting blood.Photometry absorption value during with 1: 2048 times of dilution.Vertical bar and vertical line submeter are represented the meansigma methods and the SD of isotypes absorbance value.

* Student t test is used in significant difference p＜0.05.

After the result shows common immune HA and CD40L, improved the specific IgG1 antibody of anti-HA a little, but IgG2a antibody significantly improves, show that CD40L is partial to Th1 type immunne response, Th1 type immunne response is played the part of very important role in antivirus action.

The present invention adopts influenza virus DNA vaccine adjuvant to compare with vaccine and adjuvant thereof that tradition is used for the prevention of infectious disease, and following characteristics are arranged:

(1), the present invention uses immune adjuvant can reduce the use amount of nucleic acid vaccine, reduces the security hidden danger of nucleic acid vaccine.

(2), the present invention not only strengthened the human body body fluid responsing reaction of immune animal, strengthened simultaneously cell-mediated immune response, and the latter is the important prerequisite condition that improves dna vaccination efficient.

(3), dna vaccination of the present invention produces easily, stability is strong, and is safer. Dry DNA granule is at room temperature relatively stable, does not need refrigerating equipment, and is therefore also comparatively convenient to backwoodsman use. Dna vaccination adds water at once before inoculation just can simply recover original state, and this all is favourable with the public health aspect economically.

(4) preparation method of the present invention is easy, and is cheap.Dna vaccination only needs to produce in antibacterial, makes up efficient expression plasmid, compares with common vaccine, and loaded down with trivial details time-consuming procedure such as antigen extraction and purification have been saved in the making of dna vaccination, make manufacturing cycle shorten greatly.

(5) immunne response of the present invention is lasting, because can there be the long period in vivo in exogenous gene, and constantly expresses foreign protein, it can provide stimulation to immune system constantly, therefore, very micro-antigen can stimulate body to produce strong and lasting immune response.

Generally speaking, the common immunity of CD40L and HA can strengthen the immunne response of influenza virus surface antigen HA gene, and the attack of the anti-lethal dose influenza virus that can more effectively watch for animals is a kind of influenza virus dna vaccination that broad prospect of application is arranged.

Claims

1. the dna vaccination of a flu-prevention virus is characterized in that this vaccine comprises the dna vaccination of adjuvant CD40L and from the strains of influenza viruses HA dna vaccination of HINI hypotype, the HA dna vaccination mixes with mass ratio with the dna vaccination of CD40L at 1: 1.