CN1281277C - DNA vaccine for preventing influenza virus infection - Google Patents

Abstract

The invention relates to a DNA vaccine for preventing influenza virus. The invention adopts CD40L and influenza virus DNA to immunize animals together, and the immune response induced in mice can well protect mice against lethal influenza virus attack, and the anti-influenza effect is better than that of DNA vaccine without CD40L. The DNA vaccine of the invention immunizes animals, has the advantages of simple preparation method, low price, convenient use and good immune effect, overcomes the shortcomings of known influenza prevention products, and provides a relatively safe and effective influenza prevention product.

Description

A kind of anti-influenza virus DNA vaccine

Technical field:

The invention belongs to a biological product, in particular to a vaccine for preventing influenza virus.

Background technique:

Existing influenza biological prevention product and preparation method have following kind: (1), influenza virus inactivated vaccine. The whole virus inactivated vaccine is a virus vaccine made by applying physical or chemical methods to completely inactivate (lose infectivity) the virus particles on the basis of the whole virus. Compared with other vaccines, the inactivated influenza vaccine has the advantages of simple production method and easy control of the production process, but it also has its obvious disadvantages. Since the influenza virus used for preparing the vaccine needs to be cultured and grown in the allantoic cavity of the chicken embryo, the preparation period is long, and the immunogenicity will be reduced or changed. Inactivated flu vaccines are not long-lasting. The immune response induced by it is short-lived even against homologous strains, so annual or bi-annual vaccination is required. (2) Influenza subunit vaccine. Influenza subunit vaccine refers to the effective viral surface antigen components obtained from wild influenza virus strains after cultivation, lysis and purification, mainly HA and NA vaccines. However, the production process of the traditional preparation method of subunit vaccines is relatively cumbersome, and it is difficult to obtain a large amount of antigens. Therefore, subunit vaccines are expensive and difficult to promote and apply. (3) Live attenuated influenza vaccine. Live attenuated influenza vaccines have demonstrated a broader immune response in mice. As an effective vaccine, the gene reassortant cold-adapted strain must have 2 RNA segments encoding HA and NA derived from the wild strain, while the other 6 segments must be derived from the cold-adapted donor strain. It takes about 5 weeks from obtaining the clone of the wild virus strain to preparing the live attenuated vaccine strain. Before inoculating the population, the vaccine strain must also be cultured in the allantoic cavity of special sterile eggs to further test its toxicity. The attenuation treatment of the live attenuated influenza vaccine is only empirical, and there is still the possibility of reverting to its virulence. Especially for cold-adapted vaccine strains, due to the need for multiple passages, the time is very long. When the virus is prevalent, it is not realistic to carry out cold-adapted passage of the popular strains. (4) Influenza synthetic peptide vaccine. Artificially synthesized peptides with the same amino acid sequence as the determinants of influenza virus protective antigens (HA, NA, etc.), prepared as immunogens and inoculated to animals or humans, can promote the body to produce protective antibodies. This peptide is A synthetic peptide vaccine for influenza. There are some theoretical and practical difficulties in synthetic peptide vaccines: one is how to find and construct the peptide with the best antigenic determinant; the other is that the immunogenicity of synthetic peptides is weak, and adjuvants must be used when using them. Since Freund's adjuvant cannot be used in humans, the clinical evaluation of synthetic peptides urgently requires the development of an effective adjuvant that can be used in humans. (5) Influenza genetically engineered subunit vaccine. Insert the gene encoding the influenza antigenic determinant (HA, NA, etc.) that induces a protective immune response into the expression vector DNA, and then introduce the vector into yeast, insect or mammalian cells to express the virus antigen protein, and the product is obtained after purification A genetically engineered subunit vaccine for influenza. The disadvantage of genetically engineered subunit vaccines is the heavy workload of screening positive expression clones. Expressed proteins are sometimes not properly folded or modified, which can affect immunogenicity. The process of separation and purification of recombinant proteins is sometimes relatively complicated. (6) Influenza virus DNA vaccine. DNA Vaccine (DNA Vaccine) is to directly introduce the exogenous gene encoding a certain antigenic protein into the animal body cells, and synthesize the antigenic protein through the expression system of the host cell, and induce the host to generate an immune response to the antigenic protein, so as to achieve prevention and purpose of treating disease. DNA vaccines are composed of two parts: protective antigen genes of pathogens (including viruses and bacteria, etc.) and carrier plasmids. Plasmid DNA must meet the following conditions: ①Have a bacterial replicon (Ori) to ensure that the plasmid can replicate in Escherichia coli; ②Have a resistance gene for selection, and the selection gene can be kanamycin, ampicillin or neomycin ③ eukaryotic promoters (some containing enhancers), such as CMV, SV40 promoters; ④ with Poly A sequence, can ensure the stability of mRNA in vivo, this stability factor Poly A sources vary. At present, it is believed that the better Poly A is derived from the bovine growth hormone gene (BGH). DNA vaccines are introduced in a variety of ways, including intramuscular injection, intradermal injection, intranasal drip or nasal spray, liposome method, and newly developed gene gun immunization and live electric shock immunization. Influenza viruses are still one of the main causes of human death, because influenza viruses are prone to mutations, so humans are still unable to conquer influenza. Vaccination is an effective way to prevent influenza, DNA vaccine (DNA vaccine) provides a new choice for our country. In 1993, Ulmer JB et al. injected the plasmid encoding the NP gene of influenza virus A/PR/8/34 into the quadriceps muscle of BALB/c mice, which produced a strong and specific CTL response, which could provide cross-protection. Pioneered the research on the use of nucleic acid vaccines to prevent influenza viruses [Ulmer JB et al Science.1993 Mar 19; 259(5102): 1745-9.]; Chen Ze et al cloned the HA coding gene into chicken β-actin expression In the carrier, inoculate twice at intervals of 3 weeks, using gene gun or live electric shock immunization, the inoculation dose is 1 μg DNA per mouse for gene gun, and 30 μg DNA per mouse for live electric shock immunization. Seven days after the booster immunization, challenge with the homologous virus strain. The results show that mice inoculated with HA DNA vaccine can effectively resist virus 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 HA gene should be used as an important component of influenza DNA vaccine. DNA vaccines can induce the body to produce humoral immunity and cellular immunity in the immune response. DNA vaccines have been successfully applied to animals such as mice, sheep, chickens, cats, cows, pigs, horses, monkeys and chimpanzees. The efficiency in the response is relatively low, which affects its practical application. Therefore, it is a problem to be solved to find other DNA molecules to improve the immune efficiency of nucleic acid vaccines.

Invention content:

The invention aims to develop a high-efficiency DNA vaccine for preventing influenza virus, so as to improve the immune efficiency of the DNA vaccine.

The purpose of the above invention is achieved through the following technical solutions. The vaccine of the present invention is a DNA vaccine for preventing influenza virus using CD40L as an adjuvant, and the HA DNA vaccine and CD40L are jointly immunized with a mass ratio of 1:1.

The present invention is described in further detail below.

Description of drawings:

Figure 1 is a graph showing the results of weight loss (g) and recovery after infection of virus-challenged mice.

Figure 2 is a graph showing the titration results of virus-specific serum IgG.

Figure 3 is a graph showing the results of IgG antibody subtype levels in mice after co-immunization with HA and CD40L (after booster immunization).

CD40L is a member of the tumor necrosis factor superfamily. It contains 261 amino acids and is a type II transmembrane glycoprotein located on Xq24. Its relative molecular weight (Mr) is 39000. [Eur J Immunol.1992Dec; 22(12): 3191-4]

The high-efficiency DNA vaccine for preventing influenza of the present invention is prepared through the following steps: obtaining influenza virus antigen HA and CD40L genes, constructing HA and CD40L DNA vaccines, and identifying HA and CD40L DNA vaccines.

Acquisition of Influenza Virus Antigen HA and CD40L Genes

Viral RNA was extracted from viruses propagated in 10-day-old chicken embryos. RNA was reverse-transcribed to obtain single-stranded cDNA. The HA gene was amplified by PCR using cDNA as a template. The forward primer was 5'AAC CTC GAG AAT GAA GGC AAA CCT ACT GGT CC-3', and the reverse primer was 5'AAC CCC GGG TCT CAG ATG CATATT CTG CAC TGC A-3'. The forward primer contains an Xho I restriction site, and the reverse primer contains a Sma I restriction site.

Using the cDNA library of mouse spleen cells (Clontech Company) as a template, the CD40L gene was cloned by PCR. The forward primer was 5'TTC CTC GAG CAT GAT AGA AAC ATA CAG-3', and the reverse primer was 5'TGA CCC GGG GTA TAG GGA AGA CTG CCA-3'. The forward primer contains an Xho I restriction site, and the reverse primer contains a Sma I restriction site.

Construction of HA and CD40L DNA vaccine

The low-melting-point agarose gel recovered the PCR product, connected it into the pGEM-T vector, transformed Escherichia coli, and determined whether the HA and CD40L genes had been connected into the T vector through plasmid extraction and enzyme digestion identification. The identified recombinants were then amplified in large quantities, purified and recovered. The T vector with HA and CD40L genes was digested with Xho I-Sma I, and the HA and CD40L fragments excised from the T vector were recovered at a low melting point and cloned into the expression vector pCAGGSP7 that had been treated with the same restriction enzymes to obtain the recombinant plasmid pCAGGSP7/ HA, pCAGGSP7/CD40L. The nucleotide sequences of HA and CD40L genes were confirmed by 377 DNA sequencer (Applied Biosystem.U.S.A.).

The eukaryotic expression vector pCAGGSP7 is derived from pCAGGSP7 [Niwa H et al. Gene 1991, 108: 103-200.] constructed by Niwa et al, which is the multiple cloning sites Kpnl, Xhol, Clal, EcoRV, Smal, Notl and Sacl Insertion into the EcoRl site of pCAGGS yielded pCAGGSP7. Plasmid pCAGGS contains chicken β-actin promoter component, SV40 origin of replication (Ori), ampicillin resistance gene, CMV transient enhancer, bovine growth hormone gene (BGH) poly A.

Identification of HA and CD40L DNA vaccines

Plasmids encoding HA and CD40L were respectively amplified in Escherichia coli XL1-blue and purified with QIAGEN purification kit (QIAGEN, Tip500). The concentration and purity of the plasmid were measured by ultraviolet spectrophotometry. The concentration and purity of the DNA were determined by OD260 and OD280. The plasmid DNA with a ratio of OD260/OD280 of 1.8-2.0 was selected to immunize mice.

HA and CD40L plasmids were cryopreserved at -20°C, thawed before immunizing mice and mixed with HA and CD40L plasmids according to the dosage.

Immunization experiment steps and experiment results

The HA used in the immune experiment is from the influenza virus strain of H1N1 subtype (A/PR/8/34), and its full name is hemagglutinin.

A/PR/8/34 Physiochemical properties of HA (hemagglutinin):

The major surface glycoprotein of this strain is hemagglutinin (HA). It has the property of agglutinating red blood cells of various animals.

HA is encoded by influenza virus RNA fragment 4 and is a typical type I glycoprotein, which contains four structural domains: signal peptide (leader sequence), cytoplasmic domain, transmembrane domain and extracellular domain. HA consists of about 562-566 amino acids, and there is a signal peptide consisting of 16 hydrophobic amino acids at the amino terminal of HA. Immediately following the signal peptide is the HA1 part consisting of 328 amino acid residues, and the carboxy-terminal consists of 221 amino acid residues forming HA2. There is an arginine residue between HA1 and HA2. The carboxy-terminal (185-211 amino acid region) of HA2 is mainly composed of hydrophobic amino acid residues. Most of the last 10 amino acid residues are hydrophilic.

The three-dimensional structure of HA: the HA protein of influenza virus exists on the bilayer lipid membrane in the form of trimer, that is, the HA fiber is composed of three HA monomer molecules, and the total length of the monomer is 13.4nm. It can be divided into two parts, one is the spherical head, composed of HA1, containing the receptor binding site and antigenic determinant; the other part is the stalk, composed of HA2 and part of HA1, connected with the capsule, about 7.6 nm. The distance between the amino terminus of HA2 and the carboxyl terminus of HA1 is 2.1 nm. The amino terminus of HA2 is located at the trimer interface of the molecule, 3 nm away from the viral membrane, and it is rich in glycine residues, forming an unusual helical structure extending out of the trimer interface.

As experimental animals, 8-week-old BALB/c female mice were selected.

Intraperitoneal injection of anesthesia (ketamine hydrochloride, lobeline hydrochloride mixture) to anesthetize the mouse, wipe the quadriceps muscle of the right hind leg of the mouse with alcohol, and then use a disposable syringe to absorb the solution dissolved in TE (1M Tris, 0.5M EDTA) DNA solution, insert the needle vertically into the quadriceps muscle, and slowly inject the DNA. A group of BALB/c mice were immunized with 30 μg HA (1 μg/μl) and 30 μg CD40L (1 μg/μl) in a total volume of 30 μl; a group of BALB/c mice were immunized with 30 μg HA (1 μg/μl) in a total volume of 30 μl; The mice were immunized with the empty vector of exogenous gene as negative control. Insert the two electrodes of the electric shock instrument on both sides of the intramuscular injection needle hole, with a distance of 0.5cm, and then electric shock (voltage 100v, electric shock time 50ms, positive and negative shocks three times each, with an interval of 1s), to complete one immunization. In the third week after immunization, the same dose and composition of DNA were used to boost the immunization.

Mice were anesthetized with chloroform, and blood was collected from the heart. Make the mice lie on their backs on the experimental table, insert the needle of the syringe at an angle of 15-20 degrees at the slightly left side under the xiphoid cartilage, and slowly draw blood until the blood flow stops. The collected blood was left at room temperature for about 1 hour. After coagulation, the serum is precipitated. Centrifuge at 4000 rpm for 10 minutes. The serum was aspirated under sterile conditions and stored in a -20°C refrigerator.

HA-specific IgG antibodies were detected by enzyme-linked immunosorbent assay (ELISA). _Coat 96-well ELISA plate with 10 _μg /mL inactivated vaccine, ₃₇ ° _C for 2 hours; g, Tween-20 0.454ml) was washed three times and then added with blocking solution (1 liter of solution contains 8.0g of NaCl, 0.2g of KCl, 1.15g of Na ₂ HPO ₄ , 0.2g of KH ₂ PO ₄ , 10g of bovine serum albumin), 4 ℃ overnight. After diluting the antiserum by a factor of 2 with blocking solution, add it to the microtiter plate and incubate at 37°C for 1 hour. After washing with PBS-T three times, a goat anti-mouse IgG secondary antibody labeled with Biotin was added, and goat anti-mouse IgG ₁ and IgG _2a secondary antibodies (γ-chain specific, Southern Biotechnology Associates, Inc.USA), incubated at 37°C for 1 hour. After washing with PBS-T three times, alkaline phosphatase-labeled streptavidin (Southern Biotechnology Associates, Inc. USA) was added and incubated at 37°C for 1 hour. Finally, after washing three times with PBS-T, 10 mg/ml PNPP (Southern Biotechnology Associates, Inc. USA) was added for color development. Within 30 minutes, use a microplate reader (Labsystems Multiskan Ascent produced in Finland) to measure the OD value with dual wavelengths (414nm-405nm), and finally determine the highest dilution of antibody IgG to determine the level of antibody.

The trachea and lungs of the mice were taken out, washed three times with PBS (containing 0.1% BSA). The rinsing solution was centrifuged to remove cell debris for virus titer determination. Make a 10-fold serial dilution of the lung wash solution, take 0.1ml of each dilution and co-culture with MDCK cells adsorbed on a 6-well plate for 1 hour to allow the cells and viruses to fully adsorb, and cover 6 wells with 2ml of agar powder medium, Cultivate in the _CO2 incubator for two days, plaques are formed, calculate the number of plaques, and express the amount of virus in plaque formation units per ml of virus fluid. The virus titer of each experimental group is represented by the mean±SD of the virus titer per milliliter of all mice in each experimental group.

One week after the booster immunization, the mice were challenged with a lethal dose of influenza virus (40LD ₅₀ ) by intranasal drop (17 μl of virus suspension). The survival rate of the mice was used to judge the protective effect of the new DNA vaccine within three weeks.

Mixed immunization with HA and CD40L protects mice against lethal influenza virus challenge

The mice were challenged with a lethal dose of influenza virus, and the dead mice and surviving mice were counted 3 weeks after the challenge to determine the survival rate of the mice. As shown in Table 1, the results showed that the survival rate of mice immunized with mixed HA and CD40L reached 100%, and the survival rate of mice immunized only with HA DNA vaccine could also reach 100%.

Table 1. Survival rate of mice after lethal virus challenge immune plasmid protective effect Survives/Total Attacks HA+CD40LHA empty vector 5/5 ^* 5/5 ^* 0/5

^* Significant difference (P＜0.05)

CD40L can improve mice's resistance to lethal influenza virus challenge

After the mice were challenged with a lethal dose of influenza virus, there was no difference in the protection rate of the mice, but the body weight changes of the mice after the challenge were different, as shown in Figure 1. The tested mice were immunized twice with an interval of three weeks. One week after the booster immunization, they were challenged with a lethal dose of influenza virus. Mice were monitored for body weight changes and death on days 0-14. Body weight loss in the figure is the average body weight of the mice. All the mice in the control group died on the 7th day, and all the mice in the immunized HA group and the HA+CD40L group survived. The results showed that compared with immunization with HA alone, mice co-immunized with HA and CD40L significantly reduced body weight loss and accelerated weight recovery. Although it has been proved in our previous experiments that 30 μg HA has been able to fully provide protection, after virus challenge, the mice lost more body weight, but after co-immunization with HA and CD40L, the mice lost little body weight and recovered Soon, weight loss is a very obvious clinical symptom after influenza A virus infection in mice. This shows that the amount of virus in the mice of the co-immunized HA and CD40L DNA vaccine group is lower than that of the experimental mice immunized only with the HA DNA vaccine.

Co-immunization with HA and CD40L DNA vaccines increased IgG antibody titers

IgG antibodies in serum were detected by ELISA. We found that co-immunization with HA and CD40L significantly increased the production of IgG antibodies against HA compared with immunization with HA alone (Figure 2), and the antibody level after the booster immunization was higher than that after the prime immunization antibody levels.

(Fig. 2A) Three weeks after the initial immunization of the mice, blood was taken from the tail by the tail docking method, and the IgG antibody against HA in the serum was measured, and the serum was diluted 1:200;

( FIG. 2B ) Blood was collected from the heart after the booster immunization, and the anti-HA IgG antibody in the serum was measured, and the serum was diluted 1:4096. Co-immunization with HA and CD40L enhanced the specific antibodies against HA in mice. Vertical bars and sub-tables represent mean and SD of absorbance values of isotype antigens.

*Significant difference p<0.05, using Student's t test.

Changes of IgG antibody subtypes after co-immunization with CD40L and HA

In order to detect whether the CD40L plasmid affects the change of the IgG antibody subtype, we detected the relative amount of IgG ₁ and IgG _2a in the anti-HA specific serum IgG after the booster immunization, as shown in Figure 3, 10 mice in each group, immunized Twice, with an interval of three weeks, each dose was 30 μg HA, 30 μg HA+30 μg CD40L. One week after the booster immunization, they were challenged with a lethal dose of influenza virus. Three days later, five mice in each group were taken to collect blood from the heart. The light absorbance was measured at 1:2048 times dilution. Vertical bars and sub-tables represent mean and SD of absorbance values of isotype antigens.

*Significant difference p<0.05, using Student's t test.

The results showed that after co-immunization with HA and CD40L, the anti-HA specific IgG1 antibody was slightly increased, but the IgG2a antibody was significantly increased, indicating that CD40L was biased towards Th1 type immune response, and Th1 type immune response played a very important role in the antiviral effect.

The influenza virus DNA vaccine adjuvant that the present invention adopts compares with traditional vaccine and adjuvant thereof for the prevention of infectious diseases, has the following characteristics:

(1), the use of immune adjuvants in the present invention can reduce the usage amount of nucleic acid vaccines and reduce the potential safety hazards of nucleic acid vaccines.

(2) The present invention not only enhances the body fluid response of the immunized animals, but also enhances the cell-mediated immune response, which is an important prerequisite for improving the efficiency of DNA vaccines.

(3), the DNA vaccine of the present invention is easy to produce, has strong stability and is relatively safe. Dried DNA pellets are relatively stable at room temperature and do not require refrigeration equipment, so it is more convenient to use in remote areas. DNA vaccines can be reconstituted simply by adding water immediately before vaccination, which is an economic and public health advantage.

(4) The preparation method of the present invention is simple and cheap. DNA vaccines only need to be produced in bacteria to construct high-efficiency expression plasmids. Compared with ordinary vaccines, the production of DNA vaccines eliminates the tedious and time-consuming process of antigen extraction and purification, which greatly shortens the preparation cycle.

(5) The immune response of the present invention is durable. Because the exogenous gene can exist in the body for a long time and continuously expresses the exogenous protein, it can continuously provide stimulation to the immune system. Therefore, a very small amount of antigen can stimulate the body to produce a strong immune response. And durable immune response.

In general, the co-immunization of CD40L and HA can enhance the immune response of influenza virus surface antigen HA gene, and can more effectively protect animals against lethal influenza virus attack. It is a kind of influenza virus DNA vaccine with broad application prospects.

Claims (1)

1. A DNA vaccine for preventing influenza virus is characterized in that the vaccine comprises the DNA vaccine of adjuvant CD40L and the influenza virus strain HA DNA vaccine from the HINI subtype, and the DNA vaccine of HA DNA vaccine and CD40L is in a mass ratio of 1: 1. mix.

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