What is a DNA vaccine? What are the antigenic components?

DNA vaccine, also known as naked DNA vaccine, gene vaccine, nucleic acid vaccine, polynucleotide vaccine and other related names, is a new research field derived and developed in gene therapy in recent years. It means that the recombinant eukaryotic expression vector encoding a protein antigen is directly injected into animals, so that foreign genes are expressed in vivo, and the generated antigen activates the immune system of the body, thereby inducing specific humoral immunity and cellular immune response. The vaccine not only has the advantages of attenuated vaccine. At the same time, there is no danger of reversal, so people pay more and more attention to it, and it is considered to be the third generation vaccine after traditional vaccine and genetic engineering subunit vaccine [1].

The development of DNA vaccine is another major breakthrough of genetic engineering technology in vaccine research. Recombination of antigen gene into eukaryotic expression vector, direct or packaged injection into the body to express the corresponding antigen, and induce the body to produce immune response. This is a new vaccine with great development potential [2].

DNA vaccine; veterinarian

In recent years, many viral infectious diseases of livestock and poultry can not be prevented by traditional vaccines such as inactivated vaccine and attenuated vaccine. The emergence of DNA vaccine improved this situation. Plasmid DNA encoding different kinds of antigen genes such as viruses, bacteria and parasites can cause strong and lasting immune responses in vertebrates such as mammals, birds and fish. DNA vaccine is called the "third generation vaccine" after inactivated vaccine, attenuated vaccine and subunit vaccine, and has broad development prospects.

Brief introduction of 1 DNA vaccine

DNA vaccine, also known as nucleic acid vaccine or gene vaccine, is a eukaryotic expression plasmid DNA (sometimes RNA) encoding immunogen or related to immunogen. It can enter animals through a certain way and be transcribed and translated by host cells to express antigen proteins, which can stimulate the body to produce nonspecific and specific immune responses, thus playing an immune protection role.

DNA vaccine has many advantages: ① The structure of DNA inoculation vector (such as plasmid) is simple, and the process of purifying plasmid DNA is simple, so the production cost is low and it is suitable for mass production; ②DNA molecular cloning is relatively easy, and DNA vaccine can be updated at any time as needed; ③DNA molecules are very stable and can be made into freeze-dried DNA vaccine. When in use, the original activity can be restored in salt solution, which is convenient for transportation and storage. ④ It is safer than traditional vaccine. Although DNA vaccine has the same immunogenicity as attenuated vaccine, it can activate cytotoxic T lymphocytes and induce cellular immunity. However, because the DNA sequence only encodes a single virus gene, there is basically no possibility of toxicity reversal, so there is no danger of increasing the virulence of attenuated vaccines (Davis et al., 1999), and because the antigen-related epitopes of DNA vaccines are relatively stable in the immune system, unlike attenuated vaccines or subunit vaccines, DNA vaccines will lose their epitopes (Donnelly et al.,1. ⑤ Plasmid itself can be used as adjuvant, so DNA vaccine is used without adjuvant, which not only reduces the cost but also is convenient to use (Babiuk et al.,1999); ⑥ By simply mixing a variety of plasmid DNA, antigens with similar biochemical characteristics (such as different strains from the same pathogen) or different antigens of 1 pathogen can be combined into multivalent vaccine, so that 1 DNA vaccine can induce immune protection against multiple epitopes, which greatly increases the flexibility of DNA vaccine production.

Application of DNA vaccine

2. 1. 1 Pseudorabies virus (PRV) Immunizing pigs with plasmid DNA encoding PRVgC or gD gene can induce protective antibodies and cellular immunity; It is more effective to guide immune response by mixing plasmid DNA encoding gB, gC and gD (Gerdts V et al.,1997; Rooij E M et al., 1998).

2. 1.2 The experimental results of swine influenza virus (HIV) Mackling et al. (1998) showed that the plasmid DNA encoding hemagglutinin (HA) and nucleocapsid protein (NP) of HIV/kloc-0 was coated with gold particles, and the pig epidermis was bombarded with gene gun for immunization. The HA plasmid DNA could make pigs produce mucosal immune response.

2. 1.3 The major envelope glycoprotein GP5 encoded by PRRS PRRS gene fragment ORF5 of porcine respiratory and reproductive syndrome virus (PRRS) is one of the three major structural proteins of the virus. Plasmid DNA containing ORF5 gene can induce pigs to produce specific GP5 neutralizing antibodies. And the peripheral blood mononuclear cells of immunized pigs can undergo transformation reaction in the presence of GP5 recombinant protein, which indicates that GP5-specific cellular immunity is produced (Pirzadeh B et al., 1998). Meng (2000) cloned GP5 gene into eukaryotic expression plasmid under the control of early promoter of cytomegalovirus (CMV) to prepare DNA vaccine, which can induce antibody production after immunizing piglets, and has shown good protective effect after laboratory challenge.

2. 1.4 Foot-and-mouth disease virus (FMDV) The full-length genome cDNA of FMDV was cloned into plasmid, and the DNA sequence encoding the cell binding site of nucleocapsid protein VP 1 was removed to construct plasmid DNA for intramuscular or intradermal injection. After 2-4 weeks of initial immunization, all pigs can produce specific virus neutralizing antibodies, which show partial protection in virus attack test (Ward G et al., 65438

2. 1.5 Classical swine fever virus (CSFV) Yu Xinglong et al. (2000) constructed four different eukaryotic expression plasmids of E2 gene, the main protective antigen of CSFV. The results of mouse immunization test showed that different functional regions of E2 gene had great influence on the immune response of gene vaccine. E2 gene with signal peptide sequence can induce specific immune response, E2 gene without transmembrane region sequence can induce stronger immune response than E2 gene with transmembrane region sequence, but E2 gene without signal peptide sequence can not induce specific immune response of CFSV. The results of challenge protection test showed that the immunized rabbits could resist at least 10 dose of classical swine fever attenuated vaccine (HCIV) with the lowest infection dose (MID). Immune pigs can resist lethal dose of CFSV Shimen strain.

2.2. 1 the bovine respiratory syncytial virus (BRSV) DNA vaccine constructed with BRSV G gene has stronger immune response than intradermal or intramuscular injection when calf is immunized without needle.

2.2.2 Bovine herpesvirus (BHV) is caused by BHV? The highly neutralizing antibody can be produced by immunizing cattle with plasmid DNA vaccine of 1 gD gene. After the attack test, it was found that the virus distribution in the immune group was significantly lower than that in the non-immune group (schrijver R S et al., 1997). This vaccine can induce immune response by intramuscular or intradermal injection. However, the immune response caused by intradermal injection is stronger (Meng Songshu et al., 2000). ?

2.2.3 intramuscular injection of bovine viral diarrhea virus (BVDV) expressing plasmid DNA of BVDV 1 major glycoprotein E2 can produce virus neutralizing antibody and antigen-specific cell proliferation reaction; After immunization 16 weeks, it was found that immunized cattle could produce strong memory antibody reaction against BVDV 1 and 2 serum neutralizing antibodies, which had partial immune protection for cattle (Cox G J et al., 1993). ?

2.3. 1 Newcastle disease virus (DNV) Sakaguchi et al. (1996) inserted NDV F gene into plasmid vector, and the expression of F gene was influenced by cytomegalovirus early enhancer and chicken β? Actin promoter control. After intramuscular injection of recombinant plasmid into 1 week-old chickens, 2/5 chickens injected with linear plasmid and 4/5 chickens injected with mixture of linear plasmid and liposome transfection agent produced high levels of anti-F protein antibodies, while chickens injected with closed-loop plasmid could not produce antibodies. After 9 weeks of immunization, the experimental chickens with antibodies in their bodies could resist the attack of lethal dose NDV. ?

2.3.2 infectious laryngotracheitis virus (ILT) The recombinant eukaryotic expression plasmid containing gB, gC and gD genes of Wang Gang strain of infectious laryngotracheitis virus and empty vector plasmid were injected into chicks in groups, and the immune protection effect was observed after challenge. The results showed that the recombinant plasmid could induce immune response, and the immune protection rate reached 79%. The gene vaccine can be used as 1 supplement to prevent ILV. ?

2.3.3 Avian influenza virus Robinson et al. (1993) first applied DNA vaccine to chickens. Immunizing 3-week-old chickens with plasmid (DNA) encoding hemagglutinin (HA) gene of avian influenza virus H7N9 strain through different routes (intravenous injection, intraperitoneal injection and subcutaneous injection) can provide 50% protection against intranasal attack of lethal dose of H7N9 virus. Fyna et al. (1993) code H7? HA plasmid DNA was used to immunize 3-week-old chickens by intravenous injection, intramuscular injection, subcutaneous injection, eye drop, intracapsular injection and nasal drop. The second immunization was carried out after 4 weeks. At the 6th week, when challenged with lethal dose of H7N7, the survival rate of immunized chickens was 10% ~ 63%, while that of the control group was only 2%. Among them, intravenous injection and intramuscular injection have the same immune effect, which is superior to intramuscular injection, eye drops, bursa of fabricius and nasal drops. Cody Harry et al. (1997) will code H5? Chickens were immunized with 0.25, 0.5, 65, 438+0.5 or 65, 438+00 μ g of HA plasmid DNA. Four weeks later, chickens were immunized with 100LD? 50% of Ty/lre/83 strains were attacked by intranasal immunization, as low as 0.25μg DNA, and 50% of immunized chickens could survive. The doses of 1, 5, 10 μg can completely resist the attack of lethal dose virus. DNA vaccine can also provide 95% protection for lethal dose of antigen mutant, which proves that DNA influenza vaccine has broad development prospects.

The H7 subtype hemagglutinin gene DNA vaccine developed in China can successfully induce immune protection response at a very small dose, and effectively block the infection and detoxification of homologous low-toxic avian influenza virus in vivo. H5 subtype hemagglutinin DNA vaccine has good immunogenicity, intramuscular injection can obtain immune protection against homologous virulent attack, and can effectively block the detoxification effect of immune protection of live chickens.

3 Safety of DNA Vaccine Although people have doubts about the safety of using DNA vaccine and are worried that DNA may be integrated into the chromosome of the host cell and cause insertion mutation, many research results have not found the phenomenon of insertion mutation. Plasmid DNA will degrade slowly in animals, which will not cause animals' autoimmunity (Chen Hualan et al., 1998) and will not be introduced into offspring with eggs and sperm. As the biological chain enters other species, it will also be inactivated, causing little pollution to the environment, so its risk is far lower than that of the current vaccine.