Vaccination is the process of injecting antigenic material to stimulate immune response. Conventional vaccines insert attenuated or killed infectious agent, such as virus or antigenic protein, into the body to stimulate immune system to develop immunity against the pathogen. However, this type of vaccines has some weakness. For example, attenuated virus may suddenly become very active in the body and cause disease instead of stimulating immune response. Conventional vaccines also unable to stimulate immune system to develop protection from some viruses such as African swine fever virus and Human Immunodeficiency Virus. A new approach to make vaccines is by utilizing gene cloning technique and DNA sequence. This type of vaccination is called DNA vaccination.
DNA vaccination is a method used to generate antigen-specific antibody and cell-mediated immunity. The basic concept of DNA vaccine is to clone target gene into a mammalian expression. In other words, this type of vaccination allows mammalian cells to uptake DNA sequence code for antigenic protein (such as viral RNA) and express it as protein. This protein will be recognized as foreign to cells and induce immune response. Before a vaccine is made, it is important to decide whether vaccine is to induce antibodies for protection against infection or to eliminate established infection or tumor. Vaccine for preventing infection mostly designed to induce neutralizing antibody, and the target is usually protein on the surface of pathogen in its extracellular state. For example, DNA vaccination utilize protein L1, which could self-assembled to mimic papillomavirus natural surface, to form protection against papillomavirus. On the other hand, vaccine for eliminating infection aim to induce cell-mediated immune response, and the target are antigens that are expressed intracellularly during infection. For example, the Epstein-Barr virus (EBV) nuclear antigen I (EBNA 1) have a domain that protects the protein from degraded by proteasome. Removing this domain allows EBNA 1 to be recognized by immune system. The first step of making DNA vaccines is obtaining DNA sequence of pathogen (for example virus). If the source is RNA, then RNA should be converted to DNA by reverse transcription. Next, based on DNA sequence generated from RNA, a cDNA coding for specific region of DNA is created. The cDNA requires a plasmid backbone as the vector to bring it to the host cell. An ideal vector should be safe when put into human body, easily produced, and have strong promoter to drive the expression of transgene of interest, otherwise vaccination will have no use. Usually, pcDNA3 is used as vector because it contains the cytomegalovirus (CMV) strong promoter. Strong promoter is able to drive protein expression fast, thus benefit for inducing a strong immune response. After insertion of cDNA, vector is then injected into bacterial plasmid for massive production through bacteria replication. DNA plasmid is then purified and injected into a person. Purification process is very important to remove impurities such as RNA, proteins, or process additives. Purification process have three steps: (1) cell lysis to release plasmid DNA, (2) primary purification and concentration, and (3) secondary purification. Alkaline lysis is usually used to lyse bacteria to obtain lysate containing plasmid DNA and impurities. The second process, primary purification and concentration, starts with precipitating plasmid DNA using agents. Precipitate is recovered and concentrated by filtration process using tangential-flow filtration (TFF). Obtained plasmid DNA is then purified once more using chromatography techniques to remove remaining impurities. It is important to point that chromatography …show more content…
Injection is commonly done using needle injection. However, immune response generated is weak, mainly due to inefficient uptake of plasmids by cell, resulting in lower level of antigenic protein produced. There are also some inconveniences regarding injection of vaccines using needle. To solve these problems, many new strategies such as: (1) electroporation, (2) chemical (liposome and various polymers) and mucosal delivery, and (3) needle-free injection are being tested out. Electroporation induces temporary pores to allow cell membrane uptake bulky molecules such as plasmid DNA. Liposomes and mucosal delivery are able to fuse with cell and release their content. Needle-free injection arise due to recent awareness of needle-associated transmission diseases such as HIV or hepatitis B. Needle-free injection works by forcing liquid at high speed and pressure through a micro-orifice that is held against the
DNA vaccination is a method used to generate antigen-specific antibody and cell-mediated immunity. The basic concept of DNA vaccine is to clone target gene into a mammalian expression. In other words, this type of vaccination allows mammalian cells to uptake DNA sequence code for antigenic protein (such as viral RNA) and express it as protein. This protein will be recognized as foreign to cells and induce immune response. Before a vaccine is made, it is important to decide whether vaccine is to induce antibodies for protection against infection or to eliminate established infection or tumor. Vaccine for preventing infection mostly designed to induce neutralizing antibody, and the target is usually protein on the surface of pathogen in its extracellular state. For example, DNA vaccination utilize protein L1, which could self-assembled to mimic papillomavirus natural surface, to form protection against papillomavirus. On the other hand, vaccine for eliminating infection aim to induce cell-mediated immune response, and the target are antigens that are expressed intracellularly during infection. For example, the Epstein-Barr virus (EBV) nuclear antigen I (EBNA 1) have a domain that protects the protein from degraded by proteasome. Removing this domain allows EBNA 1 to be recognized by immune system. The first step of making DNA vaccines is obtaining DNA sequence of pathogen (for example virus). If the source is RNA, then RNA should be converted to DNA by reverse transcription. Next, based on DNA sequence generated from RNA, a cDNA coding for specific region of DNA is created. The cDNA requires a plasmid backbone as the vector to bring it to the host cell. An ideal vector should be safe when put into human body, easily produced, and have strong promoter to drive the expression of transgene of interest, otherwise vaccination will have no use. Usually, pcDNA3 is used as vector because it contains the cytomegalovirus (CMV) strong promoter. Strong promoter is able to drive protein expression fast, thus benefit for inducing a strong immune response. After insertion of cDNA, vector is then injected into bacterial plasmid for massive production through bacteria replication. DNA plasmid is then purified and injected into a person. Purification process is very important to remove impurities such as RNA, proteins, or process additives. Purification process have three steps: (1) cell lysis to release plasmid DNA, (2) primary purification and concentration, and (3) secondary purification. Alkaline lysis is usually used to lyse bacteria to obtain lysate containing plasmid DNA and impurities. The second process, primary purification and concentration, starts with precipitating plasmid DNA using agents. Precipitate is recovered and concentrated by filtration process using tangential-flow filtration (TFF). Obtained plasmid DNA is then purified once more using chromatography techniques to remove remaining impurities. It is important to point that chromatography …show more content…
Injection is commonly done using needle injection. However, immune response generated is weak, mainly due to inefficient uptake of plasmids by cell, resulting in lower level of antigenic protein produced. There are also some inconveniences regarding injection of vaccines using needle. To solve these problems, many new strategies such as: (1) electroporation, (2) chemical (liposome and various polymers) and mucosal delivery, and (3) needle-free injection are being tested out. Electroporation induces temporary pores to allow cell membrane uptake bulky molecules such as plasmid DNA. Liposomes and mucosal delivery are able to fuse with cell and release their content. Needle-free injection arise due to recent awareness of needle-associated transmission diseases such as HIV or hepatitis B. Needle-free injection works by forcing liquid at high speed and pressure through a micro-orifice that is held against the