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Recombinant DNA technology in recent years has become a boon to produce new generation vaccines. These are the genetically modified pathogenic organisms (bacteria or viruses) that are made non-pathogenic and used as vaccines.
These are the genetically modified viral vectors that can be used as vaccines against certain pathogens.
As already stated, subunit recombinant vaccines are the components (proteins, peptides, DNAs) of the pathogenic organisms. India is the fourth country (after USA, France and Belgium) in the world to develop an indigenous hepatitis B vaccine.
Biotechnologists have been successful in inserting hepatitis B gene into the cells of the tomato plant. This cDNA was then digested with restriction enzymes and the fragments were cloned by using plasmid pBR322 in E. Each one of these short peptides (domains) was bound to the surface of a carrier protein (Fig.
This gene for gD was cloned in a mammalian vector and expressed in Chinese hamster ovary (CHO).
In some countries, particularly the developing ones, BCG vaccine is widely used to protect against tuberculosis.
Group B strain of meningococci, namely Neisseria meningitidis causes meningitis in adolescents and young adults. The development of two subunit vaccines, specifically the glycoproteins of HIV envelope is described here. On entering the body, the HIV binds to the host cells (T-lymphocytes) by attaching gp120 to the CD4 receptor sites on the cell surface. Biotechnologists have isolated the genes for gp120 and gp41 and inserted them into the bacterium E. The description of vaccine development against AIDS (given above), which appears attractive is not so simple. Genetic immunization by using DNA vaccines is a novel approach that came into being in 1990.
The plasmid carrying DNA vaccine normally contains a promoter site, cloning site for the DNA vaccine gene, origin of replication, a selectable marker sequence (e.g. DNA vaccine—plasmids can be administered to the animals by one of the following delivery methods.
An illustration of a DNA vaccine and the mechanism of its action in developing immunity is given in Fig.
As the antigen molecules bind to B- lymphocytes, they trigger the production of antibodies which can destroy the pathogens. The protein fragments of the antigen bound to MHC molecules can activate the cytotoxic T-lymphocytes. A novel approach for the production of antigens as well as antibodies by DNA vaccine was developed in 1997. The ultimate goal of scientists is to choose the right DNA fragment from the pathogen to serve as a vaccine for the strongest immune response against the invading pathogen. The immune response for each one of the DNA vaccines can be studied by injecting the pathogen.
Since 1990, several groups of workers world-over have been trying to develop DNA vaccines against various diseases in experimental animals.
The cut pieces of potato leaves are exposed to an antibiotic which can kill the cells that lack the new genes. In the early years of vaccine research, attenuated strains of some pathogenic organisms were prepared by prolonged cultivation — weeks, months or even years.
It is now possible to genetically engineer the organisms (bacteria or viruses) and use them as live vaccines, and such vaccines are referred to as attenuated recombinant vaccines. The advantage with attenuated vaccines is that the native conformation of the immunogenic determinants is preserved; hence the immune response is substantially high. Some of the important attenuated vaccines developed by genetic manipulations are briefly described. Cholera is an intestinal disease characterized by diarrhea, dehydration, abdominal pain and fever. By genetic engineering, it was possible to delete the DNA sequence encoding A1 peptide and create a new strain of V. The development of a new strain of Vibrio cholera that can effectively serve as an attenuated recombinant vaccine is depicted in Fig. A group workers have developed a gene altered potato containing attenuated cholera vaccine.
The different strains of Salmonella genus are responsible for causing typhoid, enteric fever, food poisoning and infant death. Aro genes encode for the enzymes responsible for the biosynthesis of aromatic compounds, while pur genes encode for enzymes of purine metabolism. The doubly deleted strains have a very restricted growth in vivo, while they can stimulate immunological response. Leishmania species are flagellated protozoan parasites and are responsible for the disease leishmaniasis. An attenuated strain of leishmania has been created and successfully used in mice to offer immunoprotection against leishmaniasis.
Vaccinia viruses is basically the vaccine that was originally used by Jenner for the eradication of smallpox.
The vaccinia virus can replicate in the host cell cytoplasm (of the infected cells) rather than the nucleus. The vaccinia viruses are generally harmless, relatively easy to cultivate and stable for years after lyophilization (freeze-drying). The advantage with vector vaccine is that it stimulates B-lymphocytes (to produce antibodies) and T-lymphocytes (to kill virus infected cells). Antigen genes for certain diseases have been successfully incorporated into vaccinia virus genome and expressed.
Most of the work on the development of live viral vaccines has been carried out on vaccinia virus. It is known that the antigens located on the surface of a bacterial cell are more immunogenic than the antigens in the cytoplasm.
Help Our Community Answer Your Questions Better By Including In Your Post: Gender, Have You Been Tested For An STD Yet. The three types are: (1) Subunit Vaccines (2) Attenuated Recombinant Vaccines and (3) Vector Recombinant Vaccines.
By this approach, some of the limitations (listed above) of traditional vaccine production could be overcome. Some of the developments made in the production of recombinant vaccines against certain diseases are briefly described. The advantages of these vaccines include their purity in preparation, stability and safe use. Immunization against hepatitis B is strongly recommended to anyone coming in contact with blood or body secretions. A formalin killed foot and mouth disease virus (FMDV) was previously used to vaccinate against this disease. This is mainly because a short peptide usually is not enough to be sufficiently immunogenic, since it may not have the same conformation as that of the original viral particle.

In addition, it also causes sexually transmitted diseases, encephalitis and severe eye infections.
It is often fatal, and as per some estimates nearly 3 million deaths occur every year due to this highly infectious disease. However, countries like United States have not approved BCG vaccination for various reasons.
Meningitis is characterized by inflammation of the membranes covering brain and spinal cord.
They identified 350 proteins (potential protective antigens) and the entire sequence of genome coding for these proteins in N. This disease is characterized by immunosuppression, neo-plasma and neurological manifestations.
Some of the B-lymphocytes become memory cells that can protect the host against future infections. In the experiments conducted in mice, researchers injected plasmids containing the genes for malarial parasite and also the genes for the antibodies against malarial parasite.
For this purpose, the pathogen’s DNA can be broken into fragments and a large number of vaccines DNA—plasmids can be prepared. By screening the DNA fragments of the pathogenic genome, it is possible to choose one or few DNA vaccines that can offer maximal immune protection. The tedious and costly procedures of purifying antigens or creating recombinant vaccines are not necessary.
In general, DNA vaccines elicit much higher immune response compared to other kinds of vaccines. DNA vaccines are more stable for temperature variations (low or high) than the conventional vaccines. The post-translational modification of the gene (DNA vaccine) product in host cells may not be the same as that found in the native antigen. The first clinical trials in humans, using a plant- derived vaccine were conducted in 1997.
Although the reasons are not known, the infectious organism would lose its ability to cause disease but retains its capability to act as an immunizing agent. Genetic manipulation of non-pathogenic organisms to carry and express antigen determinants from pathogenic organisms. The DNA sequence of A1 peptide is incorporated into a plasmid, cloned and digested with restriction enzymes (Clal and Xbal). The plasmid, containing the deleted A1 peptide sequence is transferred by conjugation into the V. Recombination can occur between the plasmid (containing a small portion of peptide A1 coding sequence) and the chromosome of V. The genetically engineered attenuated vaccines of Salmonella have been shown to be effective as oral vaccines in experimental animals (mice, cattle, sheep, and chickens). In Leishmania major, the genes encoding dihydrofolate reductase-thymidylate synthase can be replaced by the genes encoding resistance to antibiotics G-418 and hygromycin. This is possible since the vaccinia virus possesses the machinery for DNA replication, transcription-DNA polymerase, RNA polymerase etc. Thus, vector vaccines have been developed against hepatitis, influenza, herpes simplex virus, rabies, angular stomatitis virus and malaria. Now genes from hepatitis B virus, herpes simplex virus and influenza virus are added one after another and inserted into vaccinia virus genome. As the viral replication occurs, the plasmids are taken up to produce recombinant vaccinia viruses. There is a possibility of vaccinating several diseases with one recombinant vaccinia virus. There may be serious complications of using recombinant viral vaccines in immunosuppressed individuals such as AIDS patients. Other viruses such as adenovirus, poliovirus and varicella-zoster virus are also being tried as recombinant vaccines. Based on this observation, scientists have developed strategies to coat the surfaces of non-pathogenic organisms with antigens of pathogenic bacteria. A synthetic oligonucleotide encoding the epitope of cholera toxin B subunit was inserted into Salmonella flagellin gene. Therefore, pneumatic rubber fenders serves as a critical protective medium against collision when ship-to-ship (STS) transfer operations and ship-to-dock (STD) berthing and mooring operations. In addition, several new strategies, involving gene manipulation are being tried to create novel recombinant vaccines. It is however, very difficult to grow hepatitis B virus in mammalian cell culture and produce surface antigens. The HBsAg gene is expressed to produce 2nm sized particles similar to those found in patients infected with hepatitis B. All the health professionals—physicians, surgeons, medical laboratory technicians, nurses, dentists, besides police officers, firefighters etc., must get vaccinated against hepatitis B.
The day may not be far off to get immunized against hepatitis B by having a tomato with lunch!
The genome of FMDV is composed of a single— stranded RNA, covered by four viral proteins (VP1, VP2, VP3 and VP4). The recombinant vaccine for FMDV in the form of viral protein 1 was used to vaccinate animals. From the C-terminal end of VPI, amino acids 141 to 160, 151 to 160 and 200 to 213, and from N-terminal end, amino acids 9 to 24, 17 to 32 and 25 to 41 were synthesized. Among the peptides used, the one corresponding to amino acids 141 to 160 was found to be effective in immunizing guinea pigs against FMD. However, scientists continue their search for specific, inexpensive and safe synthetic peptide vaccines for various diseases.
BCG vaccine itself causes tuberculosis in some individuals (AIDS victims) and the vaccinated people respond positively for laboratory diagnosis of tuberculosis. Of about 100 such proteins, six were found to be useful (either individually or in combination) to immunize guinea pigs.
Now gp41 binds to the host cell surface and opens a passage for the entry of the virus into the cell.
These bacterial cells produce gp120 and gp41 that can be used as recombinant vaccines against AIDS. In a comparable manner, gp41 molecules also result in the production of anti-gp41 antibodies.
A DNA vaccine consists of a gene encoding an antigenic protein, inserted onto a plasmid, and then incorporated into the cells in a target animal. The plasmid vaccine carrying the DNA (gene) for antigenic protein enters the nucleus of the inoculated target cell of the host. Some of the activated T-lymphocytes become memory cells which can kill the future infecting pathogens.
The B-lymphocytes 4 of these mice performed a double duty, and produced antigens for and antibodies against malarial parasite. The generation of antigens and antibodies by using a DNA vaccine is a recent development in immunology, and is referred to as antigenic antibody approach of DNA vaccine. There is a possibility of this DNA getting integrated into the host genome and this may interrupt the normal functions. These include influenza A virus, rabies virus, hepatitis B virus, bovine herpes virus, HIV type I, and Plasmodium species (malarial parasite).

The bacterium, Agrobacterium tumefaciens is commonly used to deliver the DNA (genetic material) for bacterial or viral antigens. This enterotoxin stimulates the cells lining intestinal walls to release sodium, bicarbonate and other ions.
In this manner, the A1 peptide coding sequence is deleted (the DNA encoding for 183 of the 194 amino acids of the A1 peptide is actually removed). They cannot produce active enterotoxin but possess all other biochemical functions of the pathogen. Some workers claim that the new strain of Salmonella offers immunoprotection in humans also.
Vaccinia virus contains a double-stranded DNA (187 kb) that encodes about 200 different proteins. The foreign genes inserted into the vaccinia virus can also be expressed along with the viral genome.
The cloned foreign genes (from a pathogenic organism) can be inserted into vaccinia virus genome for encoding antigens which in turn produces antibodies against the specific disease- causing agent. Thus, vaccinia virus can provide a high level of immunoprotection against pathogenic organisms.
The plasmid insertion vector incorporates its genes into vaccinia virus genome at a place that encodes for the enzyme thymidine kinase (TK). One is that it is easy to select recombined vaccinia viruses that lack TK gene and the second is that these viruses are less infectious than the normal viruses. This enables the amplification of the antigens for their action on B-lymphocytes and T-lymphocytes.
Scientists are attracted to develop a recombinant poliovirus as it can be orally administered.
Scientists carefully evaluate the pros and cons of subunit vaccines for each disease, and proceed on the considered merits. It consists of a core containing a viral genome (DNA) surrounded by a phospholipid envelope carrying surface antigens (Fig. However, VPI vaccination was found to be less effective than that of the whole virus in protecting FMD. In addition, when two peptides were joined together (amino acids 141 to 158, and amino acids 200 to 213), they served as more efficient recombinant vaccines.
An envelope glycoprotein D (gD) of HSV that can elicit antibody production has been identified. The glycoprotein gp120 projects out of the HIV envelope while the other glycoprotein gp41 lies beneath gp120. Transgenic plants (tomato, potato) have been developed for expressing antigens derived from animal viruses (rabies virus, herpes virus).
A plasmid carrying the antigen gene and an antibiotic resistance gene are incorporated into the bacterial cells (A.
Cholera epidemics are frequently seen in developing countries where the water purification and sewage disposal systems are not well developed.
This destroys the DNA sequence encoding for A1 peptide, besides making the strain resistant to tetracycline.
The genome of this virus can accommodate stretches of foreign DNA which can be expressed along with the viral genes. Thus, the foreign DNA is under the control of the virus, and is expressed independently from the host cell genome.
Another advantage of vaccinia virus is the possibility of vaccinating individuals against different diseases simultaneously. It is argued that recombinant vaccinia virus might create life threatening complications in humans.
The recombinant vaccinia viruses, released from the cultured animal cells, can be successfully used as vaccines.
It might take many more years for the recombinant viral vaccines to become a reality for human use. These flagella-engineered bacteria, when administered to mice, raised antibodies against the cholera toxin B subunit peptide. However, during the 80s, following increases in the rates of intravenous drug use and the number of people with multiple sexual partners, syphilis rates started rising. Recombinant hepatitis B vaccine as a subunit vaccine, is produced by cloning HbsAg gene in yeast cells.
The subunit HBsAg as 22 nm particles can be isolated and used to immunize individuals against hepatitis B. In fact, vaccines are being continuously developed and field tested, although there has been no success so far.
The net result of using gp120 and gp41 vaccines is that the entry of HIV into the host cells is prevented. Despite these limitations, scientists have not lost hope, and continue their research to develop vaccines against AIDS. The antigen can act directly for developing humoral immunity or as fragments in association with major histocompatibility class (MHC) molecules for developing cellular immunity. Therefore, the progress in the development of RNA vaccines has been rather slow compared to DNA vaccines.
A selected list of recombinant vaccines against animal viruses produced in plants is given in Table 16.2.
Unfortunately, the tetracycline resistant gene is easily lost and the enterotoxin activity is restored. This can be done by a recombinant vaccinia viruses which carries genes encoding different antigens. The plasmid contains the foreign subunit genes, the natural vaccinia genes, including the promoter genes. It may be possible in future to incorporate multiple epitopes (2 or 3) into the flagellin gene to create multivalent bacterial vaccines.
Saccharomyces cerevisiae, a harmless baking and brewing yeast, is used for this purpose (Fig.
A double- stranded complementary DNA (cDNA) from the single-stranded viral RNA (genome) was synthesized.
The glycoprotein D was modified by deleting the trans membrane portion of the protein (Fig. Preliminary results indicate that this attenuated vaccine can protect about 90% of the volunteers against cholera. However, more experiments on animals have to be carried out before the leishmania attenuated vaccine goes for human trials.
The A subunit has two functional domains-the A1 peptide which possesses the toxic activity and A2 peptide that joins A subunit to B subunits. The gene for HBsAg is inserted (pMA 56) which is linked to the alcohol dehydrogenase promoter.

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