Journal of Bacteriology & Parasitology

Bacterial Plasmid DNA Enhances the Efficacy of Vaccine and their Advantages in Vaccine Development

Muruganandam M^* Department of Einsteein Bio-Engineering Research Foundation, Tamil Nadu, India
^*Correspondence: Muruganandam M, Department of Einsteein Bio-Engineering Research Foundation, Tamil Nadu, India, Email:

Received: 12-Feb-2021 Published: 03-Mar-2021, DOI: 10.35248/2155-9597.21.s9.004

Introduction

Bacteria cause many infectious diseases to humans and animals. They create mortality and morbitality to the host organism. Vaccination is one of the best method to prevention and control the infections of pathogenic bacteria [1]. Many bacterial components has antigenic properties which are suitable for vaccine development. The main components of vaccine complex such as Antigens, adjuvants, preservatives, stabilizers, etc. The adjuvants are boost up the efficacy of vaccine. The preservatives help to avoid bacterial and fungal contamination in the vaccine. The stabilizers help to maintain the conditions of vaccine. The plasmid DNA is one of the important antigenic components of bacteria which are highly useful for vaccine development [2-5].

Plasmid DNA

Plasmid DNA is an extra chromosomal genetic element. It is present in cytoplasm. They vary in size from a few to several hundred kilo bases in length. The chromosome and plasmids together constitute the bacterial genome. Plasmids are small, circular and autonomously replicating double standard DNA molecules. Plasmids are transferable to other cells. They have great use in recombinant DNA technology. A cell may contains 1-100 plasmids. There are different types of plasmids based on their function. The important types are f plasmids, v plasmids, degradative plasmids, colplasmids, etc. The f plasmids have ability to transfer chromosomal genes to other cells. They can also transfer themselves to the cells. The R plasmid has the gene for resistance to one or more antibiotics. If the virulence plasmid is present inside the bacterium it turns that bacterium into pathogen which is an agent of diseases. The degenerative plasmids help to the host bacterium to digest compounds. The col-plasmid have the ability to synthesize a toxin called colicins.

Components

Plasmid DNA has various general characteristics which are help to extent different way to existence. Plasmid DNA consist of different components that includes, Antibiotic resistance genes, Multiple cloning sites, primer binding sites, etc. The antibiotic resistance genes is one of the main components of plasmids. These genes play important role in drug resistance. Through conjugation process, plasmid DNA is capable of transferring antibiotic resistance properties to one species to another species of bacteria. A promoter region is another components of plasmid DNA which involved in transcriptional machinery. Another component is primer binding site. It is a small sequence of DNA on a single strand that is typically used for DNA sequencing. In plasmid multiple cloning site is a short sequence DNA consisting of a few sites for cleavage by restriction enzymes. As such they allows for easy insertion of DNA through ligation or restriction enzyme digestion. Plasmids have one or few origins of replication, which are specific location for replication begins [7-12]..

Isolation methods

For isolation of plasmid DNA in lab, commonly follow alkaline lysis method. In this method, bacterial cells are harvested by centrifugation from bacterial broth culture. Then isolation procedure will be started. First resuspention of the pellet in isotonic solution ethylene diamine tetra acetate which is prevents nuclease activity. Alkaline lysis process involves cell lysis by using sodium dodecylsulfate to disintegrate the lipid structure on the cell membrane. Precipitation of dissolved proteins by using a solution of acidic potassium acetate. A mixture of phenol and chloroform is used for purification of the plasmid DNA.This step removes protein content. In the precipitation step, first add ethanol for precipitation, by using centrifugation. Then wash with 70% ethanol to remove salt content. After that centrifuge 12,000 rpm for 30 minutes to sediment plasmid DNA then dissolve in TE buffer solution and store for further use.

Applications

Plasmids are used in genetic engineering to amplify or produce many copies of certain genes. Some plasmids help to resists various antibiotics. They make toxins to kill other bacteria. Some help resist environmental factors. They are also use unusual chemical compounds as nutrients. In pathogenic bacteria plasmid acts as antigens during infection. In cloning, a plasmid is a type of vector and uses in gene transfer to the cells of superior organisms to improve their resistance to diseases, growth rates and any other required traits. It is used to produce proteins and antibiotics at large scale. Plasmid are used to transfer genes into human cells as part as gene therapy. Plasmid cannot produce diseases. If it is combined with antigenic protein it will be induces immunity. So it is highly suitable for vaccine development. These vaccines induces to produces more antibodies, it will also useful in plasma therapy for viral infection[13].

Vaccine development

Major research move towards rDNA based vaccine development, For eg: Salmonella typhimurium engineered to express antigens of Vibrio cholera. So in this presentation, another alternative way ie) direct plasmid DNA based vaccines development is focused. Plasmid DNA induces immunity so it is useful to vaccine development. Plasmid DNA based different types of vaccines are as follows.

Whole plasmid DNA vaccine

In this study was done in bacterial pathogen Aeromonas hydrophila. It is mainly present in drinking and ground water of various countries.

In it mainly gives problem to immuno suppressive patient and normal host. Aeromonas hydrophila, water borne pathogen causing bacteraemia, cellulites to human has not got a main stay treatment till date.

Now there is no commercial vaccine. The aim of this work is to develop novel vaccine. In this study two experiments were carried out. In this first experiment lives attenuated vaccine with various. adjuvants such as vitamin A, C and E were tested.

In this second experiment six various types of vaccines were tested such as killed vaccines live attenuated vaccines, toxic vaccine DNA vaccines, plasmid DNA vaccines and protein vaccine and the results shows the maximum humoral and cell mediated response was observed in protein and plasmid DNA vaccines. In this study, vitamin A acts as best adjuvant.

Mutant strains’ plasmid DNA vaccine

Staphylococcus aureus cause a variety of disease in human either through toxin production or invasion. The most common cause of food poisoning is staph toxin, this bacterium grow in improperly stored food the cooking process kill them but the toxins are heat resistant and it also infect wounds.

In this experiment, the pathogen Staphylococcus aureus was collected from patients in hospital for confirmation of S. aureus all routine micro biological and Biochemical tests were done.

During these experiments five sets of spread plate culture were maintained and four sets are exposed to UV radiation with different time intervals (0, 2, 4, 6 and 8 minutes). First sets were maintained as a control after 24 hours the mutant strains were observed. Then it was isolated and put in to subculture[14].

The mutant strains plasmid DNA was isolated by alkaline, lyses method and these was used as vaccine. In this experiment albino rats was used as test animal. The same level vitamin C (200 mg) was provided as adjuvants.

After fifteen days in active pathogens as injected to all treatment including control treatment. After that blood samples of the entire albino rat were taken for analysis. The maximum, immune response was observed in 6th minute treatment strain.

In all the treatment, inactivated whole cell pathogens were tested, and also the similar result was observed. So it is concluded that this 6 minute treatment strain’s plasmid DNA and inactivated whole cell is suitable for development of S. aureus vaccine[15-18].

The second experiment, maximum immune response was observed in EcoR-I+Hind-III and Hind-III+BamH-I digested treatments. So these are suitable to develop plasmid DNA based vaccine for Staphylococcus aureus.

Cocktail plasmid DNA vaccine

Water borne diseases are caused due to intake of contaminated drinking water. The potable water is degraded due to the faecal contamination, which contain many pathogens like. Escherichia coli, Staphylococcus aureus, Salmonella typhi, etc. which causes fever, dysentery, vomiting, diarrheal, rapid pulse, cramps, etc. Still now there is no proper cocktail vaccine recognized. Here is an attempt to develop a cocktail vaccine for these diseases. In this experiment, four treatments were tested and one control was maintained. In the first treatment, the plasmid DNA was isolated from three pathogens and it was mixed then use as vaccine. In the second experiments, the plasmid DNA was isolated and all the pathogens were digested by Pst-I enzyme and in third treatment, plasmid DNA was digested by BamH-I and in fourth treatment these plasmid DNA was double digested by both enzymes used as vaccine. Albinorats were used as test animals. It was observed that double digested plasmid DNA mixture induces more immune response compared to other treatment and so it was recommended for development of cocktail vaccine for these waterborne diseases.

Plasmid DNA with proteins

Plasmid DNA with extracellular protein was tested in Staphylococcus aureus. In this study, plasmid DNA, extracellular protein and mixed of both treatment were tested. Albino rates were used as test animals. First test vaccines were injected and after 15 days, inactivated pathogens were injected. After one week, blood samples were collected for analysis. The maximum immune response was observed in mixer treatment compared to other treatment. Based on this study, plasmid DNA with other immunogenic protein produces good immune response.

Experimental conclusion

Based on these experiments, it is concluded that, Plasmid DNA act as good antigen. The plasmid DNA from the mutant pathogen are also acts as good antigens. The single and double digested plasmid DNA act as good antigen in some cases. The mixer of various pathogenic bacteria are also gives better immune response which are also help to prepare mixer vaccines for more than one diseases. Our lab studies prove that plasmid DNA of various pathogenic bacteria induces antibody production and it also induces cell mediated immunity. If plasmid DNA combined with antigenic proteins gives better immunity, it will be very much useful to produce good vaccines with least cost.

Potential merits

For plasmid DNA based vaccine development, short duration is sufficient. The plasmid DNA vaccine produces long term immunity. It is stable and easy to transport. It is also thermo liable i.e) heat resistant. It is easy to sequence. Formalin is used.

Inactivation of pathogen and preservation of amino acids in vaccines. Here formalin is not required for DNA vaccine. If plasmid DNA combined with suitable antigenic proteins, it will enhance the efficacy of vaccine. Now it is possible to change the sequence of antigenic protein and also sequence of plasmid DNA. So the production is easy and also suitable to develop advanced vaccines in future. The DNA vaccine development is easy to prepare more than one infection. It is also suitable for pain free vaccination. The plasmid DNA vaccines are highly suitable for developing countries. Because they are highly effective but it is low cost production vaccine.

Possible vaccine delivery methods

There are many methods are used in vaccination programmes in all over the world. Here the important methods are discussed.

Oral vaccination

Oral vaccination is one of the best method in needle free vaccination. It may replace vaccine injections. The primary problem with this type of delivery system is that the environment of the digestive system which is possible to destroy vaccines. Now vaccine capsule has been developed as an alternative to these problems. Oral delivery is an even more attractive route of administration. Polio virus vaccine administrated orally is the greatest current examples of a practioner and it is user friendly, economically feasible and clinically effective vaccines.

Eye drop administration

This method is mainly used in poultry farm. It is a most effective method. It ensures that the vaccine reaches the individual birds. The eye drop administration provides good protection. It need only short duration.

Intranasal vaccination

Some vaccines which protect against respiratory diseases which is to be given as drops in the nose. These vaccines generally provided faster production than those given intramuscularly or subcutaneously. Intranasal vaccines are less likely to cause allergic reactions and are more likely to provide protection.

Needle free injections

Different types of needle free injections are now developed. They are mainly used to vaccine delivery and other drug delivery functions without pain. The needle free injectors offers less pain, avoid needle stick injuries and contamination that also allows self administration and results is no needle phobia and thus strongly preferred by the patients.

Skin cream vaccinations

In our lab experiment proves that skin cream plasmid DNA vaccine also induces immunity. In that study, the skin cream is prepared by using beeswax with coconut oil during cream preparation, the plasmid DNA was dissolved in double distilled water and mixed well with cream.

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