DNA vaccines: A hope full ray in Immunology

 

Ms. Dhanashri U. Gadhave1*, Ms. Puja S. Gaikwad1, Ms. Nayana V. Pimpodkar2,

Mrs. Swati B. Udugade1

1Lecturer, College of Pharmacy (D. Pharm) Degaon, Satara.

2Principal, College of Pharmacy (D. Pharm) Degaon, Satara.

*Corresponding Author E-mail: dhanu.gadhave22@gmail.com

 

ABSTRACT:

DNA vaccines uses foreign DNA to express an endcoded protein and stimulates the body’s immune system. It represents a new approach to immunization which is potentially less expensive than the old vaccines. After inoculation into the host, DNA enters the cells, where the antigen is expressed , processed and subsequently recognized by immune system as in a natural  infection. Certain methods like gene gun and elecroporation are used to deliver DNA vaccines.DNA vaccines undergo through clinical trials to find the way to treat autoimmune diseases, hepatitis, mycobacterial diseases, allergy and malaria. This review focuses on mechanism by which DNA vaccination induces immune response, delivering method and applications of DNA vaccinations.

 

KEYWORDS: DNA vaccines, Mechanism, Delivering methods.

 

 


1. INTRODUCTION:

Vaccination is one of the most important discoveries in medical sciences. It was described over 200 years ago by Jenner and today it is widely used to prevent a reduce the infection by many pathogens. In developed nations vaccines have almost exterminated polio and small pox and a tightly controlled diseases such hepatitis A, hepatitis B, Typhus, rota virus.

 

There are three generations of vaccinations: First generation vaccines are either weekend or killed forms of whole organism, but there is a problem with first generation vaccines. The pathogens can still revert to dangerous forms and caused disease in immunocompromised vaccine recipient. Second generation vaccines are specific protein antigens, which are safer but cannot generate killer T- cell responces. Third generation vaccines are consist of recombinant plasmids that have been transformed to produced  one to two proteins from a pathogen.[1]

 

DNA vaccination is technique for protecting an organism against disease by injecting it with genetically engineered DNA to produce an immunological response. The intentinal transfer of genetic material to somatic cells for the purpose of influencing the immuno systeme[4]. DNA vaccines represent a new approach to protect against infectious disease and hence may improve human and animal welfare, reduce antibiotic usage and reduce the speed of pathogens. Vaccines may be administered in the form of genetically modified viruses and bacteria. In DNA vaccines there is a transfer of nucleic acids in ways that do not entail the use of genetically modified organisms.[2,3]

 

The DNA is injected directly into somatic cells where, through transcription and translation the proteins are created. The proteins are recognized as a foreign and processed by the cell and displayed on the cell by major histocompatibility complex (MHC). Here they raise helper T-cell and antibody immune response.

 

Potentials of DNA vaccines

·         Long term persistence of immunogen.

·         Antigen presentation by both MHC class I and class II molecules.

·         Subunit vaccination with no risks for infection.

·         Immune response focused only on antigen of interest.

·         Able to polarise T-cell help toward type I or type II.

·         Obviates need for peptide synthesis, expression and purification of recombinant proteins and the use of toxic adjuvant[5].

·         They can be made in a short time span.

·         DNA vaccines are easy to transfer and stores.

·         Ease of development and production.

·         There is a no risks to those who are making vaccines.

·         They are very cheap to make.[6]

 

Risks of DNA vaccines

·         Initial attempts to create DNA vaccines have not worked.

·         No DNA vaccines have been licensed for using humans yet.

·         Possibility of inducing antibody production against DNA.

·         Risks of affecting genes controlling cell growth.

·         Possibility of tolerance antigen(protein) produced.[6]

 

DNA vaccines elicit the best immune response when highly active expression vectors are used. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest[7]. Intron A may sometimes be included to improve mRNA stability and hence increase protein expression.[8]Plasmids also include a strong polyadenylation transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences. Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.[9]

 

Because the plasmid is the “vehicle” from which the immunogen is expressed, optimising vector design for maximal protein expression is essential[10] One way of enhancing protein expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT-content than the species being immunized, so altering the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.

 

Another consideration is the choice of promoter. The SV40 promoter was conventionally used until research showed that vectors driven by the Rous Sarcoma Virus (RSV) promoter had much higher expression rates.]

 

 

METHOD OF PREPARATION: (Fig.-1 and 2)

More recently, expression rates have been further increased by the use of the cytomegalovirus (CMV) immediate early promoter. Inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev increased envelope expression. Furthermore the CTE+rev construct was significantly more immunogenic than CTE-alone vector. Additional modifications to improve expression rates have included the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences. An example of DNA vaccine plasmid is pVAC, it uses SV40 promoter.

 

Mechanism of Plasmids

Once the plasmid inserts itself into the nucleus of the transfected cell, it starts to encode for a gene resulting in production of peptide string of a foreign antigen. The cell on its surface displays the foreign antigen with both histocompatibility complex (MHC) classes I and class II molecule. The antigen-presenting cell then travels to the lymph nodes and presents the antigen peptide and costimulatory molecule signaled by T-cell results in initiate of the immune response.

 

Vaccine insert design

Immunogens can be targeted to various cellular compartments in order to improve antibody or cytotoxic T-cell responses. Secreted or plasma membrane-bound antigens are more effective at inducing antibody responses than cytosolic antigens, while cytotoxic T-cell responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the major histocompatibility complex (MHC) class I pathway.This is usually accomplished by the addition of N-terminal ubiquitin signals.


 

Fig.1: Method of Preparation of DNA Vaccines

Source: https://www.google.co.in/search?safe=active&hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=677&q=Method+of+ Preparation+of+DNA+Vaccines&oq=Method+of+Preparation+of+DNA+Vaccines&gs_l=img.12...25.25.0.1051.1.1.0.0.0.0.221.221.2-1.1.0....0...1ac..64.img..1.0.0.2mY33hT3ZWw#imgrc=P1S-2newNIjDtM%3A

 


 

Fig.2: Method of preparation of DNA Vaccine

Source: https://www.google.co.in/search?safe=active&hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=677&q=Method+of+Preparation+of+DNA+Vaccines&oq=Method+of+Preparation+of+DNA+Vaccines&gs_l=img.12...25.25.0.1051.1.1.0.0.0.0.221.221.2-1.1.0....0...1ac..64.img..1.0.0.2mY33hT3ZWw#tbm=isch&tbs=rimg%3ACT9Uvtp3sDSIIjjwtCvUwoxrtvMCKGh0XeHUzjT4vEUJlYR9S-fy3PF8265xeEjk9Qn2iaE3bdoMpsWJ0uxr1fIZtSoSCfC0K9TCjGu2EVC5_1YqcUjNoKhIJ8wIoaHRd4dQRe-lC8xAXtV0qEgnONPi8RQmVhBFcJIJloCP2JioSCX1L5_1Lc8XzbEesUPnQv7VTbKhIJrnF4SOT1CfYR4KTwPjeyZSIqEgmJoTdt2gymxRGuyOycO_1MfeCoSCYnS7GvV8hm1EVxeLTLfO3iO&q=Method%20of%20Preparation%20of%20DNA%20Vaccines&hl=en&imgrc=wD6xpO0UoGI97M%3A

 

DELIVERING METHOD

Table 1. Summary of Plasmid DNA delivery methods

Method of Delivery

Formulation of DNA

Target Tissue

Amount of DNA

Parenteral

Injection (hypodermic needle)

Aqueous solution in saline

IM (skeletal); ID; (IV, subcutaneous and intraperitoneal with variable success)

Large amounts

(approximately 100-200 μg)

Gene Gun

DNA-coated gold beads

ED (abdominal skin); vaginal mucosa; surgically exposed muscle and other organs

Small amounts

(as little as 16 ng)

Pneumatic (Jet) Injection

Aqueous solution

ED

Very high

(as much as 300 μg)

Topical application

Aqueous solution

Ocular; intravaginal

Small amounts

(up to 100 μg)

Cytofectin-mediated

Liposomes (cationic); microspheres; recombinant adenovirus vectors; attenuated Shigella vector; aerosolised cationic lipid formulations

IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes

variable

 


 

 

The conformation of the protein can also have an effect on antibody responses, with “ordered” structures (like viral particles) being more effective than unordered structures. Strings of minigenes (or MHC class I epitopes) from different pathogens are able to raise cytotoxic T-cell responses to a number of pathogens, especially if a TH epitope is also included.[7]

 

DNA vaccines have been introduced animal tissues by a no of different methods in that most widely used methods are injection or gene gun.

·         Injection: As much as 100 to 200 microlitres of DNA are injected directly into the muscle tissue. Muscle tissue is inefficient as an uptake side so more DNA is needed to ensure that enough plasmids are taken into host cells.

 

·         Gene Gun: In this method as little as 16 nanograms of DNA are fired directly into the abdominal epidermis. This method needs so little DNA because the DNA is directly bombarded into the individual cells. This method is essentially an air gun that fires microscopic heavy metal particles covered with plasmid DNA.

 

·         Other methods includes partical bombardment or biolistic delivery and in VIVO Electroporation (EP).In VIVO EP has the key features of improved DNA uptake increase antigen expression and a local inflammation. These factors are essential to make DNA vaccine effective in a larger host. Early data from clinical trials with DNA vaccines delivered by in VIVO EP are cautiously promising.[11]

 

MECHANISM OF STIMULATING IMMUNE RESPONSE

 

Fig.3: Mechanism of Plasmid

Source: https://www.google.co.in/search?safe=active&hl=en&site=imghp&tbm=isch&source=hp&biw=1366&bih=677&q=MECHANISM+OF+STIMULATING+IMMUNE+RESPONSE&oq=MECHANISM+OF+STIMULATING+IMMUNE+RESPONSE&gs_l=img.12...1328.1328.0.2238.1.1.0.0.0.0.143.143.0j1.1.0....0...1ac.1.64.img..1.0.0.2HOPfGohI5Y#imgrc=NSBMf1_8E4g0KM%3A

DNA vaccines favours a cell mediated immune response. DNA plasmid vector vaccines carry the genetic information encoding and antigen, allowing the antigen to be produced inside the host cell leading to a cell mediated immune response via MHC-I pathway. The plasmid DNA vaccines carries genetic code for a piece of pathogen. The plasmid vector is taken up into cell and transcript in the nucleus.[12]

 

The single stranded m-RNA is translated to protein in the cytoplasm. The DNA vaccine derived protein antigen is then decreaded by proteosomes into intracellular peptides. The vaccine derived peptide binds MHC class I molecules. Peptide antigen MHC I complex are presented on cell surface binding cytotoxic CD8+ lymphocytes and inducing a cell mediated immune response.[12]

 

Applications of DNA vaccines:

DNA vaccines would be safer than live viruse vaccine especially in immunocopromised patients such as those infected with HIV. It may play a role in attacking antibiotic resistant strains of microorganisms. Potential targets for DNA vaccine technology include streptococcus species, enterococcus and pseudomonas species. DNA immunization has proven to be an effective candidate for antitumour therapy.DNA vaccine increasing a cancer victims immune response to malignant cells. Patients with melanoma, mesothelioma, renal cell cancer and breast cancer being treated with gene transfer.[13-15]

 

A veterinary DNA vaccines for use on horses to protect from West Nile Virus has been approved. Positive results were announced for vaccines against Bird Flu and multiple sclerosis.DNA immunization can also be employed in alternative human immunotherapies for the treatment of diseases such as autoimmunity, cancer, allergy or asthma.DNA vaccines are also good candidates to immunize neonates.[16-17]

 

CONCLUSION:

DNA vaccines hold promises for protection against a range of diseases caused by viruses and intracellular bacteria for which there at present are no efficient vaccines based on either live attenuated viruses or vaccines containing recombinant viral antigens.DNA vaccination is a simple, safe and cheap alternative for the induction of protective humoral and cell mediated immunity. The better understanding of mechanism responsible for the generation of immune response after DNA inoculation is leading to the design of more efficacious protocols. It is expected that DNA immunization will become the treatment of choice for both prophylactic and therapeutic protocols in the very near future.

 

REFERENCES:

1.        Alarcon JB, Waine GW, McManus DP. "DNA vaccines: technology and application as anti-parasite and anti-microbial agents", Advances in Parasitology 42: 343–41,1999.

2.        Robinson HL, Pertmer TM. "DNA vaccines for viral infections: basic studies and applications", Advances in Virus Research 55: 1–74, 2000.

3.        Tan, D, Devit M, Johnston S.A. "Genetic immunization is a simple method for eliciting an immune response", 356 (6365): 152–154, 1992.

4.        Regulation of DNA vaccines and gene therapy on animals page no-10

5.        The World Health Organisation http// www.who.int

6.        Sedegah, M, Hedstrom R, Hobar P, Hoffman S.L, "Protection against Malaria by Immunization with Plasmid DNA Encoding Circumsporozoite Protein". Proceedings of the National Academy of Sciences of the United States of America 91 (21),: 9866–9870, 1994.

7.        Mor, G.; Klinman, DM; Shapiro, S; Hagiwara, E; Sedegah, M; Norman, JA; Hoffman, SL; Steinberg, AD  "Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA". The Journal of Immunology. 1995.

8.        Leitner, W.W.; Seguin, MC; Ballou, WR; Seitz, JP; Schultz, AM; Sheehy, MJ; Lyon, JA. "Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites". The Journal of Immunology.1997. 

9.        Böhm, W.; Kuhröber, A.; Paier, T.; Mertens, T.; Reimann, J.; Schirmbeck, R. "DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection". Journal of Immunological Methods.1996.  DNA vaccination- An immunological prspective S., Moreno M., Timon Mologen Molecular Medicines, Spain. Page no 47.

10.     http//www.medscape.com

11.     Frelin L, Brass A, Ahlen G, Brenndorfer E “Electroporation: A Promising method for non viral delivery of DNA Vaccine

12.     Irsa M, Saba I, Journal of Health Science. “A Review on DNA vaccines”

13.     Barnes, Kirsty, "First positive results for DNA-based flu vaccine". In-Pharma Technologist, 2004-06-07.

14.     "Fort Dodge Animal Health Announces Approval of West Nile Virus DNA Vaccine for Horses". PR Newswire. 2005-07-18.

15.     "CDC and Fort Dodge Animal Health Achieve First Licensed DNA Vaccine". CDC. 2005-07-18.

16.     Stuve O, Eagar T.N., Frohman E.M., Cravens, P.D., "DNA Plasmid Vaccination for Multiple Sclerosis". Archives of Neurology 64 (10): 1385–6, 2007.

17.     DNA vaccines: Immunology application and optimization Annual review on immunology vol.18:927-974, Apr-2000.

 

 

 

Received on 30.04.2015          Accepted on 02.06.2015        

© Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 5(2): April-June 2015; Page 126-131

DOI: 10.5958/2231-5659.2015.00020.X