DNA Vaccines: Optimization Methods

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Table of Contents

Introduction
The DNA vaccine
Regulatory elements
Kozak sequences
Codon optimization
Conclusion
Reference list

Introduction

As the man was eradicating diseases that had been threatening his existence, new and more dangerous diseases were emerging. This led to an increased headache for doctors and other research experts in the field of medicine who were using any available methods to control the new diseases. While in the past it was easy to control diseases using the traditional methods, the diseases which have emerged are more resistant to some of those methods because they have undergone gene mutations. As a result, scientists have been forced to come up with new methods through which these diseases can be controlled. Among the methods which have emerged to protect man and other living things from these dangerous diseases, DNA vaccination has emerged as one of the best since it attacks the genes which cause these diseases. In this review, we shall go through the various methods and strategies which scientists have been using in optimizing and making the DNA vaccines as efficient as possible. These different strategies include the co-expression of stimulatory molecules, the use of either localization or secretory signals among other methods. The three optimization methods scientists have been using to optimize DNA vaccines are the use of regulatory elements, optimization of the codons, and addition of the kozak sequences.

The DNA vaccine

DNA is the gene-carrying component that carries the genetic information required in the functioning of all living organisms. Its main long-term role is the storage of genetic information. The DNA is made of two strands which are anti-parallel and attached to each strand are nitrogenous bases which translate into a particular amino acid. Each nitrogenous base is attached to a sugar (Calladine 2004). The nitrogenous sugar bases are arranged into a specific sequence of a particular amino acid which then forms a particular gene. When making DNA vaccines, genetic engineers modify or interfere with gene sequence in order to come up with a specific kind of amino acid that can function as a vaccine. A DNA vaccine is therefore described as the changed or the modified DNA sequence (Roat-Marone 2007). The DNA sequence which is created, codes for a specific antigenic protein of the pathogen. As a result, when DNA vaccines are inserted into cells of human beings, they are translated to form antigenic proteins and since these antigenic proteins are not recognizable by the body, the body creates an immune system towards it. The DNA vaccine helps the body in achieving immunity in this way. DNA vaccines are derived from bacteria that possess plasmid vectors which are usually used as the transferring agents from the vector to the body (Barrett& Stanberry 2009).

Regulatory elements

A gene is usually divided into two regions. One is the transcription region while the other is the regulatory region. The transcription region is the region that is usually transcribed into an RNA molecule. The regulatory region on the other hand is divided into two regions: the cis-regulatory region and the other which is the trans-regulatory element, (Thakurta 2006). When making DNA vaccines, scientists usually use the trans-regulatory elements which are the DNA sequences that encode the transcription factors. Studies have shown that the strength of a promoter (where gene transcription is initiated) in DNA vaccines influences how they function and thus how efficient they are. The gene promoters are more expressed when using the in vivo method than the other eukaryotic promoters when used. This brings about why the DNA vaccines have gained much reception and appreciation in the field of medicine. For example, the CMV promoter which is the strongest promoter described has been known to exhibit the highest levels of transgene expressions in high celled organisms when compared to other types of promoters. For example, in a study of a plasmid which was used to express the Human Immuno Deficiency Virus type 1, when a CMV enhancer was used for comparison with a plasmid utilizing the traditional vaccine methods of AKV murine leukemia long terminal repeat, the results showed that the CMV containing plasmid showed higher responses. Due to those results, it was then translated to mean that DNA vaccines show greater expression when compared to the traditional methods of vaccination. Table 1 below which was accessed from the Genetics Vaccines and Therapy journal shows a comparison of two studies on gene expression when using the in vitro and the in vivo methods. The table shows how superior the DNA vaccination method is when compared to other methods.

The rate of gene transcription is usually influenced by the strength of a promoter. As a result, the termination or the end of transcription can also become rate-limiting depending on the power. Since the RNA transcript process is known to vary between the existing polyadenylation sequences of different genes, the DNA sequence used within the DNA vaccine may also have various effects on transgene expression depending on the gene sequences of the living organism of interest (Garmory, Brown& Titball 2003).

Kozak sequences

The DNA sequences surrounding the AUG initiator codon play a very great role in how it is recognized. It is thus very important to understand the conditions which are required for translation of genes for the vaccine being made or being used to work efficiently and optimally (Wong 2006). As a result of these conditions, the Kozak sequence has been known to be very useful in providing the required gene through modification. Scientists have proposed that for the translation of a gene in the DNA vaccine to be efficient, the Kozak sequence should be included in the AUG initiator codon. This is in order to increase the expression of genes that might be used as vaccines but they do not possess the Kozak sequences, insertion of the sequence is usually done to increase its quality (Lorie& Whalen 2000).

Codon optimization

It is important to note that a codon bias exists across all living organisms. As a result, experts have been using selective codons in genes in order to express the gene or the vaccine efficiently (Nielson 2005). The selection of codons that maximize translational efficiency in genes enables the DNA vaccines to work more efficiently when compared to other vaccination methods (Gingold& Pilpel 2011). For example, to improve the expression of Human Immunodeficiency Virus type1 from a DNA vector, scientists generated an artificial sequence in which most of the wild codons were replaced with genes that were highly expressed in humans. After this, there was increased expression of the genes in the modified compared to the wild-type sequence. As a result of these and other studies, it has been found that increased immune responses can be also be attained when transgene sequences are optimized (Bins 2007).

Conclusion

The use of regulatory elements, kozak elements and optimization of codons are some of the optimization methods which scientists have been using to make the DNA vaccines more efficient in their functioning. By use of optimizing methods, DNA vaccines increase their efficiency when compared to the traditional methods. However, while the use of optimization methods is an important consideration for genetic engineers as they develop DNA sequences, it is important to note that other aspects of vector design other than the use of optimization methods can also influence the efficiency of the DNA vaccine.

Reference list

Barrett, A., & Stanberry, L., 2009, Vaccines for biodiesel and emerging and neglected diseases. London: Academic Press.

Bins, A., 2007, Induction and Analysis of Antigen-Specific T Cell Responses in Melanoma Patients and Animal Models. Amsterdam Netherlands: Amsterdam University Press.

Calladine, C., 2004, Understanding DNA: the molecule & how it works. 3^rd Edition. London: Academic Press.

Garmory, H, Brown, K., & Titball, R., 2003. DNA vaccines: improving expression of antigens. Genetics, Vaccines and Therapy. Vol. 1. Iss.2, pp. 1-5

Gingold, H., and Pilpel, Y. 2011, “Determinants of translation efficiency and accuracy”. Web.

Lorrie, D., & Whalen, R., 2000, DNA vaccines: methods and protocols. New Jersey: Humana Press.

Nielson, R., 2005, Statistical methods in molecular evolution. New York: Springer Publishers.

Roat-Marone, R.M., 2007, Bioinorganic chemistry: a short course. 2^nd Edition. New Jersey: John& Wiley and Sons,

Sethupathy, P., 2008, Analysis of the role of gene regulatory elements in human health and evolution. Web.

Thakurta, D., 2006. Computational identification of transcriptional regulatory elements in DNA sequence. The Nucleic acids studies journal, Vol.34, Iss.12. pp. 3585-3598.

Wong, D., 2006, The ABCs of gene cloning. 2^nd Edition. New York: Birkhäuser.

Table 1
Comparison of promoters used in DNA expression studies in vitro and in vivo
Expressed antigen	Promoters/enhancers compared	In vitro/in vivo comparison	Reference

GFP	CMV, muscle-specific creatine kinase (CKM) promoter	Consistently higher levels of GFP expression were driven by the CKM promoter compared to CMV in mice.	[5]
LacZ	CMV, glial fibrillary acidic protein (GFAP) promoter, neuron-specific enolase (NSE) promoter	Injection of mice with the constructs containing the different promoters showed that GFAP is as efficient at driving lacZ expression as CMV.	[6]
CAT	HIV-1-LTR (long terminal repeat), RSV-TAR (transactivation response element)	HIV-1-LTR could be transactivated by tat in both stimulated and unstimulated cells; RSV-TAR was only transactivated in unstimulated cells.	[7]
CAT	CMV, RSV, SV40, murine leukemia virus (SL3-3) promoter	The CMV promoter was found to be stronger than any of the other promoters tested in muscle.	[8]
CAT	CMV, SV2	The CMV promoter was found to have greatest transcriptional activity.	[9]
Luciferase	CMV, RSV, SV40, PGK, hybrid β-actin promoter/CMV enhancer, CMV/IA (intron A)	The hybrid β-actin/CMV promoter/enhancer showed greater luciferase expression than RSV, SV40, PGK or CMV. CMV/IA also showed 2–6 fold in vitro and 1.5–3 fold in vivo higher luciferase expression than CMV.	[10]
Hepatitis B surface antigen (HBsAg)	CMV, desmin	The promoters performed equally well in vitro, and CTL and Th1 serum antibody responses against HbsAg in mice were of similar magnitude.	[11]
Hepatitis B envelope proteins	CMV, desmin	Greater in vitro expression of antigen was attributed to the desmin promoter. However, comparable humoral and cytotoxic immune responses were stimulated following i.m. injection of mice.	[12]
Rabies virus G protein	CMV, SV40	Comparable G antigen-specific antibody titres were stimulated in mice. Slightly higher T cell responses were observed from the CMV construct.	[13]
Influenza virus H5 hemagglutinin (HA)	CMV, β-actin	Constructs containing the CMV or β-actin promoters provided comparable protection against influenza in chickens.	[14]
Influenza virus H5 hemagglutinin (HA)	CMV, β-actin, RSV, SV40	Similar in vitro expression of HA. The greatest HA-specific antibody and protection against influenza in chickens was provided with the CMV construct.	[15]
Bovine herpesvirus glycoprotein D (gD)	RSV, CMV/IA	CMV/IA construct produced higher neutralising antibody titres against gD in i.d. injected cattle.	[16]
HIV-1 gag/env	CMV, AKV murine leukemia viral long terminal repeat	CMV showed 10–20 fold greater activity than AKV in vitro. Immunised macaques developed high humoral responses with the CMVconstruct only.	[17]
SV40 large tumour antigen	CMV, SV40	The CMV construct induced higher levels of antibody and protection in the murine experimental metastasis model than the SV40 construct.	[18]
M. tuberculosis apa + pro proteins	CMV, UbC	The CMV promoter was the most efficient tested.	[19]
Adenovirus E4 ORF3	CMV, RSV, SV40, UbC, EF-1α	Following i.n. dosing to mice, constructs containing the UbC and EF-1α promoters stimulated the most stable expression of antigen	[20]

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