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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 1  |  Issue : 3  |  Page : 192-197

DNA vaccine construct formation using Mycobacterium-specific gene Inh-A


1 The Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan
2 Department of Biochemistry, The Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

Date of Submission17-May-2022
Date of Decision20-Jul-2022
Date of Acceptance10-Aug-2022
Date of Web Publication18-Sep-2022

Correspondence Address:
Dr. Mirza Imran Shahzad
Department of Biochemistry, The Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpdtsm.jpdtsm_63_22

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  Abstract 


BACKGROUND: Tuberculosis (TB) remains a highly contagious disease and a leading cause of death worldwide. It is caused by Mycobacterium tuberculosis and Mycobacterium bovis. TB infection is still uncontrolled because of the unavailability of an effective vaccine, co-infection with HIV, lengthy treatment, and the emergence of resistant forms of M. tb like multi/extreme drug resistance strains. TB is mainly a disease of underdeveloped countries because of inadequate health facilities. The development of the new state of modern art vaccine-like DNA vaccine is a promising approach to control TB. The DNA vaccine can be used alone or in combination with Bacille Calmette Guerin (BCG). The objective of the current study is to develop an M. tb gene inh-A based DNA vaccine.
METHODS: The immunodominant gene (Rv1484/INH-A) was amplified using sequence-specific primers. The amplified product was cloned into Topo 2.1 polymerase chain reaction vector, confirmed through restriction digestion and sequence analysis. Finally, subcloned into mammalian expression pVAX1 vector.
RESULTS: The inh/A-pVAX1 construct was again confirmed through restriction digestion and sequence analysis. The rightly oriented constructs were selected, and these will be used for in Vivo DNA vaccine immunization studies.
CONCLUSITONS: DNAvaccine can be used alone or in combination with BCG. DNA vaccines have enough potential to be used with TB treatment and reduce the treatment time in future.

Keywords: DNA vaccine, genetic vectors, inh-A gene, Mycobacterium tuberculosis


How to cite this article:
Anwar S, Qureshi JA, Shahzad MI, Zaman MM, Jilani A. DNA vaccine construct formation using Mycobacterium-specific gene Inh-A. J Prev Diagn Treat Strategies Med 2022;1:192-7

How to cite this URL:
Anwar S, Qureshi JA, Shahzad MI, Zaman MM, Jilani A. DNA vaccine construct formation using Mycobacterium-specific gene Inh-A. J Prev Diagn Treat Strategies Med [serial online] 2022 [cited 2022 Oct 1];1:192-7. Available from: http://www.jpdtsm.com/text.asp?2022/1/3/192/356295




  Introduction Top


Tuberculosis (TB) is one of the primary causes of death worldwide and is caused by the Mycobacterium tuberculosis complex (MTBC). The MTBC consists of Mycobacterium africanum, Mycobacterium pinnipedi, Mycobacterium caprae, Mycobacterium Canetti, and Mycobacterium bovis, Mycobacterium microti, and M. tuberculosis.[1] According to WHO (Global TB Report 2021), TB killed 1.7 million individuals, while TB has infected 10.4 million people globally. Further, 161,000 multi-drug resistance TB cases were reported.[2] In Pakistan, 525,000 new TB cases have been diagnosed annually, and 44,000 people died every year from TB and HIV co-infection. In high burden carrying countries, Pakistan stands at the fifth position in multi-drug-resistant TB.[3]

There are almost seven million new TB cases per year and the WHO warned that if a situation is not controlled, more cases would be reported. It is alarming for the entire world because TB does not respect national boundaries. Bacille Calmette Guerin (BCG) is the only vaccine available for TB and prepared from the attenuation of M. bovis.[4],[5] It has been reported that the BCG vaccine does not provide enough protection to achieve complete protective immunity in adults. Its efficacy ranges from 0 to 80% in a different area of the world.[6] Moreover, BCG is not recommended in most countries of Europe and America due to its involvement in misleading results of purified protein derivative test.[7]

Generally, the treatment of TB requires a long course of treatment for about 6–9 months and is limited to a few numbers of antibiotics, most commonly with rifampin (Rif) and isoniazid (INH).[8] Multi-drug resistance is increasing because of inadequate protection provided by first-line treatment.[9] Developing new tools and knowledge for the current study is essential to achieving the desired goals.[10],[11] The latest TB vaccine approaches include antigen-based subunit vaccines and recombinant vaccines using conventional and recombinant DNA technologies. Some subunit vaccines are in clinical trials, and efforts have been made to search for new approaches.[12] A new vaccination method has been introduced: a plasmid containing gene sequence encoding antigen into muscle cells that stimulates a natural defense system, but depends on the action in the target antigen's muscle, named the DNA vaccine. The DNA vaccine's advantages over traditional methods, including better vaccine stability, activation of B, and T-cell based responses.[13] DNA vaccine (s) development is a promising area since few DNA vaccines have been developed in the last 30 years and many DNA vaccines are in the pipeline, especially against viruses, cancers, diabetes, and some other infectious diseases. Recently a few DNA vaccines have been approved by the FDA.[14]

Mycobacterium is a slow-growing organism due to its great thick, waxy cell wall. Its unique cell wall is rich in mycolic acids and lipids that protect it against phagocytosis.[15] According to a recent study, NADH-dependent enoyl-(ACP) reductase/inh-A inhibits mycolic acid biosynthesis and causes cell death of M. tb. Therefore, this gene is highlighted as a good target for the development of inh-A based DNA vaccine.[16] Therefore, the present study is designed to use the Rv1484/Inh-A gene to develop a DNA vaccine to control or reduce M. tb infection.


  Methods Top


The present study was conducted at the Department of Biochemistry, Institute of Biochemistry, Biotechnology, and Bioinformatics, The Islamia University of Bahawalpur, Bahawalpur and some work is done at Institute of Molecular Biology and Biochemistry, The University of Lahore, Lahore.

Bacterial strain, restriction enzyme, vectors, and primers

The Bacterial Strain, Restriction enzymes, Vectors, and primers used in the present study are presented in [Table 1].
Table 1: Bacterial strain, restriction enzyme, vectors and primers

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Bacterial growth

Generally, all the bacteria (transformed and untransformed) were grown in the Luria Bertani (LB) media with and without specific antibiotics. The Escherichia coli strain TOP10 was grown in the LB broth and incubated for 16 h at 37°C overnight in a shaking incubator. After transformation with Topo T/A cloning vector, the bacteria were grown in the ampicillin+ (100 μg/ml) LB broth medium, and with pVa × 1 expression vector, the bacteria were grown in the Kanamycin+ (50 μg/ml) LB broth medium.

Preparation of competent cells

Competent cells of E. coli strain (TOP10), was prepared by cold treatment of 0.1M CaCl2/MgCl2 solution as described by.[17]

Polymerase chain reaction for amplification of gene

Polymerase chain reaction (PCR) conditions were optimized by changing different parameters like annealing time and temperature, MgCl2, primer concentration, etc., PCR master mix (Catlog# K0172) was used for amplification purposes.

Cloning experiments

The M. tb gene inh-A was cloned into Topo pCR 2.1 cloning vector and further subcloned into pVAX1 vector using standard molecular biology procedures like DNA ligation, transformation, and clones selection.

DNA ligation

In this experiment, 810 bp long amplified Rv1484/inh-A gene bearing BglII, and XbaI restriction sites were cloned into Topo pCR 2.1 (Thermo Scientific™, USA) cloning vector using T4 DNA ligase provided in the ligation kit (Invitrogen, USA). Moreover, subsequently subcloned into the pVAXI expression vector (Invitrogen, USA). A schematic description of the cloning strategy is presented in [Figure 1].
Figure 1: Cloning scheme of Rv1484/inh-A gene to develop DNA vaccine construct: The amplified DNA fragment 810 bp coding inh-A gene was cloned into Topo Pcr 2.1 T/A vector using BglII and XbaI restriction sites to form Recombinant plasmid I (pSIJ). The Rv1484/inh-A gene was further subcloned into the pVAXI expression vector using HindIII and XbaI restriction sites to obtain Recombinant plasmid II (PSIJ). PCR: Polymerase chain reaction

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Transformation

The chemically competent cells (E. coli TOP 10) were transformed with recombinant vectors using the heat shock method.[18]

Selection of clones

A single bacterial colony was selected from the antibiotic-containing agar medium. Then transferred to LB medium containing ampicillin+ (100 μg/ml) for inh/A-Topo construct, and Kanamycin+ (50 μg/ml) for the inh/A-pVAX1 construct. The clones were incubated at 37°C overnight in a shaking incubator.[19]

Recombinant plasmid DNA extraction

All the Plasmid DNAs were extracted using the alkaline lysis method as described by.[18]

Restriction analysis

The clones were confirmed through restriction digestion using BglII, HindIII, and XbaI (ER0082, ER0502, ER0682, Thermo Scientific, USA) restriction enzymes.

Agarose gel electrophoresis

All DNA samples were analyzed agarose gel electrophoresis. 0.9%–1.2% agarose gels were made in 1X TAE Buffers in the different experiments, and finally, all gels were stained with ethidium bromide (10 mg/ml) before taking the picture in gel doc (Kryndushkin et al., 2003).[1],[7] The DNA bands were visualized with a UV Transilluminator 100V–240 V (Vilber lourmat, 0722130, EU) at 320 nm UV light.

DNA Sequencing

All the constructs were confirmed through sequence analysis, and the clones with the right sequence were selected, and their glycerol stocks were made.


  Results Top


The PCR conditions for inh-A gene was optimized by changing different parameters like primer concentrations, annealing timer and temperature and MgCl2 concentrations. The optimized PCR product was further used in ligation studies [Figure 2] and [Figure 3].
Figure 2: Development of recombinant Plasmid I and its confirmation: (a) Lane 1: 1kb DNA size marker (SM0311, ThermoFisher Scientific, USA). Lane 2: A 4710bp fragment of recombinant plasmid I (topo/inh-A). (b) Enzymatic digestion of recombinant plasmid I with BglII and XbaI restriction enzymes. Lane 1: 1kb DNA size marker (SM0311, ThermoFisher Scientific, USA). Lane 2: A 3900bp amplicon of plasmid digested with BglII and XbaI and an 810bp amplicon of inh-A. (c) Enzymatic digestion of the plasmid with HindIII and XbaI restriction enzymes. Lane 1:1kb DNA size marker (SM0311, ThermoFisher Scientific, USA). Lane 2 and 3: A 3900bp amplicon of plasmid digested with HindIII and XbaI and an 810bp fragment of inh-A

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Figure 3: Development of recombinant plasmid II and its confirmation: Lane 1: (a) 1kb DNA size marker (SM0311, ThermoFisher Scientific, USA). Lane 2: A 3810bp fragment of Recombinant Plasmid II. (b) Confirmation of the final construct. Lane 1: 1kb DNA size marker (SM0311, ThermoFisher Scientific, USA). Lane 2-3: The 810bp fragment of confirm the inh-A gene from recombinant plasmid II

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Confirmation of inh/A-topo construct

The inh/A-Topo construct was confirmed through restriction digestion, PCR, and sequence analysis. [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d represents the results of these experiments. [Figure 3]a inh/A-Topo construct without restriction. The band at 4710 bp confirms the presence of the inh-A gene in TOPO pCR 2.1 vectors. [Figure 3]a, [Figure 3]b, [Figure 3]c confirms the clone through PCR, and the band at 810 bp shows the presence of the gene. [Figure 3]b and [Figure 3]c confirms the clone through double digest with BglII and XbaI as well as HindIII and XbaI. The bands at 3900 bp and 810 bp confirm the clone.

Confirmation of inh/A-pVAX1 construct

[Figure 3]a, [Figure 3]b represents the inh/A-PVAX1 construct with and without restriction. The band at 3810 bp confirms the presence of an inh-A gene in the pVAX1 vector. The clone confirms through double digest with HindIII and XbaI. The bands at 3000 bp and 810 bp confirm the pVAX1 vector. All the constructs were confirmed through sequence analysis using commercially available T7 sequencing primers.


  Discussion Top


There is an urgent need for a novel vaccine to control and eradicate TB. One of the current strategies to control TB is the preparation of modern and effective DNA-based vaccines. A DNA vaccine is the best candidate because the animal model shows that it can stimulate both humoral and cell-mediated immunity and can provide prolonged protection.[20] Researchers are continuously trying different and better strategies to improve DNA vaccines. The present study uses similar strategies to control TB through DNA vaccine using Rv1484/inh-A gene.

The scientist reported functions of expressed inh-A in living yeast cells and observed its role through various biochemical and physiological tests.[21],[22] Described the biochemical function of Inh-A in Saccharomyces cerevisiae with lipoic acid synthesis, and Inh-A showed functional homology with yeast. The Rv1484/Inh-A gene, similar to Rv3485, Rv3559, Rv3530, and Rv 0927 proteins, has been activated in S. cerevisia, the synthesis of fatty acids, glycerol development, and the reductase activity existence.[23] According to published data, whole-sequencing the genome of M. tuberculosis, TB comprises the entire complement of FASII components. Inh-A is a significant component of fatty acid biosynthesis.[24] Another study revealed that FAS-II reductase is represented by Inh-A that completes the fatty acid final stage elongation process.[25] The data indicate that the inh-A gene is a component of developing very-long-chain fatty acids, mycolic acids. So, Inh-A is part of a mechanism of fatty acid elongation intermediates of molecules' biosynthesis.

In another study, a recombinant Mpt63 based construct was tested on the guinea pig model and found effective in provoking immune response against M. tb. Considerable antibody response was seen in model animals. Further, this study has proved that the Mpt63 gene lacks cross-reactive epitopes and does not react with other mycobacterium species.[22] In another recombinant DNA vaccine, an in-frame fused, the single construct of two M. tb genes ESAT-6 and Ag85B was made with tissue plasminogen activator sequence. The fused construct has stimulated the cell-mediated and humoral immune response in the mice models. This study further concludes that the Kozak sequence and other elements in TOPO and pVAX1 vectors have substantially increased the cloning and expression efficiency of fused genes.[26] In another study, numbers of esat6 gene constructs were made to get a suitable construction to be used as a promising DNA vaccine. All these constructs were tested in 293T human embryonic kidney cell lines for expression first, and none of the constructs gave a detectable level of expression on Western blot (WB). The tpa-esat6-pND14 construct was selected as a DNA vaccine candidate and injected intramuscularly and intradermal in balb/c mice along with controls. Animals were tested 9 weeks postvaccination and found positive against tpa-esat6-pND14 vaccine through WB and multiplex microbeads immunoassay.[27]

The report showed that the DNA vaccine as interleukin-2 expression and HSP65 fusion gene had been reported in another research. The immunogenicity, therapeutic and preventive effects of the HSP65/DNA vaccine in mice have been improved against TB. The Th1-type response was improved in mice.[28] The mammalian expression vector pVAX1 containing the gene of interest has a high expression rate, and immune responses against the gene's antigen are maximum. Vaccines based on pVAX1-ESAT6 generates the highest level of antigen-specific antibodies, and a suitable amount of INF-γ shows that this vaccine can also generate cell-mediated immune responses. The booster effect of ESAT6/DNA vaccine in mice targets developing a TB vaccine.[29]

In a recent study, pVAX-Rv1419 DNA is injected into mice. Three doses were given 2 weeks intervals. The high levels of CD4+ T-cells and IFN-γ production was recorded from vaccinated animals through flow cytometry. This result supports the pVAX-Rv1419 construct immunogenicity and effectiveness to be used as a DNA vaccine. Minimum side effects are additional benefits of this vaccine.[30]

In another DNA vaccine study, Mpt32 and Bfrb genes of M. tb were selected, amplified, and cloned into the pcDNA vector. These clones were confirmed through restriction digestion and sequence analysis and tested on the mice model. Antibody production against both antigens was observed and confirmed through the dot blot and Agar Gel Immuno Diffusion test. The study supports the idea that both Mpt32 and Bfrb genes are good candidates for DNA vaccines against TB and can be used alone or in DNA vaccine cocktails.[31]

In recent studies, Mpt63 was used and proved the importance of bacterial pathogenesis and DNA vaccine. Mpt63 includes three highly quantified secretory proteins found in the culture filtrate of M. tb. This protein is only reported from members of the M. tb complex. Mpt63 has a suggested role in necrosis and cavitation, but its exact role is unknown. However, the studies support the idea that this gene is immunogenic.[32]

The high numbers of fatal cases have dramatically increased multiple-drug tolerance, and nearly 40% of TB victims in some countries are affected by INH and r Rif-resistant strains. These drugs are extensively used for many centuries, and only two drugs available as antituberculosis. Inh-A is the main objective of INH.[33] The importance of Inh-A increases to one of the essential pathogen proteins in medicine. Therefore, this is vital in using current scientific methods to research the inh-A gene to evaluate new ways and procedures to develop a DNA vaccine. The results of our observation that Inh-A can lead to overcoming primary drug resistance as a DNA vaccine against TB. Therefore, DNA vaccines are a newly emerging strategy, and it can play an essential role in eradication TB. In a recent study, the Rv1484/inh-A gene fragment was successfully cloned into mammalian expression vector pVAX1. This construct may be used in animal models as a DNA vaccine to stimulate immune responses in future experiments.


  Conclusitons Top


The DNA vaccine holds a promising poteintial to control TB alone or in combination with Bacillus Calmette–Guérin (BCG) vaccine and current antibiotic regime.

Limitation of study

There is a minor risk associated with integration of recombinant plasmids with host chromosomal DNA. Therefor, as possible as low dose of DNA should be used in vaccination strategy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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