Hepatitis B Antigen in Coleus forskohlii Expression | InformativeBD

 

Neha Guleria, Paramanahally Hanumanthe Gowda Ramanjini Gowda, Satish Kumar Kariyaiah, and Renu Pasricha, from the different institute of the  india, wrote a research article about, Hepatitis B Antigen in Coleus forskohlii Expression. entitled, Expression of Hepatitis B Surface Antigen in Coleus forskohlii. This research paper published by the International Journal of Biomolecules and Biomedicine | IJBB. an open access scholarly research journal on Biomedicine. under the affiliation of the International Network For Natural Sciences | INNSpub.an open access multidisciplinary research journal publisher.

Abstract

Hepatitis B, a common viral infection, affecting millions of people every year, leads to disability and death. It has become one of the alarming diseases in the world. Vaccination is the best possible way to prevent this deadly viral infection and its consequences. Unaffordability of the mass vaccination program due to low health budgets especially in developing countries have demanded the economical, effective and easily available vaccine production against hepatitis B virus. The expression of subunit vaccines and recombinant proteins in plants is a convenient, safe and potentially economical platform technology. Hence, development of a plant-based vaccine could be promising. Therefore, the present investigation focused on expression and large- scale production of hepatitis B surface antigen (HBsAg) in Coleus forskohlii for the development of vaccine. Eight transformed C. forskohlii plants were generated via Agrobacterium -mediated transformation method. The integration of 681bp of HBsAg gene into the plant genome was confirmed using PCR. SDS-PAGE showed the presence of ~48kDa dimer and ~24kDa monomer form of HBsAg protein and the expression of recombinant protein was further confirmed by western blot. C. forskohlii expressed HBs Ag was recorded 260µg/g leaf fresh weight as measured by ELISA. Transformed plants of HBsAg showed the accumulation of Virus Like Particles of 22 nm size using transmission electron microscopy. This study offers a great potential for the large -scale production of hepatitis B vaccine in C. forskohlii which provides a strategy for contributing a means to achieve global immunization for the hepatitis B prevention and eradication.

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Introduction

Hepatitis B virus (HBV) causes transient and chronic infection of the liver ultimately leads to untreatable liver cancer (Yogendra et al., 2009). In the HBV- infected people, the virus persists for the rest of their lives and can be passed on to others (Goldstein et al., 2005). HBV infection has a worldwide distribution. It is estimated that more than 2 billion people have been infected. Of these, approximately 400 million are chronically infected and at risk of serious illness and death from cirrhosis and hepatocellular carcinoma (HCC), diseases that are estimated to cause 650 000–780 000 deaths each year worldwide (WHO, 2015). Therefore, Hepatitis B has become a global public health problem.Vaccination against this virus was introduced over 25 years ago (Czyzet al., 2014), a proven strategy to control HBV infection. The current subunit vaccines e.g. Shanvac B, Engerix B, Genevac-B, Biovac-B etc. produced in Pichia pastoris (methylotrophic yeast) which express the major surface antigen of the Hepatitis B virus, is used for vaccination. But HBV is still responsible for significant morbidity and mortality in poor countries (Kong et al., 2001). The situation is deteriorating, also in developing countries due to limited budget for health care as large- scale vaccination programs are required (Smith et al., 2003). Hence, there is a need for large-scale production and use of relatively cheap and effective hepatitis B vaccine.

Earlier, the majority of work was focused on the production of recombinant proteins and subunit vaccines in prokaryotes, mainly in Escherichia coli because it offers low cost and short production timescale (Ma et al., 2003). With the time, limitations of prokaryotes came into light such as low product yield and lack of post-translational modifications. Therefore, then researchers turned to eukaryotic systems: yeast, mammalian cells, insects and transgenic animals. These systems were also proven inefficient in terms of cost, production, safety, authenticity and scalability (Balen and Rasol, 2007; Ahmad, 2014). The plants obtained in recent decades synthesize great number of valuable proteins such as human serum proteins, growth regulators, antibodies, vaccines, industrial enzymes, biopolymers, and reagents for molecular biology and biochemistry.

Plant cells possess enzymatic systems of post-translational modification, which are necessary for the assembly of synthesized monomeric proteins of vaccine into immunogenic multimers (Rukavtsova et al., 2015). The target antigens causing active immune response can also be synthesized in plant cells (Mason and Arntzen, 1995). Thus, now the plants have received the most attention in molecular pharming community for the production of subunit vaccines. The expression of subunit vaccines and recombinant proteins in plants has emerged as a convenient, safe and potentially economical platforms technology (Thomas et al., 2011; Xu et al., 2012). Similarly, several studies have revealed that plant expression system produce many biologically active complex human proteins (Merle et al., 2002; Peeters et al., 2001). Therefore, with the time extensive technology advancements for glycan modification makes plants the most versatile platform for the production of recombinant protein and subunit vaccines.

Hepatitis B surface antigen (HBsAg) is a transmembrane protein consisting of S, M and L-HBsAg. All HBsAg proteins are encoded within a single reading frame and contain a common S domain. The S-HBsAg contains only the S domain and form strongly immunogenic determinant (Pniewski, 2013; Rybicki, 2014). The hydrophobic S-HBsAg carries all the necessary inform-ation for membrane translocation, the component of virus envelope and assembled into immunogenic structures known as Virus Like Particles (VLPs). These particles are self- assembled protein structures, devoid of viral DNA, unable to replicate and non-infectious (Hyun et al., 2014). It has been known that the lipid molecules closely associated with S-HBsAg are responsible for the antigenicity, protein conformation and stability of the VLPs (Jadwiga et al., 2014). VLPs possess repetitive high-density displays of viral surface proteins and are potentially effective in eliciting strong humoral and cellular immune responses. Hence, these characteristics provide a great platform for VLPs as an effective vaccine candidate (Natasha et al., 2012).

In 1986, Federal Drug Administration (USA) approved the first recombinant protein-based Hepatitis B vaccine for humans. It is based on a recombinant HBV surface antigen (HBsAg), which upon production in yeast or mammalian cells forms 22-nm spherical VLPs (Greiner et al., 2012). Similarly the structure authenticity and function of plant produced VLPs is first explained by Mason et al., in 1992 who studied expression and production of HBsAg VLPs in tobacco. The same group studied the immunogenic response of VLPs produced in transgenic potato on mice. The study emphasized the importance and effectiveness of VLP vaccine. Therefore, it would be safe to say that VLPs can represent one of the most exciting new technologies to rapidly produce effective vaccines with long lasting protection (Rybicki, 2014).

The present report is the first study on the expression of HBsAg in C. forskohlii for the development of injection HBsAg vaccine. C. forskohlii is animportant medicinal herb in Indian Ayurvedic medicine considered endangered medicinal plant, the C. forskohlii, a member of the Lamiaceae family is valued for the production of labdane diterpenoid forskolin (produced only in roots) from its tuberous roots used for relief of cough, eczema, skin infections, heart failures and certain type of cancers (Boby and Bagyaraj, 2003).

The novelty and priority of the research is that C. forskohlii is a potential candidate to produce vaccine, as this crop is vegetatively propagated with high biomass production and can be grown in the field without any chances of genetic contamination (does not set seeds), so it offers good platform to produce novel compounds. It means, large quantities of recombinant protein can be produced quite rapidly, thereby significantly reducing the cost of production. When we have a plant which does not carry threat of gene pollution then biosafety laws of any given country can accommodate a programme to grow recombinant protein producing plant (Lou et al., 2007).

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