Introduction to Molecular Vaccinology

Question: Discuss about the Introduction to Molecular Vaccinology. Answer: The vaccine developed against smallpox is vaccinia virus. Experiments were conducted with this replication competent virus and the results showed effectiveness. Therefore, with further development of Recombinant DNA Technology, recombinant vaccinia vaccines were generated that express the desired foreign genes (Monath, 2005). This vaccine system is developed to express recombinant proteins in mammalian cells. The major advantage offered by this expression system is "post-translational modification". (Giese, 2016) Studies showed that recombinant vaccinia system is also effective against diseases other than smallpox (Monath, 2015). Clinical trials include a use of recombinant vaccinia system in cancer, parasitic and viral diseases. The paper will discuss the details of expression of vaccinia vector vaccine.' In order to determine whether the gene has expressed in the cell or organism the gene that was incorporated in recombinant DNA is replaced with the reporter gene at the coding portion. Later the gene expression can be monitored by assessing the fluorescence or enzyme activity of the gene product. The DNA sequences present at the upstream of the coding region regulate the transcription of the reporter gene and drive its expression as shown in the figure 1. The recombinant DNA molecule is then introduced in the cell. Figure: 1 Gene expression using reporter gene (Source: Jang et al., 2014) A) In this example the coding sequence forproteinX is replaced by the coding sequence for protein Y. (B) Various fragments ofDNAcontaining candidate regulatory sequences are added in combinations. Therecombinant DNAmolecules are then tested forexpressionafter theirtransfectioninto a variety of different types of mammalian cells, (C). For experiments in eucaryotic cells, two commonly used reporter proteins are the enzymes -galactosidase(-gal)andgreen fluorescent protein Initially a vector for expression for influenza virus is constructed as discussed in the previous assignment. The vector was transformed to host E.coli that was grown in LB media containing ampicillin and chloramphenical. The cells were cultured upto O.D of 0.65 at 600 nm. 1 mM IPTG was added in the medium and protein expression was induced for the duration of six hours at the temperature of 30C. The cell lysate was centrifuged to obtain pellet fraction which will be used for performing SDS-PAGE (Jang et al., 2014). Protein bands were visualised by staining with coomassie brilliant blue as shown in figure 2. Figure: 2 Analysis of protein profiles via SDS-PAGE. (Source: Jang et al., 2014) The immunological studies conducted by Harrington et al., (2002) describe in details the immune response in mice to introduction with Recombinant vaccinia viruses (rVV). rVV were manufactured from the P13 clone of smallpox. rVV were introduced in the mice after infection with vaccinia virus that expresses the cytotoxic T lymphocyte epitopes from lymphocytic choriomeningitis virus (LCMV). The rise in CTLs was analysed by staining cytokines before which it was stimulated with specific peptide. Mice were inoculated with different doses of VV to evaluate the antibody response. It was found by performing ELISA and Western blotting that there was the increase in antibody response specifically to gE and gB genes of VV. References Giese, M. (2016). Types of Recombinant Vaccines. InIntroduction to Molecular Vaccinology(pp. 199-232). Springer International Publishing. Harrington, L. E., van der Most, R., Whitton, J. L., Ahmed, R. (2002). Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope.Journal of virology,76(7), 3329-3337. Jang, Y. H., Cho, S. H., Son, A., Lee, Y. H., Lee, J., Lee, K. H., Seong, B. L. (2014). High-yield soluble expression of recombinant influenza virus antigens from Escherichia coli and their potential uses in diagnosis.Journal of virological methods,196, 56-64. Monath, T. P. (2005). Yellow fever vaccine.Expert review of vaccines,4(4), 553-574. Monath, T. P., Seligman, S. J., Robertson, J. S., Guy, B., Hayes, E. B., Condit, R. C., ... Brighton Collaboration Viral Vector Vaccines Safety Working Group. (2015). Live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment.Vaccine,33(1), 62-72.