Optimization, characterization, comparison of self-assembly VLP of capsid protein L1 in yeast and reverse vaccinology design against human papillomavirus type 52.

BACKGROUND: Vaccination is the one of the agendas of many countries to reduce cervical cancer caused by the Human papillomavirus. Currently, VLP-based vaccine is the most potent vaccine against HPV, which could be produced by a variety of expression systems. Our study focuses on a comparison of recombinant protein expression L1 HPV52 using two common yeasts, Pichia pastoris and Hansenula polymorpha that have been used for vaccine production on an industrial scale. We also applied bioinformatics approach using reverse vaccinology to design alternative multi-epitope vaccines in recombinant protein and mRNA types. RESULTS: Our study found that P. pastoris relatively provided higher level of L1 protein expression and production efficiency compared to H. polymorpha in a batch system. However, both hosts showed self-assembly VLP formation and stable integration during protein induction. The vaccine we have designed exhibited high immune activation and safe in computational prediction. It is also potentially suitable for production in a variety of expression systems. CONCLUSION: By monitoring the overall optimization parameter assessment, this study can be used as the basis reference for large-scale production of the HPV52 vaccine.

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3.

BACKGROUND: A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. RESULTS: The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs. CONCLUSION: Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.