Expression and scale-up production of recombinant human papillomavirus type 52 L1 protein in methylotrophic yeast Hansenula polymorpha.

BACKGROUND: Human papillomavirus (HPV) vaccination is one of the crucial national vaccination programs aimed at reducing the prevalence of the diseases associated with HPV infections, which continue to pose a global health concern. However, a significant disparity exists in the distribution of HPV vaccine, particularly in low-middle income countries where the cost of HPV vaccine becomes a major obstacle. Thus, it is essential to ensure the availability of an economically feasible HPV vaccine, necessitating immediate efforts to enhance the cost-effectiveness of vaccine production. This study aimed to develop an efficient production system for the recombinant HPV type 52 L1 protein as HPV vaccine material using methylotrophic yeast Hansenula polymorpha expression system. RESULTS: This study presents an in-depth examination of the expression and scale-up production of HPV type 52 L1 protein using DASGIP® parallel bioreactor system. The pHIPX4 plasmid, which is regulated by the MOX promoter, generates stable clones that express the target protein. Cultivation employing the synthetic medium SYN6(10) with controlled parameters (e.g. temperature, pH, feeding strategy, and aeration) produces 0.15 µg/mL of HPV type 52 L1 protein, suggesting a possibility for scaling up to a higher production level. CONCLUSION: The scale-up production of HPV type 52 L1 protein using Hansenula polymorpha expression system described in this study provides an opportunity for an economical manufacturing platform for the development of the HPV vaccine.

Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115.

BACKGROUND: Cervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein. RESULT: The recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 µg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP. CONCLUSION: The recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.

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.

Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system.

Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.