Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles

Abstract

Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.

Chimeric Human Papillomavirus-16 Virus-like Particles Presenting P18I10 and T20 Peptides from HIV-1 Envelope Induce HPV16 and HIV-1-Specific Humoral and T Cell-Mediated Immunity in BALB/c Mice

“…It could be attributed to the negative stain reagent that we used in this study. In our recent study published in previous study [ 37 ] and another ongoing paper under peer review (data not shown), we have got good quality and resolution of electron micrographs when yeast- and baculovirus-derived L1:P18I10 VLPs (the same chimeric construct) were equilibrated in PBS and negative-stained with phosphotungstic acid (PTA). Because we used the different VLP production and purification system in this study, mammalian cell-derived L1:P18I10 and L1:T20 VLPs were equilibrated with Tris-HCl and negative-stained with uranyl acetate.…”

“…The HPV:HIV (L1:P18I10 and L1:T20) VLP samples were serially purified using cation exchange (Capto SP ImpRes, GE, Boston, MA, USA), size exclusion (Capto Core 700, GE) and affinity (HiTrap Heparin HP, GE) chromatography. The chromatographic protocols were described in our previous studies [ 36 , 37 ] and following the manufacturer’s protocol [ 38 ]. The L1 protein signal in each purification step was characterized by Western blot analysis and probed with anti-HPV16 L1 antibody CAMVIR-1 [ 39 ].…”

Virus-like Particles as Antiviral Vaccine: Mechanism, Design, and Application

“…This clinical trial has achieved partial protection, inducing a low protective efficacy of 31.2% against HIV-1 infection in Thailand [89,92]. The P18I10 peptide (RGPGRAFVTI) form the V3 loop of HIV-1 gp120 is inserted to the D-E loop of the HPV L1 capsid, the resulting chimeric VLPs vaccine named L1P18 is expressed in yeast aiming to elicit protective effect against both HPV and HIV [93]. Further efficiency test of this construct will be conducted in future study.…”

Challenges and Opportunities in the Process Development of Chimeric Vaccines

“…The intracellularly expressed chimeric L1P18 protein was purified via size exclusion chromatography, ultracentrifugation, and ultrafiltration with 96% purity and a recovery yield of 9.23%. The approach highlights bivalent vaccines’ easy and reliable production against major global pathogens using an alternative yeast-based expression platform [ 95 ]. In another study, researchers developed a multi-component malarial vaccine consisting of distinct parasite ligands of Plasmodium falciparum , namely, PfAMA-1 (domain III of apical membrane ag-1), PfMSP1 (merozoite surface protein), and PfEBA-175 (binding domain for glycophorin A on erythrocytes).…”