Conformational and Thermal Stability Improvements for the Large-Scale Production of Yeast-Derived Rabbit Hemorrhagic Disease Virus-Like Particles as Multipurpose Vaccine

Abstract

Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans.

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

“…Optimization of the induction phase methanol concentration and the batch phase time, as well as inclusion of a glycerol transition phase, resulted in the maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L −1 . Previous work in the area of optimization of recombinant VLP production reported yields ranging from 0.18 g L −1 in E. coli to 0.3 g L −1 in P. pastoris . The almost three‐fold increase in yield presented here represents a major advance compared to these previous reports.…”

Design and development of a chimeric vaccine candidate against zoonotic hepatitis E and foot-and-mouth disease

“…In other words, Seq 8-P222 seems to be stable for more than 6 weeks without cold chain requirements and this represents a very attractive property for a vaccine. Moreover, the proteins were tested without any formulation and thus we could speculate that these periods could be extended with an adequate adjuvant formulation as reported previously [ 36 , 37 ]. It is worth noting that the instability of Seq 8-P216 could be due to the fluctuations in the Seq 8 subunit predicted in the flexibility analysis (Fig.…”

Sneaking Out for Happy Hour: Yeast-Based Approaches to Explore and Modulate Immune Response and Immune Evasion

“…These particles are suitable for mucosal immunization due to their resistance to proteolytic degradation and compatibility with mucosal environment [129]. Yeast VLP scaffolds have been used to display immunogenic antigens from pathogens such as porcine circovirus [130], rabbit hemorrhagic disease virus [131], coxsackievirus A6 [132], hepatitis B virus [133], Zika virus [128], chikungunya virus [134], dengue virus [135], enterovirus [127,136].…”