Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: Advantages for immunization strategies and vaccine implementation

Farnós, Omar; Fernández, Erlinda; Chiong, Maylin; Parra, Francisco; Joglar, Marisdania; Méndez, Lídice; Rodríguez, Elsa Rodríguez; Moya, Galina; Rodríguez, Dorian; Lleonart, Ricardo; González, Ernesto; Alonso, Alena; Alfonso, P; Suárez, Marisela; Rodríguez, María Pilar; Toledo, Jorge R.

doi:10.1016/j.antiviral.2008.08.007

Cited by 16 publications

(9 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Pichia pastoris PVP12 strain [22] allowed the production of the recombinant VP1 multimers. VP1 obtained associated to the yeast membranous system of P. pastoris PVP11 strain [28] was used for comparative purposes in Western blot analyses.…”

Section: Methodsmentioning

confidence: 99%

“…The study of particles integrity in equilibrium sucrose density gradients was conducted in duplicate as previously described [22].…”

Section: Methodsmentioning

confidence: 99%

“…The morphology and size of RHDV VLP in samples purified at different pH values or subjected to thermal stresses was examined by transmission electron microscopy (JEOL JEM 2000 EX, Japan) as already described [22].…”

Section: Methodsmentioning

confidence: 99%

“…Preceded by successful examples of subunit vaccine production and application in human and veterinary massive vaccination programs [25]–[27] one Pichia pastoris mutant strain was selected for the expression at high levels of the capsid protein from RHDV [22], [28]. The antigen was obtained forming soluble virus-like particles (VLP) inside the yeast cells with high antigenic resemblance to RHDV and attractive levels for a simple and inexpensive production process.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2013

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

“…The study of particles integrity in equilibrium sucrose density gradients was conducted in duplicate as previously described [22].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…Therefore, it is important to prevent and control the disease with effective vaccines. Unfortunately, the lack of an efficient in vitro system for propagating RHDV has hindered production of the virus as a source of vaccine antigens [7]. Currently, commercially available vaccines are produced from tissues collected from experimentally infected rabbits.…”

Section: Discussionmentioning

confidence: 99%

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression inSpodoptera frugiperda9 (Sf9) cells

Gao

Chen

et al. 2013

J Vet Sci

View full text Add to dashboard Cite

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.

show abstract

Expression and Purification of Recombinant Proteins for Vaccine Applications

Zambonelli

Martinez‐Guzmann

Srivastava

2011

Development of Vaccines

View full text Add to dashboard Cite

Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: Advantages for immunization strategies and vaccine implementation

Cited by 16 publications

References 61 publications

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

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

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression inSpodoptera frugiperda9 (Sf9) cells

Expression and Purification of Recombinant Proteins for Vaccine Applications

Contact Info

Product

Resources

About