Back to Search
Start Over
Pichia pastoris-Expressed Dengue 2 Envelope Forms Virus-Like Particles without Pre-Membrane Protein and Induces High Titer Neutralizing Antibodies
- Source :
- PLoS ONE, PLoS ONE, Vol 8, Iss 5, p e64595 (2013)
- Publication Year :
- 2013
- Publisher :
- The University of North Carolina at Chapel Hill University Libraries, 2013.
-
Abstract
- Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+)-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (p
- Subjects :
- Viral Diseases
Mouse
viruses
Yeast and Fungal Models
Dengue virus
Antibodies, Viral
medicine.disease_cause
Pichia
Dengue fever
Dengue
Mice
Viral Envelope Proteins
Mice, Inbred BALB C
Vaccines
Multidisciplinary
Attenuated vaccine
Viral Vaccine
Vaccination
virus diseases
Animal Models
Recombinant Proteins
Infectious Diseases
Medicine
Research Article
Biotechnology
Mice, 129 Strain
medicine.drug_class
Science
Molecular Sequence Data
Immunology
Biology
Monoclonal antibody
Virus
Cell Line
Pichia pastoris
Model Organisms
Vaccine Development
medicine
Animals
Humans
Amino Acid Sequence
Dengue vaccine
Virion
Immunity
Viral Vaccines
Dengue Virus
medicine.disease
biology.organism_classification
Antibodies, Neutralizing
Virology
Clinical Immunology
Protein Multimerization
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- PLoS ONE, PLoS ONE, Vol 8, Iss 5, p e64595 (2013)
- Accession number :
- edsair.doi.dedup.....11715f4cb0188fed2642e458c7955f3e
- Full Text :
- https://doi.org/10.17615/mswa-v172