Dataset Information

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.

ABSTRACT: VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant protein secretion from Pichia pastoris and if domain boundaries were applicable across different VAR2CSA alleles.VAR2CSA sequences were bioinformatically analysed to identify the predicted C11 and C12 cysteine residues at the C-termini of DBL domains and revised N- and C-termimal domain boundaries were predicted in VAR2CSA. Multiple construct boundaries were systematically evaluated for protein secretion in P. pastoris and secreted proteins were tested as immunogens.From a total of 42 different VAR2CSA constructs, 15 proteins (36%) were secreted. Longer construct boundaries, including the predicted C11 and C12 cysteine residues, generally improved expression of poorly or non-secreted domains and permitted expression of all six VAR2CSA DBL domains. However, protein secretion was still highly empiric and affected by subtle differences in domain boundaries and allelic variation between VAR2CSA sequences. Eleven of the secreted proteins were used to immunize rabbits. Antibodies reacted with CSA-binding infected erythrocytes, indicating that P. pastoris recombinant proteins possessed native protein epitopes.These findings strengthen emerging data for a revision of DBL domain boundaries in var-encoded proteins and may facilitate pregnancy malaria vaccine development.

SUBMITTER: Avril M

PROVIDER: S-EPMC2714522 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.

Avril Marion M Hathaway Marianne J MJ Cartwright Megan M MM Gose Severin O SO Narum David L DL Smith Joseph D JD

Malaria journal 20090629

<h4>Background</h4>VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant ...[more]

PMID: 19563628

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Project description:BACKGROUND: Placental malaria is a disease linked to the sequestration of Plasmodium falciparum infected red blood cells (IRBC) in the placenta, leading to reduced materno-fetal exchanges and to local inflammation. One of the virulence factors of P. falciparum involved in cytoadherence to chondroitin sulfate A, its placental receptor, is the adhesive protein VAR2CSA. Its localisation on the surface of IRBC makes it accessible to the immune system. VAR2CSA contains six DBL domains. The DBL6epsilon domain is the most variable. High variability constitutes a means for the parasite to evade the host immune response. The DBL6epsilon domain could constitute a very attractive basis for a vaccine candidate but its reported variability necessitates, for antigenic characterisations, identifying and classifying commonalities across isolates. METHODOLOGY/PRINCIPAL FINDINGS: Local alignment analysis of the DBL6epsilon domain had revealed that it is not as variable as previously described. Variability is concentrated in seven regions present on the surface of the DBL6epsilon domain. The main goal of our work is to classify and group variable sequences that will simplify further research to determine dominant epitopes. Firstly, variable sequences were grouped following their average percent pairwise identity (APPI). Groups comprising many variable sequences sharing low variability were found. Secondly, ELISA experiments following the IgG recognition of a recombinant DBL6epsilon domain, and of peptides mimicking its seven variable blocks, allowed to determine an APPI cut-off and to isolate groups represented by a single consensus sequence. CONCLUSIONS/SIGNIFICANCE: A new sequence approach is used to compare variable regions in sequences that have extensive segmental gene relationship. Using this approach, the VAR2CSA DBL6 domain is composed of 7 variable blocks with limited polymorphism. Each variable block is composed of a limited number of consensus types. Based on peptide based ELISA, variable blocks with 85% or greater sequence identity are expected to be recognized equally well by antibody and can be considered the same consensus type. Therefore, the analysis of the antibody response against the classified small number of sequences should be helpful to determine epitopes.

Dataset Information

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.

Publications

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.

Similar Datasets

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