Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

Abstract

Background: To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope.

Methods: A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot.

Results: The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n=38) and a clinical specificity of 100% (n=24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice.

Conclusions: The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

Figure 1

Nucleotide differences between the original (ICB10ori, GenBank access no. AF435594) and the modified (MLCICB10) nucleotide sequences of P. vivax MSP-1.

Figure 2

(A) Gene construction scheme for the expression of the MLC chimeric recombinant protein. LB and RB indicate the left and right borders of the T-DNA region, which is stably integrated into nuclear chromosomal DNA during Agrobacterium-mediated DNA delivery. TSS represents the translation start site, and SEKDEV (Serine-Glutamate-Lysine-Aspartate-Glutamate-Valine) stands for secretion signal. Additionally, this signal promotes exact oligomerization. The selectable marker used for plant transformation is the nptII gene, which confers kanamycin resistance. The GUS reporter protein was used as a marker to enable rapid screening in selective steps of plant regeneration. (B) Nucleotide and deduced amino acid sequence of the MLC chimeric recombinant gene. It is comprised of a modified PvMSP-1 (PvMSP1_ICB10, AF435594), a linker (Pro-Gly repeat, underlined), and PvCSP (PvCSP_TR, AF316582).

Figure 3

Detection of GUS activity in explants of regenerated B. napus plants. (A) GUS reporter activity as measured by color change. (B) GUS reporter activity was quantified by measuring sample optical densities at 595 nm.

Figure 4

(A) Confirmation of transformed B. napus by PCR amplification of the MLC gene (I) and the nptII gene (II). Lanes 1, 3-6, transformed explants; Lane 2, non-transformed explant. (B) Expression and confirmation of the MLC chimeric recombinant protein in B. napus. Lane 1, wild type plant; Lane 2, pHSS2-MLC transgenic plant; Lane 3, reacted with anti-PvMSP antibodies; Lane 4, reacted with anti-PvCSP antibodies.

Figure 5

Scattergram of absorbances measured by ELISA using MLC chimeric recombinant protein expressed in B. napus. Sera from healthy individuals and malaria patients infected with P. vivax were used.

Figure 6

Antibody induction in BALB/c mice orally immunized with transgenic B. napus that expresses the MLC chimeric recombinant protein. (A) BALB/c mice fed PBS only; (B), BALB/c mice fed 200 μg of chimeric recombinant protein; (C), BALB/c mice fed 1 g of chimeric recombinant protein; (D), positive control-reacted with anti-his antibody.

Figure 7

Cytokine expression in the spleens of BALB/c mice orally immunized with transgenic B. napus that expresses the MLC chimeric recombinant protein. Concentrations of IL-2, IL-12(p40), IFN-γ, and TNF-α were measured. Group 1, BALB/c mice fed PBS only; Group 2, BALB/c mice fed non-transformed plant; Group 3, BALB/c mice fed MLC chimeric recombinant protein. Values represent the mean ± SD (n = 3). * Significant difference between Groups 1 and 3 (P < 0.05). ^†Significant difference between Groups 2 and 3 (P < 0.05).