Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis

Abstract

Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.

Immunogenicity analysis of conserved fragments in Plasmodium ovale species merozoite surface protein 4

“…This suggests complete conservation of PoMSP4, which is consistent with previous findings, especially in P. falciparum orthologs [16,33,34]. Only one mutation was observed in the EGF-like domain at the carboxyl terminus in one P. o. wallikeri isolate (from Uganda); however, this single mutation could not affect the antigenicity of MSP4 proteins because EGF domains have shown relatively poor immunogenicity [30,31,37].…”

“…PoMSP4-predicted open reading frames (ORFs) without the EGF-like domain consisting of 1-119 (PocMSP4) and 1-97 (PowMSP4) amino acids, for which a P. falciparum ortholog has been reported as highly immunogenic [30,31,37], were selected. Genomic DNA from P. ovale isolates was used as template for PCR amplification of pomsp4 ORFs.…”

“…This finding suggested that MSP4 is a potential malaria vaccine candidate, especially in combination with other antigens. MSP4 immunogenicity has been associated with protection in natural infections with P. falciparum [30][31][32]. In addition, MSP4 shows a high degree of conservation among P. falciparum isolates [16,33,34], supporting its potential consideration as a subunit component for malaria vaccine formulations.…”

Immunogenicity analysis of conserved fragments in Plasmodium ovale species merozoite surface protein 4

“…falciparum [30][31][32]. In addition, MSP4 shows a high degree of conservation among P. falciparum isolates [16,33,34], supporting its potential consideration as a subunit component for malaria vaccine formulations.…”

Expression of Stichopus japonicus Lysozyme Gene in Bacillus subtilis WB600

“…The other advantages of B. subtilis include direct secretion of the functional proteins into the medium, no significant bias in codon usage, its well-known genetics, simple fermentation processes, and the development of simplified downstream processing procedures [16,22]. These further make the B. subtilis a potential bacillus in genetic engineering and industrial application [4,11].…”