Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum

Abstract Background The production of recombinant proteins with proper conformation, appropriate post-translational modifications in an easily scalable and cost-effective system is challenging. Lactococcus lactis has recently been identified as an efficient Gram positive cell factory for the production of recombinant protein. We and others have used this expression host for the production of selected malaria vaccine candidates. The safety of this production system has been confirmed in multiple clinical trials. Here we have explored L. lactis cell factories for the production of 31 representative Plasmodium falciparum antigens with varying sizes (ranging from 9 to 90 kDa) and varying degree of predicted structural complexities including eleven antigens with multiple predicted structural disulfide bonds, those which are considered difficult-to-produce proteins. Results Of the 31 recombinant constructs attempted in the L. lactis expression system, the initial expression efficiency was 55% with 17 out of 31 recombinant gene constructs producing high levels of secreted recombinant protein. The majority of the constructs which failed to produce a recombinant protein were found to consist of multiple intra-molecular disulfide-bonds. We found that these disulfide-rich constructs could be produced in high yields when genetically fused to an intrinsically disorder protein domain (GLURP-R0). By exploiting the distinct biophysical and structural properties of the intrinsically disordered protein region we developed a simple heat-based strategy for fast purification of the disulfide-rich protein domains in yields ranging from 1 to 40 mg/l. Conclusions A novel procedure for the production and purification of disulfide-rich recombinant proteins in L. lactis is described

Neutrophils dominate in opsonic phagocytosis of<i> P. falciparum</i> blood-stage merozoites and protect against febrile malaria

Antibody-mediated opsonic phagocytosis (OP) of Plasmodium falciparum blood-stage merozoites has been associated with protection against malaria. However, the precise contribution of different peripheral blood phagocytes in the OP mechanism remains unknown. Here, we developed an in vitro OP assay using peripheral blood leukocytes that allowed us to quantify the contribution of each phagocytic cell type in the OP of merozoites. We found that CD14 (+) (+)CD16(−) monocytes were the dominant phagocytic cells at very low antibody levels and Fc gamma receptor (FcγR) IIA plays a key role. At higher antibody levels however, neutrophils were the main phagocytes in the OP of merozoites with FcγRIIIB acting synergistically with FcγRIIA in the process. We found that OP activity by neutrophils was strongly associated with protection against febrile malaria in longitudinal cohort studies performed in Ghana and India. Our results demonstrate that peripheral blood neutrophils are the main phagocytes of P. falciparum blood-stage merozoites

Human blood neutrophils generate ROS through FcγR-signaling to mediate protection against febrile P. falciparum malaria

Abstract Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria

Naturally acquired antibodies from Beninese infants promote Plasmodium falciparum merozoite-phagocytosis by human blood leukocytes: implications for control of asymptomatic malaria infections

Abstract Background Immunoglobulin G (IgG) antibodies are thought to play important roles in the protection against Plasmodium falciparum (P. falciparum) malaria. A longitudinal cohort study performed in the Southern part of Benin, identified a group of infants who were able to control asymptomatic malaria infections (CAIG). Methods IgG antibodies against distinct merozoite antigens were quantified in plasma from Beninese infants. Functionality of these antibodies was assessed by the merozoite-phagocytosis assay using THP-1 cells and primary neutrophils as effector cells. Gm allotypes were determined by a serological method of haemagglutination inhibition. Results Purified IgG from infants in CAIG promoted higher levels of merozoite-phagocytosis than did IgG from children who were unable to control asymptomatic infections (Ologit multivariate regression model, Coef. = 0.06, 95% CI 0.02;0.10, P = 0.002). High level of merozoite-phagocytosis activity was significantly associated with high levels of IgG against AMA1 (Coef. = 1.76, 95% CI 0.39;3.14, P = 0.012) and GLURP-R2 (Coef. = 12.24, 95% CI 1.35;23.12, P = 0.028). Moreover, infants of the G3m5,6,10,11,13,14,24 phenotype showed higher merozoite-phagocytosis activity (Generalized linear model multivariate regression, Coef. = 7.46, 95% CI 0.31;14.61, P = 0.041) than those presenting other G3m phenotypes. Conclusion The results of the present study confirm the importance of antibodies to merozoite surface antigens in the control of asymptomatic malaria infection in Beninese infants. The study also demonstrated that G3m phenotypes impact the functional activity of IgG. This last point could have a considerable impact in the research of candidate vaccines against malaria parasites or other pathogens