Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

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

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.

Susceptibility to malaria in fulani, Bariba, Otamari and gando individuals living in sympatry in Benin: Role of opsonizing antibodies to Plasmodium falciparum merozoites.

Objectives: Fulani in Africa are known to be less susceptible to Plasmodium falciparum (Pf) malaria. This study explored a potential involvement of antibody-mediated merozoite phagocytosis mechanism in this natural protection against malaria. Methods: Before the start of the malaria transmission season (MTS) in Benin, the functionality of antibodies against Pf merozoites was determined by the opsonic phagocytosis (OP) assay in plasma samples from Fulani, Bariba, Otamari and Gando groups. These individuals were actively followed-up for malaria detection from the beginning to the end of MTS. Anti-GLURP Immunoglobulin G antibody quantification, malaria Rapid Diagnostic Test (RDT) and spleen palpation were performed before and after MTS. Results: In Bariba, Otamari and Gando, but not in Fulani, plasma from adults promoted higher levels of OP than the children (P = 0.003; P = 0.012; P = 0.031 and P = 0.122). A high proportion of Fulani children had higher OP and anti-GLURP (P < 0.0001) antibody levels as compared to non-Fulani children; whereas this was not observed for Fulani adults (P = 0.223). High OP levels before MTS were significantly related to negative RDT after MTS (P = 0.011). Conclusion: Our results highlight the ability of opsonizing antibodies to potentially enhance natural protection of young Fulani individuals against Pf malaria in Benin.

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

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.

Neutrophils dominate in opsonic phagocytosis of P. falciparum 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.

Peripheral Merozoite Surface Proteins Are Targets of Naturally Acquired Immunity against Malaria in both India and Ghana.

Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a high priority. Immune-epidemiological studies are effective tools for the identification of antigenic targets of naturally acquired immunity (NAI) against malaria. However, differences in study design and methodology may compromise interstudy comparisons. Here, we assessed antibody responses against intact merozoites and a panel of 24 recombinant merozoite antigens in longitudinal cohort studies of Ghanaian (n = 115) and Indian (n = 121) populations using the same reagents and statistical methods. Anti-merozoite antibodies were associated with NAI in both the Indian (hazard ratio [HR] = 0.41, P = 0.020) and the Ghanaian (HR = 0.17, P < 0.001) participants. Of the 24 antigen-specific antibodies quantified, 12 and 8 were found to be protective in India and Ghana, respectively. Using least absolute shrinkage and selection operator (LASSO) regression, a powerful variable subselection technique, we identified subsets of four (MSP6, MSP3.7, MSPDBL2, and Pf12) and five (cMSP33D7, MSP3.3, MSPDBL1, GLURP-R2, and RALP-1) antigens that explained NAI better than the individual antibodies in India (HR = 0.18, P < 0.001) and Ghana (HR = 0.31, P < 0.001), respectively. IgG1 and/or IgG3 subclasses against five antigens from these subsets were associated with protection. Through this comparative study, maintaining uniformity of reagents and methodology, we demonstrate that NAI across diverse geographic regions may result from antibodies to multiple antigenic targets that constitute the peripheral merozoite surface protein complexes.

Breadth of Functional Antibodies Is Associated With Plasmodium falciparum Merozoite Phagocytosis and Protection Against Febrile Malaria.

BACKGROUND: The specific targets of functional antibodies against Plasmodium falciparum merozoites remain largely unexplored and, more importantly, their relevance to naturally acquired immunity in longitudinal cohort studies (LCSs) is yet to be tested. METHODS: Functionality of immunoglobulin G (IgG) antibodies against 24 merozoite antigens was determined at the baseline of an LCS in Ghana using a bead-based opsonic phagocytosis assay (BPA). Antigen-specific IgG3 subclass antibodies were quantified in the same samples by the Luminex multiplex system. RESULTS: A wide range of BPA activity was observed across the different antigens. High BPA responses of nMSP3K1, GLURP-R2, MSP23D7, MSP119k, and PfRh2-2030 coupled beads were significantly associated with a higher probability of children not experiencing febrile malaria. Children with high breadth of functional antibodies against these antigens together with cMSP33D7 had a significantly reduced risk of febrile malaria (adjusted hazard ratio, 0.36 [95% confidence interval, .18-.72]; P = .004). Five of the 6 BPA activities significantly (likelihood ratio rest, P ≤ .05) contributed to the protective immunity observed with the IgG3 antibodies. CONCLUSIONS: The development of BPA allowed profiling of functional antibodies in an LCS. Identification of targets of opsonic phagocytosis may have implications in the development of a subunit malaria vaccine.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria.

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [Kd ], 7.24 × 10-6 M) than recombinant HNA-1bb (Kd , 1.15 × 10-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala78Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.

Cytophilic Antibodies Against Key Plasmodium falciparum Blood Stage Antigens Contribute to Protection Against Clinical Malaria in a High Transmission Region of Eastern India.

Background: The collection of clinical data from a tribal population in a malaria-endemic area of India suggests the occurrence of naturally acquired immunity (NAI) against Plasmodium falciparum malaria. Methods: Quantity and functionality of immunoglobulin G (IgG) antibodies against intact merozoites and recombinant proteins were assessed in a 13-month longitudinal cohort study of 121 individuals, 3-60 years of age. Results: Opsonic phagocytosis of merozoites activity was strongly associated (hazard ratio [HR] = 0.34; 95% confidence interval [CI] = .18-.66; P = .0013) with protection against febrile malaria. Of the different IgG subclasses, only IgG3 antibodies against intact whole merozoites was significantly associated with protection against febrile malaria (HR = 0.47; 95% CI = .26-.86; P = .01). Furthermore, a combination of IgG3 antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 was strongly associated with protection against febrile malaria (HR = 0.15; 95% CI, .06-.37; P = .0001). Conclusions: These data suggest that NAI may, at least in part, be explained by opsonic phagocytosis of merozoites and IgG3 responses against whole merozoites, and in particular to a combination of 4 antigens is critical in this population. These results may have implications in the development of a subunit malaria vaccine. Opsonic phagocytosis of Plasmodium falciparum merozoites was associated with protection against clinical malaria in an India population. Antibody profiling identified four merozoite antigens (Pf12, MSP3.7, MSP3.3, and MSP2) as targets of protective Immunoglobuline G3 antibodies.

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

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.

Naturally Acquired Antibodies Target the Glutamate-Rich Protein on Intact Merozoites and Predict Protection Against Febrile Malaria.

Background: Plasmodium species antigens accessible at the time of merozoite release are likely targets of biologically functional antibodies. Methods: Immunoglobulin G (IgG) antibodies against intact merozoites were quantified in the plasma of Ghanaian children from a longitudinal cohort using a novel flow cytometry-based immunofluorescence assay. Functionality of these antibodies, as well as glutamate-rich protein (GLURP)-specific affinity-purified IgG from malaria hyperimmune Liberian adults, was assessed by the opsonic phagocytosis (OP) assay. Results: Opsonic phagocytosis activity was strongly associated (hazard ratio [HR] = 0.46; 95% confidence interval [CI] = .30-.73; P = .0008) with protection against febrile malaria. Of the antimerozoite-specific antibodies, only IgG3 was significantly associated with both OP and protection (HR = 0.53; 95% CI = .34-.84; Pcorrected = .03) against febrile malaria. Similarly, GLURP-specific antibodies previously shown to be protective against febrile malaria in this same cohort were significantly associated with OP activity in this study. GLURP-specific antibodies recognized merozoites and also mediated OP activity. Conclusions: These findings support previous studies that found OP of merozoites to be associated with protection against malaria and further shows IgG3 and GLURP antibodies are key in the OP mechanism, thus giving further impetus for the development of malaria vaccines targeting GLURP.