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Long-lasting and sterile homologous protection against malaria can be achieved by the exposure of malaria-naive volunteers under chemoprophylaxis to Plasmodium falciparum-infected mosquitoes (chemoprophylaxis and sporozoite [CPS] immunization). While CPS-induced antibodies neutralize sporozoite infectivity in vitro and in vivo, antibody-mediated effector mechanisms are still poorly understood. Here, we investigated whether complement contributes to CPS-induced preerythrocytic immunity. Sera collected before and after CPS immunization in the presence of active or inactive complement were assessed for the recognition of homologous NF54 and heterologous NF135.C10 sporozoites, complement fixation, sporozoite lysis, and possible subsequent effects on in vitro sporozoite infectivity in human hepatocytes. CPS immunization induced sporozoite-specific IgM (P < 0.0001) and IgG (P = 0.001) antibodies with complement-fixing capacities (P < 0.0001). Sporozoite lysis (P = 0.017), traversal (P < 0.0001), and hepatocyte invasion inhibition (P < 0.0001) by CPS-induced antibodies were strongly enhanced in the presence of active complement. Complement-mediated invasion inhibition in the presence of CPS-induced antibodies negatively correlated with cumulative parasitemia during CPS immunizations (P = 0.013). While IgG antibodies similarly recognized homologous and heterologous sporozoites, IgM binding to heterologous sporozoites was reduced (P = 0.023). Although CPS-induced antibodies did not differ in their abilities to fix complement, lyse sporozoites, or inhibit the traversal of homologous and heterologous sporozoites, heterologous sporozoite invasion was more strongly inhibited in the presence of active complement (P = 0.008). These findings demonstrate that CPS-induced antibodies have complement-fixing activity, thereby significantly further enhancing the functional inhibition of homologous and heterologous sporozoite infectivity in vitro. The combined data highlight the importance of complement as an additional immune effector mechanism in preerythrocytic immunity after whole-parasite immunization against Plasmodium falciparum malaria.

Additional file 1: Figure S1. Association between gametocyte densities and oocystâ prevalence and density. A. Overview of gametocyte densities and infection rates of experiments of Figs. 1, 2, 3, 4 and Additional file 2: Figure S2. B. Overview of gametocyte densities and mean oocysts of experiments of Figs. 1, 2, 3, 4 and Additional file 2: Figure S2.

Protocol. Malaria antigen-specific IgG ELISA (S1). Isolation and cultivation of primary human hepatocytes (S2). In vitro sporozoite infectivity assay of primary human hepatocytes (S3). Immunofluorescent analysis of P. falciparum-infected primary human hepatocytes (S4). In vitro PBMC restimulation with PfRBCs and flow cytometry staining (S5) [11, 12, 14, 17, 42]. (DOCX 20 kb)

Additional file 1: Table S1. Observations with different buffers for mosquito homogenisation. Table S2. Experimental infection data CT values by qPCR vs Optical Density in CSP-ELISA. Table S3. Overview of processing time of the four methods compared in seconds per 80 mosquitoes. Figure S1. Protocol for mixed infection detection using the same 50Â Âľl volume. Homogenate is transferred from the prior experiments to the subsequent ones. It is important that every step is timed and thought ahead on when to start the different steps. Figure S2. Processing of mosquitoes by CS-ELISA when tested for both P. falciparum and P. vivax.

Protocol. Malaria antigen-specific IgG ELISA (S1). Isolation and cultivation of primary human hepatocytes (S2). In vitro sporozoite infectivity assay of primary human hepatocytes (S3). Immunofluorescent analysis of P. falciparum-infected primary human hepatocytes (S4). In vitro PBMC restimulation with PfRBCs and flow cytometry staining (S5) [11, 12, 14, 17, 42]. (DOCX 20 kb)

First-wave parasitemia after challenge. Parasitemia on day 7 post-challenge in immunized (open circles) and control (closed circles) volunteers. The line and error bars show the geometric mean and 95% CI interval. Figure S2. Inhibition of in vitro homologous and heterologous intra-hepatic sporozoite development in primary human hepatocytes by mAb 2A10. P. falciparum NF54 (blue), NF135.C10 (orange) and NF166.C8 (green) sporozoites in the presence of 10% heat-inactivated naive human control serum were pre-incubated with 3-fold serial dilutions of the 2A10 monoclonal antibody (0.027–20 μg/mL), targeting the repeat region of the circumsporozoite protein (CSP), and added to primary human hepatocyte cultures. Six days post-infection, the number of P. falciparum-infected hepatocytes was assessed as described in Additional file 1: S4 and S5. Figure S3. Amino acid changes in CSP. Table S1. Whole-genome sequencing statistics. Table S2. Mosquito salivary gland infectivity and sporozoite load of the three clones. Mean mosquito salivary gland infectivity and sporozoite load determined 1 day prior to challenge infection by dissecting a sample of 10 mosquitoes per strain. (DOCX 427 kb)

Additional file 2: Figure S2. Effect of different flow-rate MACS on gametocyte infectivity. Number of oocysts per mosquito after membrane-feeding assay (MIDI cages). 1 600 µL of gametocyte-infected whole blood from starting material, fed directly. 2 9000 µL of gametocyte-infected whole blood from starting material added once over MACS column with 23G needle, eluted and reconstituted in 600 µL of uninfected blood before feed. 3 9000 µL of gametocyte-infected whole blood from starting material added once over MACS column with 21G needle, eluted and reconstituted in 600 µL of uninfected blood before feed. Vertical bars indicate mean with SEM.

Additional file 1: Table S1. Observations with different buffers for mosquito homogenisation. Table S2. Experimental infection data CT values by qPCR vs Optical Density in CSP-ELISA. Table S3. Overview of processing time of the four methods compared in seconds per 80 mosquitoes. Figure S1. Protocol for mixed infection detection using the same 50Â Âľl volume. Homogenate is transferred from the prior experiments to the subsequent ones. It is important that every step is timed and thought ahead on when to start the different steps. Figure S2. Processing of mosquitoes by CS-ELISA when tested for both P. falciparum and P. vivax.

Additional file 1: Figure S1. Association between gametocyte densities and oocystâ prevalence and density. A. Overview of gametocyte densities and infection rates of experiments of Figs. 1, 2, 3, 4 and Additional file 2: Figure S2. B. Overview of gametocyte densities and mean oocysts of experiments of Figs. 1, 2, 3, 4 and Additional file 2: Figure S2.

Additional file 2: Figure S2. Effect of different flow-rate MACS on gametocyte infectivity. Number of oocysts per mosquito after membrane-feeding assay (MIDI cages). 1 600 µL of gametocyte-infected whole blood from starting material, fed directly. 2 9000 µL of gametocyte-infected whole blood from starting material added once over MACS column with 23G needle, eluted and reconstituted in 600 µL of uninfected blood before feed. 3 9000 µL of gametocyte-infected whole blood from starting material added once over MACS column with 21G needle, eluted and reconstituted in 600 µL of uninfected blood before feed. Vertical bars indicate mean with SEM.

First-wave parasitemia after challenge. Parasitemia on day 7 post-challenge in immunized (open circles) and control (closed circles) volunteers. The line and error bars show the geometric mean and 95% CI interval. Figure S2. Inhibition of in vitro homologous and heterologous intra-hepatic sporozoite development in primary human hepatocytes by mAb 2A10. P. falciparum NF54 (blue), NF135.C10 (orange) and NF166.C8 (green) sporozoites in the presence of 10% heat-inactivated naive human control serum were pre-incubated with 3-fold serial dilutions of the 2A10 monoclonal antibody (0.027–20 μg/mL), targeting the repeat region of the circumsporozoite protein (CSP), and added to primary human hepatocyte cultures. Six days post-infection, the number of P. falciparum-infected hepatocytes was assessed as described in Additional file 1: S4 and S5. Figure S3. Amino acid changes in CSP. Table S1. Whole-genome sequencing statistics. Table S2. Mosquito salivary gland infectivity and sporozoite load of the three clones. Mean mosquito salivary gland infectivity and sporozoite load determined 1 day prior to challenge infection by dissecting a sample of 10 mosquitoes per strain. (DOCX 427 kb)