The Complement System Contributes to Functional Antibody-Mediated Responses Induced by Immunization with Plasmodium falciparum Malaria Sporozoites.

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.

Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children.

BACKGROUND: Antibodies targeting Plasmodium falciparum sporozoites play a key role in human immunity to malaria. However, antibody mechanisms that neutralize sporozoites are poorly understood. This has been a major constraint in developing highly efficacious vaccines, as we lack strong correlates of protective immunity. METHODS: We quantified the ability of human antibodies from malaria-exposed populations to interact with human complement, examined the functional effects of complement activity against P. falciparum sporozoites in vitro, and identified targets of functional antibodies. In children and adults from malaria-endemic regions, we determined the acquisition of complement-fixing antibodies to sporozoites and their relationship with antibody isotypes and subclasses. We also investigated associations with protective immunity in a longitudinal cohort of children (n = 206) residing in a malaria-endemic region. RESULTS: We found that antibodies to the major sporozoite surface antigen, circumsporozoite protein (CSP), were predominately IgG1, IgG3, and IgM, and could interact with complement through recruitment of C1q and activation of the classical pathway. The central repeat region of CSP, included in leading vaccines, was a key target of complement-fixing antibodies. We show that antibodies activate human complement on P. falciparum sporozoites, which consequently inhibited hepatocyte cell traversal that is essential for establishing liver-stage infection, and led to sporozoite death in vitro. The natural acquisition of complement-fixing antibodies in malaria-exposed populations was age-dependent, and was acquired more slowly to sporozoite antigens than to merozoite antigens. In a longitudinal cohort of children, high levels of complement-fixing antibodies were significantly associated with protection against clinical malaria. CONCLUSIONS: These novel findings point to complement activation by antibodies as an important mechanism of anti-sporozoite human immunity, thereby enabling new strategies for developing highly efficacious malaria vaccines. We also present evidence that complement-fixing antibodies may be a valuable correlate of protective immunity in humans.

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590 .

Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum.

BACKGROUND: Long-lasting and sterile protective immunity against Plasmodium falciparum can be achieved by immunization of malaria-naive human volunteers under chloroquine prophylaxis with sporozoites delivered by mosquito bites (CPS-immunization). Protection is mediated by sporozoite/liver-stage immunity. In this study, the capacity of CPS-induced antibodies to interfere with sporozoite functionality and development was explored. METHODS: IgG was purified from plasma samples obtained before and after CPS-immunization from two separate clinical trials. The functionality of these antibodies was assessed in vitro in gliding and human hepatocyte traversal assays, and in vivo in a human liver-chimeric mouse model. RESULTS: Whereas pre-treatment of sporozoites with 2 mg/ml IgG in the majority of the volunteers did not have an effect on in vitro sporozoite gliding motility, CPS-induced IgG showed a distinct inhibitory effect in the sporozoite in vitro traversal assay. Pre-treatment of P. falciparum sporozoites with post-immunization IgG significantly inhibited sporozoite traversal through hepatocytes in 9/9 samples when using 10 and 1 mg/ml IgG, and was dose-dependent, resulting in an average 16% and 37% reduction with 1 mg/ml IgG (p = 0.003) and 10 mg/ml IgG (p = 0.002), respectively. In vivo, CPS-induced IgG reduced liver-stage infection and/or development after a mosquito infection in the human liver-chimeric mouse model by 91.05% when comparing 11 mice receiving post-immunization IgG to 11 mice receiving pre-immunization IgG (p = 0.0008). CONCLUSIONS: It is demonstrated for the first time that CPS-immunization induces functional antibodies against P. falciparum sporozoites, which are able to reduce parasite-host cell interaction by inhibiting parasite traversal and liver-stage infection. These data highlight the functional contribution of antibody responses to pre-erythrocytic immunity after whole-parasite immunization against P. falciparum malaria.

Human antibodies against noncircumsporozoite proteins block Plasmodium falciparum parasite development in hepatocytes.

Sporozoite-based approaches currently represent the most effective vaccine strategies for induction of sterile protection against Plasmodium falciparum (Pf) malaria. Clinical development of subunit vaccines is almost exclusively centered on the circum-sporozoite protein (CSP), an abundantly expressed protein on the sporozoite membrane. Anti-CSP antibodies are able to block sporozoite invasion and development in human hepatocytes and subsequently prevent clinical malaria. Here, we have investigated whether sporozoite-induced human antibodies with specificities different from CSP can reduce Pf-liver stage development. IgG preparations were obtained from 12 volunteers inoculated with a protective immunization regime of whole sporozoites under chloroquine prophylaxis. These IgGs were depleted for CSP specificity by affinity chromatography. Recovered non-CSP antibodies were tested for sporozoite membrane binding and for functional inhibition of sporozoite invasion of a human hepatoma cell line and hepatocytes both in vitro and in vivo. Postimmunization IgGs depleted for CS specificity of 9 of 12 donors recognized sporozoite surface antigens. Samples from 5 of 12 donors functionally reduced parasite-liver cell invasion or development using the hepatoma cell line HC-04 and FRG-huHep mice containing human liver cells. The combined data provide clear evidence that non-CSP proteins, as yet undefined, do represent antibody targets for functional immunity against Pf parasites responsible for malaria.

Evaluation of immunity against malaria using luciferase-expressing Plasmodium berghei parasites.

BACKGROUND: Measurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic Plasmodium berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter luciferase. This study explores the benefit of using these parasites for the evaluation of immunity against malaria, compared to qRT-PCR techniques in vivo and in vitro. METHODS: Mice were immunized with either radiation attenuated sporozoites (RAS) or wildtype sporozoites under chloroquine prophylaxis (CPS) and challenged with PbGFP-Luccon. The in vitro transgenic sporozoites neutralization assay (TSNA) was adapted by replacing PbCS(Pf) parasites for PbGFP-Luccon parasites. RESULTS: Application of PbGFP-Luccon transgenic parasites provides live quantitative visual information about the relation between parasite liver load and protection. Moreover, fast and reproducible results are obtained by using these parasites in the transgenic sporozoites neutralization assay, measuring functional antibody-mediated immune responses. CONCLUSIONS: PbGFP-Luccon parasites are a straightforward and valuable tool for comprehension of the biological and immunological principles underlying protection against malaria.

Outcomes of controlled human malaria infection after BCG vaccination.

Recent evidence suggests that certain vaccines, including Bacillus-Calmette Guérin (BCG), can induce changes in the innate immune system with non-specific memory characteristics, termed 'trained immunity'. Here we present the results of a randomised, controlled phase 1 clinical trial in 20 healthy male and female volunteers to evaluate the induction of immunity and protective efficacy of the anti-tuberculosis BCG vaccine against a controlled human malaria infection. After malaria challenge infection, BCG vaccinated volunteers present with earlier and more severe clinical adverse events, and have significantly earlier expression of NK cell activation markers and a trend towards earlier phenotypic monocyte activation. Furthermore, parasitemia in BCG vaccinated volunteers is inversely correlated with increased phenotypic NK cell and monocyte activation. The combined data demonstrate that BCG vaccination alters the clinical and immunological response to malaria, and form an impetus to further explore its potential in strategies for clinical malaria vaccine development.

Functional antibodies against Plasmodium falciparum sporozoites are associated with a longer time to qPCR-detected infection among schoolchildren in Burkina Faso.

Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure. Methods: We cleared schoolchildren living in an area (health district of Saponé, Burkina Faso) with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1). In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed using Plasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by 18S quantitative PCR were studied. Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median inhibition, 19.4% [IQR 15.2-40.9%]), and there were positive correlations between invasion inhibition and gliding inhibition (P=0.005, Spearman's ρ=0.67) and between invasion inhibition and CSP-specific antibodies (P=0.002, Spearman's ρ=0.76). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority.

Humoral protection against mosquito bite-transmitted Plasmodium falciparum infection in humanized mice.

A malaria vaccine that prevents infection will be an important new tool in continued efforts of malaria elimination, and such vaccines are under intense development for the major human malaria parasite Plasmodium falciparum (Pf). Antibodies elicited by vaccines can block the initial phases of parasite infection when sporozoites are deposited into the skin by mosquito bite and then target the liver for further development. However, there are currently no standardized in vivo preclinical models that can measure the inhibitory activity of antibody specificities against Pf sporozoite infection via mosquito bite. Here, we use human liver-chimeric mice as a challenge model to assess prevention of natural Pf sporozoite infection by antibodies. We demonstrate that these mice are consistently infected with Pf by mosquito bite and that this challenge can be combined with passive transfer of either monoclonal antibodies or polyclonal human IgG from immune serum to measure antibody-mediated blocking of parasite infection using bioluminescent imaging. This methodology is useful to down-select functional antibodies and to investigate mechanisms or immune correlates of protection in clinical trials, thereby informing rational vaccine optimization.

Behavior of neuropathic pain in mice following chronic constriction injury comparing silk and catgut ligatures.

INTRODUCTION: Neuropathic pain is defined as pain arising as a direct consequence of a lesion or disease affecting the somatosensory system and is common after surgery. Neuropathic pain can persist without an obvious injury. In this study we aim to validate a murine chronic constriction injury model as a model for neuropathic pain research and determine if silk or catgut ligatures induced most stable neuropathic pain behavior. METHODS: In this study mice underwent chronic constriction or sham surgery. Mice were tested on cutaneous hyperalgesia with the cumulative reaction time in the acetone test, on allodynia with the cumulative reaction time and number of lifts in the cold plate test and the maximal force before withdrawal in von Frey test. RESULTS: In the acetone test neuropathic pain was seen in CCI mice, but not in sham mice. Hyperalgesia was present postoperatively in CCI mice compared with preoperatively. In the cold plate test cumulative reaction time and number of lifts were higher in the ipsilateral hind paw than in the contralateral hind paw and sham mice. Postoperative measurements were higher than preoperatively. In the von Frey test the postoperative measurements were lower in the ipsilateral hind paw than preoperatively, while the contralateral hind paw showed an increase in maximal force before withdrawal. The contralateral hind paw showed more difference with sham mice than the ipsilateral hind paw. Silk ligatures showed more stable neuropathic pain behavior. In the acetone test, the cold plate test and the von Frey test the mice scored higher on neuropathic pain having silk ligatures, compared with catgut ligatures. CONCLUSION: In this study we validated a murine CCI model for neuropathic pain behavior. In the murine CCI model it appears that silk ligatures demonstrate more stable neuropathic pain behaviors than catgut ligatures in de CCI model.

Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate--Tackling the Cocktail Challenge.

Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 µg/ml), the blood stage (40-60 µg/ml) and the sexual stage (1.75 µg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy.

Sporozoite immunization of human volunteers under mefloquine prophylaxis is safe, immunogenic and protective: a double-blind randomized controlled clinical trial.

Immunization of healthy volunteers with chloroquine ChemoProphylaxis and Sporozoites (CPS-CQ) efficiently and reproducibly induces dose-dependent and long-lasting protection against homologous Plasmodium falciparum challenge. Here, we studied whether chloroquine can be replaced by mefloquine, which is the only other licensed anti-malarial chemoprophylactic drug that does not affect pre-erythrocytic stages, exposure to which is considered essential for induction of protection by CPS immunization. In a double blind randomized controlled clinical trial, volunteers under either chloroquine prophylaxis (CPS-CQ, n = 5) or mefloquine prophylaxis (CPS-MQ, n = 10) received three sub-optimal CPS immunizations by bites from eight P. falciparum infected mosquitoes each, at monthly intervals. Four control volunteers received mefloquine prophylaxis and bites from uninfected mosquitoes. CPS-MQ immunization is safe and equally potent compared to CPS-CQ inducing protection in 7/10 (70%) versus 3/5 (60%) volunteers, respectively. Furthermore, specific antibody levels and cellular immune memory responses were comparable between both groups. We therefore conclude that mefloquine and chloroquine are equally effective in CPS-induced immune responses and protection. Trial registration: ClinicalTrials.gov NCT01422954.