Characterisation of Plasmodium Sexual Stage Antigens as Targets of Transmission Blocking Immunity

A promising strategy to achieve malaria elimination is by interrupting transmission using transmission-blocking vaccines (TBVs). TBVs target the sexual or mosquito stages of the malaria parasite to inhibit parasite development within the mosquito. To date, however, there are only three lead TBV candidate antigens Pfs230, Pfs48/45 and Pfs25 in various stages of clinical development. This project sought to identify novel antigens expressed on gametocytes, gametes and ookinetes with potential as TBV candidates. Furthermore, naturally acquired immune responses (NAI) to gametocytes have been described and have the potential to guide the development and the implementation of TBVs Therefore, this work also sought to improve our understanding of NAI to gametocytes. This was achieved by (1) carrying out a systematic review and meta-analysis of studies investigating NAI to the lead gametocyte-stage TBV candidates, and (2) assessing the changing patterns of gametocyte carriage at the Kenyan coast over time. Key indicators of gametocytaemia and anti-gametocyte immunity were identified and evaluated against a novel panel of gametocyte antigens. These antigens, together with a separate set of gamete and ookinete stage antigens, were identified as potential TBV candidates using a combination of bioinformatic tools and laboratory investigations. Immunoprofiling of the identified gametocyte candidates provided evidence that stable responses can be generated to sexual stage antigens. Moreover, antigens that could serve as serological markers of recent gametocyte exposure, in particular PEB-P (PF3D7_0303900), were also identified. The ability of the sexual stage antigens to induce transmission-blocking immunity was also assessed. Promising candidates identified included PBCPP2 (PBANKA_0719100), a novel conserved and uncharacterised P. berghei protein), and SOAP (PBANKA_1037800). Further characterisation of these antigens may yield new candidates to add to the TBV development pipeline

Corrigendum: Immune Responses to Gametocyte Antigens in a Malaria Endemic Population-The African falciparum Context: A Systematic Review and Meta-Analysis.

[This corrects the article DOI: 10.3389/fimmu.2019.02480.]

Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria.Wellcome Trust (084535, 077092, 084538, and 098635)This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/IAI.00772-1

Detection of Plasmodium falciparum infected Anopheles gambiae using near-infrared spectroscopy

Background: Large-scale surveillance of mosquito populations is crucial to assess the intensity of vector-borne disease transmission and the impact of control interventions. However, there is a lack of accurate, cost-effective and high-throughput tools for mass-screening of vectors. Methods: A total of 750 Anopheles gambiae (Keele strain) mosquitoes were fed Plasmodium falciparum NF54 gametocytes through standard membrane feeding assay (SMFA) and afterwards maintained in insectary conditions to allow for oocyst (8 days) and sporozoite development (14 days). Thereupon, each mosquito was scanned using near infra-red spectroscopy (NIRS) and processed by quantitative polymerase chain reaction (qPCR) to determine the presence of infection and infection load. The spectra collected were randomly assigned to either a training dataset, used to develop calibrations for predicting oocyst- or sporozoite-infection through partial least square regressions (PLS); or to a test dataset, used for validating the calibration’s prediction accuracy. Results: NIRS detected oocyst- and sporozoite-stage P. falciparum infections with 88% and 95% accuracy, respectively. This study demonstrates proof-of-concept that NIRS is capable of rapidly identifying laboratory strains of human malaria infection in African mosquito vectors. Conclusions: Accurate, low-cost, reagent-free screening of mosquito populations enabled by NIRS could revolutionize surveillance and elimination strategies for the most important human malaria parasite in its primary African vector species. Further research is needed to evaluate how the method performs in the field following adjustments in the training datasets to include data from wild-caught infected and uninfected mosquitoes

Differential Plasmodium falciparum surface antigen expression among children with Malarial Retinopathy

Retinopathy provides a window into the underlying pathology of life-threatening malarial coma (“cerebral malaria”), allowing differentiation between 1) coma caused by sequestration of Plasmodium falciparum-infected erythrocytes in the brain and 2) coma with other underlying causes. Parasite sequestration in the brain is mediated by PfEMP1; a diverse parasite antigen that is inserted into the surface of infected erythrocytes and adheres to various host receptors. PfEMP1 sub-groups called “DC8” and “DC13” have been proposed to cause brain pathology through interactions with endothelial protein C receptor. To test this we profiled PfEMP1 gene expression in parasites from children with clinically defined cerebral malaria, who either had or did not have accompanying retinopathy. We found no evidence for an elevation of DC8 or DC13 PfEMP1 expression in children with retinopathy. However, the proportional expression of a broad subgroup of PfEMP1 called “group A” was elevated in retinopathy patients suggesting that these variants may play a role in the pathology of cerebral malaria. Interventions targeting group A PfEMP1 may be effective at reducing brain pathology

Plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of Kenyan adults with varying natural exposure.

BACKGROUND: The PfEMP1 family of Plasmodium falciparum antigens play a key role in pathogenesis of severe malaria through their insertion into the surface of parasite infected erythrocytes, and adhesion to host cells. Previous studies have suggested that parasites expressing PfEMP1 subclasses group A and DC8, associated with severe malaria, may have a growth advantage in immunologically naïve individuals. However, this idea has not been tested in longitudinal studies. METHODS: Here we assessed expression of the var genes encoding PfEMP1, in parasites sampled from volunteers with varying prior exposure to malaria, following experimental infection by sporozoites (PfSPZ). Using qPCR, we tested for associations between the expression of various var subgroups in surviving parasite populations from each volunteer and 1) the levels of participants' antibodies to infected erythrocytes before challenge infection and 2) the apparent in vivo parasite multiplication rate. RESULTS: We show that 1) expression of var genes encoding for group A and DC8-like PfEMP1 were associated with low levels of antibodies to infected erythrocytes (αIE) before challenge, and 2) expression of a DC8-like CIDRα1.1 domain was associated with higher apparent parasite multiplication rate in a manner that was independent of levels of prior antibodies to infected erythrocytes. CONCLUSIONS: This study provides insight into the role of antibodies to infected erythrocytes surface antigens in the development of naturally acquired immunity and may help explain why specific PfEMP1 variants may be associated with severe malaria. TRIAL REGISTRATION: Pan African Clinical Trial Registry: PACTR201211000433272 . Date of registration: 10th October 2012

Exploring Plasmodium falciparum Var Gene Expression to Assess Host Selection Pressure on Parasites During Infancy

In sub-Saharan Africa, children below 5 years bear the greatest burden of severe malaria because they lack naturally acquired immunity that develops following repeated exposure to infections by Plasmodium falciparum. Antibodies to the surface of P. falciparum infected erythrocytes (IE) play an important role in this immunity. In children under the age of 6 months, relative protection from severe malaria is observed and this is thought to be partly due to trans-placental acquired protective maternal antibodies. However, the protective effect of maternal antibodies has not been fully established, especially the role of antibodies to variant surface antigens (VSA) expressed on IE. Here, we assessed the immune pressure on parasites infecting infants using markers associated with the acquisition of naturally acquired immunity to surface antigens. We hypothesized that, if maternal antibodies to VSA imposed a selection pressure on parasites, then the expression of a relatively conserved subset of var genes called group A var genes in infants should change with waning maternal antibodies. To test this, we compared their expression in parasites from children between 0 and 12 months and above 12 months of age. The transcript quantity and the proportional expression of group A var subgroup, including those containing domain cassette 13, were positively associated with age during the first year of life, which contrasts with above 12 months. This was accompanied by a decline in infected erythrocyte surface antibodies and an increase in parasitemia during this period. The observed increase in group A var gene expression with age in the first year of life, when the maternal antibodies are waning and before acquisition of naturally acquired antibodies with repeated exposure, is consistent with the idea that maternally acquired antibodies impose a selection pressure on parasites that infect infants and may play a role in protecting these infants against severe malaria

Plasmodium falciparum adapts its investment into replication versus transmission according to the host environment

The malaria parasite life cycle includes asexual replication in human blood, with a proportion of parasites differentiating to gametocytes required for transmission to mosquitoes. Commitment to differentiate into gametocytes, which is marked by activation of the parasite transcription factor ap2-g, is known to be influenced by host factors but a comprehensive model remains uncertain. Here we analyze data from 828 children in Kilifi, Kenya with severe, uncomplicated, and asymptomatic malaria infection over 18 years of falling malaria transmission. We examine markers of host immunity and metabolism, and markers of parasite growth and transmission investment. We find that inflammatory responses associated with reduced plasma lysophosphatidylcholine levels are associated with markers of increased investment in parasite sexual reproduction (i.e., transmission investment) and reduced growth (i.e., asexual replication). This association becomes stronger with falling transmission and suggests that parasites can rapidly respond to the within-host environment, which in turn is subject to changing transmission

Characterization of Naturally Acquired Immunity to a Panel of Antigens Expressed in Mature P. falciparum Gametocytes.

INTRODUCTION: Naturally acquired immune responses against antigens expressed on the surface of mature gametocytes develop in individuals living in malaria-endemic areas. Evidence suggests that such anti-gametocyte immunity can block the development of the parasite in the mosquito, thus playing a role in interrupting transmission. A better comprehension of naturally acquired immunity to these gametocyte antigens can aid the development of transmission-blocking vaccines and improve our understanding of the human infectious reservoir. METHODS: Antigens expressed on the surface of mature gametocytes that had not previously been widely studied for evidence of naturally acquired immunity were identified for protein expression alongside Pfs230-C using either the mammalian HEK293E or the wheat germ cell-free expression systems. Where there was sequence variation in the candidate antigens (3D7 vs a clinical isolate PfKE04), both variants were expressed. ELISA was used to assess antibody responses against these antigens, as well as against crude stage V gametocyte extract (GE) and AMA1 using archived plasma samples from individuals recruited to participate in malaria cohort studies. We analyzed antibody levels (estimated from optical density units using a standardized ELISA) and seroprevalence (defined as antibody levels greater than three standard deviations above the mean levels of a pool of malaria naïve sera). We described the dynamics of antibody responses to these antigens by identifying factors predictive of antibody levels using linear regression models. RESULTS: Of the 25 antigens selected, seven antigens were produced successfully as recombinant proteins, with one variant antigen, giving a total of eight proteins for evaluation. Antibodies to the candidate antigens were detectable in the study population (N = 216), with seroprevalence ranging from 37.0% (95% CI: 30.6%, 43.9%) for PSOP1 to 77.8% (95% CI: 71.6%, 83.1%) for G377 (3D7 variant). Responses to AMA1 and GE were more prevalent than those to the gametocyte proteins at 87.9% (95% CI: 82.8%, 91.9%) and 88.3% (95% CI: 83.1%, 92.4%), respectively. Additionally, both antibody levels and breadth of antibody responses were associated with age and concurrent parasitaemia. CONCLUSION: Age and concurrent parasitaemia remain important determinants of naturally acquired immunity to gametocyte antigens. Furthermore, we identify novel candidates for transmission-blocking activity evaluation