Binding of Plasmodium falciparum Merozoite Surface Proteins DBLMSP and DBLMSP2 to Human Immunoglobulin M Is Conserved among Broadly Diverged Sequence Variants.

Diversity at pathogen genetic loci can be driven by host adaptive immune selection pressure and may reveal proteins important for parasite biology. Population-based genome sequencing of Plasmodium falciparum, the parasite responsible for the most severe form of malaria, has highlighted two related polymorphic genes called dblmsp and dblmsp2, which encode Duffy binding-like (DBL) domain-containing proteins located on the merozoite surface but whose function remains unknown. Using recombinant proteins and transgenic parasites, we show that DBLMSP and DBLMSP2 directly and avidly bind human IgM via their DBL domains. We used whole genome sequence data from over 400 African and Asian P. falciparum isolates to show that dblmsp and dblmsp2 exhibit extreme protein polymorphism in their DBL domain, with multiple variants of two major allelic classes present in every population tested. Despite this variability, the IgM binding function was retained across diverse sequence representatives. Although this interaction did not seem to have an effect on the ability of the parasite to invade red blood cells, binding of DBLMSP and DBLMSP2 to IgM inhibited the overall immunoreactivity of these proteins to IgG from patients who had been exposed to the parasite. This suggests that IgM binding might mask these proteins from the host humoral immune system.

Vaccine candidate discovery for the next generation of malaria vaccines.

Although epidemiological observations, IgG passive transfer studies and experimental infections in humans all support the feasibility of developing highly effective malaria vaccines, the precise antigens that induce protective immunity remain uncertain. Here, we review the methodologies applied to vaccine candidate discovery for Plasmodium falciparum malaria from the pre- to post-genomic era. Probing of genomic and cDNA libraries with antibodies of defined specificities or functional activity predominated the former, whereas reverse vaccinology encompassing high throughput in silico analyses of genomic, transcriptomic or proteomic parasite data sets is the mainstay of the latter. Antibody-guided vaccine design spanned both eras but currently benefits from technological advances facilitating high-throughput screening and downstream applications. We make the case that although we have exponentially increased our ability to identify numerous potential vaccine candidates in a relatively short space of time, a significant bottleneck remains in their validation and prioritization for evaluation in clinical trials. Longitudinal cohort studies provide supportive evidence but results are often conflicting between studies. Demonstration of antigen-specific antibody function is valuable but the relative importance of one mechanism over another with regards to protection remains undetermined. Animal models offer useful insights but may not accurately reflect human disease. Challenge studies in humans are preferable but prohibitively expensive. In the absence of reliable correlates of protection, suitable animal models or a better understanding of the mechanisms underlying protective immunity in humans, vaccine candidate discovery per se may not be sufficient to provide the paradigm shift necessary to develop the next generation of highly effective subunit malaria vaccines.

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.

Multiple clinical episodes of Plasmodium falciparum malaria in a low transmission intensity setting: exposure versus immunity.

BACKGROUND: Epidemiological studies indicate that some children experience many more episodes of clinical malaria than their age mates in a given location. Whether this is as a result of the micro-heterogeneity of malaria transmission with some children effectively getting more exposure to infectious mosquitoes than others, or reflects a failure in the acquisition of immunity needs to be elucidated. Here, we investigated the determinants of increased susceptibility to clinical malaria by comparing the intensity of exposure to Plasmodium falciparum and the acquisition of immunity in children at the extreme ends of the over-dispersed distribution of the incidence of clinical malaria. METHODS: The study was nested within a larger cohort in an area where the intensity of malaria transmission was low. We identified children who over a five-year period experienced 5 to 16 clinical malaria episodes (children at the tail-end of the over-dispersed distribution, n = 35), remained malaria-free (n = 12) or had a single episode (n = 26). We quantified antibodies against seven Plasmodium falciparum merozoite antigens in plasma obtained at six cross-sectional surveys spanning these five years. We analyzed the antibody responses to identify temporal dynamics that associate with disease susceptibility. RESULTS: Children experiencing multiple episodes of malaria were more likely to be parasite positive by microscopy at cross-sectional surveys (X (2) test for trend 14.72 P = 0.001) and had a significantly higher malaria exposure index, than those in the malaria-free or single episode groups (Kruskal-Wallis test P = 0.009). In contrast, the five-year temporal dynamics of anti-merozoite antibodies were similar in the three groups. Importantly in all groups, antibody levels were below the threshold concentrations previously observed to be correlated with protective immunity. CONCLUSIONS: We conclude that in the context of a low malaria transmission setting, susceptibility to clinical malaria is not accounted for by anti-merozoite antibodies but appears to be a consequence of increased parasite exposure. We hypothesize that intensive exposure is a prerequisite for protective antibody concentrations, while little to modest exposure may manifest as multiple clinical infections with low levels of antibodies. These findings have implications for interventions that effectively lower malaria transmission intensity.

Cysticercosis and epilepsy in rural Tanzania: a community-based case-control and imaging study.

OBJECTIVE: To assess the contribution of neurocysticercosis (NCC) to the burden of epilepsy in a rural Tanzanian population. METHODS: We identified adult people with epilepsy (PWE) in a door-to-door study in an established demographic surveillance site. PWE and community controls were tested for antibodies to Taenia solium, the causative agent of NCC, and all PWE were offered a computed tomography (CT) head scan. Data on household occupancy and sanitation, pig-keeping and pork consumption were collected from PWE and controls and associations with epilepsy were assessed using chi-square or Fisher's exact tests. RESULTS: Six of 218 PWE had antibodies to T. solium (2.8%; 95% CI 0.6-4.9), compared to none of 174 controls (Fisher's exact test, P = 0.04). Lesions compatible with NCC were seen in eight of 200 CT scans (4.0%; 95% CI 1.3-6.7). A total of 176 PWE had both investigations of whom two had positive serology along with NCC-compatible lesions on CT (1.1%; 95% 0.3-4.0). No associations between epilepsy and any risk factors for NCC were identified. CONCLUSIONS: Neurocysticercosis is present in this population but at a lower prevalence than elsewhere in Tanzania and sub-Saharan Africa. Insights from low-prevalence areas may inform public health interventions designed to reduce the burden of preventable epilepsy.

Clinical features, proximate causes, and consequences of active convulsive epilepsy in Africa.

PURPOSE: Epilepsy is common in sub-Saharan Africa (SSA), but the clinical features and consequences are poorly characterized. Most studies are hospital-based, and few studies have compared different ecological sites in SSA. We described active convulsive epilepsy (ACE) identified in cross-sectional community-based surveys in SSA, to understand the proximate causes, features, and consequences. METHODS: We performed a detailed clinical and neurophysiologic description of ACE cases identified from a community survey of 584,586 people using medical history, neurologic examination, and electroencephalography (EEG) data from five sites in Africa: South Africa; Tanzania; Uganda; Kenya; and Ghana. The cases were examined by clinicians to discover risk factors, clinical features, and consequences of epilepsy. We used logistic regression to determine the epilepsy factors associated with medical comorbidities. KEY FINDINGS: Half (51%) of the 2,170 people with ACE were children and 69% of seizures began in childhood. Focal features (EEG, seizure types, and neurologic deficits) were present in 58% of ACE cases, and these varied significantly with site. Status epilepticus occurred in 25% of people with ACE. Only 36% received antiepileptic drugs (phenobarbital was the most common drug [95%]), and the proportion varied significantly with the site. Proximate causes of ACE were adverse perinatal events (11%) for onset of seizures before 18 years; and acute encephalopathy (10%) and head injury prior to seizure onset (3%). Important comorbidities were malnutrition (15%), cognitive impairment (23%), and neurologic deficits (15%). The consequences of ACE were burns (16%), head injuries (postseizure) (1%), lack of education (43%), and being unmarried (67%) or unemployed (57%) in adults, all significantly more common than in those without epilepsy. SIGNIFICANCE: There were significant differences in the comorbidities across sites. Focal features are common in ACE, suggesting identifiable and preventable causes. Malnutrition and cognitive and neurologic deficits are common in people with ACE and should be integrated into the management of epilepsy in this region. Consequences of epilepsy such as burns, lack of education, poor marriage prospects, and unemployment need to be addressed.

Standardized immunological assays for assessing COVID-19 vaccines by the CEPI-Centralized Laboratory Network.

The CEPI Centralized Laboratory Network implemented key steps in the transfer and monitoring of the developed immunological SARS-CoV-2 assays to ensure standardization across all the facilities of the network. This comprehensive evaluation reinforces the reliability of the generated data and establishes a solid foundation for a standardized approach, enabling precise inter-laboratory comparisons and contributing to the overall integrity of our network's clinical results. Herein, we will provide a brief elaboration on the specific measures and procedures implemented to standardize the transfer of assays across our network.

Candidate antibody reference reagents for Chlamydia trachomatis serology.

Chlamydia trachomatis (Ct) serology is an important tool for monitoring infection and disease burden but there are currently no formal reference reagents to harmonize results reporting. Our objective was to develop a panel of candidate reference reagents with reactivity against the major outer membrane protein (MOMP) and virulence factor (pgp3) antigens. Plasma packs from females (20-40 years old) were screened against MOMP and pgp3 antigens and selected positive and negative samples pooled to create a panel of candidate antibody reference reagents that were tested in two laboratories. Antigen specificity and internal quality assurance were also evaluated. Suitable candidate materials have been selected to produce Ct reference reagents.

Global evaluation of lineage-specific human papillomavirus capsid antigenicity using antibodies elicited by natural infection.

Human Papillomavirus (HPV) type variants have been classified into lineages and sublineages based upon their whole genome sequence. Here we have examined the specificity of antibodies generated following natural infection with lineage variants of oncogenic types (HPV16, 18, 31, 33, 45, 52 and 58) by testing serum samples assembled from existing archives from women residing in Africa, The Americas, Asia or Europe against representative lineage-specific pseudoviruses for each genotype. We have subjected the resulting neutralizing antibody data to antigenic clustering methods and created relational antigenic profiles for each genotype to inform the delineation of lineage-specific serotypes. For most genotypes, there was evidence of differential recognition of lineage-specific antigens and in some cases of a sufficient magnitude to suggest that some lineages should be considered antigenically distinct within their respective genotypes. These data provide compelling evidence for a degree of lineage specificity within the humoral immune response following natural infection with oncogenic HPV.

Driving consistency: CEPI-Centralized Laboratory Network's conversion factor initiative for SARS-CoV-2 clinical assays used for efficacy assessment of COVID vaccines.

To date, thousands of SARS-CoV-2 samples from many vaccine developers have been tested within the CEPI-Centralized Laboratory Network. To convert data from each clinical assay to international standard units, the WHO international standard and the CEPI standard generated by the Medicines and Healthcare products Regulatory Agency were run in multiple facilities to determine the conversion factor for each assay. Reporting results in international units advances global understanding of SARS-CoV-2 immunity and vaccine efficacy, enhancing the quality, reliability, and utility of clinical assay data.

Analysis of antibodies to newly described Plasmodium falciparum merozoite antigens supports MSPDBL2 as a predicted target of naturally acquired immunity.

Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed (surf) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [n = 497] and Ngerenya [n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children <11 years of age during 6 months follow-up surveillance. Antibodies to the polymorphic central region of MSPDBL2 were associated with reduced risk of malaria in both cohorts, with statistical significance remaining for the 3D7 allelic type after adjustment for individuals' ages in years and antibody reactivity to whole-schizont extract (Chonyi, risk ratio, 0.51, and 95% confidence interval [CI], 0.28 to 0.93; Ngerenya, risk ratio, 0.38, and 95% CI, 0.18 to 0.82). For the MSPDBL1 Palo Alto allelic-type antigen, there was a protective association in one cohort (Ngerenya, risk ratio, 0.53, and 95% CI, 0.32 to 0.89), whereas the other antigens showed no protective associations after adjustment. These findings support the prediction that antibodies to the polymorphic region of MSPDBL2 contribute to protective immunity.

Anti-merozoite antibodies induce natural killer cell effector function and are associated with immunity against malaria.

Natural killer (NK) cells are potent immune effectors that can be activated via antibody-mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that NK cells degranulate and release IFN-γ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK (Ab-NK) activity was largely strain transcending and enhanced invasion inhibition into erythrocytes. Ab-NK was associated with the successful control of parasitemia after experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent P. falciparum infections, and was associated with a lower risk of clinical episodes of malaria. Nine of the 14 vaccine candidates tested induced Ab-NK, including some less well-characterized antigens: P41, P113, MSP11, RHOPH3, and Pf_11363200. These data highlight an important role of Ab-NK activity in immunity against malaria and provide a potential mechanism for evaluating vaccine candidates.

Strain Specific Variations in Acinetobacter baumannii Complement Sensitivity.

The complement system is required for innate immunity against Acinetobacter baumannii, an important cause of antibiotic resistant systemic infections. A. baumannii strains differ in their susceptibility to the membrane attack complex (MAC) formed from terminal complement pathway proteins, but the reasons for this variation remain poorly understood. We have characterized in detail the complement sensitivity phenotypes of nine A. baumannii clinical strains and some of the factors that might influence differences between strains. Using A. baumannii laboratory strains and flow cytometry assays, we first reconfirmed that both opsonization with the complement proteins C3b/iC3b and MAC formation were inhibited by the capsule. There were marked differences in C3b/iC3b and MAC binding between the nine clinical A. baumannii strains, but this variation was partially independent of capsule composition or size. Opsonization with C3b/iC3b improved neutrophil phagocytosis of most strains. Importantly, although C3b/iC3b binding and MAC formation on the bacterial surface correlated closely, MAC formation did not correlate with variations between A. baumannii strains in their levels of serum resistance. Genomic analysis identified only limited differences between strains in the distribution of genes required for serum resistance, but RNAseq data identified three complement-resistance genes that were differentially regulated between a MAC resistant and two MAC intermediate resistant strains when cultured in serum. These data demonstrate that clinical A. baumannii strains vary in their sensitivity to different aspects of the complement system, and that the serum resistance phenotype was influenced by factors in addition to the amount of MAC forming on the bacterial surface.

Distinct kinetics of antibodies to 111 Plasmodium falciparum proteins identifies markers of recent malaria exposure.

Strengthening malaria surveillance is a key intervention needed to reduce the global disease burden. Reliable serological markers of recent malaria exposure could improve current surveillance methods by allowing for accurate estimates of infection incidence from limited data. We studied the IgG antibody response to 111 Plasmodium falciparum proteins in 65 adult travellers followed longitudinally after a natural malaria infection in complete absence of re-exposure. We identified a combination of five serological markers that detect exposure within the previous three months with >80% sensitivity and specificity. Using mathematical modelling, we examined the antibody kinetics and determined that responses informative of recent exposure display several distinct characteristics: rapid initial boosting and decay, less inter-individual variation in response kinetics, and minimal persistence over time. Such serological exposure markers could be incorporated into routine malaria surveillance to guide efforts for malaria control and elimination.

Sequential Vaccination With Heterologous Acinetobacter baumannii Strains Induces Broadly Reactive Antibody Responses.

Antibody therapy may be an alternative treatment option for infections caused by the multi-drug resistant (MDR) bacterium Acinetobacter baumannii. As A. baumannii has multiple capsular serotypes, a universal antibody therapy would need to target conserved protein antigens rather than the capsular polysaccharides. We have immunized mice with single or multiple A. baumannii strains to induce antibody responses to protein antigens, and then assessed whether these responses provide cross-protection against a collection of genetically diverse clinical A. baumannii isolates. Immunized mice developed antibody responses to multiple protein antigens. Flow cytometry IgG binding assays and immunoblots demonstrated improved recognition of both homologous and heterologous clinical strains in sera from mice immunized with multiple strains compared to a single strain. The capsule partially inhibited bacterial recognition by IgG and the promotion of phagocytosis by human neutrophils. However, after immunization with multiple strains, serum antibodies to protein antigens promoted neutrophil phagocytosis of heterologous A. baumannii strains. In an infection model, mice immunized with multiple strains had lower bacterial counts in the spleen and liver following challenge with a heterologous strain. These data demonstrate that antibodies targeting protein antigens can improve immune recognition and protection against diverse A. baumannii strains, providing support for their use as an antibody therapy.

protGear: A protein microarray data pre-processing suite.

Protein microarrays are versatile tools for high throughput study of the human proteome, but systematic and non-systematic sources of bias constrain optimal interpretation and the ultimate utility of the data. Published guidelines to limit technical variability whilst maintaining important biological variation favour DNA-based microarrays that often differ fundamentally in their experimental design. Rigorous tools to guide background correction, the quantification of within-sample variation, normalisation, and batch correction specifically for protein microarrays are limited, require extensive investigation and are not centrally accessible. Here, we develop a generic one-stop-shop pre-processing suite for protein microarrays that is compatible with data from the major protein microarray scanners. Our graphical and tabular interfaces facilitate a detailed inspection of data and are coupled with supporting guidelines that enable users to select the most appropriate algorithms to systematically address bias arising in customized experiments. The localization and distribution of background signal intensities determine the optimal correction strategy. A novel function overcomes the limitations in the interpretation of the coefficient of variation when signal intensities are at the lower end of the detection threshold. We demonstrate essential considerations in the experimental design and their impact on a range of algorithms for normalization and minimization of batch effects. Our user-friendly interactive web-based platform eliminates the need for prowess in programming. The open-source R interface includes illustrative examples, generates an auditable record, enables reproducibility, and can incorporate additional custom scripts through its online repository. This versatility will enhance its broad uptake in the infectious disease and vaccine development community.

Plasmodium falciparum Merozoite Associated Armadillo Protein (PfMAAP) Is Apically Localized in Free Merozoites and Antibodies Are Associated With Reduced Risk of Malaria.

Understanding the functional role of proteins expressed by Plasmodium falciparum is an important step toward unlocking potential targets for the development of therapeutic or diagnostic interventions. The armadillo (ARM) repeat protein superfamily is associated with varied functions across the eukaryotes. Therefore, it is important to understand the role of members of this protein family in Plasmodium biology. The Plasmodium falciparum armadillo repeats only (PfARO; Pf3D7_0414900) and P. falciparum merozoite organizing proteins (PfMOP; Pf3D7_0917000) are armadillo-repeat containing proteins previously characterized in P. falciparum. Here, we describe the characterization of another ARM repeat-containing protein in P. falciparum, which we have named the P. falciparum Merozoites-Associated Armadillo repeats protein (PfMAAP). Antibodies raised to three different synthetic peptides of PfMAAP show apical staining of free merozoites and those within the mature infected schizont. We also demonstrate that the antibodies raised to the PfMAAP peptides inhibited invasion of erythrocytes by merozoites from different parasite isolates. In addition, naturally acquired human antibodies to the N- and C- termini of PfMAAP are associated with a reduced risk of malaria in a prospective cohort analysis.

Serological Profiling for Malaria Surveillance Using a Standard ELISA Protocol.

The enzyme-linked immunosorbent assay (ELISA) is a reliable and relatively low-cost method for measuring soluble ligands such as antibodies and proteins in biological samples. For analysis of specific antibodies in serum, a capture antigen is immobilized onto a solid polystyrene surface from which it can capture the antibodies. The captured antibodies are subsequently detected using a secondary antibody conjugated to an enzyme. Detection is accomplished by addition of a colorimetric substrate, and the readout is absorbance (optical density). Here, we provide a detailed standardized ELISA protocol for the quantification of antibodies against malaria antigens.

KILchip v1.0: A Novel Plasmodium falciparum Merozoite Protein Microarray to Facilitate Malaria Vaccine Candidate Prioritization.

Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high-throughput multiplexed antibody detection from individual samples. The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal-Wallis H test for trend: p < 0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over a duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA [median (range) Spearman's R of 0.84 (0.65-0.95)]. KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next generation of malaria vaccines.

Changes in Serological Immunology Measures in UK and Kenyan Adults Post-controlled Human Malaria Infection.

Background: The timing of infection is closely determined in controlled human malaria infection (CHMI) studies, and as such they provide a unique opportunity to dissect changes in immunological responses before and after a single infection. The first Kenyan Challenge Study (KCS) (Pan African Clinical Trial Registry: PACTR20121100033272) was performed in 2013 with the aim of establishing the CHMI model in Kenya. This study used aseptic, cryopreserved, attenuated Plasmodium falciparum sporozoites administered by needle and syringe (PfSPZ Challenge) and was the first to evaluate parasite dynamics post-CHMI in individuals with varying degrees of prior exposure to malaria. Methods: We describe detailed serological and functional immunological responses pre- and post-CHMI for participants in the KCS and compare these with those from malaria-naïve UK volunteers who also underwent CHMI (VAC049) (ClinicalTrials.gov NCT01465048) using PfSPZ Challenge. We assessed antibody responses to three key blood-stage merozoite antigens [merozoite surface protein 1 (MSP1), apical membrane protein 1 (AMA1), and reticulocyte-binding protein homolog 5 (RH5)] and functional activity using two candidate measures of anti-merozoite immunity; the growth inhibition activity (GIA) assay and the antibody-dependent respiratory burst activity (ADRB) assay. Results:Clear serological differences were observed pre- and post-CHMI by ELISA between malaria-naïve UK volunteers in VAC049, and Kenyan volunteers who had prior malaria exposure. Antibodies to AMA1 and schizont extract correlated with parasite multiplication rate (PMR) post-CHMI in KCS. Serum from volunteer 110 in KCS, who demonstrated a dramatically reduced PMR in vivo, had no in vitro GIA prior to CHMI but the highest level of ADRB activity. A significant difference in ADRB activity was seen between KCS volunteers with minimal and definite prior exposure to malaria and significant increases were seen in ADRB activity post-CHMI in Kenyan volunteers. Quinine and atovaquone/proguanil, previously assumed to be removed by IgG purification, were identified as likely giving rise to aberrantly high in vitro GIA results. Conclusions: The ADRB activity assay is a promising functional assay that warrants further investigation as a measure of prior exposure to malaria and predictor of control of parasite growth. The CHMI model can be used to evaluate potential measures of naturally-acquired immunity to malaria.