Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

Age-dependent acquisition of IgG antibodies to Shigella serotypes-a retrospective analysis of seroprevalence in Kenyan children with implications for infant vaccination.

Background: Shigellosis mainly affects children under 5 years of age living in low- and middle-income countries, who are the target population for vaccination. There are, however, limited data available to define the appropriate timing for vaccine administration in this age group. Information on antibody responses following natural infection, proxy for exposure, could help guide vaccination strategies. Methods: We undertook a retrospective analysis of antibodies to five of the most prevalent Shigella serotypes among children aged <5 years in Kenya. Serum samples from a cross-sectional serosurvey in three Kenyan sites (Nairobi, Siaya, and Kilifi) were analyzed by standardized ELISA to measure IgG against Shigella sonnei and Shigella flexneri 1b, 2a, 3a, and 6. We identified factors associated with seropositivity to each Shigella serotype, including seropositivity to other Shigella serotypes. Results: A total of 474 samples, one for each participant, were analyzed: Nairobi (n = 169), Siaya (n = 185), and Kilifi (n = 120). The median age of the participants was 13.4 months (IQR 7.0-35.6), and the male:female ratio was 1:1. Geometric mean concentrations (GMCs) for each serotype increased with age, mostly in the second year of life. The overall seroprevalence of IgG antibodies increased with age except for S. flexneri 6 which was high across all age subgroups. In the second year of life, there was a statistically significant increase of antibody GMCs against all five serotypes (p = 0.01-0.0001) and a significant increase of seroprevalence for S. flexneri 2a (p = 0.006), S. flexneri 3a (p = 0.006), and S. sonnei (p = 0.05) compared with the second part of the first year of life. Among all possible pairwise comparisons of antibody seropositivity, there was a significant association between S. flexneri 1b and 2a (OR = 6.75, 95% CI 3-14, p < 0.001) and between S. flexneri 1b and 3a (OR = 23.85, 95% CI 11-54, p < 0.001). Conclusion: Children living in low- and middle-income settings such as Kenya are exposed to Shigella infection starting from the first year of life and acquire serotype-specific antibodies against multiple serotypes. The data from this study suggest that Shigella vaccination should be targeted to infants, ideally at 6 or at least 9 months of age, to ensure children are protected in the second year of life when exposure significantly increases.

Characterization of a novel Plasmodium falciparum merozoite surface antigen and potential vaccine target.

Introduction: Detailed analyses of genetic diversity, antigenic variability, protein localization and immunological responses are vital for the prioritization of novel malaria vaccine candidates. Comprehensive approaches to determine the most appropriate antigen variants needed to provide broad protection are challenging and consequently rarely undertaken. Methods: Here, we characterized PF3D7_1136200, which we named Asparagine-Rich Merozoite Antigen (ARMA) based on the analysis of its sequence, localization and immunogenicity. We analyzed IgG and IgM responses against the common variants of ARMA in independent prospective cohort studies in Burkina Faso (N = 228), Kenya (N = 252) and Mali (N = 195) using a custom microarray, Div-KILCHIP. Results: We found a marked population structure between parasites from Africa and Asia. African isolates shared 34 common haplotypes, including a dominant pair although the overall selection pressure was directional (Tajima's D = -2.57; Fu and Li's F = -9.69; P < 0.02). ARMA was localized to the merozoite surface, IgG antibodies induced Fc-mediated degranulation of natural killer cells and strongly inhibited parasite growth in vitro. We found profound serological diversity, but IgG and IgM responses were highly correlated and a hierarchical clustering analysis identified only three major serogroups. Protective IgG and IgM antibodies appeared to target both cross-reactive and distinct epitopes across variants. However, combinations of IgG and IgM antibodies against selected variants were associated with complete protection against clinical episodes of malaria. Discussion: Our systematic strategy exploits genomic data to deduce the handful of antigen variants with the strongest potential to induce broad protection and may be broadly applicable to other complex pathogens for which effective vaccines remain elusive.

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.

Serum immunoglobulin G and mucosal immunoglobulin A antibodies from prepandemic samples collected in Kilifi, Kenya, neutralize SARS-CoV-2 in vitro.

OBJECTIVES: Many regions of Africa have experienced lower COVID-19 morbidity and mortality than Europe. Pre-existing humoral responses to endemic human coronaviruses (HCoV) may cross-protect against SARS-CoV-2. We investigated the neutralizing capacity of SARS-CoV-2 spike reactive and nonreactive immunoglobulin (Ig)G and IgA antibodies in prepandemic samples. METHODS: To investigate the presence of pre-existing immunity, we performed enzyme-linked immunosorbent assay using spike antigens from reference SARS-CoV-2, HCoV HKU1, OC43, NL63, and 229E using prepandemic samples from Kilifi in coastal Kenya. In addition, we performed neutralization assays using pseudotyped reference SARS-CoV-2 to determine the functionality of the identified reactive antibodies. RESULTS: We demonstrate the presence of HCoV serum IgG and mucosal IgA antibodies, which cross-react with the SARS-CoV-2 spike. We show pseudotyped reference SARS-CoV-2 neutralization by prepandemic serum, with a mean infective dose 50 of 1: 251, which is 10-fold less than that of the pooled convalescent sera from patients with COVID-19 but still within predicted protection levels. The prepandemic naso-oropharyngeal fluid neutralized pseudo-SARS-CoV-2 at a mean infective dose 50 of 1: 5.9 in the neutralization assay. CONCLUSION: Our data provide evidence for pre-existing functional humoral responses to SARS-CoV-2 in Kilifi, coastal Kenya and adds to data showing pre-existing immunity for COVID-19 from other regions.

Malaria attributable fractions with changing transmission intensity: Bayesian latent class vs logistic models.

BACKGROUND: Asymptomatic carriage of malaria parasites is common in high transmission intensity areas and confounds clinical case definitions for research studies. This is important for investigations that aim to identify immune correlates of protection from clinical malaria. The proportion of fevers attributable to malaria parasites is widely used to define different thresholds of parasite density associated with febrile episodes. The varying intensity of malaria transmission was investigated to check whether it had a significant impact on the parasite density thresholds. The same dataset was used to explore an alternative statistical approach, using the probability of developing fevers as a choice over threshold cut-offs. The former has been reported to increase predictive power. METHODS: Data from children monitored longitudinally between 2005 and 2017 from Junju and Chonyi in Kilifi, Kenya were used. Performance comparison of Bayesian-latent class and logistic power models in estimating malaria attributable fractions and probabilities of having fever given a parasite density with changing malaria transmission intensity was done using Junju cohort. Zero-inflated beta regressions were used to assess the impact of using probabilities to evaluate anti-merozoite antibodies as correlates of protection, compared with multilevel binary regression using data from Chonyi and Junju. RESULTS: Malaria transmission intensity declined from over 49% to 5% between 2006 and 2017, respectively. During this period, malaria attributable fraction varied between 27-59% using logistic regression compared to 10-36% with the Bayesian latent class approach. Both models estimated similar patterns of fevers attributable to malaria with changing transmission intensities. The Bayesian latent class model performed well in estimating the probabilities of having fever, while the latter was efficient in determining the parasite density threshold. However, compared to the logistic power model, the Bayesian algorithm yielded lower estimates for both attributable fractions and probabilities of fever. In modelling the association of merozoite antibodies and clinical malaria, both approaches resulted in comparable estimates, but the utilization of probabilities had a better statistical fit. CONCLUSIONS: Malaria attributable fractions, varied with an overall decline in the malaria transmission intensity in this setting but did not significantly impact the outcomes of analyses aimed at identifying immune correlates of protection. These data confirm the statistical advantage of using probabilities over binary data.

Phagocytosis of Plasmodium falciparum ring-stage parasites predicts protection against malaria.

Ring-infected erythrocytes are the predominant asexual stage in the peripheral circulation but are rarely investigated in the context of acquired immunity against Plasmodium falciparum malaria. Here we compare antibody-dependent phagocytosis of ring-infected parasite cultures in samples from a controlled human malaria infection (CHMI) study (NCT02739763). Protected volunteers did not develop clinical symptoms, maintained parasitaemia below a predefined threshold of 500 parasites/µl and were not treated until the end of the study. Antibody-dependent phagocytosis of both ring-infected and uninfected erythrocytes from parasite cultures was strongly correlated with protection. A surface proteomic analysis revealed the presence of merozoite proteins including erythrocyte binding antigen-175 and -140 on ring-infected and uninfected erythrocytes, providing an additional antibody-mediated protective mechanism for their activity beyond invasion-inhibition. Competition phagocytosis assays support the hypothesis that merozoite antigens are the key mediators of this functional activity. Targeting ring-stage parasites may contribute to the control of parasitaemia and prevention of clinical malaria.

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.

Characterization of Anopheles gambiae D7 salivary proteins as markers of human-mosquito bite contact.

BACKGROUND: Malaria is transmitted when infected Anopheles mosquitoes take a blood meal. During this process, the mosquitoes inject a cocktail of bioactive proteins that elicit antibody responses in humans and could be used as biomarkers of exposure to mosquito bites. This study evaluated the utility of IgG responses to members of the Anopheles gambiae D7 protein family as serological markers of human-vector contact. METHODS: The D7L2, D7r1, D7r2, D7r3, D7r4 and SG6 salivary proteins from An. gambiae were expressed as recombinant antigens in Escherichia coli. Antibody responses to the salivary proteins were compared in Europeans with no prior exposure to malaria and lifelong residents of Junju in Kenya and Kitgum in Uganda where the intensity of malaria transmission is moderate and high, respectively. In addition, to evaluate the feasibility of using anti-D7 IgG responses as a tool to evaluate the impact of vector control interventions, we compared responses between individuals using insecticide-treated bednets to those who did not in Junju, Kenya where bednet data were available. RESULTS: We show that both the long and short forms of the D7 salivary gland antigens elicit a strong antibody response in humans. IgG responses against the D7 antigens reflected the transmission intensities of the three study areas, with the highest to lowest responses observed in Kitgum (northern Uganda), Junju (Kenya) and malaria-naïve Europeans, respectively. Specifically, the long form D7L2 induced an IgG antibody response that increased with age and that was lower in individuals who slept under a bednet, indicating its potential as a serological tool for estimating human-vector contact and monitoring the effectiveness of vector control interventions. CONCLUSIONS: This study reveals that D7L2 salivary antigen has great potential as a biomarker of exposure to mosquito bites and as a tool for assessing the efficacy of vector control strategies such as bednet use.

Comparative performance of WANTAI ELISA for total immunoglobulin to receptor binding protein and an ELISA for IgG to spike protein in detecting SARS-CoV-2 antibodies in Kenyan populations.

Many SARS-CoV-2 antibody detection assays have been developed but their differential performance is not well described. In this study we compared an in-house (KWTRP) ELISA which has been used extensively to estimate seroprevalence in the Kenyan population with WANTAI, an ELISA which has been approved for widespread use by the WHO. Using a wide variety of sample sets including pre-pandemic samples (negative gold standard), SARS-CoV-2 PCR positive samples (positive gold standard) and COVID-19 test samples from different periods (unknowns), we compared performance characteristics of the two assays. The overall concordance between WANTAI and KWTRP was 0.97 (95% CI, 0.95-0.98). For WANTAI and KWTRP, sensitivity was 0.95 (95% CI 0.90-0.98) and 0.93 (95% CI 0.87-0.96), respectively. Specificity for WANTAI was 0.98 (95% CI, 0.96-0.99) and 0.99 (95% CI 0.96-1.00) while KWTRP specificity was 0.99 (95% CI, 0.98-1.00) and 1.00 using pre-pandemic blood donors and pre-pandemic malaria cross-sectional survey samples respectively. Both assays show excellent characteristics to detect SARS-CoV-2 antibodies.

Hepcidin regulation in Kenyan children with severe malaria and non-typhoidal Salmonella bacteremia.

Malaria and invasive non-typhoidal Salmonella (NTS) are life-threatening infections that often co-exist in African children. The iron-regulatory hormone hepcidin is highly upregulated during malaria and controls the availability of iron, a critical nutrient for bacterial growth. We investigated the relationship between Plasmodium falciparum malaria and NTS bacteremia in all pediatric admissions aged <5 years between August 1998 and October 2019 (n=75,034). We then assayed hepcidin and measures of iron status in five groups: (1) children with concomitant severe malarial anemia (SMA) and NTS (SMA+NTS, n=16); and in matched children with (2) SMA (n=33); (3) NTS (n=33); (4) cerebral malaria (CM, n=34); and (5) community-based children. SMA and severe anemia without malaria were associated with a 2-fold or more increased risk of NTS bacteremia, while other malaria phenotypes were not associated with increased NTS risk. Children with SMA had lower hepcidin/ferritin ratios (0.10; interquartile range [IQR]: 0.03-0.19) than those with CM (0.24; IQR: 0.14-0.69; P=0.006) or asymptomatic malaria (0.19; IQR: 0.09-0.46; P=0.01) indicating suppressed hepcidin levels. Children with SMA+NTS had lower hepcidin levels (9.3 ng/mL; IQR: 4.7-49.8) and hepcidin/ferritin ratios (0.03; IQR: 0.01-0.22) than those with NTS alone (105.8 ng/mL; IQR: 17.3-233.3; P=0.02 and 0.31; IQR: 0.06-0.66; P=0.007, respectively). Since hepcidin degrades ferroportin on the Salmonella-containing vacuole, we hypothesize that reduced hepcidin in children with SMA might contribute to NTS growth by modulating iron availability for bacterial growth. Further studies are needed to understand how the hepcidin-ferroportin axis might mediate susceptibility to NTS in severely anemic children.

Seven-year kinetics of RTS, S/AS01-induced anti-CSP antibodies in young Kenyan children.

BACKGROUND: RTS,S/AS01, the leading malaria vaccine has been recommended by the WHO for widespread immunization of children at risk. RTS,S/AS01-induced anti-CSP IgG antibodies are associated with the vaccine efficacy. Here, the long-term kinetics of RTS,S/AS01-induced antibodies was investigated. METHODS: 150 participants were randomly selected from the 447 children who participated in the RTS,S/AS01 phase IIb clinical trial in 2007 from Kilifi-Kenya. Cumulatively, the retrospective follow-up period was 93 months with annual plasma samples collection. The levels of anti-CSP IgM, total IgG, IgG1, IgG2, IgG3, and IgG4 antibodies were then determined using an enzyme-linked immunosorbent assay. RESULTS: RTS,S/AS01 induced high levels of anti-CSP IgG antibodies which exhibited a rapid waning over 6.5 months post-vaccination, followed by a slower decay over the subsequent years. RTS,S/AS01-induced anti-CSP IgG antibodies remained elevated above the control group levels throughout the 7 years follow-up period. The anti-CSP IgG antibodies were mostly IgG1, IgG3, IgG2, and to a lesser extent IgG4. IgG2 predominated in later timepoints. RTS,S/AS01 also induced high levels of anti-CSP IgM antibodies which increased above the control group levels by month 3. The controls exhibited increasing levels of the anti-CSP IgM antibodies which caught up with the RTS,S/AS01 vaccinees levels by month 21. In contrast, there were no measurable anti-CSP IgG antibodies among the controls. CONCLUSION: RTS,S/AS01-induced anti-CSP IgG antibodies kinetics are consistent with long-lived but waning vaccine efficacy. Natural exposure induces anti-CSP IgM antibodies in children, which increases with age, but does not induce substantial levels of anti-CSP IgG antibodies.

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.

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.

Gametocyte carriage in an era of changing malaria epidemiology: A 19-year analysis of a malaria longitudinal cohort.

Background: Interventions to block malaria transmission from humans to mosquitoes are currently in development. To be successfully implemented, key populations need to be identified where the use of these transmission-blocking and/or reducing strategies will have greatest impact. Methods: We used data from a longitudinally monitored cohort of children from Kilifi county located along the Kenyan coast collected between 1998-2016 to describe the distribution and prevalence of gametocytaemia in relation to transmission intensity, time and age. Data from 2,223 children accounting for 9,134 person-years of follow-up assessed during cross-sectional surveys for asexual parasites and gametocytes were used in logistic regression models to identify factors predictive of gametocyte carriage in this cohort. Results: Our analysis showed that children 1-5 years of age were more likely to carry microscopically detectable gametocytes than their older counterparts. Carrying asexual parasites and recent episodes of clinical malaria were also strong predictors of gametocyte carriage. The prevalence of asexual parasites and of gametocyte carriage declined over time, and after 2006, when artemisinin combination therapy (ACT) was introduced, recent episodes of clinical malaria ceased to be a predictor of gametocyte carriage.  Conclusions: Gametocyte carriage in children in Kilifi has fallen over time.  Previous episodes of clinical malaria may contribute to the development of carriage, but this appears to be mitigated by the use of ACTs highlighting the impact that gametocidal antimalarials can have in reducing the overall prevalence of gametocytaemia when targeted on acute febrile illness.

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.

Age, Spatial, and Temporal Variations in Hospital Admissions with Malaria in Kilifi County, Kenya: A 25-Year Longitudinal Observational Study.

BACKGROUND: Encouraging progress has been seen with reductions in Plasmodium falciparum malaria transmission in some parts of Africa. Reduced transmission might lead to increasing susceptibility to malaria among older children due to lower acquired immunity, and this has implications for ongoing control strategies. METHODS AND FINDINGS: We conducted a longitudinal observational study of children admitted to Kilifi County Hospital in Kenya and linked it to data on residence and insecticide-treated net (ITN) use. This included data from 69,104 children aged from 3 mo to 13 y admitted to Kilifi County Hospital between 1 January 1990 and 31 December 2014. The variation in malaria slide positivity among admissions was examined in logistic regression models using the following predictors: location of the residence, calendar time, the child's age, ITN use, and the enhanced vegetation index (a proxy for soil moisture). The proportion of malaria slide-positive admissions declined from 0.56 (95% confidence interval [CI] 0.54-0.58) in 1998 to 0.07 (95% CI 0.06-0.08) in 2009 but then increased again through to 0.24 (95% CI 0.22-0.25) in 2014. Older children accounted for most of the increase after 2009 (0.035 [95% CI 0.030-0.040] among young children compared to 0.22 [95% CI 0.21-0.23] in older children). There was a nonlinear relationship between malaria risk and prevalence of ITN use within a 2 km radius of an admitted child's residence such that the predicted malaria positive fraction varied from ~0.4 to <0.1 as the prevalence of ITN use varied from 20% to 80%. In this observational analysis, we were unable to determine the cause of the decline in malaria between 1998 and 2009, which pre-dated the dramatic scale-up in ITN distribution and use. CONCLUSION: Following a period of reduced transmission, a cohort of older children emerged who have increased susceptibility to malaria. Further reductions in malaria transmission are needed to mitigate the increasing burden among older children, and universal ITN coverage is a promising strategy to achieve this goal.