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.

How much does effective health facility inspection cost? An analysis of the economic costs of Kenya's Joint Health Inspection innovations.

BACKGROUND: In most low- and middle-income countries, health facility regulation is fragmented, ineffective and under-resourced. The Kenyan Government piloted an innovative regulatory regime involving Joint Health Inspections (JHI) which synthesized requirements across multiple regulatory agencies; increased inspection frequency; digitized inspection tools; and introduced public display of regulatory results. The pilot significantly improved regulatory compliance. We calculated the costs of the development and implementation of the JHI pilot and modelled the costs of national scale-up in Kenya. METHODS: We calculated the economic costs of three phases: JHI checklist development, start-up activities, and first year of implementation, from the providers' perspective in three pilot counties. Data collection involved extraction from expenditure records and key informant interviews. The annualized costs of JHI were calculated by adding annualized development and start-up costs to annual implementation costs. National level scale-up costs were also modelled and compared to those of current standard inspections. RESULTS: The total economic cost of the JHI pilot was USD 1,125,600 (2017 USD), with the development phase accounting for 19%, start-up 43% and the first year of implementation 38%. The annualized economic cost was USD 519,287, equivalent to USD 206 per health facility visit and USD 311 per inspection completed. Scale up to the national level, while replacing international advisors with local staff, was estimated to cost approximately USD 4,823,728, equivalent to USD 103 per health facility visit and USD 155 per inspection completed. This compares to an estimated USD 86,997 per year (USD 113 per inspection completed) spent on a limited number of inspections prior to JHI. CONCLUSION: Information on costs is essential to consider affordability and value for money of regulatory interventions. This is the first study we are aware of costing health facility inspections in sub-Saharan Africa. It has informed debates on appropriate inspection design and potential efficiency gains. It will also serve as an important benchmark for future studies, and a key input into cost-effectiveness analyses.

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.

What Lies Behind Successful Regulation? A Qualitative Evaluation of Pilot Implementation of Kenya's Health Facility Inspection Reforms.

BACKGROUND: Health facility regulation in low- and middle-income countries (LMICs) is generally weak, with potentially serious consequences for safety and quality. Innovative regulatory reforms were piloted in three Kenyan counties including: a Joint Health Inspection Checklist (JHIC) synthesizing requirements across multiple regulatory agencies; increased inspection frequency; allocating facilities to compliance categories which determined warnings, sanctions and/or time to re-inspection; and public display of regulatory results. The reforms substantially increased inspection scores compared with control facilities. We developed lessons for future regulatory policy from this pilot by identifying key factors that facilitated or hindered its implementation. METHODS: We conducted a qualitative study to understand views and experiences of actors involved in the one-year pilot. We interviewed 77 purposively selected staff from the national, county and facility levels. Data were analyzed using the framework approach, identifying facilitating/hindering factors at the facility, inspection system, and health system levels. RESULTS: The joint health inspections (JHIs) were generally viewed as fair, objective and transparent, which enhanced their perceived legitimacy. Interactions with inspectors were described as friendly and supportive, in contrast to the punitive culture of previous inspections when bribery had been common. Inspector training and use of an electronic checklist were strongly praised. However, practical challenges with transport, route planning and budgets highlighted the critical nature of strong logistical management. The effectiveness of inspection in improving compliance was hampered by limitations in related systems, particularly facility licensing, enforcement of closures and, in the public sector, control of funds. However, an inclusive reform development process had led to high buy-in across regulatory agencies which was key to the system's success. CONCLUSION: Effective facility inspection involves more than "hardware" such as checklists, protocols and training. Cultural, relational and institutional "software" are also crucial for legitimacy, feasibility of implementation and enforceability, and should be carefully integrated into regulatory reforms.

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.

Author Correction: Airway response to respiratory syncytial virus has incidental antibacterial effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Airway response to respiratory syncytial virus has incidental antibacterial effects.

RSV infection is typically associated with secondary bacterial infection. We hypothesise that the local airway immune response to RSV has incidental antibacterial effects. Using coordinated proteomics and metagenomics analysis we simultaneously analysed the microbiota and proteomes of the upper airway and determined direct antibacterial activity in airway secretions of RSV-infected children. Here, we report that the airway abundance of Streptococcus was higher in samples collected at the time of RSV infection compared with samples collected one month later. RSV infection is associated with neutrophil influx into the airway and degranulation and is marked by overexpression of proteins with known antibacterial activity including BPI, EPX, MPO and AZU1. Airway secretions of children infected with RSV, have significantly greater antibacterial activity compared to RSV-negative controls. This RSV-associated, neutrophil-mediated antibacterial response in the airway appears to act as a regulatory mechanism that modulates bacterial growth in the airways of RSV-infected children.

Effective coverage and budget implications of skill-mix change to improve neonatal nursing care: an explorative simulation study in Kenya.

INTRODUCTION: Neonatal mortality is an urgent policy priority to improve global population health and reduce health inequality. As health systems in Kenya and elsewhere seek to tackle increased neonatal mortality by improving the quality of care, one option is to train and employ neonatal healthcare assistants (NHCAs) to support professional nurses by taking up low-skill tasks. METHODS: Monte-Carlo simulation was performed to estimate the potential impact of introducing NHCAs in neonatal nursing care in four public hospitals in Nairobi on effectively treated newborns and staff costs over a period of 10 years. The simulation was informed by data from 3 workshops with >10 stakeholders each, hospital records and scientific literature. Two univariate sensitivity analyses were performed to further address uncertainty. RESULTS: Stakeholders perceived that 49% of a nurse full-time equivalent could be safely delegated to NHCAs in standard care, 31% in intermediate care and 20% in intensive care. A skill-mix with nurses and NHCAs would require ~2.6 billionKenyan Shillings (KES) (US$26 million) to provide quality care to 58% of all newborns in need (ie, current level of coverage in Nairobi) over a period of 10 years. This skill-mix configuration would require ~6 billion KES (US$61 million) to provide quality of care to almost all newborns in need over 10 years. CONCLUSION: Changing skill-mix in hospital care by introducing NHCAs may be an affordable way to reduce neonatal mortality in low/middle-income countries. This option should be considered in ongoing policy discussions and supported by further evidence.

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.