Controlled Human Malaria Infection in Semi-Immune Kenyan Adults (CHMI-SIKA): a study protocol to investigate in vivo Plasmodium falciparum malaria parasite growth in the context of pre-existing immunity [version 2; peer review: 2 approved]

Malaria remains a major public health burden despite approval for implementation of a partially effective pre-erythrocytic malaria vaccine. There is an urgent need to accelerate development of a more effective multi-stage vaccine. Adults in malaria endemic areas may have substantial immunity provided by responses to the blood stages of malaria parasites, but field trials conducted on several blood-stage vaccines have not shown high levels of efficacy. We will use the controlled human malaria infection (CHMI) models with malaria-exposed volunteers to identify correlations between immune responses and parasite growth rates in vivo. Immune responses more strongly associated with control of parasite growth should be prioritized to accelerate malaria vaccine development. We aim to recruit up to 200 healthy adult volunteers from areas of differing malaria transmission in Kenya, and after confirming their health status through clinical examination and routine haematology and biochemistry, we will comprehensively characterize immunity to malaria using >100 blood-stage antigens. We will administer 3,200 aseptic, purified, cryopreserved Plasmodium falciparum sporozoites (PfSPZ Challenge) by direct venous inoculation. Serial quantitative polymerase chain reaction to measure parasite growth rate in vivo will be undertaken. Clinical and laboratory monitoring will be undertaken to ensure volunteer safety. In addition, we will also explore the perceptions and experiences of volunteers and other stakeholders in participating in a malaria volunteer infection study. Serum, plasma, peripheral blood mononuclear cells and whole blood will be stored to allow a comprehensive assessment of adaptive and innate host immunity. We will use CHMI in semi-immune adult volunteers to relate parasite growth outcomes with antibody responses and other markers of host immunity. / Registration: ClinicalTrials.gov identifier NCT02739763

Dynamics and role of antibodies to Plasmodium falciparum merozoite antigens in children living in two settings with differing malaria transmission intensity.

Young infants have reduced susceptibility to febrile malaria compared with older children, but the mechanism for this remains unclear. There are conflicting data on the role of passively acquired antibodies. Here, we examine antibody titres to merozoite surface antigens in the protection of children in their first two years of life in two settings with differing malaria transmission intensity and compare these titres to previously established protective thresholds.Two cohorts of children aged four to six weeks were recruited in Banfora, Burkina and Keur Soce, Senegal and followed up for two years. Malaria infections were detected by light microscopic examination of blood smears collected at active and passive case detection visits. The titres of antibodies to the Plasmodium falciparum recombinant merozoite proteins (AMA1-3D7, MSP1-19, MSP2-Dd2, and MSP3-3D7) were measured by enzyme-linked immunosorbent assay at 1-6, 9, 12, 15 and 18 months of age and compared with the protective thresholds established in Kenyan children.Antibody titres were below the protective thresholds throughout the study period and we did not find any association with protection against febrile malaria. Antibodies to AMA1 and MSP1-19 appeared to be markers of exposure in the univariate analysis (and so associated with increasing risk) and adjusting for exposure reduced the strength and significance of this association.The antibody levels we measured are unlikely to be responsible for the apparent protection against febrile malaria seen in young infants. Further work to identify protective antibody responses might include functional assays and a wider range of antigens

Cytomegalovirus viraemia is associated with poor growth and T-cell activation with an increased burden in HIV-exposed uninfected infants

Objective: Factors associated with poor health in HIV-exposed-uninfected (HEU) infants are poorly defined. We describe the prevalence and correlates of CMV viraemia in HEU and HIV-unexposed-uninfected (HUU) infants, and quantify associations with anthropometric, haematological and immunological outcomes. Design: Cross-sectional, including HEU and HUU infants from rural coastal Kenya. Methods: Infants aged 2-8 months were studied. The primary outcome was CMV viraemia and viral load, determined by quantitative PCR. Correlates were tested by logistic and linear regression; coefficients were used to describe associations between CMV viraemia and clinical/immunological parameters. Results: 42/65 (64.6%) infants had CMV viraemia (median viral load, 3.0 [IQR: 2.7-3.5] log10 IU/mL). Compared to community controls, HEU infants had 6-fold increased odds of being viraemic (adjusted OR 5.95 [95% CI: 1.82-19.36], P=0.003). Age, but not HEU/HUU status, was a strong correlate of CMV viral load (coeff = -0.15, P = 0.009). CMV viral load associated negatively with weight-for-age z-score (coeff. = -1.06, P = 0.008) and head circumference-for-age z-score (coeff. = -1.47, P = 0.012) and positively with CD8 T cell co-expression of CD38/HLADR (coeff. = 15.05, P = 0.003). Conclusions: The odds of having CMV viraemia was six-fold greater in HEU than HUU infants when adjusted for age. CMV viral load was associated with adverse growth and heightened CD8 T cell immune activation. Longitudinal assessments of the clinical effects of primary CMV infection and associated immunomodulation in early life in HEU and HUU populations are warranted

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

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

Dynamics in multiplicity of Plasmodium falciparum infection among children with asymptomatic malaria in central Ghana

Abstract Background The determinants of malaria parasite virulence is not entirely known, but the outcome of malaria infection (asymptomatic or symptomatic) has been associated with carriage of distinct parasite genotypes. Alleles considered important for erythrocyte invasion and selected as candidate targets for malaria vaccine development are increasingly being shown to have distinct characteristics in infection outcomes. Any unique/distinct patterns or alleles linked to infection outcome should be reproducible for a given malaria-cohort regardless of location, time or intervention. This study compared merozoite surface protein 2 (MSP2) genotypes from children with asymptomatic malaria at same geographical location, from two time periods. Results As the prevalence and incidence of malaria (measured for other studies) significantly reduced between 2004 (time point one) and 2009 (time point two), MSP2 multiplicity of infections (MOI) also reduced significantly from 2.3 at time point (TP) one to 1.9 at TP two. IC/3D7 genotypes out-numbered FC27 genotypes at both time points. At TP2 however, FC27 allele diversity was more than the IC/3D7 allele diversity. A decrease in the IC/3D7:FC27 genotype proportions from 2:1 at TP1 to 1:1 at TP2, seemed to be driven mainly by a decrease in carriage of IC/3D7 alleles. MOI was higher in the dry season than in the subsequent wet season, but the decrease was not significant at TP2. Conclusion MSP2 MOI was higher in the dry season than in the subsequent wet season, while the carriage of IC/3D7 alleles decreased over this time period. It may be that decreases in transmission are related specifically to the IC/3D7 allelic family. The influence of transmission on MSP2 allele diversity needs to be clearly deciphered in studies which should include the use of sensitive methods for the detection of polymorphic parasite markers for both symptomatic and asymptomatic malaria. Such studies will enable better understanding of associations between allelic variants, MOI, transmission, malaria infection and disease