Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field

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

Assessment of humoral immune responses to blood-stage malaria antigens following ChAd63-MVA immunization, controlled human malaria infection and natural exposure.

The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the Plasmodium falciparum human malaria parasite--MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors--ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI) with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i) ChAd63-MVA immunization, ii) immunization and CHMI, and iii) primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i) total IgG responses before and after CHMI, ii) responses to allelic variants of MSP1 and AMA1, iii) functional growth inhibitory activity (GIA), iv) IgG avidity, and v) isotype responses (IgG1-4, IgA and IgM). These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other diseases targets, these data should help to guide further immuno-monitoring studies of vaccine-induced human antibody responses

Analysis of human B‐cell responses following ChAd63‐MVA MSP1 and AMA1 immunization and controlled malaria infection

Acquisition of non‐sterilizing natural immunity to Plasmodium falciparum malaria has been shown in low transmission areas following multiple exposures. However, conflicting data from endemic areas suggest that the parasite may interfere with the induction of effective B‐cell responses. To date, the impact of blood‐stage parasite exposure on antigen‐specific B cells has not been reported following controlled human malaria infection (CHMI). Here we analysed human B‐cell responses in a series of Phase I/IIa clinical trials, which include CHMI, using candidate virus‐vectored vaccines encoding two blood‐stage antigens: merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1). Previously vaccinated volunteers show boosting of pre‐existing antigen‐specific memory B‐cell (mBC) responses following CHMI. In contrast, unvaccinated malaria‐naive control volunteers developed an mBC response against MSP1 but not AMA1. Serum IgG correlated with the mBC response after booster vaccination but this relationship was less well maintained following CHMI. A significant reduction in peripheral MSP1‐specific mBC was observed at the point of diagnosis of blood‐stage infection. This was coincident with a reduction in peripheral blood B‐cell subsets expressing CXCR3 and elevated serum levels of interferon‐γ and CXCL9, suggesting migration away from the periphery. These CHMI data confirm that mBC and antibody responses can be induced and boosted by blood‐stage parasite exposure, in support of epidemiological studies on low‐level parasite exposure

Unmanned aircraft systems as a new source of disturbance for wildlife: A systematic review.

The use of small Unmanned Aircraft Systems (UAS; also known as "drones") for professional and personal-leisure use is increasing enormously. UAS operate at low altitudes (<500 m) and in any terrain, thus they are susceptible to interact with local fauna, generating a new type of anthropogenic disturbance that has not been systematically evaluated. To address this gap, we performed a review of the existent literature about animals' responses to UAS flights and conducted a pooled analysis of the data to determine the probability and intensity of the disturbance, and to identify the factors influencing animals' reactions towards the small aircraft. We found that wildlife reactions depended on both the UAS attributes (flight pattern, engine type and size of aircraft) and the characteristics of animals themselves (type of animal, life-history stage and level of aggregation). Target-oriented flight patterns, larger UAS sizes, and fuel-powered (noisier) engines evoked the strongest reactions in wildlife. Animals during the non-breeding period and in large groups were more likely to show behavioral reactions to UAS, and birds are more prone to react than other taxa. We discuss the implications of these results in the context of wildlife disturbance and suggest guidelines for conservationists, users and manufacturers to minimize the impact of UAS. In addition, we propose that the legal framework needs to be adapted so that appropriate actions can be undertaken when wildlife is negatively affected by these emergent practices

Safety and Immunogenicity of ChAd63 and MVA ME-TRAP in West African Children and Infants.

Malaria remains a significant global health burden and a vaccine would make a substantial contribution to malaria control. Chimpanzee Adenovirus 63 Modified Vaccinia Ankara Multiple epitope thrombospondin adhesion protein (ME-TRAP) and vaccination has shown significant efficacy against malaria sporozoite challenge in malaria-naive European volunteers and against malaria infection in Kenyan adults. Infants are the target age group for malaria vaccination; however, no studies have yet assessed T-cell responses in children and infants. We enrolled 138 Gambian and Burkinabe children in four different age-groups: 2-6 years old in The Gambia; 5-17 months old in Burkina Faso; 5-12 months old, and also 10 weeks old, in The Gambia; and evaluated the safety and immunogenicity of Chimpanzee Adenovirus 63 Modified Vaccinia Ankara ME-TRAP heterologous prime-boost immunization. The vaccines were well tolerated in all age groups with no vaccine-related serious adverse events. T-cell responses to vaccination peaked 7 days after boosting with Modified Vaccinia Ankara, with T-cell responses highest in 10 week-old infants. Heterologous prime-boost immunization with Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara ME-TRAP was well tolerated in infants and children, inducing strong T-cell responses. We identify an approach that induces potent T-cell responses in infants, which may be useful for preventing other infectious diseases requiring cellular immunity

Viral Vector Malaria Vaccines Induce High-Level T Cell and Antibody Responses in West African Children and Infants.

Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8+ T cell-mediated immunity to malaria sporozoite challenge in European malaria-naive and Kenyan semi-immune adults. This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8+ and CD4+ T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine

Demonstration of the Blood-Stage Plasmodium falciparum Controlled Human Malaria Infection Model to Assess Efficacy of the P. falciparum Apical Membrane Antigen 1 Vaccine, FMP2.1/AS01

We study whether the relationship between the state unemployment rate at the time of conception and infant health, infant mortality and maternal characteristics in the United States has changed over the years 1980-2004. We use microdata on births and deaths for years 1980-2004 and find that the relationship between the state unemployment rate at the time of conception and infant mortality and birthweight changes over time and is stronger for blacks than whites. For years 1980-1989 increases in the state unemployment rate are associated with a decline in infant mortality among blacks, an effect driven by mortality from gestational development and birth weight, and complications of placenta while in utero. In contrast, state economic conditions are unrelated to black infant mortality in years 1990-2004 and white infant mortality in any period, although effects vary by cause of death. We explore potential mechanisms for our findings and, including mothers younger than 18 in the analysis, uncover evidence of age-related maternal selection in response to the business cycle. In particular, in years 1980-1989 an increase in the unemployment rate at the time of conception is associated with fewer babies born to young mothers. The magnitude and direction of the relationship between business cycles and infant mortality differs by race and period. Age-related selection into motherhood in response to the business cycle is a possible explanation for this changing relationship