Clinical and immunological evaluation of new malaria vaccines encoding the Thrombospondin Related Adhesion Protein

Malaria causes around 2 million deaths per year, and the mortality appears to be rising. A prophylactic vaccination is urgently needed. A viral vectored vaccination regimen has been developed in Oxford to induce T cell responses reactive to pre-erythrocytic antigens of Plasmodium falciparum malaria. I conducted a series of studies to evaluate the clinical outcome and immunological response to the vaccine. I analysed PCR data from experimental sporozoite challenge studies in the UK, and showed that vaccination reduced the numbers of parasites completing their pre-erythrocytic development. I conducted preliminary studies to explore the safety and immunogenicity of vaccination in adults and then children in Kilifi, Kenya. Antigen insert, multiple versus single priming, different batches of vaccine (in one instance) and pre-vaccination immunity influenced the reactogenicity and immunogenicity of vaccination. Enhanced memory responses were seen with novel alternating vector regimens. Having established immunogenicity, safety, and preliminary evidence of efficacy, I then conducted a large efficacy trial in the field, randomizing 406 children to either active vaccination or control. The primary outcome was time to first episode of febrile malaria. Although the regimen was immunogenic, the magnitude of T cell responses was lower than in previous studies. 346 children were vaccinated according to protocol. Episodes of febrile malaria were more frequent in the FFM ME-TRAP group (52/171 vs 401175 among controls), but this was not statistically significant (95% confidence interval 0.83 to 2.08, P=0.55 by logrank). Among children vaccinated with FFM ME-TRAP, there was no correlation between immunogenicity and malaria incidence. I examined factors in the cohort of children that may have influenced immunogenicity. Malaria was more immunosuppressive than other parasitic infections, and is therefore the factor most likely to impact the development of investigational T cell inducing vaccines in malaria endemic areas

Incorporating a quiz into informed consent processes: Qualitative study of participants' reactions

<p>Abstract</p> <p>Background</p> <p>Formal checks of participant understanding are now widely recommended to improve informed consent processes. However, the views of the participants these assessments are designed to protect are rarely considered. In this paper the findings of a qualitative study aimed at documenting community reactions to a semi-structured questionnaire ('quiz') are reported. The quiz was administered to 189 mothers after consenting for their children to participate in a malaria vaccine trial on the Kenyan Coast.</p> <p>Methods</p> <p>Once the malaria vaccine trial was underway, focus group discussions were held with some of these mothers (nine groups; 103 mothers), and with community-based field staff attached to the malaria vaccine trial (two groups of five workers). Individual interviews with other trial staff were also held.</p> <p>Results</p> <p>The quiz prompted community members to voice concerns about blood sampling and vaccine side-effects, thereby encouraging additional discussions and interactions between the research team and potential study participants. However, it also caused significant upset and concern. Some of the quiz questions, or the way in which they were asked, appeared to fuel misconceptions and fears, with potentially negative consequences for both the study and community members.</p> <p>Conclusion</p> <p>Formal approaches to checking study understanding should be employed with sensitivity and caution. They are influenced by and impact upon complex social relationships between and among researchers and community members. Adequate consideration of these contexts in assessments of understanding, and in responding to the issues raised, requires strong social science capacity.</p

Longitudinal analyses of immune responses to Plasmodium falciparum derived peptides corresponding to novel blood stage antigens in coastal Kenya.

We have recently described 95 predicted alpha-helical coiled-coil peptides derived from putative Plasmodium falciparum erythrocytic stage proteins. Seventy peptides recognized with the highest level of prevalence by sera from three endemic areas were selected for further studies. In this study, we sequentially examined antibody responses to these synthetic peptides in two cohorts of children at risk of clinical malaria in Kilifi district in coastal Kenya, in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Antibody levels from 268 children in the first cohort (Chonyi) were assayed against 70 peptides. Thirty-nine peptides were selected for further study in a second cohort (Junju). The rationale for the second cohort was to confirm those peptides identified as protective in the first cohort. The Junju cohort comprised of children aged 1-6 years old (inclusive). Children were actively followed up to identify episodes of febrile malaria in both cohorts. Of the 70 peptides examined, 32 showed significantly (p<0.05) increased antibody recognition in older children and 40 showed significantly increased antibody recognition in parasitaemic children. Ten peptides were associated with a significantly reduced odds ratio (OR) for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and AS202.11) were associated with a significantly reduced OR in both cohorts. LR146 is derived from hypothetical protein PFB0145c in PlasmoDB. Previous work has identified this protein as a target of antibodies effective in antibody dependent cellular inhibition (ADCI). The current study substantiates further the potential of protein PFB0145c and also identifies protein PF11_0424 as another likely target of protective antibodies against P. falciparum malaria

Efficacy of RTS,S malaria vaccines: individual-participant pooled analysis of phase 2 data.

BACKGROUND: The efficacy of RTS,S/AS01 as a vaccine for malaria is being tested in a phase 3 clinical trial. Early results show significant, albeit partial, protection against clinical malaria and severe malaria. To ascertain variations in vaccine efficacy according to covariates such as transmission intensity, choice of adjuvant, age at vaccination, and bednet use, we did an individual-participant pooled analysis of phase 2 clinical data. METHODS: We analysed data from 11 different sites in Africa, including 4453 participants. We measured heterogeneity in vaccine efficacy by estimating the interactions between covariates and vaccination in pooled multivariable Cox regression and Poisson regression analyses. Endpoints for measurement of vaccine efficacy were infection, clinical malaria, severe malaria, and death. We defined transmission intensity levels according to the estimated local parasite prevalence in children aged 2-10 years (PrP₂₋₁₀), ranging from 5% to 80%. Choice of adjuvant was either AS01 or AS02. FINDINGS: Vaccine efficacy against all episodes of clinical malaria varied by transmission intensity (p=0·001). At low transmission (PrP₂₋₁₀ 10%) vaccine efficacy was 60% (95% CI 54 to 67), at moderate transmission (PrP₂₋₁₀ 20%) it was 41% (21 to 57), and at high transmission (PrP₂₋₁₀ 70%) the efficacy was 4% (-10 to 22). Vaccine efficacy also varied by adjuvant choice (p<0·0001)--eg, at low transmission (PrP₂₋₁₀ 10%), efficacy varied from 60% (95% CI 54 to 67) for AS01 to 47% (14 to 75) for AS02. Variations in efficacy by age at vaccination were of borderline significance (p=0·038), and bednet use and sex were not significant covariates. Vaccine efficacy (pooled across adjuvant choice and transmission intensity) varied significantly (p<0·0001) according to time since vaccination, from 36% efficacy (95% CI 24 to 45) at time of vaccination to 0% (-38 to 38) after 3 years. INTERPRETATION: Vaccine efficacy against clinical disease was of limited duration and was not detectable 3 years after vaccination. Furthermore, efficacy fell with increasing transmission intensity. Outcomes after vaccination cannot be gauged accurately on the basis of one pooled efficacy figure. However, predictions of public-health outcomes of vaccination will need to take account of variations in efficacy by transmission intensity and by time since vaccination. FUNDING: Medical Research Council (UK); Bill & Melinda Gates Foundation Vaccine Modelling Initiative; Wellcome Trust

Correlations between three ELISA protocols measurements of RTS,S/AS01-induced anti-CSP IgG antibodies

Background RTS,S/AS01 induced anti-circumsporozoite protein (CSP) IgG antibodies are associated with the vaccine efficacy. There is currently no international standardisation of the assays used in the measurement of anti-CSP IgG antibody concentrations for use in evaluations of the vaccine’s immunogenicity and/or efficacy. Here, we compared the levels of RTS,S/AS01 induced anti-CSP IgG antibodies measured using three different enzyme-Linked ImmunoSorbent Assays (ELISA). Methods 196 plasma samples were randomly selected from the 447 samples collected during the RTS,S/AS01 phase IIb trial in 2007 from Kenyan children aged between 5–17 months. The vaccine-induced anti-CSP IgG antibodies were then measured using two independently developed ELISA protocols (‘Kilifi-RTS,S’ and ‘Oxford-R21’) and compared to the results from the reference ‘Ghent-RTS,S’ protocol for the same participants. For each pair of protocols, a deming regression model was fitted. Linear equations were then derived to aid in conversions into equivalent ELISA units. The agreement was assessed using Bland and Altman method. Findings The anti-CSP IgG antibodies measured from the three ELISA protocols were in agreement, and were positively and linearly correlated; ‘Oxford’ and ‘Kilifi’ r = 0.93 (95% CI 0.91–0.95), ‘Oxford’ and ‘Ghent’ r = 0.94 (95% CI: 0.92–0.96), and ‘Kilifi’ and ‘Ghent’ r = 0.97 (95% CI: 0.96–0.98), p<0.0001 for all correlations. Conclusions With the linearity, agreement and correlations established between the assays, conversion equations can be applied to convert results into equivalent units, enabling comparisons of immunogenicities across different vaccines of the same CSP antigens. This study highlights the need for the international harmonisation of anti-CSP antibody measurements

Corrigendum: Immune Responses to Gametocyte Antigens in a Malaria Endemic Population-The African falciparum Context: A Systematic Review and Meta-Analysis.

[This corrects the article DOI: 10.3389/fimmu.2019.02480.]

Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density

BACKGROUND: Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge. METHODS: The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia. RESULTS: Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities. CONCLUSION: It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density

Clearance of Asymptomatic P. falciparum Infections Interacts with the Number of Clones to Predict the Risk of Subsequent Malaria in Kenyan Children

BACKGROUND: Protective immunity to malaria is acquired after repeated infections in endemic areas. Asymptomatic multiclonal P. falciparum infections are common and may predict host protection. Here, we have investigated the effect of clearing asymptomatic infections on the risk of clinical malaria. METHODS: Malaria episodes were continuously monitored in 405 children (1-6 years) in an area of moderate transmission, coastal Kenya. Blood samples collected on four occasions were assessed by genotyping the polymorphic P. falciparum merozoite surface protein 2 using fluorescent PCR and capillary electrophoresis. Following the second survey, asymptomatic infections were cleared with a full course of dihydroartemisinin. RESULTS: Children who were parasite negative by PCR had a lower risk of subsequent malaria regardless of whether treatment had been given. Children with ≥ 2 clones had a reduced risk of febrile malaria compared with 1 clone after clearance of asymptomatic infections, but not if asymptomatic infections were not cleared. Multiclonal infection was associated with an increased risk of re-infection after drug treatment. However, among the children who were re-infected, multiclonal infections were associated with a shift from clinical malaria to asymptomatic parasitaemia. CONCLUSION: The number of clones was associated with exposure as well as blood stage immunity. These effects were distinguished by clearing asymptomatic infection with anti-malarials. Exposure to multiple P. falciparum infections is associated with protective immunity, but there appears to be an additional effect in untreated multiclonal infections that offsets this protective effect

Defining childhood severe falciparum malaria for intervention studies.

Background Clinical trials of interventions designed to prevent severe falciparum malaria in children require a clear endpoint. The internationally accepted definition of severe malaria is sensitive, and appropriate for clinical purposes. However, this definition includes individuals with severe nonmalarial disease and coincident parasitaemia, so may lack specificity in vaccine trials. Although there is no “gold standard” individual test for severe malaria, malaria-attributable fractions (MAFs) can be estimated among groups of children using a logistic model, which we use to test the suitability of various case definitions as trial endpoints. Methods and Findings A total of 4,583 blood samples were taken from well children in cross-sectional surveys and from 1,361 children admitted to a Kenyan District hospital with severe disease. Among children under 2 y old with severe disease and over 2,500 parasites per microliter of blood, the MAFs were above 85% in moderate- and low-transmission areas, but only 61% in a high-transmission area. HIV and malnutrition were not associated with reduced MAFs, but gastroenteritis with severe dehydration (defined by reduced skin turgor), lower respiratory tract infection (clinician's final diagnosis), meningitis (on cerebrospinal fluid [CSF] examination), and bacteraemia were associated with reduced MAFs. The overall MAF was 85% (95% confidence interval [CI] 83.8%–86.1%) without excluding these conditions, 89% (95% CI 88.4%–90.2%) after exclusions, and 95% (95% CI 94.0%–95.5%) when a threshold of 2,500 parasites/μl was also applied. Applying a threshold and exclusion criteria reduced sensitivity to 80% (95% CI 77%–83%). Conclusions The specificity of a case definition for severe malaria is improved by applying a parasite density threshold and by excluding children with meningitis, lower respiratory tract infection (clinician's diagnosis), bacteraemia, and gastroenteritis with severe dehydration, but not by excluding children with HIV or malnutrition