Measuring naturally acquired immune responses to candidate malaria vaccine antigens in Ghanaian adults

<p>Abstract</p> <p>Background</p> <p>To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Assessing T cell responses is especially challenging, given genetic restriction, low responses observed in endemic areas, their variability over time, potential suppression by parasitaemia and the intrinsic variability of the assays.</p> <p>Methods</p> <p>In Part A of this study, antibody titres were measured in adults from urban and rural communities in Ghana to recombinant <it>Plasmodium falciparum </it>CSP, SSP2/TRAP, LSA1, EXP1, MSP1, MSP3 and EBA175 by ELISA, and to sporozoites and infected erythrocytes by IFA. Positive ELISA responses were determined using two methods. T cell responses to defined CD8 or CD4 T cell epitopes from CSP, SSP2/TRAP, LSA1 and EXP1 were measured by <it>ex vivo </it>IFN-γ ELISpot assays using HLA-matched Class I- and DR-restricted synthetic peptides. In Part B, the reproducibility of the ELISpot assay to CSP and AMA1 was measured by repeating assays of individual samples using peptide pools and low, medium or high stringency criteria for defining positive responses, and by comparing samples collected two weeks apart.</p> <p>Results</p> <p>In Part A, positive antibody responses varied widely from 17%-100%, according to the antigen and statistical method, with blood stage antigens showing more frequent and higher magnitude responses. ELISA titres were higher in rural subjects, while IFA titres and the frequencies and magnitudes of e<it>x vivo </it>ELISpot activities were similar in both communities. DR-restricted peptides showed stronger responses than Class I-restricted peptides. In Part B, the most stringent statistical criteria gave the fewest, and the least stringent the most positive responses, with reproducibility slightly higher using the least stringent method when assays were repeated. Results varied significantly between the two-week time-points for many participants.</p> <p>Conclusions</p> <p>All participants were positive for at least one malaria protein by ELISA, with results dependent on the criteria for positivity. Likewise, ELISpot responses varied among participants, but were relatively reproducible by the three methods tested, especially the least stringent, when assays were repeated. However, results often differed between samples taken two weeks apart, indicating significant biological variability over short intervals.</p

Haematological and Biochemical Reference Values for Healthy Adults in the Middle Belt of Ghana

BACKGROUND: Reference values are very important in clinical management of patients, screening participants for enrollment into clinical trials and for monitoring the onset of adverse events during these trials. The aim of this was to establish gender-specific haematological and biochemical reference values for healthy adults in the central part of Ghana. METHODS: A total of 691 adults between 18 and 59 years resident in the Kintampo North Municipality and South District in the central part of Ghana were randomly selected using the Kintampo Health and Demographic Surveillance System and enrolled in this cross-sectional survey. Out of these, 625 adults made up of 316 males and 309 females were assessed by a clinician to be healthy. Median values and nonparametric 95% reference values for 16 haematology and 22 biochemistry parameters were determined for this population based on the Clinical Laboratory and Standards Institute guidelines. Values established in this study were compared with the Caucasian values being used currently by our laboratory as reference values and also with data from other African and western countries. RESULTS: REFERENCE VALUES ESTABLISHED INCLUDE: haemoglobin 113-164 g/L for males and 88-144 g/L for females; total white blood cell count 3.4-9.2 × 10(9)/L; platelet count 88-352 × 10(9)/L for males and 89-403 × 10(9)/L for females; alanine aminotransferase 8-54 U/L for males and 6-51 U/L for females; creatinine 56-119 µmol/L for males and 53-106 µmol/L for females. Using the haematological reference values based on the package inserts would have screened out up to 53% of potential trial participants and up to 25% of the population using the biochemical parameters. CONCLUSION: We have established a panel of locally relevant reference parameters for commonly used haematological and biochemical tests. This is important as it will help in the interpretation of laboratory results both for clinical management of patients and safety monitoring during a trial

Safety and Immunogenicity of EBA-175 RII-NG Malaria Vaccine Administered Intramuscularly in Semi-Immune Adults: A Phase 1, Double-Blinded Placebo Controlled Dosage Escalation Study

<div><p>The erythrocyte binding antigen region II (EBA-175 RII) is a <i>Plasmodium falciparum</i> ligand that mediates erythrocyte invasion and is considered an important malaria vaccine candidate. A phase Ia trial in malaria naïve adults living in the United States found the recombinant non-glycosylated vaccine antigen, EBA-175 RII-NG adjuvanted with aluminium phosphate to be safe, immunogenic and capable of inducing biologically active antibodies that can inhibit parasite growth <i>in vitro</i>. The aim of the current study was to assess the safety and immunogenicity of this vaccine in malaria exposed semi-immune healthy adults living in a malaria endemic country, Ghana. In this double-blinded, placebo controlled, dose escalation phase I trial, eighteen subjects per group received ascending dose concentrations (5 μg, 20 μg or 80 μg) of the vaccine intramuscularly at 0, 1 and 6 months, while 6 subjects received placebo (normal saline). The primary end point was the number of subjects experiencing Grade 3 systemic or local adverse events within 14 days post-vaccination. Serious adverse events were assessed throughout the study period. Blood samples for immunological analyses were collected at days 0, 14, 28, 42, 180 and 194. A total of 52 subjects received three doses of the vaccine in the respective groups. No serious adverse events were reported. The majority of all adverse events reported were mild to moderate in severity, with local pain and tenderness being the most common. All adverse events, irrespective of severity, resolved without any sequelae. Subjects who received any of the EBA-175 RII-NG doses had high immunoglobulin G levels which moderately inhibited <i>P</i>. <i>falciparum</i> growth <i>in vitro</i>, compared to those in the placebo group. In conclusion, the EBA-175 RII-NG vaccine was safe, well tolerated and immunogenic in malaria semi-immune Ghanaian adults. Its further development is recommended.</p><p>Trial registration</p><p>ClinicalTrials.gov. Identifier: <a href="https://clinicaltrials.gov/ct2/show/NCT01026246" target="_blank">NCT01026246</a></p></div

Proportion of subjects with 4-fold increase from baseline in anti-EBA175 antibody response.

<p>Proportion of subjects with 4-fold increase from baseline in anti-EBA175 antibody response.</p

Trial profile.

<p>^a Vaccination was discontinued in one subject due to blood phobia. ^b One subject was excluded from further vaccinations due to severe anemia. ^c One subject migrated out of the country without prior notification. ^d One subject voluntarily withdrew to relocate to another town. ^e One subject discontinued the study and was lost to follow up for safety evaluation after receiving all three vaccinations. All attempts to find this subject failed. Abbreviation: EBA-175 RII-NG–Erythrocyte binding antigen 175 region II non-glycosylated.</p

Observed laboratory adverse events for all dose escalation groups.

<p><b>Panel A</b> is for screening prior to enrolment; <b>Panels B</b> and <b>C</b> are for vaccination 1, Day 0 and Day 14 respectively; <b>Panels D</b> and <b>E</b> are for vaccination 2, Day 0 and Day 14 respectively and <b>Panels F, G</b> and <b>H</b> are for vaccination 3, Day 0, Day 14 and Day 28 respectively. Abbreviations: ALT—Alanine Transaminase; AST—Alanine Aminotransferase; CR–Creatinine; BG–Blood glucose; K–Potassium; Na–Sodium; Hb–Hemoglobin; PLT–Platelets; WBC–White Blood Cells.</p

Demographic and baseline characteristics by dose of study population.

<p>Demographic and baseline characteristics by dose of study population.</p

Anti-EBA-175 RII-NG IgG antibody levels (ELISA units) and growth inhibition activity against <i>P</i>. <i>falciparum</i> 3D7 parasite.

<p><b>Panel A:</b> Geometric mean IgG antibody levels (ELISA units) to the vaccine antigen EBA-175 RII-NG measured in each treatment group on different days (D0 to D194) post vaccination. <b>Panel B:</b> The left y-axis represents mean percentage <i>P</i>. <i>falciparum</i> 3D7 parasite growth inhibition measured for each treatment group on the different days post vaccination plotted as vertical bars. The right y-axis represents mean EBA175 ELISA units (on log 10 scale) used per growth inhibition assay well for each treatment group on the different days post vaccination plotted as lines. One subject in the placebo group recorded the highest anti-EBA-175 RII-NG IgG levels at baseline which persisted throughout the study. Both the high mean EBA 175 ELISA units/GIA well and mean GIA values observed in the placebo group were largely due to this subject. One ELISA unit is the reciprocal of the dilution required to give an optical density = 1 in the standardized assay. Any data point less than the minimal detectable level was assigned as 5 ELISA units in the growth inhibition assay well in the analysis. Vaccinations occurred on Days 0, 28 and 180. Blood samples were drawn for immunogenicity prior to vaccination. Abbreviations: EBA-175 RII-NG–Erythrocyte binding antigen 175 region II non-glycosylated; IgG–Immunoglobulin G; GIA–Growth inhibition assay.</p

Adverse events by severity vaccine dose and frequency of vaccinations.

<p>Adverse events by severity vaccine dose and frequency of vaccinations.</p

Haematology reference values for Kintampo.

<p>Med: Median.</p>*<p>Mann-Whitney test for differences in between males and females.</p