Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of Africa.

Background: Ps48/45, a Plasmodium gametocyte surface protein, is a promising candidate for malaria transmission-blocking (TB) vaccine. Due to its relevance for a multispecies vaccine, we explored the cross-reactivity and TB activity of a recombinant P. vivax Ps48/45 protein (rPvs48/45) with sera from P. falciparum-exposed African donors. Methods: rPvs48/45 was produced in Chinese hamster ovary cell lines and tested by ELISA for its cross-reactivity with sera from Burkina Faso, Tanzania, Mali, and Nigeria - In addition, BALB/c mice were immunized with the rPvs48/45 protein formulated in Montanide ISA-51 and inoculated with a crude extract of P. falciparum NF-54 gametocytes to evaluate the parasite-boosting effect on rPvs48/45 antibody titers. Specific anti-rPvs48/45 IgG purified from African sera was used to evaluate the ex vivo TB activity on P. falciparum, using standard mosquito membrane feeding assays (SMFA). Results: rPvs48/45 protein showed cross-reactivity with sera of individuals from all four African countries, in proportions ranging from 94% (Tanzania) to 40% (Nigeria). Also, the level of cross-reactive antibodies varied significantly between countries (p<0.0001), with a higher antibody level in Mali and the lowest in Nigeria. In addition, antibody levels were higher in adults (≥ 17 years) than young children (≤ 5 years) in both Mali and Tanzania, with a higher proportion of responders in adults (90%) than in children (61%) (p<0.0001) in Mali, where male (75%) and female (80%) displayed similar antibody responses. Furthermore, immunization of mice with P. falciparum gametocytes boosted anti-Pvs48/45 antibody responses, recognizing P. falciparum gametocytes in indirect immunofluorescence antibody test. Notably, rPvs48/45 affinity-purified African IgG exhibited a TB activity of 61% against P. falciparum in SMFA. Conclusion: African sera (exposed only to P. falciparum) cross-recognized the rPvs48/45 protein. This, together with the functional activity of IgG, warrants further studies for the potential development of a P. vivax and P. falciparum cross-protective TB vaccine.

Normal Clinical Laboratory Ranges by Age and Sex, and Impact on Study Screening Outcomes in Rural Mali.

The interpretation of a laboratory test result requires an appropriate reference range established in healthy subjects, and normal ranges may vary by factors such as geographic region, sex, and age. We examined hematological and clinical chemistry parameters in healthy residents at two rural vaccine trial sites: Bancoumana and Doneguebougou in Mali, West Africa. During screening of clinical studies in 2018 and 2019, peripheral blood samples from 1,192 apparently healthy individuals age 6 months to 82 years were analyzed at a laboratory accredited by the College of American Pathologists for a complete blood count, and creatinine and/or alanine aminotransferase levels. Based on manufacturers' reference range values, which are currently used in Malian clinical laboratories, abnormal values were common in this healthy population. In fact, 30.4% of adult participants had abnormal neutrophil levels and 19.8% had abnormal hemoglobin levels. Differences by sex were observed in those who were older, but not in those younger than 10 years, for several parameters, including hemoglobin, platelet, and absolute neutrophil counts in hematology, and creatinine in biochemistry. The site-specific reference intervals we report can be used in malaria vaccine clinical trials and other interventional studies, as well as in routine clinical care, to identify abnormalities in hematological and biochemical parameters among healthy Malian trial participants.

Malaria transmission-blocking vaccines Pfs230D1-EPA and Pfs25-EPA in Alhydrogel in healthy Malian adults; a phase 1, randomised, controlled trial.

BACKGROUND: Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians. METHODS: We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4·5, and 16·5 of either 47 µg Pfs25, 40 µg Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 µg Pfs25 plus 40 µg Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462. FINDINGS: Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 µg), Pfs230D1-EPA/Alhydrogel (15 µg) or comparator, followed by Pfs25-EPA/Alhydrogel (16 µg) plus Pfs230D1-EPA/Alhydrogel (15 µg) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 µg Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 µg Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 µg Pfs25-EPA/Alhydrogel plus 40 µg Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77·8 [95% CI 56·9-106·3], 146·4 [108·3-198·0], and 410·2 [301·6-558·0]; Pfs25 geometric mean 177·7 [130·3-242·4] and 315·7 [209·9-474·6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74·5% [66·6-82·5]) and Pfs25 plus Pfs230D1 (68·6% [57·3-79·8]), after the third dose and after the fourth dose (88·9% [81·7-96·2] for Pfs230D1 and 85·0% [78·4-91·5] Pfs25 plus Pfs230D1) but not for Pfs25 (58·2% [49·1-67·3] after the third dose and 58·2% [48·5-67·9] after the fourth dose). Pfs230D1 transmission-reducing activity (73·7% [64·1-83·3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0·013 to 0·014], Pfs230D1 22 [2%] of 954 [-0·005 to 0·027], and combination 11 [1%] of 940 [-0·024 to 0·002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0·024 to 0·001). INTERPRETATION: Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness. FUNDING: Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.

Seroreactivity of the Severe Acute Respiratory Syndrome Coronavirus 2 Recombinant S Protein, Receptor-Binding Domain, and Its Receptor-Binding Motif in COVID-19 Patients and Their Cross-Reactivity With Pre-COVID-19 Samples From Malaria-Endemic Areas.

Despite the global interest and the unprecedented number of scientific studies triggered by the COVID-19 pandemic, few data are available from developing and low-income countries. In these regions, communities live under the threat of various transmissible diseases aside from COVID-19, including malaria. This study aims to determine the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroreactivity of antibodies from COVID-19 and pre-COVID-19 samples of individuals in Mali (West Africa). Blood samples from COVID-19 patients (n = 266) at Bamako Dermatology Hospital (HDB) and pre-COVID-19 donors (n = 283) from a previous malaria survey conducted in Dangassa village were tested by ELISA to assess IgG antibodies specific to the full-length spike (S) protein, the receptor-binding domain (RBD), and the receptor-binding motif (RBM436-507). Study participants were categorized by age, gender, treatment duration for COVID-19, and comorbidities. In addition, the cross-seroreactivity of samples from pre-COVID-19, malaria-positive patients against the three antigens was assessed. Recognition of the SARS-CoV-2 proteins by sera from COVID-19 patients was 80.5% for S, 71.1% for RBD, and 31.9% for RBM (p < 0.001). While antibody responses to S and RBD tended to be age-dependent, responses to RBM were not. Responses were not gender-dependent for any of the antigens. Higher antibody levels to S, RBD, and RBM at hospital entry were associated with shorter treatment durations, particularly for RBD (p < 0.01). In contrast, higher body weights negatively influenced the anti-S antibody response, and asthma and diabetes weakened the anti-RBM antibody responses. Although lower, a significant cross-reactive antibody response to S (21.9%), RBD (6.7%), and RBM (8.8%) was detected in the pre-COVID-19 and malaria samples. Cross-reactive antibody responses to RBM were mostly associated (p < 0.01) with the absence of current Plasmodium falciparum infection, warranting further study.

Safety and efficacy of a three-dose regimen of Plasmodium falciparum sporozoite vaccine in adults during an intense malaria transmission season in Mali: a randomised, controlled phase 1 trial.

BACKGROUND: WHO recently approved a partially effective vaccine that reduces clinical malaria in children, but increased vaccine activity is required to pursue malaria elimination. A phase 1 clinical trial was done in Mali, west Africa, to assess the safety, immunogenicity, and protective efficacy of a three-dose regimen of Plasmodium falciparum sporozoite (PfSPZ) Vaccine (a metabolically active, non-replicating, whole malaria sporozoite vaccine) against homologous controlled human malaria infection (CHMI) and natural P falciparum infection. METHODS: We recruited healthy non-pregnant adults aged 18-50 years in Donéguébougou, Mali, and surrounding villages (Banambani, Toubana, Torodo, Sirababougou, Zorokoro) for an open-label, dose-escalation pilot study and, thereafter, a randomised, double-blind, placebo-controlled main trial. Pilot study participants were enrolled on an as-available basis to one group of CHMI infectivity controls and three staggered vaccine groups receiving: one dose of 4·5 × 105, one dose of 9 × 105, or three doses of 1·8 × 106 PfSPZ via direct venous inoculation at approximately 8 week intervals, followed by homologous CHMI 5 weeks later with infectious PfSPZ by direct venous inoculation (PfSPZ Challenge). Main cohort participants were stratified by village and randomly assigned (1:1) to receive three doses of 1·8 × 106 PfSPZ or normal saline at 1, 13, and 19 week intervals using permuted block design by the study statistician. The primary outcome was safety and tolerability of at least one vaccine dose; the secondary outcome was vaccine efficacy against homologous PfSPZ CHMI (pilot study) or against naturally transmitted P falciparum infection (main study) measured by thick blood smear. Combined artesunate and amodiaquine was administered to eliminate pre-existing parasitaemia. Outcomes were analysed by modified intention to treat (mITT; including all participants who received at least one dose of investigational product; safety and vaccine efficacy) and per protocol (vaccine efficacy). This trial is registered with ClinicalTrials.gov, number NCT02627456. FINDINGS: Between Dec 20, 2015, and April 30, 2016, we enrolled 56 participants into the pilot study (five received the 4·5 × 105 dose, five received 9 × 105, 30 received 1·8 × 106, 15 were CHMI controls, and one withdrew before vaccination) and 120 participants into the main study cohort with 60 participants assigned PfSPZ Vaccine and 60 placebo in the main study. Adverse events and laboratory abnormalities post-vaccination in all dosing groups were few, mainly mild, and did not differ significantly between vaccine groups (all p>0·05). Unexpected severe transaminitis occured in four participants: one participant in pilot phase that received 1·8 × 106 PfSPZ Vaccine, one participant in main phase that received 1·8 × 106 PfSPZ Vaccine, and two participants in the main phase placebo group. During PfSPZ CHMI, approximately 5 weeks after the third dose of 1·8 × 106 PfSPZ, none of 29 vaccinees and one of 15 controls became positive on thick blood smear; subsequent post-hoc PCR analysis for submicroscopic blood stage infections detected P falciparum parasites in none of the 29 vaccine recipients and eight of 15 controls during CHMI. In the main trial, 32 (58%) of 55 vaccine recipients and 42 (78%) of 54 controls became positive on thick blood smear during 24-week surveillance after vaccination. Vaccine efficacy (1-hazard ratio) was 0·51 per protocol (95% CI 0·20-0·70; log-rank p=0·0042) and 0·39 by mITT (0·04-0·62; p=0·033); vaccine efficacy (1-risk ratio) was 0·24 per-protocol (0·02-0·41; p=0·031) and 0·22 mITT (0·01-0·39; p=0·041). INTERPRETATION: A three-dose regimen of PfSPZ Vaccine was safe, well tolerated, and conferred 51% vaccine efficacy against intense natural P falciparum transmission, similar to 52% vaccine efficacy reported for a five-dose regimen in a previous trial. FUNDING: US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Sanaria. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Ultra-sensitive RDT performance and antigen dynamics in a high-transmission Plasmodium falciparum setting in Mali.

BACKGROUND: The recent expansion of tools designed to accurately quantify malaria parasite-produced antigens has enabled us to evaluate the performance of rapid diagnostic tests (RDTs) as a function of the antigens they detect-typically histidine rich protein 2 (HRP2) or lactate dehydrogenase (LDH). METHODS: For this analysis, whole blood specimens from a longitudinal study in Bancoumana, Mali were used to evaluate the performance of the ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT). The samples were collected as part of a transmission-blocking vaccine trial in a high transmission region for Plasmodium falciparum malaria. Furthermore, antigen dynamics after successful anti-malarial drug treatment were evaluated in these samples using the Q-Plex Human Malaria Array (4-Plex) to quantify antigen concentrations. RESULTS: The uRDT had a 50% probability of a positive result at 207 pg/mL HRP2 [95% credible interval (CrI) 160-268]. Individuals with symptomatic infection remained positive by uRDT for a median of 33 days [95% confidence interval (CI) 28-47] post anti-malarial drug treatment. Biphasic exponential decay models accurately captured the population level post-treatment dynamics of both HRP2 and Plasmodium LDH (pLDH), with the latter decaying more rapidly. Motivated by these differences in rates of decay, a novel algorithm that used HRP2:pLDH ratios to predict if an individual had active versus recently cleared P. falciparum infection was developed. The algorithm had 77.5% accuracy in correctly classifying antigen-positive individuals as those with and without active infection. CONCLUSIONS: These results characterize the performance of the ultra-sensitive RDT and demonstrate the potential for emerging antigen-quantifying technologies in the field of malaria diagnostics to be helpful tools in distinguishing between active versus recently cleared malaria infections.

Ex-vivo Sensitivity of Plasmodium falciparum to Common Anti-malarial Drugs: The Case of Kéniéroba, a Malaria Endemic Village in Mali.

BACKGROUND: In 2006, the National Malaria Control Program in Mali recommended artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. Since the introduction of artemisinin-based combination therapy, few reports are available on the level of resistance of Plasmodium falciparum to the most common anti-malarial drugs in Mali. METHODS: From 2016 to 2017, we assessed the ex-vivo drug sensitivity of P. falciparum isolates in Kéniéroba, a village located in a rural area of southern Mali. We collected P. falciparum isolates from malaria-infected children living in Kéniéroba. The isolates were tested for ex-vivo sensitivity to commonly used anti-malarial drugs, namely chloroquine, quinine, amodiaquine, mefloquine, lumefantrine, dihydroartermisinin, and piperaquine. We used the 50% inhibitory concentration determination method, which is based on the incorporation of SYBR® Green into the parasite's genetic material. RESULTS: Plasmodium falciparum isolates were found to have a reduced ex-vivo sensitivity to quinine (25.7%), chloroquine (12.2%), amodiaquine (2.7%), and mefloquine (1.3%). In contrast, the isolates were 100% sensitive to lumefantrine, dihydroartermisinin, and piperaquine. A statistically significant correlation was found between 50% inhibitory concentration values of quinine and amodiaquine (r = 0.80; p < 0.0001). CONCLUSIONS: Plasmodium falciparum isolates were highly sensitive to dihydroartermisinin, lumefantrine, and piperaquine and less sensitive to amodiaquine (n = 2), mefloquine (n = 1), and quinine (n = 19). Therefore, our data support the previously reported increasing trend in chloroquine sensitivity in Mali.

A comprehensive analysis of drug resistance molecular markers and Plasmodium falciparum genetic diversity in two malaria endemic sites in Mali.

BACKGROUND: Drug resistance is one of the greatest challenges of malaria control programme in Mali. Recent advances in next-generation sequencing (NGS) technologies provide new and effective ways of tracking drug-resistant malaria parasites in Africa. The diversity and the prevalence of Plasmodium falciparum drug-resistance molecular markers were assessed in Dangassa and Nioro-du-Sahel in Mali, two sites with distinct malaria transmission patterns. Dangassa has an intense seasonal malaria transmission, whereas Nioro-du-Sahel has an unstable and short seasonal malaria transmission. METHODS: Up to 270 dried blood spot samples (214 in Dangassa and 56 in Nioro-du-Sahel) were collected from P. falciparum positive patients in 2016. Samples were analysed on the Agena MassARRAY® iPLEX platform. Specific codons were targeted in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, Pfarps10, Pfferredoxin, Pfexonuclease and Pfmdr2 genes. The Sanger's 101-SNPs-barcode method was used to assess the genetic diversity of P. falciparum and to determine the parasite species. RESULTS: The Pfcrt_76T chloroquine-resistance genotype was found at a rate of 64.4% in Dangassa and 45.2% in Nioro-du-Sahel (p = 0.025). The Pfdhfr_51I-59R-108N pyrimethamine-resistance genotype was 14.1% and 19.6%, respectively in Dangassa and Nioro-du-Sahel. Mutations in the Pfdhps_S436-A437-K540-A581-613A sulfadoxine-resistance gene was significantly more prevalent in Dangassa as compared to Nioro-du-Sahel (p = 0.035). Up to 17.8% of the isolates from Dangassa vs 7% from Nioro-du-Sahel harboured at least two codon substitutions in this haplotype. The amodiaquine-resistance Pfmdr1_N86Y mutation was identified in only three samples (two in Dangassa and one in Nioro-du-Sahel). The lumefantrine-reduced susceptibility Pfmdr1_Y184F mutation was found in 39.9% and 48.2% of samples in Dangassa and Nioro-du-Sahel, respectively. One piperaquine-resistance Exo_E415G mutation was found in Dangassa, while no artemisinin resistance genetic-background were identified. A high P. falciparum diversity was observed, but no clear genetic aggregation was found at either study sites. Higher multiplicity of infection was observed in Dangassa with both COIL (p = 0.04) and Real McCOIL (p = 0.02) methods relative to Nioro-du-Sahel. CONCLUSIONS: This study reveals high prevalence of chloroquine and pyrimethamine-resistance markers as well as high codon substitution rate in the sulfadoxine-resistance gene. High genetic diversity of P. falciparum was observed. These observations suggest that the use of artemisinins is relevant in both Dangassa and Nioro-du-Sahel.

Safety and immunogenicity of Pfs25H-EPA/Alhydrogel, a transmission-blocking vaccine against Plasmodium falciparum: a randomised, double-blind, comparator-controlled, dose-escalation study in healthy Malian adults.

BACKGROUND: Pfs25H-EPA is a protein-protein conjugate transmission-blocking vaccine against Plasmodium falciparum that is safe and induces functional antibodies in malaria-naive individuals. In this field trial, we assessed Pfs25H-EPA/Alhydrogel for safety and functional immunogenicity in Malian adults. METHODS: This double-blind, randomised, comparator-controlled, dose-escalation trial in Bancoumana, Mali, was done in two staggered phases, an initial pilot safety assessment and a subsequent main phase. Healthy village residents aged 18-45 years were eligible if they had normal laboratory results (including HIV, hepatitis B, hepatitis C tests) and had not received a previous malaria vaccine or recent immunosuppressive drugs, vaccines, or blood products. Participants in the pilot safety cohort and the main cohort were assigned (1:1) by block randomisation to a study vaccine group. Participants in the pilot safety cohort received two doses of Pfs25H-EPA/Alhydrogel 16 µg or Euvax B (comparator vaccine), and participants in the main cohort received Pfs25H-EPA/Alhydrogel 47 µg or comparator vaccine (Euvax B for the first, second, and third vaccinations and Menactra for the fourth vaccination). Participants and investigators were masked to group assignment, and randomisation codes in sealed envelopes held by a site pharmacist. Vials with study drug for injection were covered by opaque tape and labelled with a study identification number. Group assignments were unmasked at final study visit. The primary outcomes were safety and tolerability for all vaccinees. The secondary outcome measure was immunogenicity 14 days after vaccination in the per-protocol population, as confirmed by the presence of antibodies against Pfs25H measured by ELISA IgG and antibody functionality assessed by standard membrane feeding assays and by direct skin feeding assays. This trial is registered with ClinicalTrials.gov, number NCT01867463. FINDINGS: Between May 15, and Jun 16, 2013, 230 individuals were screened for eligibility. 20 individuals were enrolled in the pilot safety cohort; ten participants were assigned to receive Pfs25H-EPA/Alhydrogel 16 µg, and ten participants were assigned to receive comparator vaccine. 100 individuals were enrolled in the main cohort; 50 participants were assigned to receive Pfs25H-EPA/Alhydrogel 47 µg, and 50 participants were assigned to receive comparator vaccine. Compared with comparator vaccinees, Pfs25H vaccinees had more solicited adverse events (137 events vs 86 events; p=0·022) and treatment-related adverse events (191 events vs 126 events, p=0·034), but the number of other adverse events did not differ between study vaccine groups (792 vs 683). Pfs25H antibody titres increased with each dose, with a peak geometric mean of 422·3 ELISA units (95% CI 290-615) after the fourth dose, but decreased relatively rapidly thereafter, with a half-life of 42 days for anti-Pfs25H and 59 days for anti-EPA (median ratio of titres at day 600 to peak, 0·19 for anti-Pfs25H vs 0·29 for anti-EPA; p=0·009). Serum transmission-reducing activity was greater for Pfs25H than for comparator vaccine after the fourth vaccine dose (p<0·001) but not after the third dose (p=0·09). Repeated direct skin feeds were well tolerated, but the number of participants who infected at least one mosquito did not differ between Pfs25H and comparator vaccinees after the fourth dose (p=1, conditional exact). INTERPRETATION: Pfs25H-EPA/Alhydrogel was well tolerated and induced significant serum activity by standard membrane feeding assays but transmission blocking activity was not confirmed by weekly direct skin feed. This activity required four doses, and titres decreased rapidly after the fourth dose. Alternative antigens or combinations should be assessed to improve activity. FUNDING: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Safety and efficacy of PfSPZ Vaccine against Plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in Mali: a randomised, double-blind phase 1 trial.

BACKGROUND: Plasmodium falciparum sporozite (PfSPZ) Vaccine is a metabolically active, non-replicating, whole malaria sporozoite vaccine that has been reported to be safe and protective against P falciparum controlled human malaria infection in malaria-naive individuals. We aimed to assess the safety and protective efficacy of PfSPZ Vaccine against naturally acquired P falciparum in malaria-experienced adults in Mali. METHODS: After an open-label dose-escalation study in a pilot safety cohort, we did a double-blind, randomised, placebo-controlled trial based in Donéguébougou and surrounding villages in Mali. We recruited 18-35-year-old healthy adults who were randomly assigned (1:1) in a double-blind manner, with stratification by village and block randomisation, to receive either five doses of 2·7 × 105 PfSPZ or normal saline at days 0, 28, 56, 84, and 140 during the dry season (January to July inclusive). Participants and investigators were masked to group assignments, which were unmasked at the final study visit, 6 months after receipt of the last vaccination. Participants received combined artemether and lumefantrine (four tablets, each containing 20 mg artemether and 120 mg lumefantrine, given twice per day over 3 days for a total of six doses) to eliminate P falciparum before the first and last vaccinations. We collected blood smears every 2 weeks and during any illness for 24 weeks after the fifth vaccination. The primary outcome was the safety and tolerability of the vaccine, assessed as local and systemic reactogenicity and adverse events. The sample size was calculated for the exploratory efficacy endpoint of time to first P falciparum infection beginning 28 days after the fifth vaccination. The safety analysis included all participants who received at least one dose of investigational product, whereas the efficacy analyses included only participants who received all five vaccinations. This trial is registered at ClinicalTrials.gov, number NCT01988636. FINDINGS: Between Jan 18 and Feb 24, 2014, we enrolled 93 participants into the main study cohort with 46 participants assigned PfSPZ Vaccine and 47 assigned placebo, all of whom were evaluable for safety. We detected no significant differences in local or systemic adverse events or laboratory abnormalities between the PfSPZ Vaccine and placebo groups, and only grade 1 (mild) local or systemic adverse events occurred in both groups. The most common solicited systemic adverse event in the vaccine and placebo groups was headache (three [7%] people in the vaccine group vs four [9%] in the placebo group) followed by fatigue (one [2%] person in the placebo group), fever (one [2%] person in the placebo group), and myalgia (one [2%] person in each group). The exploratory efficacy analysis included 41 participants from the vaccine group and 40 from the placebo group. Of these participants, 37 (93%) from the placebo group and 27 (66%) from the vaccine group developed P falciparum infection. The hazard ratio for vaccine efficacy was 0·517 (95% CI 0·313-0·856) by time-to-infection analysis (log-rank p=0·01), and 0·712 (0·528-0·918) by proportional analysis (p=0·006). INTERPRETATION: PfSPZ Vaccine was well tolerated and safe. PfSPZ Vaccine showed significant protection in African adults against P falciparum infection throughout an entire malaria season. FUNDING: US National Institutes of Health Intramural Research Program, Sanaria.

Promoting good clinical laboratory practices and laboratory accreditation to support clinical trials in sub-Saharan Africa.

Laboratory capacity in the developing world frequently lacks quality management systems (QMS) such as good clinical laboratory practices, proper safety precautions, and adequate facilities; impacting the ability to conduct biomedical research where it is needed most. As the regulatory climate changes globally, higher quality laboratory support is needed to protect study volunteers and to accurately assess biological parameters. The University of Bamako and its partners have undertaken a comprehensive QMS plan to improve quality and productivity using the Clinical and Laboratory Standards Institute standards and guidelines. The clinical laboratory passed the College of American Pathologists inspection in April 2010, and received full accreditation in June 2010. Our efforts to implement high-quality standards have been valuable for evaluating safety and immunogenicity of malaria vaccine candidates in Mali. Other disease-specific research groups in resource-limited settings may benefit by incorporating similar training initiatives, QMS methods, and continual improvement practices to ensure best practices.

Anaemia in a phase 2 study of a blood stage falciparum malaria vaccine.

BACKGROUND: A Phase 1-2b study of the blood stage malaria vaccine AMA1-C1/Alhydrogel was conducted in 336 children in Donéguébougou and Bancoumana, Mali. In the Phase 2 portion of the study (n = 300), no impact on parasite density or clinical malaria was seen; however, children who received the study vaccine had a higher frequency of anaemia (defined as haemoglobin < 8.5 g/dL) compared to those who received the comparator vaccine (Hiberix). This effect was one of many tested and was not significant after adjusting for multiple comparisons. METHODS: To further investigate the possible impact of vaccination on anaemia, additional analyses were conducted including patients from the Phase 1 portion of the study and controlling for baseline haemoglobin, haemoglobin types S or C, alpha-thalassaemia, G6PD deficiency, and age. A multiplicative intensity model was used, which generalizes Cox regression to allow for multiple events. Frailty effects for each subject were used to account for correlation of multiple anaemia events within the same subject. Intensity rates were calculated with reference to calendar time instead of time after randomization in order to account for staggered enrollment and seasonal effects of malaria incidence. Associations of anaemia with anti-AMA1 antibody were further explored using a similar analysis. RESULTS: A strong effect of vaccine on the incidence of anaemia (risk ratio [AMA1-C1 to comparator (Hiberix)]= 2.01, 95% confidence interval [1.26,3.20]) was demonstrated even after adjusting for baseline haemoglobin, haemoglobinopathies, and age, and using more sophisticated statistical models. Anti-AMA1 antibody levels were not associated with this effect. CONCLUSIONS: While these additional analyses show a robust effect of vaccination on anaemia, this is an intensive exploration of secondary results and should, therefore, be interpreted with caution. Possible mechanisms of the apparent adverse effect on haemoglobin of vaccination with AMA1-C1/Alhydrogel and implications for blood stage vaccine development are discussed. The potential impact on malaria-associated anaemia should be closely evaluated in clinical trials of AMA1 and other blood stage vaccines in malaria-exposed populations.

A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA1-C1/Alhydrogel + CPG 7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults.

A double blind, randomized and controlled Phase 1 clinical trial was conducted to assess the safety and immunogenicity in malaria-exposed adults of the Plasmodium falciparum blood stage vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with and without the novel adjuvant CPG 7909. Participants were healthy adults 18-45 years old living in the village of Donéguébougou, Mali. A total of 24 participants received 2 doses one month apart of either 80 microg AMA1-C1/Alhydrogel or 80 microg AMA1-C1/Alhydrogel + 564 microg CPG 7909. The study started in October 2007 and completed follow up in May 2008. Both vaccines were well tolerated, with only mild local adverse events and no systemic adverse events judged related to vaccination. The difference in antibody responses were over 2-fold higher in the group receiving CPG 7909 for all time points after second vaccination and the differences are statistically significant (all p<0.05). This is the first use of the novel adjuvant CPG 7909 in a malaria-exposed population.

A randomized controlled phase 2 trial of the blood stage AMA1-C1/Alhydrogel malaria vaccine in children in Mali.

A double blind, randomized, controlled Phase 2 clinical trial was conducted to assess the safety, immunogenicity, and biologic impact of the vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1), adjuvanted with Alhydrogel. Participants were healthy children 2-3 years old living in or near the village of Bancoumana, Mali. A total of 300 children received either the study vaccine or the comparator. No impact of vaccination was seen on the primary endpoint, the frequency of parasitemia measured as episodes >3000/microL/day at risk. There was a negative impact of vaccination on the hemoglobin level during clinical malaria, and mean incidence of hemoglobin <8.5 g/dL, in the direction of lower hemoglobin in the children who received AMA1-C1, although these differences were not significant after correction for multiple tests. These differences were not seen in the second year of transmission.