The effectiveness of long-lasting insecticidal nets (LLIN) depends largely on the biting behaviour of the mosquito vectors. Therefore, understanding vector bionomics in a given epidemiological setting is essential for evaluating the impact of these malaria vector control tools. This study presents the entomological profile of malaria in two ecologically distinct settings in Cameroon, Mfou, a forest region, and Tibati, a savannah region. Adult mosquitoes were collected both outdoors and indoors in randomly selected households between April and July 2019 using Human Landing Catches (HLCs). After morphological identification of the mosquitoes, a subset of anopheline mosquitoes from each species was dissected to determine parity status. DNA from the head and thorax was used for molecular analysis to identify individual mosquitoes to the An. gambiae complex and An. funestus group, and to detect Plasmodium infection. Entomological Inoculation Rate (EIR) was estimated based on Plasmodium infection rate as revealed by qPCR. A total of 3,651 Anopheles mosquitoes were collected (1,817 in Mfou and 1,834 in Tibati) and identified as belonging to 9 vector species: Anopheles gambiae sensu stricto (s.s.), An. coluzzii, An. funestus s.s., An. leesoni, An. moucheti, An. nili, An. ziemanni, An. paludis and An. pharoensis. The predominant vector was Anopheles gambiae s.l. in Mfou (68.96

Malaria remains a major public health concern in Cameroon, with Plasmodium falciparum responsible for most morbidity and mortality, particularly among children under five. In response to rising cases, Cameroon began implementing the RTS,S/AS01 malaria vaccine in early 2024. Given the vaccine’s strain-specific efficacy, understanding haplotypic diversity is critical for evaluating long-term impact. We analyzed 100 P. falciparum-positive dried blood spots collected in Mapoussere, Kaele Health District (2022–2023). Using the 4CAST amplicon sequencing assay, we targeted four genes: csp (circumsporozoite protein), ama1 (apical membrane antigen 1), sera2 (serine repeat antigen 2), and trap (thrombospondin-related anonymous protein). Haplotypes were identified using SeekDeep, and diversity metrics including heterozygosity (He), nucleotide diversity (π), and selection statistics (Tajima’s D, Fu and Li’s D*, F*) were computed. We successfully genotyped csp in 35% of samples, identifying 22 haplotypes (He = 0.908; π = 0.021). The vaccine-matched haplotype was present in 20% of genotyped infections. The T cell TH2 and TH3 epitopes of csp showed signs consistent with balancing selection. Both ama1 and sera2 exhibited higher genotyping success and diversity, with ama showing significant Tajima’s D values. Within-participant haplotype counts were highest for ama1 (mean = 2.8), followed by sera2 (2.1), csp (1.3), and trap (1.2). This study provides a baseline of P. falciparum haplotype diversity in a vaccine-targeted region. The presence of vaccine-matched strains and high diversity in TH2/TH3 epitopes in csp may influence vaccine efficacy. Continued molecular surveillance is essential to monitor antigenic shifts and guide future strategies.

Malaria remains a major public health concern in Cameroon, with Plasmodium falciparum responsible for most morbidity and mortality, particularly among children under five. In response to rising cases, Cameroon began implementing the RTS,S/AS01 malaria vaccine in early 2024. Given the vaccine's strain-specific efficacy, understanding antigenic diversity and complexity of infection (COI) is critical for evaluating long-term impact. We analyzed 100 P. falciparum-positive dried blood spots collected in Mapoussere, Kaele Health District (2022-2023). Using the 4CAST amplicon sequencing assay, we targeted four genes: csp (circumsporozoite protein), ama (apical membrane antigen 1), sera2 (serine repeat antigen 2), and trap (thrombospondin-related anonymous protein). Haplotypes were identified using SeekDeep, and diversity metrics including heterozygosity (He), nucleotide diversity ( π ), and selection statistics (Tajima's D, Fu and Li's D*, F*) were computed. We successfully genotyped csp in 35% of samples, identifying 22 haplotypes (He = 0.908; π = 0.021 ). The vaccine-matched haplotype was present in 20% of genotyped infections. The T cell TH2 and TH3 epitopes of csp showed signs of balancing selection. Both ama and sera2 exhibited higher genotyping success and diversity, with ama showing significant Tajima's D values. COI was highest for ama (mean COI = 2.8), followed by sera2 (2.1), csp (1.3), and trap (1.2). This study provides a baseline of P. falciparum antigenic diversity and COI in a vaccine-targeted region. The presence of vaccine-matched strains and high diversity in TH2/TH3 epitopes in csp may influence vaccine efficacy. Continued molecular surveillance is essential to monitor antigenic shifts and guide future strategies.

Sickle hemoglobin (HbS) confers strong protection against symptomatic malaria caused by Plasmodium falciparum . Recently, several P. falciparum alleles (termed Pfsa+ ) were found to be enriched in HbS carriers with malaria, suggesting that parasites may be evolving to overcome HbS protection. However, it is unclear whether HbS places selective pressure on parasites since it has little to no effect against asymptomatic parasitemia, which accounts for the majority of infections. Here, we consider the effect of sickle cell status on Pfsa genotype in asymptomatic infections among 2,277 children in a high malaria transmission region of Cameroon. Consistent with previous studies, we found no significant difference in asymptomatic infection rate according to HbS status. However, HbS was strongly associated with parasite genotype at both Pfsa loci tested. Strong linkage disequilibrium and evidence for within-infection competition further support selection for Pfsa+ alleles in HbS carriers. Our findings reveal that distinct parasites underlie the equal infection rates of HbS and non-HbS carriers, such that HbS has a protective effect prior to malaria symptoms against Pfsa− parasites and Pfsa+ alleles may contribute to HbS carriers’ lower conversion rate to symptomatic disease. A revised evolutionary model for HbS protection against malarial disease is needed involving both resistance and tolerance and ongoing co-evolution with P. falciparum parasites. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences of the National Institutes of Health, R35GM147709 Fondation Pierre Fabre under the IMPAS project American Heart Association, 24POST1200601 National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, U24DK126127

Despite global malaria control efforts, the disease caused 263 million cases and 597,000 deaths in 2023. While Plasmodium falciparum accounts for most cases in Africa, non-falciparum species, such as P. ovale spp. and P. vivax, can cause relapse infections and are increasingly recognized as significant contributors to human disease. In particular, the highlands of West Cameroon have previously been reported to have high P. vivax infection rates. This study presents preliminary results from the Relapsing Malaria in Africa (ReMA) study, conducted in Dschang, Cameroon, to assess the prevalence and epidemiology of P. vivax and P. ovale spp. A cross-sectional survey of 3,661 participants from 871 households across seven health areas identified a low prevalence of P. vivax (0.1%) and P. ovale spp. (0.64%) using quantitative real time PCR (qPCR), while P. falciparum remained prevalent at 8.1%. Co-infections of P. ovale spp. with P. falciparum were observed in 23.1% of P. ovale spp. cases. While gametocytemia was common among falciparum infections, leveraging a new ovale gametocyte assay, we found that gametocytemia was uncommon among the qPCR- positive ovale infections. Spatial analysis found P. vivax and P. ovale spp. infections concentrated in Penka-Michel and Baleveng, the former having higher Anopheles spp. abundance than other areas assessed. Risk factor analysis revealed children and those with recent domestic travel had higher odds of P. falciparum infection, but no significant associations were found for P. ovale spp. infections. Entomological surveys confirmed high densities of Anopheles gambiae sensu lato (s.l.) and An. funestus (s.l.), with significantly higher human landing capture rates for An. gambiae s.l compared to other mosquito species. While these findings suggest that the malaria species which cause relapse are not as widespread as previously thought in West Cameroon, understanding factors driving their persistent transmission, especially without prevalent gametocytemia, will be important for disease control.

The high genetic diversity of Plasmodium falciparum parasites is one of the challenges for malaria control and elimination in endemic areas. A better knowledge of parasite genotypes circulating in different disease endemic areas could help to optimize local malaria interventions. This study aimed at determining P. falciparum genetic diversity from isolates collected in forest (Mfou) and humid savanna (Tibati) eco-epidemiological settings in Cameroon. Dried blood spots collected from asymptomatic individuals in 2018 and 2019 were used to determine the Plasmodium infection status and distinguish the Plasmodium spp. by real-time PCR. Allelic polymorphism of the msp2 gene was assessed in the P. falciparum positive samples by nested PCR followed by capillary electrophoresis for revelation of the fragment allelic size. Multiplicity of infection (MOI) was defined as the number of coinfecting genotypes within an infection. General linear mixed models were fitted to evaluate the impact of study site, participant age, gender and bed net ownership on genetic diversity. Malaria prevalence among the asymptomatic individuals reached 59.2

Asymptomatic carriage of infected red blood cells (iRBCs) can be prevalent in communities regardless of transmission patterns and can occur with infection of different Plasmodium species. Clinical immunity dampens the inflammatory responses leading to disease symptoms in malaria. The aim of this study was to define the immunological correlates of asymptomatic carriage of Plasmodium falciparum in a highly exposed population. 142 asymptomatic Plasmodium-infected individuals greater than 2 years of age without fever (body temperature <37.5 ℃) were followed weekly for 10 weeks before being treated with artemisinin-based combination therapy (ACT). Plasma levels of 38 cytokines were measured at baseline by Luminex and the quantity and growth inhibitory activities of circulating parasite-reactive antibodies measured. The Plasmodium antigen tested included P. falciparum merozoite extract (ME) and schizont extract (SE), and the recombinant proteins erythrocyte binding antigen 175 (EBA-175) and merozoite surface protein 1 (MSP-119). Median levels of IgG against P. falciparum EBA-175 and MSP-119 at baseline were significantly higher in those older than 20 years of age compared with the younger age group and appeared to correlate with better parasite control. Amongst all participants there were no discernible changes in IgG levels over time. Parasite density was higher in the younger age group and associated with IL-10, TNF and MCP-1 levels. A balanced IL-10:TNF ratio was associated with asymptomatic malaria regardless of age, and balanced ratios of IL-10/TNF and IL-10/IFN-γ were the only significant correlate of maintenance of asymptomatic malaria over the course of the study in individuals 20 years of age and younger. The above findings indicate that asymptomatic carriage of P. falciparum in children living in a hyperendemic area occurs independently of IgG but is associated with a balanced inflammatory cytokine ratio.

Africa and the United States are both large, heterogeneous geographies with a diverse range of ecologies, climates and mosquito species diversity which contribute to disease transmission and nuisance biting. In the United States, mosquito control is nationally, and regionally coordinated and in so much as the Centers for Disease Control (CDC) provides guidance, the Environmental Protection Agency (EPA) provides pesticide registration, and the states provide legal authority and oversight, the implementation is usually decentralized to the state, county, or city level. Mosquito control operations are organized, in most instances, into fully independent mosquito abatement districts, public works departments, local health departments. In some cases, municipalities engage independent private contractors to undertake mosquito control within their jurisdictions. In sub-Saharan Africa (SSA), where most vector-borne disease endemic countries lie, mosquito control is organized centrally at the national level. In this model, the disease control programmes (national malaria control programmes or national malaria elimination programmes (NMCP/NMEP)) are embedded within the central governments' ministries of health (MoHs) and drive vector control policy development and implementation. Because of the high disease burden and limited resources, the primary endpoint of mosquito control in these settings is reduction of mosquito borne diseases, primarily, malaria. In the United States, however, the endpoint is mosquito control, therefore, significant (or even greater) emphasis is laid on nuisance mosquitoes as much as disease vectors. The authors detail experiences and learnings gathered by the delegation of African vector control professionals that participated in a formal exchange programme initiated by the Pan-African Mosquito Control Association (PAMCA), the University of Notre Dame, and members of the American Mosquito Control Association (AMCA), in the United States between the year 2021 and 2022. The authors highlight the key components of mosquito control operations in the United States and compare them to mosquito control programmes in SSA countries endemic for vector-borne diseases, deriving important lessons that could be useful for vector control in SSA.

Abstract Background Sickle cell trait (SCT) refers to the carriage of one abnormal copy of the β-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas. Methods The study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source. Results Among the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29–1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype. Conclusion Plasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite’s enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells.

Background Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele.Methods Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele.Findings Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele.Interpretation Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine.Funding Toulouse Institute for Infectious and Inflammatory Diseases.Copyright (c) 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.

Anopheles coluzzii is one of the primary vectors of human malaria in sub-Saharan Africa. Recently, it has spread into the main cities of Central Africa threatening vector control programs. The adaptation of An. coluzzii to urban environments partly results from an increased tolerance to organic pollution and insecticides. Some of the molecular mechanisms for ecological adaptation are known, but the role of transposable elements (TEs) in the adaptive processes of this species has not been studied yet. As a first step toward assessing the role of TEs in rapid urban adaptation, we sequenced using long reads six An. coluzzii genomes from natural breeding sites in two major Central Africa cities. We de novo annotated TEs in these genomes and in an additional high-quality An. coluzzii genome, and we identified 64 new TE families. TEs were nonrandomly distributed throughout the genome with significant differences in the number of insertions of several superfamilies across the studied genomes. We identified seven putatively active families with insertions near genes with functions related to vectorial capacity, and several TEs that may provide promoter and transcription factor binding sites to insecticide resistance and immune-related genes. Overall, the analysis of multiple high-quality genomes allowed us to generate the most comprehensive TE annotation in this species to date and identify several TE insertions that could potentially impact both genome architecture and the regulation of functionally relevant genes. These results provide a basis for future studies of the impact of TEs on the biology of An. coluzzii.

Malaria control relies on passive case detection, and this strategy fails detecting asymptomatic infections. In addition, infections in endemic areas harbor multiple parasite genotypes that could affect case management and malaria epidemiology. Here, we performed AmpSeq genotyping to capture polymorphisms associated with antimalarial resistance and the genetic diversity within natural Plasmodium falciparum infections. Known genetic polymorphisms associated with altered drug susceptibility were screened for the five most common marker genes, pfdhfr, pfdhps, pfmdr1, pfcrt, and pfK13, and genetic diversity was established from two known AmpSeq markers, cpmp and csp. Relative abundance of the different genotypes within mixed infections was calculated from the number of reads per genotype. Genotyping was performed on 117 samples, 63 from asymptomatic and 54 from symptomatic individuals. We identified up to 15 genotypes within an infection, and the median multiplicity of infection was higher in asymptomatic infections (median MOI = 5 in asymptomatics versus median MOI = 2 in symptomatics, P < 0.001). No genetic differentiation on parasites from asymptomatic and symptomatic individuals was found. No mutation associated with ART resistance was identified. Prevalence of the P. falciparum chloroquine resistance wild-type genotype (CVMNK) reached 80%, confirming a return to chloroquine (CQ) sensitive parasites in Cameroon. In addition, the CQ-associated resistant genotype (CVIET) was present at very low density in polyclonal infections. Persistence of low-density chloroquine resistant parasites indicates competition-survival trade-offs may contribute to maintaining genetic diversity in natura. Thus, monitoring the expansion of these low-density genotypes in different immune backgrounds will be critical to evaluate drug policy changes.

Background Anopheles coluzzii is one of the primary vectors of human malaria in sub-Saharan Africa. Recently, it has colonized the main cities of Central Africa threatening vector control programs. The adaptation of An. coluzzii to urban environments is partly due to an increased tolerance to organic pollution and insecticides. While some of the molecular mechanisms for ecological adaptation, including chromosome rearrangements and introgressions, are known, the role of transposable elements (TEs) in the adaptive processes of this species has not been studied yet. To assess the role of TEs in rapid urban adaptation, the first step is to accurately annotate TE insertions in the genomes of natural populations collected in urban settings. Results We sequenced using long-reads six An. coluzzii genomes from natural breeding sites in two major Central Africa cities. We de novo annotated the complete set of TEs in these genomes and in an additional high-quality An. coluzzii genome available and identified 64 previously undescribed TE families. TEs were non-randomly distributed throughout the genome with significant differences in the number of insertions of several superfamilies RESEARCH ARTICLE Cite as: Vargas-Chavez, C., Longo Pendy, N. M., Nsango, S., Aguilera, L., Ayala, D., and González, J. (2021). Uncovering transposable element variants and their potential adaptive impact in urban populations of the malaria vector Anopheles coluzzii. bioRxiv, 2020.11.22.393231, ver. 3 peer-reviewed and recommended by Peer community in Genomics.

Asymptomatic malarial parasitemia is highly prevalent in Plasmodium falciparum endemic areas and often associated with increased prevalence of mild to moderate anemia. The aim of this study was to assess the prevalence of anemia during asymptomatic malaria parasitemia and its interplay with persistent infection in highly exposed individuals. A household-based longitudinal survey was undertaken in a malaria hyperendemic area in Cameroon using multiplex nested polymerase chain reaction to detect plasmodial infections. Residents with P. falciparum asymptomatic parasitemia were monitored over a 3-week period with the aid of structured questionnaires and weekly measurements of axillary temperatures. Of the 353 individuals included (median age: 26 years, range 2-86 years, male/female sex ratio 0.9), 328 (92.9%) were positive for malaria parasitemia of whom 266 (81.1%) were asymptomatic carriers. The prevalence of anemia in the study population was 38.6%, of which 69.2% were asymptomatic. Multivariate analyses identified high parasitemia (> 327 parasites/µL) and female gender as associated risk factors of asymptomatic malarial anemia in the population. Furthermore, risk analyses revealed female gender and anemia at the time of enrolment as key predictors of early development of febrile illness (< 3 weeks post enrolment) among the asymptomatic individuals. Together, the data reveal an extremely high prevalence of asymptomatic malaria parasitemia and anemia in the study area, unveiling for the first time the association of asymptomatic malarial anemia with early clinical conversion from asymptomatic to symptomatic infection. Furthermore, these findings underscore the negative impact of asymptomatic malaria parasitemia on individual health, necessitating the development of appropriate control and preventive measures.

The emergence of resistance to antimalarials has prompted the steady switch to novel therapies for decades. Withdrawal of antimalarials, such as chloroquine in sub-Saharan Africa in the late 1990s, led to rapid declines in the prevalence of resistance markers after a few years, raising the possibility of reintroducing them for malaria treatment. Here, we provide evidence that the mosquito vector plays a crucial role in maintaining parasite genetic diversity. We followed the transmission dynamics of Plasmodium falciparum parasites through its vector in natural infections from gametocytes contained in the blood of asymptomatic volunteers until sporozoites subsequently developed in the mosquito salivary glands. We did not find any selection of the mutant or wild-type pfcrt 76 allele during development in the Anopheles mosquito vector. However, microsatellite genotyping indicated that minority genotypes were favored during transmission through the mosquito. The analysis of changes in the proportions of mutant and wild-type pfcrt 76 alleles showed that, regardless of the genotype, the less-represented allele in the gametocyte population was more abundant in mosquito salivary glands, indicating a selective advantage of the minority allele in the vector. Selection of minority genotypes in the vector would explain the persistence of drug-resistant alleles in the absence of drug pressure in areas with high malaria endemicity and high genetic diversity. Our results may have important epidemiological implications, as they predict the rapid re-emergence and spread of resistant genotypes if antimalarials that had previously selected resistant parasites are reintroduced for malaria prevention or treatment. IMPORTANCE Drug selection pressure in malaria patients is the cause of the emergence of resistant parasites. Resistance imposes a fitness cost for parasites in untreated infections, so withdrawal of the drug leads to the return of susceptible parasites. Little is known about the role of the malaria vector in this phenomenon. In an experimental study conducted in Cameroon, an area of high malaria transmission, we showed that the vector did not favor the parasites based on sensitivity or resistance criteria, but it did favor the selection of minority clones. This finding shows that the vector increases the diversity of plasmodial populations and could play an important role in falciparum malaria epidemiology by maintaining resistant clones despite the absence of therapeutic pressure.

Transposable elements (TEs) are mobile DNA sequences that can increase their copy number and move from one location to another within the genome [1]. Because of their transposition dynamics, TEs constitute a significant fraction of eukaryotic genomes. TEs are also known to play an important functional role and a wealth of studies has now reported how TEs may influence single host traits [e.g. 2–4]. Given that TEs are more likely than classical point mutations to cause extreme changes in gene expression and phenotypes, they might therefore be especially prone to produce the raw diversity necessary for individuals to respond to challenging environments [5,6] such as the ones found in urban area.

Background: Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia. Objectives: This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine. Patients and methods: Dihydroartemisinin/piperaquine efficacy in 42days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n=150) and plasmepsin2 gene amplification (n=148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays. Results: The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmepsin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n=87) were susceptible to dihydroartemisinin and piperaquine. Conclusions: Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaounde, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin- and piperaquine-resistant parasites, unlike Southeast Asia.

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

Malaria parasite transmission to mosquitoes relies on the uptake of sexual stage parasites during a blood meal and subsequent formation of oocysts on the mosquito midgut wall. Transmission-blocking vaccines (TBVs) and monoclonal antibodies (mAbs) target sexual stage antigens to interrupt human-to-mosquito transmission and may form important tools for malaria elimination. Although most epitopes of these antigens are considered highly conserved, little is known about the impact of natural genetic diversity on the functional activity of transmission-blocking antibodies. Here we measured the efficacy of three mAbs against leading TBV candidates (Pfs48/45, Pfs25 and Pfs230) in transmission assays with parasites from naturally infected donors compared to their efficacy against the strain they were raised against (NF54). Transmission-reducing activity (TRA) was measured as reduction in mean oocyst intensity. mAb 45.1 (α-Pfs48/45) and mAb 4B7 (α-Pfs25) reduced transmission of field parasites from almost all donors with IC 80 values similar to NF54. Sequencing of oocysts that survived high mAb concentrations did not suggest enrichment of escape genotypes. mAb 2A2 (α-Pfs230) only reduced transmission of parasites from a minority of the donors, suggesting that it targets a non-conserved epitope. Using six laboratory-adapted strains, we revealed that mutations in one Pfs230 domain correlate with mAb gamete surface binding and functional TRA. Our findings demonstrate that, despite the conserved nature of sexual stage antigens, minor sequence variation can significantly impact the efficacy of transmission-blocking mAbs. Since mAb 45.1 shows high potency against genetically diverse strains, our findings support its further clinical development and may inform Pfs48/45 vaccine design.