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In this article from Southeast Asia and Africa, the emergence of widespread decreased susceptibility of Plasmodium falciparum to artemisinins, detected throughout Southeast Asia, is associated with a point mutation in the kelch protein on chromosome 13. Artemisinin derivatives are highly potent, rapidly eliminated antimalarial drugs with a broad stage specificity of action. They clear parasitemia more rapidly than all other currently available antimalarial agents. In the 1990s, resistance to available antimalarial drugs such as chloroquine and sulfadoxine–pyrimethamine worsened across areas of the world where malaria is endemic. As a direct consequence, morbidity and mortality associated with malaria increased, especially among African children, who account for most deaths from malaria. 1 The artemisinin-based combination therapies were introduced in the mid-1990s, when there was an imminent prospect of untreatable malaria in Southeast Asia, where resistance to all available antimalarial . . .

Background Malaria is a major public health problem in Cameroon. The study of the genetic diversity within parasite population is essential for understanding the mechanism underlying malaria pathology and to determine parasite clones profile in an infection, for proper malaria control strategies. The objective of this study was to perform a molecular characterization of highly polymorphic genetic markers of Plasmodium falciparum , and to determine allelic distribution with their influencing factors valuable to investigate malaria transmission dynamics in Cameroon. Methods A total of 350 P. falciparum clinical isolates were characterized by genotyping block 2 of msp - 1 , block 3 of msp - 2 , and region II of glurp gene using nested PCR and DNA sequencing between 2012 and 2013. Results A total of 5 different genotypes with fragment sizes ranging from 597 to 817 bp were recorded for GLURP. Overall, 16 MSP-1 genotypes, including K1, MAD20 and RO33 were identified, ranging from 153 to 335 bp. A peculiarity about this study is the RO33 monomorphic pattern revealed among the Pfmsp - 1 allelic type. Again, this study identified 27 different Pfmsp - 2 genotypes, ranging from 140 to 568 bp in size, including 15 belonging to the 3D7-type and 12 to the FC27 allelic families. The analysis of the MSP-1 and MSP-2 peptides indicates that the region of the alignment corresponding K1 polymorphism had the highest similarity in the MSP1and MSP2 clade followed by MAD20 with 93% to 100% homology. Therefore, population structure of P. falciparum isolates is identical to that of other areas in Africa, suggesting that vaccine developed with K1 and MAD20 of Pfmsp1 allelic variant could be protective for Africa children but these findings requires further genetic and immunological investigations. The multiplicity of infection (MOI) was significantly higher (P < 0.05) for Pfmsp - 2 loci (3.82), as compare with Pfmsp - 1 (2.51) and heterozygotes ranged from 0.55 for Pfmsp - 1 to 0.96 for Pfmsp - 2 . Conclusion High genetic diversity and allelic frequencies in P. falciparum isolates indicate a persisting high level of transmission. This study advocate for an intensification of the malaria control strategies in Cameroon. Trial registration This study was approved by Cameroon National Ethics Committee. It is a randomized controlled trial retrospectively registered in NIH U.S. National Library of Medicine, ClinicalTrials.gov on the 28/11/2016 at https://clinicaltrials.gov/ct2/show/NCT02974348 with the registration number NCT02974348

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity.

Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection. ClinicalTrials.govNCT00870987.

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02 A , a bloodstage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02 A had the highest efficacy against AMA1 alíeles that were identical to the 3D7 vaccine-type alíele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alíeles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of alíele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alíeles of this extremely polymorphic antigen.

This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems. After a preliminary safety evaluation of low dose AMA-1/AS01B (10 microg/0.5 mL) in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 microg/0.5 mL) of AMA-1/AS01B (n = 15) or AMA-1/AS02A (n = 15), followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs) with 95% Confidence Intervals (CIs) were high: low dose AMA-1/AS01B 196 microg/mL (103-371 microg/mL), full dose AMA-1/AS01B 279 microg/mL (210-369 microg/mL) and full dose AMA-1/AS02A 216 microg/mL (169-276 microg/mL) with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA), against homologous but not against heterologous (FVO) parasites as well as demonstrable interferon-gamma (IFN-gamma) responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR). However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements. All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali. www.clinicaltrials.gov NCT00385047.

Dihydroartemisinin-piperaquine has been adopted as first-line artemisinin combination therapy (ACT) for multidrug-resistant Plasmodium falciparum malaria in Cambodia because of few remaining alternatives. We aimed to assess the efficacy of standard 3 day dihydroartemisinin-piperaquine treatment of uncomplicated P falciparum malaria, with and without the addition of primaquine, focusing on the factors involved in drug resistance. In this observational cohort study, we assessed 107 adults aged 18–65 years presenting to Anlong Veng District Hospital, Oddar Meanchey Province, Cambodia, with uncomplicated P falciparum or mixed P falciparum/Plasmodium vivax infection of between 1000 and 200 000 parasites per μL of blood, and participating in a randomised clinical trial in which all had received dihydroartemisinin-piperaquine for 3 days, after which they had been randomly allocated to receive either primaquine or no primaquine. The trial was halted early due to poor dihydroartemisinin-piperaquine efficacy, and we assessed day 42 PCR-corrected therapeutic efficacy (proportion of patients with recurrence at 42 days) and evidence of drug resistance from the initial cohort. We did analyses on both the intention to treat (ITT), modified ITT (withdrawals, losses to follow-up, and those with secondary outcomes [eg, new non-recrudescent malaria infection] were censored on the last day of follow-up), and per-protocol populations of the original trial. The original trial was registered with ClinicalTrials.gov, number NCT01280162. Between Dec 10, 2012, and Feb 18, 2014, we had enrolled 107 patients in the original trial. Enrolment was voluntarily halted on Feb 16, 2014, before reaching planned enrolment (n=150) because of poor efficacy. We had randomly allocated 50 patients to primaquine and 51 patients to no primaquine groups. PCR-adjusted Kaplan-Meier risk of P falciparum 42 day recrudescence was 54% (95% CI 45–63) in the modified ITT analysis population. We found two kelch13 propeller gene mutations associated with artemisinin resistance—a non-synonymous Cys580Tyr substitution in 70 (65%) of 107 participants, an Arg539Thr substitution in 33 (31%), and a wild-type parasite in four (4%). Unlike Arg539Thr, Cys580Tyr was accompanied by two other mutations associated with extended parasite clearance (MAL10:688956 and MAL13:1718319). This combination triple mutation was associated with a 5·4 times greater risk of treatment failure (hazard ratio 5·4 [95% CI 2·4–12]; p<0·0001) and higher piperaquine 50% inhibitory concentration (triple mutant 34 nM [28–41]; non-triple mutant 24 nM [1–27]; p=0·003) than other infections had. The drug was well tolerated, with gastrointestinal symptoms being the most common complaints. The dramatic decline in efficacy of dihydroartemisinin-piperaquine compared with what was observed in a study at the same location in 2010 was strongly associated with a new triple mutation including the kelch13 Cys580Tyr substitution. 3 days of artemisinin as part of an artemisinin combination therapy regimen might be insufficient. Strict regulation and monitoring of antimalarial use, along with non-pharmacological approaches to malaria resistance containment, must be integral parts of the public health response to rapidly accelerating drug resistance in the region. Armed Forces Health Surveillance Center/Global Emerging Infections Surveillance and Response System, Military Infectious Disease Research Program, National Institute of Allergy and Infectious Diseases, and American Society of Tropical Medicine and Hygiene/Burroughs Wellcome Fund.

Models of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5)-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers. The NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). Group 1 (n = 6) healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct) and Group 2 (n = 6) a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million) than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045) group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003), but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7-10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites. As found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a malaria vaccine eliciting strong CD8+ T cell IFN-γ responses. ClinicalTrials.govNCT00392015.

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge. NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at 1 x 1010 particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected. The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection. ClinicalTrials.gov NCT00392015.

In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment ofPlasmodium falciparuminfections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation inpfcrtencoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout theP. falciparumpopulation. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQRpfcrtallele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance.