Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

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.

Erratum: Comparative study of nanostructured carriers of calcium phosphate and magnesium phosphate loaded with SRT1720 for the protection of H2O2-induced senescent endothelium.

[This corrects the article on p. 2068 in vol. 10, PMID: 30093944.].

Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity.

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))-which kill liver-stage and blood-stage parasites, respectively-and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 104 to 2 × 105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.

Placental malaria vaccine candidate antigen VAR2CSA displays atypical domain architecture in some Plasmodium falciparum strains.

Two vaccines based on Plasmodium falciparum protein VAR2CSA are currently in clinical evaluation to prevent placental malaria (PM), but a deeper understanding of var2csa variability could impact vaccine design. Here we identified atypical extended or truncated VAR2CSA extracellular structures and confirmed one extended structure in a Malian maternal isolate, using a novel protein fragment assembly method for RNA-seq and DNA-seq data. Extended structures included one or two additional DBL domains downstream of the conventional NTS-DBL1X-6ɛ domain structure, with closest similarity to DBLɛ in var2csa and non-var2csa genes. Overall, 4/82 isolates displayed atypical VAR2CSA structures. The maternal isolate expressing an extended VAR2CSA bound to CSA, but its recombinant VAR2CSA bound less well to CSA than VAR2CSANF54 and showed lower reactivity to naturally acquired parity-dependent antibody. Our protein fragment sequence assembly approach has revealed atypical VAR2CSA domain architectures that impact antigen reactivity and function, and should inform the design of VAR2CSA-based vaccines.

Comparative study of nanostructured carriers of calcium phosphate and magnesium phosphate loaded with SRT1720 for the protection of H2O2-induced senescent endothelium.

Nanostructured calcium phosphate (CaP) and magnesium phosphate (MgP) are promising for the application as the nanocarriers in drug delivery. However, the difference between CaP and MgP nanocarriers in drug delivery is rarely investigated. In this work, we comparatively investigated nanostructured CaP, MgP and calcium magnesium phosphate (CMP) for the delivery of SRT1720, which is a silent information regulator (SIRT1) specific activator with pro-angiogenic and anti-aging properties in response to hydrogen peroxide (H2O2)-induced endothelial senescence. The protection of SRT1720-loaded CaP nanospheres, MgP nanosheets and CMP microspheres on the H2O2-induced senescent endothelium was examined by using human umbilical vein endothelial cells (HUVECs), demonstrating the improved cell viability, anti-aging, tube formation and migration. In addition, the SRT1720-loaded CaP nanospheres, MgP nanosheets and CMP microspheres can rescue the impaired angiogenic potential of HUVECs via activation of Akt/eNOS/VEGF pathway. The SRT1720-loaded MgP nanosheets and CMP microspheres have a similar protective effect compared with the pure SRT1720, while the SRT1720-loaded CaP nanospheres decrease the protective capability of SRT1720. These results lead us to figure out both MgP nanosheets and CMP microspheres are suitable and effective delivery for SRT1720 and this system can be further applied in vivo treatment.

Erratum: Comparative study of nanostructured carriers of calcium phosphate and magnesium phosphate loaded with SRT1720 for the protection of H2O2-induced senescent endothelium.

[This corrects the article on p. 2068 in vol. 10, PMID: 30093944.].

Mutation in the Plasmodium falciparum CRT protein determines the stereospecific activity of antimalarial cinchona alkaloids.

The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (-)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (-)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC(50) ratio of (-)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (-) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.

Lack of allele-specific efficacy of a bivalent AMA1 malaria vaccine.

BACKGROUND: Extensive genetic diversity in vaccine antigens may contribute to the lack of efficacy of blood stage malaria vaccines. Apical membrane antigen-1 (AMA1) is a leading blood stage malaria vaccine candidate with extreme diversity, potentially limiting its efficacy against infection and disease caused by Plasmodium falciparum parasites with diverse forms of AMA1. METHODS: Three hundred Malian children participated in a Phase 2 clinical trial of a bivalent malaria vaccine that found no protective efficacy. The vaccine consists of recombinant AMA1 based on the 3D7 and FVO strains of P. falciparum adjuvanted with aluminum hydroxide (AMA1-C1). The gene encoding AMA1 was sequenced from P. falciparum infections experienced before and after immunization with the study vaccine or a control vaccine. Sequences of ama1 from infections in the malaria vaccine and control groups were compared with regard to similarity to the vaccine antigens using several measures of genetic diversity. Time to infection with parasites carrying AMA1 haplotypes similar to the vaccine strains with respect to immunologically important polymorphisms and the risk of infection with vaccine strain haplotypes were compared. RESULTS: Based on 62 polymorphic AMA1 residues, 186 unique ama1 haplotypes were identified among 315 ama1 sequences that were included in the analysis. Eight infections had ama1 sequences identical to 3D7 while none were identical to FVO. Several measures of genetic diversity showed that ama1 sequences in the malaria vaccine and control groups were comparable both at baseline and during follow up period. Pre- and post-immunization ama1 sequences in both groups all had a similar degree of genetic distance from FVO and 3D7 ama1. No differences were found in the time of first clinical episode or risk of infection with an AMA1 haplotype similar to 3D7 or FVO with respect to a limited set of immunologically important polymorphisms found in the cluster 1 loop of domain I of AMA1. CONCLUSION: This Phase 2 trial of a bivalent AMA1 malaria vaccine found no evidence of vaccine selection or strain-specific efficacy, suggesting that the extreme genetic diversity of AMA1 did not account for failure of the vaccine to provide protection.

Population structure of the genes encoding the polymorphic Plasmodium falciparum apical membrane antigen 1: implications for vaccine design.

Immunization with the highly polymorphic Plasmodium falciparum apical membrane antigen 1 (PfAMA1) induces protection in animals but primarily against parasites that express the same or similar alleles. One strategy to overcome the obstacle of polymorphism is to combine PfAMA1 proteins representing major haplotypes into one vaccine. To determine the minimum number of haplotypes that would confer broad protection, we sequenced the coding region of PfAMA1 from 97 clones from around the world and 61 isolates from Mali, identifying 150 haplotypes for domains 1 to 3 that included previous sequences. A clustering algorithm grouped the 150 haplotypes into six populations that were independent of geographic location. Each of the six populations contained haplotypes predominantly of that population (predominant haplotypes) and haplotypes that were a mixture of haplotypes represented in other populations (admixed haplotypes). To determine the biological relevance of the populations identified through the clustering algorithm, antibodies induced against one predominant haplotype of population 1 (3D7) and one admixed haplotype of population 5 (FVO) were tested for their ability to block parasite invasion of erythrocytes. Parasites expressing PfAMA1s belonging to population 1 were efficiently inhibited by 3D7-specific antibodies, whereas parasites expressing PfAMA1s belonging to other populations were not. For FVO-specific antibodies, we observed growth inhibition against itself as well as isolates belonging to populations 3 and 6. Our data suggests that the inclusion of PfAMA1 sequences from each of the six populations may result in a vaccine that induces protective immunity against a broad range of malaria parasites.

Genome-wide variation and identification of vaccine targets in the Plasmodium falciparum genome.

One goal in sequencing the Plasmodium falciparum genome, the agent of the most lethal form of malaria, is to discover vaccine and drug targets. However, identifying those targets in a genome in which approximately 60% of genes have unknown functions is an enormous challenge. Because the majority of known malaria antigens and drug-resistant genes are highly polymorphic and under various selective pressures, genome-wide analysis for signatures of selection may lead to discovery of new vaccine and drug candidates. Here we surveyed 3,539 P. falciparum genes ( approximately 65% of the predicted genes) for polymorphisms and identified various highly polymorphic loci and genes, some of which encode new antigens that we confirmed using human immune sera. Our collections of genome-wide SNPs ( approximately 65% nonsynonymous) and polymorphic microsatellites and indels provide a high-resolution map (one marker per approximately 4 kb) for mapping parasite traits and studying parasite populations. In addition, we report new antigens, providing urgently needed vaccine candidates for disease control.

Disruption of a Plasmodium falciparum gene linked to male sexual development causes early arrest in gametocytogenesis.

A male gametocyte defect in the Plasmodium falciparum Dd2 parasite was previously discovered through the observation that all progeny clones in a Dd2 x HB3 genetic cross were the result of fertilization events between Dd2 female and HB3 male gametes. A determinant linked to the defect in Dd2 was subsequently mapped to an 800-kb segment on chromosome 12. Here, we report further mapping of the determinant to an 82-kb region and the identification of a candidate gene, P. falciparum male development gene 1 (pfmdv-1), that is expressed at a lower level in Dd2 compared with the wild-type normal male gametocyte-producing ancestor W2. Pfmdv-1 protein is sexual-stage specific and is located on the gametocyte plasma membrane, parasitophorous vacuole membrane, and the membranes of cleft-like structures within the erythrocyte. Disruption of pfmdv-1 results in a dramatic reduction in mature gametocytes, especially functional male gametocytes, with the majority of sexually committed parasites developmentally arrested at stage I. The pfmdv-1-knockout parasites show disturbed membrane structures, particularly multimembrane vesicles/tubes that likely derive from deformed cleft-like structures. Mosquito infectivity of the knockout parasites was also greatly reduced but not completely lost. The results suggest that pfmdv-1 plays a key role in gametocyte membrane formation and integrity.

Recombination hotspots and population structure in Plasmodium falciparum.

Understanding the influences of population structure, selection, and recombination on polymorphism and linkage disequilibrium (LD) is integral to mapping genes contributing to drug resistance or virulence in Plasmodium falciparum. The parasite's short generation time, coupled with a high cross-over rate, can cause rapid LD break-down. However, observations of low genetic variation have led to suggestions of effective clonality: selfing, population admixture, and selection may preserve LD in populations. Indeed, extensive LD surrounding drug-resistant genes has been observed, indicating that recombination and selection play important roles in shaping recent parasite genome evolution. These studies, however, provide only limited information about haplotype variation at local scales. Here we describe the first (to our knowledge) chromosome-wide SNP haplotype and population recombination maps for a global collection of malaria parasites, including the 3D7 isolate, whose genome has been sequenced previously. The parasites are clustered according to continental origin, but alternative groupings were obtained using SNPs at 37 putative transporter genes that are potentially under selection. Geographic isolation and highly variable multiple infection rates are the major factors affecting haplotype structure. Variation in effective recombination rates is high, both among populations and along the chromosome, with recombination hotspots conserved among populations at chromosome ends. This study supports the feasibility of genome-wide association studies in some parasite populations.

Host switch leads to emergence of Plasmodium vivax malaria in humans.

The geographical origin of Plasmodium vivax, the most widespread human malaria parasite, is controversial. Although genetic closeness to Asian primate malarias has been confirmed by phylogenetic analyses, genetic similarities between P. vivax and Plasmodium simium, a New World primate malaria, suggest that humans may have acquired P. vivax from New World monkeys or vice versa. Additionally, the near fixation of the Duffy-negative blood type (FY x B(null)/FY x B(null)) in West and Central Africa, consistent with directional selection, and the association of Duffy negativity with complete resistance to vivax malaria suggest a prolonged period of host-parasite coevolution in Africa. Here we use Bayesian and likelihood methods in conjunction with cophylogeny mapping to reconstruct the genetic and coevolutionary history of P. vivax from the complete mitochondrial genome of 176 isolates as well as several closely related Plasmodium species. Taken together, a haplotype network, parasite migration patterns, demographic history, and cophylogeny mapping support an Asian origin via a host switch from macaque monkeys.

Polymorphism in the Plasmodium falciparum erythrocyte-binding ligand JESEBL/EBA-181 alters its receptor specificity.

The malaria parasite lives within erythrocytes and depends on the binding of parasite ligands to host cell surface receptors for invasion. The most virulent human malaria parasite, Plasmodium falciparum, uses multiple ligands, including EBA-175, BAEBL, and JESEBL of the Duffy-binding-like (DBL) family of erythrocyte-binding proteins, for invasion of human erythrocytes. Region II of these parasite ligands is the erythrocyte-binding domain. Previously, we had shown that polymorphism in region II of BAEBL leads to different erythrocyte-binding specificities. We have now identified and characterized the binding specificity of six JESEBL variants. We sequenced region II of JESEBL from 20 P. falciparum clones collected from various parts of the world where malaria is endemic. We observed eight JESEBL variants that contained amino acid polymorphisms at five positions among all clones. Seven of the eight variants could be connected by a single base change that led to an amino acid change. We investigated the functional significance of these polymorphisms by transiently expressing region II from six of JESEBL variants on the surface of Chinese hamster ovary cells. We observed four erythrocyte-binding patterns to enzyme-treated erythrocytes. Thus, P. falciparum DBL ligands JESEBL and BAEBL can recognize multiple receptors on the erythrocyte surface. In contrast to Plasmodium vivax, which has disappeared from West Africa because of the Duffy-negative blood group, P. falciparum may have been successful in endemic areas because it has mutated the ligands of the DBL family to create multiple pathways of invasion, thus making selection of refractory erythrocytes unlikely.

Multiple transporters associated with malaria parasite responses to chloroquine and quinine.

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.

Chromosome-wide SNPs reveal an ancient origin for Plasmodium falciparum.

The Malaria's Eve hypothesis, proposing a severe recent population bottleneck (about 3,000-5,000 years ago) of the human malaria parasite Plasmodium falciparum, has prompted a debate about the origin and evolution of the parasite. The hypothesis implies that the parasite population is relatively homogeneous, favouring malaria control measures. Other studies, however, suggested an ancient origin and large effective population size. To test the hypothesis, we analysed single nucleotide polymorphisms (SNPs) from 204 genes on chromosome 3 of P. falciparum. We have identified 403 polymorphic sites, including 238 SNPs and 165 microsatellites, from five parasite clones, establishing chromosome-wide haplotypes and a dense map with one polymorphic marker per approximately 2.3 kilobases. On the basis of synonymous SNPs and non-coding SNPs, we estimate the time to the most recent common ancestor to be approximately 100,000-180,000 years, significantly older than the proposed bottleneck. Our estimated divergence time coincides approximately with the start of human population expansion, and is consistent with a genetically complex organism able to evade host immunity and other antimalarial efforts.