Evaluation of naturally acquired immune responses against novel pre-erythrocytic Plasmodium vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin.

BACKGROUND: Plasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity. METHODS: Twelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used. RESULTS: In those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58-99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9-35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses. CONCLUSIONS: These results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.

Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response.

T cells expressing CD19-targeting chimeric antigen receptors (CARs) reveal high efficacy in the treatment of B cell malignancies. Here, we report that T cell receptor fusion constructs (TRuCs) comprising an antibody-based binding domain fused to T cell receptor (TCR) subunits can effectively reprogram an intact TCR complex to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells kill tumor cells as potently as second-generation CAR-T cells, but at significant lower cytokine release and despite the absence of an extra co-stimulatory domain. TRuC-T cells demonstrate potent anti-tumor activity in both liquid and solid tumor xenograft models. In several models, TRuC-T cells are more efficacious than respective CAR-T cells. TRuC-T cells are shown to engage the signaling capacity of the entire TCR complex in an HLA-independent manner.

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection.

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi.

BACKGROUND: The mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America. METHODS: To determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 µg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated. RESULTS: IVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions. CONCLUSION: In conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

Ivermectin susceptibility, sporontocidal effect, and inhibition of time to re-feed in the Amazonian malaria vector Anopheles darlingi.

BACKGROUND: Outdoor malaria transmission hinders malaria elimination efforts in the Amazon region and novel vector control tools are needed. Ivermectin mass drug administration (MDA) to humans kills wild Anopheles, targets outdoor-feeding vectors, and can suppress malaria parasite transmission. Laboratory investigations were performed to determine ivermectin susceptibility, sporontocidal effect and inhibition of time to re-feed for the primary Amazonian malaria vector, Anopheles darlingi. METHODS: To assess ivermectin susceptibility, various concentrations of ivermectin were mixed in human blood and fed to An. darlingi. Mosquito survival was monitored daily for 7 days and a non-linear mixed effects model with Probit analysis was used to calculate lethal concentrations of ivermectin that killed 50% (LC50), 25% (LC25) and 5% (LC5) of mosquitoes. To examine ivermectin sporonticidal effect, Plasmodium vivax blood samples were collected from malaria patients and offered to mosquitoes without or with ivermectin at the LC50, LC25 or LC5. To assess ivermectin inhibition of mosquito time to re-feed, concentrations of ivermectin predicted to occur after a single oral dose of 200 µg/kg ivermectin were fed to An. darlingi. Every day for 12 days thereafter, individual mosquitoes were given the opportunity to re-feed on a volunteer. Any mosquitoes that re-blood fed or died were removed from the study. RESULTS: Ivermectin significantly reduced An. darlingi survivorship: 7-day-LC50 = 43.2 ng/ml [37.5, 48.6], -LC25 = 27.8 ng/ml [20.4, 32.9] and -LC5 = 14.8 ng/ml [7.9, 20.2]. Ivermectin compound was sporontocidal to P. vivax in An. darlingi at the LC50 and LC25 concentrations reducing prevalence by 22.6 and 17.1%, respectively, but not at the LC5. Oocyst intensity was not altered at any concentration. Ivermectin significantly delayed time to re-feed at the 4-h (48.7 ng/ml) and 12-h (26.9 ng/ml) concentrations but not 36-h (10.6 ng/ml) or 60-h (6.3 ng/ml). CONCLUSIONS: Ivermectin is lethal to An. darlingi, modestly inhibits sporogony of P. vivax, and delays time to re-feed at concentrations found in humans up to 12 h post drug ingestion. The LC50 value suggests that a higher than standard dose (400-µg/kg) is necessary to target An. darlingi. These results suggest that ivermectin MDA has potential in the Amazon region to aid malaria elimination efforts.

Molecular Epidemiology of Trypanosomatids and Trypanosoma cruzi in Primates from Peru.

We determined the prevalence rate and risk of infection of Trypanosoma cruzi and other trypanosomatids in Peruvian non-human primates (NHPs) in the wild (n = 126) and in different captive conditions (n = 183). Blood samples were collected on filter paper, FTA cards, or EDTA tubes and tested using a nested PCR protocol targeting the 24Sα rRNA gene. Main risk factors associated with trypanosomatid and T. cruzi infection were genus and the human-animal context (wild vs captive animals). Wild NHPs had higher prevalence of both trypanosomatids (64.3 vs 27.9%, P < 0.001) and T. cruzi (8.7 vs 3.3%, P = 0.057), compared to captive NHPs, suggesting that parasite transmission in NHPs occurs more actively in the sylvatic cycle. In terms of primate family, Pitheciidae had the highest trypanosomatid prevalence (20/22, 90.9%) and Cebidae had the highest T. cruzi prevalence (15/117, 12.8%). T. cruzi and trypanosomatids are common in Peruvian NHPs and could pose a health risk to human and animals that has not been properly studied.

Prevalence of Trypanosoma cruzi and Other Trypanosomatids in Frequently-Hunted Wild Mammals from the Peruvian Amazon.

To better understand the ecology of Trypanosoma cruzi in the northeastern Peruvian Amazon, we evaluated the prevalence of T. cruzi and other trypanosomatids in four orders of wild mammals hunted and consumed by inhabitants of three remote indigenous communities in the Peruvian Amazon. Of 300 wild mammals sampled, 115 (38.3%) were infected with trypanosomatids and 15 (5.0%) with T. cruzi. The prevalence of T. cruzi within each species was as follows: large rodents (Cuniculus paca, 5.5%; Dasyprocta spp., 2.6%), edentates (Dasypus novemcinctus, 4.2%), and carnivores with higher prevalence (Nasua nasua, 18.8%). The high prevalence of T. cruzi and other trypanosomatids in frequently hunted wild mammals suggests a sizeable T. cruzi sylvatic reservoir in remote Amazonian locations.

A malaria vaccine protects Aotus monkeys against virulent Plasmodium falciparum infection.

The Plasmodium falciparum protein, apical membrane antigen 1 forms a complex with another parasite protein, rhoptry neck protein 2, to initiate junction formation with the erythrocyte and is essential for merozoite invasion during the blood stage of infection. Consequently, apical membrane antigen 1 has been a target of vaccine development but vaccination with apical membrane antigen 1 alone in controlled human malaria infections failed to protect and showed limited efficacy in field trials. Here we show that vaccination with AMA1-RON2L complex in Freund's adjuvant protects Aotus monkeys against a virulent Plasmodium falciparum infection. Vaccination with AMA1 alone gave only partial protection, delaying infection in one of eight animals. However, the AMA1-RON2L complex vaccine completely protected four of eight monkeys and substantially delayed infection (>25 days) in three of the other four animals. Interestingly, antibodies from monkeys vaccinated with the AMA1-RON2L complex had significantly higher neutralizing activity than antibodies from monkeys vaccinated with AMA1 alone. Importantly, we show that antibodies from animals vaccinated with the complex have significantly higher neutralization activity against non-vaccine type parasites. We suggest that vaccination with the AMA1-RON2L complex induces functional antibodies that better recognize AMA1 as it appears complexed with RON2 during merozoite invasion. These data justify progression of this next generation AMA1 vaccine towards human trials.

Eosinophil-derived IL-4 drives progression of myocarditis to inflammatory dilated cardiomyopathy.

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in children and young adults. DCMi develops in up to 30% of myocarditis patients, but the mechanisms involved in disease progression are poorly understood. Patients with eosinophilia frequently develop cardiomyopathies. In this study, we used the experimental autoimmune myocarditis (EAM) model to determine the role of eosinophils in myocarditis and DCMi. Eosinophils were dispensable for myocarditis induction but were required for progression to DCMi. Eosinophil-deficient ΔdblGATA1 mice, in contrast to WT mice, showed no signs of heart failure by echocardiography. Induction of EAM in hypereosinophilic IL-5Tg mice resulted in eosinophilic myocarditis with severe ventricular and atrial inflammation, which progressed to severe DCMi. This was not a direct effect of IL-5, as IL-5TgΔdblGATA1 mice were protected from DCMi, whereas IL-5-/- mice exhibited DCMi comparable with WT mice. Eosinophils drove progression to DCMi through their production of IL-4. Our experiments showed eosinophils were the major IL-4-expressing cell type in the heart during EAM, IL-4-/- mice were protected from DCMi like ΔdblGATA1 mice, and eosinophil-specific IL-4 deletion resulted in improved heart function. In conclusion, eosinophils drive progression of myocarditis to DCMi, cause severe DCMi when present in large numbers, and mediate this process through IL-4.

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.

Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE: Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.

Unstable Malaria Transmission in the Southern Peruvian Amazon and Its Association with Gold Mining, Madre de Dios, 2001-2012.

The reemergence of malaria in the last decade in Madre de Dios, southern Peruvian Amazon basin, was accompanied by ecological, political, and socioeconomic changes related to the proliferation of illegal gold mining. We conducted a secondary analysis of passive malaria surveillance data reported by the health networks in Madre de Dios between 2001 and 2012. We calculated the number of cases of malaria by year, geographic location, intensity of illegal mining activities, and proximity of health facilities to the Peru-Brazil Interoceanic Highway. During 2001-2012, 203,773 febrile cases were identified in Madre de Dios, of which 30,811 (15.1%) were confirmed cases of malaria; all but 10 cases were due to Plasmodium vivax Cases of malaria rose rapidly between 2004 and 2007, reached 4,469 cases in 2005, and then declined after 2010 to pre-2004 levels. Health facilities located in areas of intense illegal gold mining reported 30-fold more cases than those in non-mining areas (ratio = 31.54, 95% confidence interval [CI] = 19.28, 51.60). Finally, health facilities located > 1 km from the Interoceanic Highway reported significantly more cases than health facilities within this distance (ratio = 16.20, 95% CI = 8.25, 31.80). Transmission of malaria in Madre de Dios is unstable, geographically heterogeneous, and strongly associated with illegal gold mining. These findings highlight the importance of spatially oriented interventions to control malaria in Madre de Dios, as well as the need for research on malaria transmission in illegal gold mining camps.

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax.

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Symptoms and Immune Markers in Plasmodium/Dengue Virus Co-infection Compared with Mono-infection with Either in Peru.

BACKGROUND: Malaria and dengue are two of the most common vector-borne diseases in the world, but co-infection is rarely described, and immunologic comparisons of co-infection with mono-infection are lacking. METHODOLOGY AND PRINCIPAL FINDINGS: We collected symptom histories and blood specimens from subjects in a febrile illness surveillance study conducted in Iquitos and Puerto Maldonado, Peru, between 2002-2011. Nineteen symptoms and 18 immune markers at presentation were compared among those with co-infection with Plasmodium/dengue virus (DENV), Plasmodium mono-infection, and DENV mono-infection. Seventeen subjects were identified as having Plasmodium/DENV co-infection and were retrospectively matched with 51 DENV mono-infected and 44 Plasmodium mono-infected subjects. Those with Plasmodium mono-infection had higher levels of IL-4, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, and MIP1-α/CCL3 compared with DENV mono-infection or co-infection; those with Plasmodium mono-infection had more cough than those with DENV mono-infection. Subjects with DENV mono-infection had higher levels of TGF-ß1 and more myalgia than those with Plasmodium mono-infection. No symptom was more common and no immune marker level was higher in the co-infected group, which had similar findings to the DENV mono-infected subjects. CONCLUSIONS/SIGNIFICANCE: Compared with mono-infection with either pathogen, Plasmodium/DENV co-infection was not associated with worse disease and resembled DENV mono-infection in both symptom frequency and immune marker level.

Comparative genomic analysis of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis.

BACKGROUND: The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some. Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations. RESULTS: We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes. Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference. Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays. CONCLUSIONS: The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species. The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.

Outbreak of Cutaneous Leishmaniasis in Peruvian Military Personnel Undertaking Training Activities in the Amazon Basin, 2010.

Military personnel deployed to the Amazon Basin are at high risk for cutaneous leishmaniasis (CL). We responded to an outbreak among Peruvian Army personnel returning from short-term training in the Amazon, conducting active case detection, lesion sample collection, and risk factor assessment. The attack rate was 25% (76/303); the incubation period was 2-36 weeks (median = 8). Most cases had one lesion (66%), primarily ulcerative (49%), and in the legs (57%). Real-time polymerase chain reaction (PCR) identified Leishmania (Viannia) braziliensis (59/61 = 97%) and L. (V.) guyanensis (2/61 = 3%). Being male (risk ratio [RR] = 4.01; P = 0.034), not wearing long-sleeve clothes (RR = 1.71; P = 0.005), and sleeping in open rooms (RR = 1.80; P = 0.009) were associated with CL. Sodium stibogluconate therapy had a 41% cure rate, less than previously reported in Peru (~70%; P < 0.001). After emphasizing pre-deployment education and other basic prevention measures, trainees in the following year had lower incidence (1/278 = 0.4%; P < 0.001). Basic prevention can reduce CL risk in deployed militaries.

Molecular Epidemiology of Plasmodium falciparum Malaria Outbreak, Tumbes, Peru, 2010-2012.

During 2010-2012, an outbreak of 210 cases of malaria occurred in Tumbes, in the northern coast of Peru, where no Plasmodium falciparum malaria case had been reported since 2006. To identify the source of the parasite causing this outbreak, we conducted a molecular epidemiology investigation. Microsatellite typing showed an identical genotype in all 54 available isolates. This genotype was also identical to that of parasites isolated in 2010 in the Loreto region of the Peruvian Amazon and closely related to clonet B, a parasite lineage previously reported in the Amazon during 1998-2000. These findings are consistent with travel history of index case-patients. DNA sequencing revealed mutations in the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr1 loci, which are strongly associated with resistance to chloroquine and sulfadoxine/pyrimethamine, and deletion of the Pfhrp2 gene. These results highlight the need for timely molecular epidemiology investigations to trace the parasite source during malaria reintroduction events.

A PfRH5-based vaccine is efficacious against heterologous strain blood-stage Plasmodium falciparum infection in aotus monkeys.

Antigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans.

Next-Generation Sequencing of Plasmodium vivax Patient Samples Shows Evidence of Direct Evolution in Drug-Resistance Genes.

Understanding the mechanisms of drug resistance in Plasmodium vivax, the parasite that causes the most widespread form of human malaria, is complicated by the lack of a suitable long-term cell culture system for this parasite. In contrast to P. falciparum, which can be more readily manipulated in the laboratory, insights about parasite biology need to be inferred from human studies. Here we analyze the genomes of parasites within 10 human P. vivax infections from the Peruvian Amazon. Using next-generation sequencing we show that some P. vivax infections analyzed from the region are likely polyclonal. Despite their polyclonality we observe limited parasite genetic diversity by showing that three or fewer haplotypes comprise 94% of the examined genomes, suggesting the recent introduction of parasites into this geographic region. In contrast we find more than three haplotypes in putative drug-resistance genes, including the gene encoding dihydrofolate reductase-thymidylate synthase and the P. vivax multidrug resistance associated transporter, suggesting that resistance mutations have arisen independently. Additionally, several drug-resistance genes are located in genomic regions with evidence of increased copy number. Our data suggest that whole genome sequencing of malaria parasites from patients may provide more insight about the evolution of drug resistance than genetic linkage or association studies, especially in geographical regions with limited parasite genetic diversity.

Cardiac fibroblasts mediate IL-17A-driven inflammatory dilated cardiomyopathy.

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra(-/-) mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/MΦ) cardiac infiltrates. Depletion of Ly6Chi MO/MΦ also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/MΦ in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A-fibroblast-GM-CSF-MO/MΦ axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.

Plasmodium vivax hospitalizations in a monoendemic malaria region: severe vivax malaria?

Severe malaria caused by Plasmodium vivax is no longer considered rare. To describe its clinical features, we performed a retrospective case control study in the subregion of Luciano Castillo Colonna, Piura, Peru, an area with nearly exclusive vivax malaria transmission. Severe cases and the subset of critically ill cases were compared with a random set of uncomplicated malaria cases (1:4). Between 2008 and 2009, 6,502 malaria cases were reported, including 106 hospitalized cases, 81 of which fit the World Health Organization definition for severe malaria. Of these 81 individuals, 28 individuals were critically ill (0.4%, 95% confidence interval = 0.2-0.6%) with severe anemia (57%), shock (25%), lung injury (21%), acute renal failure (14%), or cerebral malaria (11%). Two potentially malaria-related deaths occurred. Compared with uncomplicated cases, individuals critically ill were older (38 versus 26 years old, P < 0.001), but similar in other regards. Severe vivax malaria monoinfection with critical illness is more common than previously thought.