Profiling the antibody response of humans protected by immunization with Plasmodium vivax radiation-attenuated sporozoites.

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.

Genomics of Plasmodium vivax in Colombia reveals evidence of local bottle-necking and inter-country connectivity in the Americas.

Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation.

Seroprevalence of viral and bacterial pathogens among malaria patients in an endemic area of southern Venezuela.

BACKGROUND: Malaria remains a leading public health problem worldwide. Co-infections with other pathogens complicate its diagnosis and may modify the disease's clinical course and management. Similarities in malaria clinical presentation with other infections and overlapping endemicity result in underdiagnosis of co-infections and increased mortality. Thus, the aim of this study was to determine the seroprevalence of viral and bacterial pathogens among diagnosed malaria patients in malaria-endemic areas in Venezuela. METHODS: A cross-sectional study was conducted on malaria patients attending three reference medical centres in Ciudad Bolivar, Venezuela. Clinical evaluation and laboratory tests for dengue virus (DENV), chikungunya virus (CHIKV), viral hepatitis [hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV)], and leptospirosis (LEP) were performed by enzyme-linked immunosorbent assays. Previous exposure to these pathogens was defined by the presence of specific immunoglobulin (Ig) G, and co-infection or recent exposure (CoRE) was determined by the presence of specific IgM alone or IgM + IgG. Data analysis considered descriptive statistics. Parameter distribution was statistically evaluated using Kolmogorov-Smirnov test and the necessary comparison tests. Odds ratio (OR) for complications was determined according to CoRE presence with a 95% confidence interval (CI). RESULTS: A total of 161 malaria patients were studied, 66% infected with Plasmodium vivax, 27% with P. falciparum, and 7.5% harboured P. vivax/P. falciparum mixed infection. Previous exposure to DENV (60%) and CHIKV (25%) was frequent. CoRE was confirmed in 55 of the 161 malaria patients (34%) and were more frequent in P. falciparum (49%) than in P. vivax (29%) and mixed malaria patients (25%) (OR = 2.43, 95% CI: 1.39-4.25, P = 0.018). The most frequent CoRE was DENV (15%), followed by HAV (12%), HBV (6.2%), CHIKV (5.5%), and LEP (3.7%); HCV CoRE was absent. Complicated malaria was significantly more frequent in patients with CoRE (56%) than those without CoRE (36%; OR = 2.31, 95% CI: 1.18-4.92, P = 0.013). CONCLUSIONS: We found high CoRE prevalence in malaria patients as determined by serology in the study region; cases were associated with a worse clinical outcome. Further prospective studies with samples from different infection sites and the use of molecular tools are needed to determine the clinical significance of these findings.

A SARS-CoV-2 Spike Receptor Binding Motif Peptide Induces Anti-Spike Antibodies in Mice andIs Recognized by COVID-19 Patients.

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM436-507) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production.

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

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

Randomized clinical trial to assess the protective efficacy of a Plasmodium vivax CS synthetic vaccine.

A randomized, double-blind, controlled vaccine clinical trial was conducted to assess, as the primary outcome, the safety and protective efficacy of the Plasmodium vivax circumsporozoite (CS) protein in healthy malaria-naïve (phase IIa) and semi-immune (phase IIb) volunteers. Participants (n = 35) were randomly selected from a larger group (n = 121) and further divided into naïve (n = 17) and semi-immune (n = 18) groups and were immunized at months 0, 2, and 6 with PvCS formulated in Montanide ISA-51 adjuvant or placebo (adjuvant alone). Specific antibodies and IFN-γ responses to PvCS were determined as secondary outcome; all experimental volunteers developed specific IgG and IFN-γ. Three months after the last immunization, all participants were subjected to controlled human malaria infection. All naive controls became infected and drastic parasitemia reduction, including sterile protection, developed in several experimental volunteers in phase IIa (6/11) (54%, 95% CI 0.25-0.84) and phase IIb (7/11) (64%, 95% CI 0.35-0.92). However, no difference in parasitemia was observed between the phase IIb experimental and control subgroups. In conclusion, this study demonstrates significant protection in both naïve and semi-immune volunteers, encouraging further PvCS vaccine clinical development. Trial registration number NCT02083068. This trial was funded by Colciencias (grant 529-2009), NHLBI (grant RHL086488 A), and MVDC/CIV Foundation (grant 2014-1206).

Immunogenicity of full-length P. vivax rPvs48/45 protein formulations in BALB/c mice.

BACKGROUND: Pvs48/45 is a Plasmodium vivax gametocyte surface protein involved in the parasite fertilization process. Previous studies showed that Pvs48/45 proteins expressed in Escherichia coli (E. coli) and Chinese hamster ovary (CHO) cells were highly immunoreactive with sera from malaria-endemic areas and highly immunogenic in animal models. Here the immunogenicity in mice of three different vaccine formulations was compared. METHODS: Recombinant (r) Pvs48/45 proteins were expressed in E. coli and CHO, purified, formulated in Alhydrogel, GLA-SE and Montanide ISA-51 adjuvants and used to immunize BALB/c mice. Animals were immunized on days 0, 20 and 40, and serum samples were collected for serological analyses of specific antibody responses using ELISA and immunofluorescence (IFAT). Additionally, ex-vivo transmission-reducing activity (TRA) of sera on P. vivax gametocyte-infected human blood fed to Anopheles albimanus in direct membrane feeding assays (DMFA) was evaluated. RESULTS: Most immunized animals seroconverted after the first immunization, and some developed antibody peaks of 106 with all adjuvants. However, the three adjuvant formulations induced different antibody responses and TRA efficacy. While GLA-SE formulations of both proteins induced similar antibody profiles, Montanide ISA-51 formulations resulted in higher and longer-lasting antibody titers with CHO-rPvs48/45 than with the E. coli formulation. Although the CHO protein formulated in Alhydrogel generated a high initial antibody peak, antibody responses to both proteins rapidly waned. Likewise, anti-Pvs48/45 antibodies displayed differential recognition of the parasite proteins in IFAT and ex vivo blockade of parasite transmission to mosquitoes. The CHO-rPvs48/45 formulated in Montanide ISA-51 induced the most effective ex vivo parasite blockage. CONCLUSIONS: Three out of six vaccine formulations elicited antibodies with ex vivo TRA. The CHO-rPvs48/45 Montanide ISA-51 formulation induced the most stable antibody response, recognizing the native protein and the most robust ex vivo TRA. These results encourage further testing of the vaccine potential of this protein.

Immunoreactivity of Sera From Low to Moderate Malaria-Endemic Areas Against Plasmodium vivax rPvs48/45 Proteins Produced in Escherichia coli and Chinese Hamster Ovary Systems.

P48/45 is a conserved gametocyte antigen involved in Plasmodium parasite fertilization. A recombinant Plasmodium vivax P48/45 (Pvs48/45) protein expressed in Escherichia coli (E. coli) was highly antigenic and immunogenic in experimental animals and elicited specific transmission-blocking (TB) antibodies in a previous pilot study. Here, a similar Pvs48/45 gene was expressed in Chinese Hamster Ovary (CHO) cells and we compared its immunoreactivity with the E. coli product. Specific antibody titers were determined using plasma from Colombian individuals (n=227) living in endemic areas where both P. vivax and P. falciparum are prevalent and from Guatemala (n=54) where P. vivax is highly prevalent. In Colombia, plasma seroprevalence to CHO-rPvs48/45 protein was 46.3%, while for E. coli-rPvs48/45 protein was 36.1% (p<0.001). In Guatemala, the sero prevalence was 24.1% and 14.8% (p<0.001), respectively. Reactivity index (RI) against both proteins showed an age-dependent increase. IgG2 was the predominant subclass and the antibody avidity index evaluated by ELISA ranged between 4-6 mol/L. Ex vivo P. vivax mosquito direct membrane feeding assays (DMFA) performed in presence of study plasmas, displayed significant parasite transmission-blocking (TB), however, there was no direct correlation between antibody titers and oocysts transmission reduction activity (%TRA). Nevertheless, DMFA with CHO rPvs48/45 affinity purified IgG showed a dose response; 90.2% TRA at 100 µg/mL and 71.8% inhibition at 10 µg/mL. In conclusion, the CHO-rPvs48/45 protein was more immunoreactive in most of the malaria endemic places studied, and CHO-rPvs48/45 specific IgG showed functional activity, supporting further testing of the protein vaccine potential.

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

Cytokine signatures of Plasmodium vivax infection during pregnancy and delivery outcomes.

Plasmodium vivax malaria is a neglected disease, particularly during pregnancy. Severe vivax malaria is associated with inflammatory responses but in pregnancy immune alterations make it uncertain as to what cytokine signatures predominate, and how the type and quantity of blood immune mediators influence delivery outcomes. We measured the plasma concentrations of a set of thirty-one biomarkers, comprising cytokines, chemokines and growth factors, in 987 plasma samples from a cohort of 572 pregnant women from five malaria-endemic tropical countries and related these concentrations to delivery outcomes (birth weight and hemoglobin levels) and malaria infection. Samples were collected at recruitment (first antenatal visit) and at delivery (periphery, cord and placenta). At recruitment, we found that P. vivax-infected pregnant women had higher plasma concentrations of proinflammatory (IL-6, IL-1ß, CCL4, CCL2, CXCL10) and TH1-related cytokines (mainly IL-12) than uninfected women. This biomarker signature was essentially lost at delivery and was not associated with birth weight nor hemoglobin levels. Antiinflammatory cytokines (IL-10) were positively associated with infection and poor delivery outcomes. CCL11 was the only biomarker to show a negative association with P. vivax infection and its concentration at recruitment was positively associated with hemoglobin levels at delivery. Birth weight was negatively associated with peripheral IL-4 levels at delivery. Our multi-biomarker multicenter study is the first comprehensive one to characterize the immunological signature of P. vivax infection in pregnancy thus far. In conclusion, data show that while TH1 and pro-inflammatory responses are dominant during P. vivax infection in pregnancy, antiinflammatory cytokines may compensate excessive inflammation avoiding poor delivery outcomes, and skewness toward a TH2 response may trigger worse delivery outcomes. CCL11, a chemokine largely neglected in the field of malaria, emerges as an important marker of exposure or mediator in this condition.

Blood cytokine, chemokine and growth factor profiling in a cohort of pregnant women from tropical countries.

The immune status of women changes during and after pregnancy, differs between blood compartments at delivery and is affected by environmental factors particularly in tropical areas endemic for multiple infections. We quantified the plasma concentration of a set of thirty-one TH1, TH2, TH17 and regulatory cytokines, pro-inflammatory and anti-inflammatory cytokines and chemokines, and growth factors (altogether biomarkers), in a cohort of 540 pregnant women from five malaria-endemic tropical countries. Samples were collected at recruitment (first antenatal visit), delivery (periphery, cord and placenta) and postpartum, allowing a longitudinal analysis. We found the lowest concentration of biomarkers at recruitment and the highest at postpartum, with few exceptions. Among them, IL-6, HGF and TGF-ß had the highest levels at delivery, and even higher concentrations in the placenta compared to peripheral blood. Placental concentrations were generally higher than peripheral, except for eotaxin that was lower. We also compared plasma biomarker concentrations between the tropical cohort and a control group from Spain at delivery, presenting overall higher biomarker levels the tropical cohort, particularly pro-inflammatory cytokines and growth factors. Only IL-6 presented lower levels in the tropical group. Moreover, a principal component analysis of biomarker concentrations at delivery showed that women from Spain grouped more homogenously, and that IL-6 and IL-8 clustered together in the tropical cohort but not in the Spanish one. Plasma cytokine concentrations correlated with Plasmodium antibody levels at postpartum but not during pregnancy. This basal profiling of immune mediators over gestation and in different compartments at delivery is important to subsequently understand response to infections and clinical outcomes in mothers and infants in tropical areas.

Prevalence of dengue antibodies in healthy children and adults in different Colombian endemic areas.

OBJECTIVES: Colombia is a dengue hyperendemic country; however, the prevalence of antibodies against dengue in the general population including the inhabitants of rural areas is unknown. This study aimed to determine the prevalence of dengue IgM and IgG antibodies in healthy children and adults in urban and rural areas of seven different endemic regions in Colombia between 2013 and 2015. DESIGN OR METHOD: Blood samples from healthy volunteers (1,318) were processed by serology (by indirect IgG and capture IgM and IgG ELISA) and molecular tests to detect viral RNA and circulating serotypes. RESULTS: The seroprevalence of IgG for dengue were 85% in children and over 90% for adults. In addition to the high IgM positive rate (14.9%) and secondary recent infection marker rate (capture IgG, 16%), 8.4% of the healthy volunteers were positive for dengue virus (DENV) RNA. CONCLUSION: This study confirmed the broad and permanent circulation of DENV in Colombia and the high rates of infection and reinfection suffered by its inhabitants. This information can be used by the health authorities to strengthen vector control and vaccine policies and review the algorithms of diagnosis and disease management in children and adults.

Malaria y coinfecciones en una región endémica de Venezuela

Introducción: La malaria continúa siendo un importante problema de salud pública en todo el mundo. Las coinfecciones son un factor de riesgo que incrementa la mortalidad de esta enfermedad. En Venezuela no existen estudios que describan la presencia de coinfecciones en pacientes con malaria. Nosotros determinamos las características clínicas y epidemiológicas de los pacientes con malaria y la presencia de coinfecciones en Ciudad Bolívar, Estado Bolívar, Venezuela. Metodología: Se realizó un estudio descriptivo, correlacional y transversal, que incluyó pacientes diagnosticados con malaria por prueba rápida y/o gota gruesa y extendido de sangre periférica que consultaron en tres centros médicos de Ciudad Bolívar, estado Bolívar, entre junio y noviembre de 2018. Se realizó una evaluación clínica y de laboratorio de cada paciente, las coinfecciones con Dengue (VD), Hepatitis viral (HV) (A, B y C), Leptospirosis (LP), y Chikungunya (VCHIK) fueron evaluadas mediante la técnica de ELISA. Resultados: Un total de 161 pacientes fueron estudiados, 106 (65,8 %) presentaron infección por P. vivax, 43 (26,7 %) por P. falciparum y 12 (7,4 %) tenían malaria mixta (Pf/Pv). La media de edad fue 33,8 (±13,43) años; 103 (63,9 %) fueron hombres, la raza más frecuente fue mestiza (94,4 %); la mayoría de los pacientes (37,3 %) practicaban la minería ilegal. Los síntomas más frecuentes fueron fiebre, escalofríos y cefalea. Anemia leve, trombocitopenia moderada, y compromiso de la función hepática fueron los hallazgos de laboratorio más relevantes en todas las especies parasitarias. Se encontró coinfección en 55/161 (34,2 %) pacientes, siendo más frecuente entre los pacientes con P. falciparum (48,8 %). La coinfección más frecuente fue con VD (14,9 %), seguida de VHA (11,8 %)VHB (6,2 %), VCHIK (5,5 %) y LP (3,7 %). En el grupo de coinfectados fue más frecuente la malaria complicada (56,36 %) que la no complicada (43,63 %) con una diferencia estadísticamente significativa (P=0,018). Conclusión: Se encontró una alta prevalencia de coinfecciones en los pacientes con malaria, y su asociación con la severidad de la Malaria, estos datos epidemiológicos influyen de manera directa en el curso clínico, así como en la mortalidad de la enfermedad. Estos hallazgos deben darse a conocer al personal de salud para la identificación oportuna de coinfecciones en estos pacientes.

Introduction: Malaria continues to be a major public health problem worldwide. Co-infections are a risk factor that increases the mortality of this disease. In Venezuela there are no studies describing the presence of coinfections in patients with malaria. We determine the clinical and epidemiological characteristics of patients with malaria and the presence of coinfections in Ciudad Bolívar, Bolívar state, Venezuela. Methodology: A descriptive, correlational and cross-sectional study was carried out, which included patients diagnosed with malaria by rapid test and / or thick and extended peripheral blood drop that they consulted in three medical centers in Ciudad Bolívar, Bolívar state, between June and November 2018 A clinical and laboratory evaluation of each patient was performed, coinfections with Dengue (DV), viral hepatitis (HV) (A, B and C), Leptospirosis (LP), and Chikungunya (VCHIK) were evaluated using the technique of ELISA Results: A total of 161 patients were studied, 106 (65.8 %) had P. vivax infection, 43 (26.7 %) due to P. falciparum and 12 (7.4 %) had mixed malaria (Pf / Pv ). The mean age was 33.8 (± 13.43) years; 103 (63.9 %) were men, the most frequent race was mestizo (94.4 %); the majority of patients (37.3 %) practiced illegal mining. The most frequent symptoms were fever, chills and headache. Mild anemia, moderate thrombocytopenia, and hepatic function impairment were the most relevant laboratory findings in all parasitic species. Coinfection was found in 55/161 (34.2 %) patients, being more frequent among patients with P. falciparum (48.8 %). The most frequent coinfection was with RV (14.9 %), followed by HAV (11.8 %) HBV (6.2 %), HCV (5.5 %) and LP (3.7 %). Complicated malaria (56.36 %) was more frequent than uncomplicated (43.63 %) with a statistically significant difference (P = 0.018). Conclusion: A high prevalence of coinfections was found in patients with malaria, and its association with the severity of Malaria, these epidemiological data directly influence the clinical course, as well as the mortality of the disease. These findings should be made known to health personnel for the timely identification of coinfections in these patients.

A Multi-Stage Plasmodium vivax Malaria Vaccine Candidate Able to Induce Long-Lived Antibody Responses Against Blood Stage Parasites and Robust Transmission-Blocking Activity.

Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4+ T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.

Limited differentiation among Plasmodium vivax populations from the northwest and to the south Pacific Coast of Colombia: A malaria corridor?

BACKGROUND: Malaria remains endemic in several countries of South America with low to moderate transmission intensity. Regional human migration through underserved endemic areas may be responsible for significant parasite dispersion making the disease resilient to interventions. Thus, the genetic characterization of malarial parasites is an important tool to assess how endemic areas may connect via the movement of infected individuals. Here, four sites in geographically separated areas reporting 80% of the malaria morbidity in Colombia were studied. The sites are located on an imaginary transect line of 1,500 km from the northwest to the south Pacific Coast of Colombia with a minimal distance of 500 km between populations that display noticeable ethnic, economic, epidemiological, and ecological differences. METHODOLOGY/PRINCIPAL FINDINGS: A total of 624 Plasmodium vivax samples from the four populations were genotyped by using eight microsatellite loci. Although a strong geographic structure was expected between these populations, only moderate evidence of genetic differentiation was observed using a suite of population genetic analyses. High genetic diversity, shared alleles, and low linkage disequilibrium were also found in these P. vivax populations providing no evidence for a bottleneck or clonal expansions as expected from recent reductions in the transmission that could have been the result of scaling up interventions or environmental changes. These patterns are consistent with a disease that is not only endemic in each site but also imply that there is gene flow among these populations across 1,500 km. CONCLUSION /SIGNIFICANCE: The observed patterns in P. vivax are consistent with a "corridor" where connected endemic areas can sustain a high level of genetic diversity locally and can restore parasite-subdivided populations via migration of infected individuals even after local interventions achieved a substantial reduction of clinical cases. The consequences of these findings in terms of control and elimination are discussed.

Malaria systems immunology: Plasmodium vivax induces tolerance during primary infection through dysregulation of neutrophils and dendritic cells.

OBJECTIVES: To dissect the transcriptional networks underpinning immune cells responses during primary Plasmodium vivax infection of healthy human adults. METHODS: We conducted network co-expression analysis of next-generation RNA sequencing data from whole blood from P. vivax and P. falciparum controlled human malaria infection (CHMI) of healthy naïve and malaria-exposed volunteers. Single cell transcription signatures were used to deconvolute the bulk RNA-Seq data into cell-specific signals. RESULTS: Initial exposure to P. vivax induced activation of innate immunity, including efficient antigen presentation and complement activation. However, this effect was accompanied by strong immunosuppression mediated by dendritic cells via the induction of Indoleamine 2,3-Dioxygenase 1(IDO1) and Lymphocyte Activation Gene 3 (LAG3). Additionally, P. vivax induced depletion of neutrophil populations associated with down regulation of 3G-protein coupled receptors, CRXCR1, CXCR2 and CSF3R. Accordingly, in malaria-exposed volunteers the inflammatory response was attenuated, with a decreased class II antigen presentation in dendritic cells. While the immunosuppressive signalling was maintained between plasmodium species, response to P. falciparum was significantly more immunogenic. CONCLUSIONS: In silico analyses suggest that primary infection with P. vivax induces potent immunosuppression mediated by dendritic cells, conditioning subsequent anti-malarial immune responses. Targeting immune evasion mechanisms could be an effective alternative for improving vaccine efficacy.

Individualized Transcriptional Resolution of Complicated Malaria in a Colombian Study.

To evaluate whether recovery from complicated malaria follows a common trajectory in terms of immunological mechanism or, rather, is highly individualized for each patient, we performed longitudinal gene expression profiling of whole blood. RNA sequencing (RNAseq) was performed on blood samples obtained from eight patients on four consecutive days between hospital admission and discharge. Six patients were infected with Plasmodium falciparum, and two with Plasmodium vivax; one patient was a pregnant woman infected with P. falciparum, who was hospitalized for several weeks. The characterization of blood transcript modules (BTM) and blood informative transcripts (BIT) revealed that patients' responses showed little commonality, being dominated by the balance of gene activity relating to lymphocyte function, inflammation, and interferon responses specific to each patient. Only weak correlations with specific complicated malaria symptoms such as jaundice, thrombocytopenia, or anemia were observed. The differential expression of individual genes, including transcripts derived from the human leukocyte antigen (HLA) complex, generally reflected differences in the underlying immune processes. Although the results of this pilot study do not point to any single process that might provide a target for complicated malaria treatment or prevention or personalized medical strategies, larger patient series and more extensive blood sampling may allow the classification of patients according to their type of response in order to develop novel therapeutic approaches.

Patients' costs, socio-economic and health system aspects associated with malaria in pregnancy in an endemic area of Colombia.

Malaria in pregnancy threatens birth outcomes and the health of women and their newborns. This is also the case in low transmission areas, such as Colombia, where Plasmodium vivax is the dominant parasite species. Within the Colombian health system, which underwent major reforms in the 90s, malaria treatment is provided free of charge to patients. However, patients still incur costs, such as transportation and value of time lost due to the disease. We estimated such costs among 40 pregnant women with clinical malaria (30% Plasmodium falciparum, 70% Plasmodium vivax) in the municipality of Tierralta, Northern Colombia. In a cross-sectional study, women were interviewed after an outpatient or inpatient laboratory confirmed malaria episode. Women were asked to report all types of cost incurred before (including prevention), during and immediately after the contact with the health facility. Median total cost was over 16US$ for an outpatient visit, rising to nearly 30US$ if other treatments were sought before reaching the health facility. Median total inpatient cost was 26US$ or 54US$ depending on whether costs incurred prior to admission were excluded or included. For both outpatients and inpatients, direct costs were largely due to transportation and indirect costs constituted the largest share of total costs. Estimated costs are likely to represent only one of the constraints that women face when seeking treatment in an area characterized, at the time of the study, by armed conflict, displacement, and high vulnerability of indigenous women, the group at highest risk of malaria. Importantly, the Colombian peace process, which culminated with the cease-fire in August 2016, may have a positive impact on achieving universal access to healthcare in conflict areas. The current study can inform malaria elimination initiatives in Colombia.

IgG Responses to the Plasmodium falciparum Antigen VAR2CSA in Colombia Are Restricted to Pregnancy and Are Not Induced by Exposure to Plasmodium vivax.

Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.

Integrative metabolomics and transcriptomics signatures of clinical tolerance to Plasmodium vivax reveal activation of innate cell immunity and T cell signaling.

Almost invariably, humans become ill during primary infections with malaria parasites which is a pathology associated with oxidative stress and perturbations in metabolism. Importantly, repetitive exposure to Plasmodium results in asymptomatic infections, which is a condition defined as clinical tolerance. Integration of transcriptomics and metabolomics data provides a powerful way to investigate complex disease processes involving oxidative stress, energy metabolism and immune cell activation. We used metabolomics and transcriptomics to investigate the different clinical outcomes in a P. vivax controlled human malaria infection trial. At baseline, the naïve and semi-immune subjects differed in the expression of interferon related genes, neutrophil and B cell signatures that progressed with distinct kinetics after infection. Metabolomics data indicated differences in amino acid pathways and lipid metabolism between the two groups. Top pathways during the course of infection included methionine and cysteine metabolism, fatty acid metabolism and urea cycle. There is also evidence for the activation of lipoxygenase, cyclooxygenase and non-specific lipid peroxidation products in the semi-immune group. The integration of transcriptomics and metabolomics revealed concerted molecular events triggered by the infection, notably involving platelet activation, innate immunity and T cell signaling. Additional experiment confirmed that the metabolites associated with platelet activation genes were indeed enriched in the platelet metabolome.