Vanessa Cristina Nicolete

Background: Relatively few Amazonian infants have clinical malaria diagnosed, treated and notified before their first birthday, either because they are little exposed to an infection or remain asymptomatic once infected. Here we measure the proportion of children who have experienced Plasmodium vivax infection and malaria by 2 years of age in the main transmission hotspot of Amazonian Brazil. Methods: We measured IgG antibodies to 3 blood-stage P. vivax antigens at the 1- and 2-year follow-up assessment of 435 participants in a population-based birth cohort. Children’s malaria case notifications were retrieved from the electronic database of the Ministry of Health. We used multiple Poisson regression models to identify predictors of serologically proven P. vivax infection and clinical vivax malaria during the first 2 years of life. Results: Overall, 23 [5.3%; 95% confidence interval (CI): 3.5–7.8%) children had antibodies to ≥2 antigens detected during at least one follow-up assessment, consistent with past P. vivax infection(s). Fifteen (3.4%; 95% CI: 2.1–5.6%) children had clinical vivax episodes notified during the first 2 years of life; 7 of them were seronegative. We estimate that half of the infections remained unnotified. Children born to women who experienced P. vivax infection during pregnancy were more likely to be infected and develop clinical vivax malaria, while those breast-fed for ≥12 months had their risk of being P. vivax-seropositive (which we take as evidence of blood-stage P. vivax infection during the first 2 years of life) decreased by 79.8% (95% CI: 69.3–86.7%). Conclusion: P. vivax infections in early childhood are underreported in the Amazon, are associated with anemia at 2 years of age, and appear to be partially prevented by prolonged breastfeeding.

Background: Malaria causes significant morbidity and mortality in children under 5 years of age in sub-Saharan Africa and the Asia-Pacific region. Neonates and young infants remain relatively protected from clinical disease and the transplacental transfer of maternal antibodies is hypothesized as one of the protective factors. The adverse health effects of Plasmodium vivax malaria in early childhood–traditionally viewed as a benign infection–remain largely neglected in relatively low-endemicity settings across the Amazon. Methodology/Principal findings: Overall, 1,539 children participating in a birth cohort study in the main transmission hotspot of Amazonian Brazil had a questionnaire administered, and blood sampled at the two-year follow-up visit. Only 7.1% of them experienced malaria confirmed by microscopy during their first 2 years of life– 89.1% of the infections were caused by P. vivax. Young infants appear to be little exposed to, or largely protected from infection, but children >12 months of age become as vulnerable to vivax malaria as their mothers. Few (1.4%) children experienced ≥4 infections during the 2-year follow-up, accounting for 43.4% of the overall malaria burden among study participants. Antenatal malaria diagnosed by microscopy during pregnancy or by PCR at delivery emerged as a significant correlate of subsequent risk of P. vivax infection in the offspring (incidence rate ratio, 2.58; P = 0.002), after adjusting for local transmission intensity. Anti-P. vivax antibodies measured at delivery do not protect mothers from subsequent malaria; whether maternal antibodies transferred to the fetus reduce early malaria risk in children remains undetermined. Finally, recent and repeated vivax malaria episodes in early childhood are associated with increased risk of anemia at the age of 2 years in this relatively low-endemicity setting. Conclusions/Significance: Antenatal infection increases the risk of vivax malaria in the offspring and repeated childhood P. vivax infections are associated with anemia at the age of 2 years.

The Plasmodium vivax Duffy-binding protein (DBP) is a prime target of the protective immune response and a promising vaccine candidate for P. vivax malaria. Naturally acquired immunity (NAI) protects against malaria in adults residing in infection-endemic regions, and the passive transfer of malarial immunity confers protection. A vaccine that replicates NAI will effectively prevent disease. Here, we report the structures of DBP region II in complex with human-derived, neutralizing monoclonal antibodies obtained from an individual in a malaria-endemic area with NAI. We identified protective epitopes using X-ray crystallography, hydrogen–deuterium exchange mass spectrometry, mutational mapping and P. vivax invasion studies. These approaches reveal that naturally acquired human antibodies neutralize P. vivax by targeting the binding site for Duffy antigen receptor for chemokines (DARC) and the dimer interface of P. vivax DBP. Antibody binding is unaffected by polymorphisms in the vicinity of epitopes, suggesting that the antibodies have evolved to engage multiple polymorphic variants of DBP. The human antibody epitopes are broadly conserved and are distinct from previously defined epitopes for broadly conserved murine monoclonal antibodies. A library of globally conserved epitopes of neutralizing human antibodies offers possibilities for rational design of strain-transcending DBP-based vaccines and therapeutics against P. vivax.

Plasmodium vivax invasion of reticulocytes relies on distinct receptor-ligand interactions between the parasite and host erythrocytes. Engagement of the highly polymorphic domain II of the P. vivax Duffy-binding protein (DBPII) with the erythrocyte’s Duffy Ag receptor for chemokines (DARC) is essential. Some P. vivax–exposed individuals acquired Abs to DBPII that block DBPII-DARC interaction and inhibit P. vivax reticulocyte invasion, and Ab levels correlate with protection against P. vivax malaria. To better understand the functional characteristics and fine specificity of protective human Abs to DBPII, we sorted single DBPII-specific IgG⁺ memory B cells from three individuals with high blocking activity to DBPII. We identified 12 DBPII-specific human mAbs from distinct lineages that blocked DBPII-DARC binding. All mAbs were P. vivax strain transcending and targeted known binding motifs of DBPII with DARC. Eleven mAbs competed with each other for binding, indicating recognition of the same or overlapping epitopes. Naturally acquired blocking Abs to DBPII from individuals with high levels residing in different P. vivax–endemic areas worldwide competed with mAbs, suggesting broadly shared recognition sites. We also found that mAbs inhibited P. vivax entry into reticulocytes in vitro. These findings suggest that IgG⁺ memory B cell activity in individuals with P. vivax strain–transcending Abs to DBPII display a limited clonal response with inhibitory blocking directed against a distinct region of the molecule. Key Points P. vivax DPB:DARC interaction is essential for RBC invasion. vivax–exposed individuals acquire Abs that block this interaction. Human-derived monoclonal Abs recognize a globally conserved blocking epitope.

Antibodies to the cysteine-rich domain II of Plasmodium vivax Duffy binding protein (PvDBP) can inhibit binding of this parasite ligand to its receptor on red blood cells, the Duffy antigen/receptor for chemokines. These binding-inhibitory antibodies (BIAbs) also inhibit P. vivax invasion of reticulocytes in vitro. To investigate whether naturally acquired anti-PvDBP antibodies are associated with reduced risk of clinical malaria in a population exposed to low levels of P. vivax transmission, we measured total levels of immunoglobulin G antibodies to 5 PvDBP variants and used a functional in vitro assay to quantify their binding-inhibitory activity in a cohort of 466 rural Amazonians followed up for up to 37 months. No association between total immunoglobulin G antibody responses to any PvDBP variant and risk of symptomatic, laboratory-confirmed vivax malaria was observed in this cohort. However, a Cox proportional hazards model, adjusted for age, sex, and genotype for the Duffy antigen/receptor for chemokines, showed a >40% decrease in the prospective risk of clinical vivax malaria in subjects with the strongest BIAb responses (upper and middle terciles). High BIAb responses were mostly PvDBP variant transcending and stable over time. Strong naturally acquired BIAb responses are associated with a reduced risk of clinical P. vivax malaria in rural Amazonians.

New frontier settlements across the Amazon Basin pose a major challenge for malaria elimination in Brazil. Here we describe the epidemiology of malaria during the early phases of occupation of farming settlements in Remansinho area, Brazilian Amazonia. We examine the relative contribution of low-density and asymptomatic parasitemias to the overall Plasmodium vivax burden over a period of declining transmission and discuss potential hurdles for malaria elimination in Remansinho and similar settings. Eight community-wide cross-sectional surveys, involving 584 subjects, were carried out in Remansinho over 3 years and complemented by active and passive surveillance of febrile illnesses between the surveys. We used quantitative PCR to detect low-density asexual parasitemias and gametocytemias missed by conventional microscopy. Mixed-effects multiple logistic regression models were used to characterize independent risk factors for P. vivax infection and disease. P. vivax prevalence decreased from 23.8% (March–April 2010) to 3.0% (April–May 2013), with no P. falciparum infections diagnosed after March–April 2011. Although migrants from malaria-free areas were at increased risk of malaria, their odds of having P. vivax infection and disease decreased by 2–3% with each year of residence in Amazonia. Several findings indicate that low-density and asymptomatic P. vivax parasitemias may complicate residual malaria elimination in Remansinho: (a) the proportion of subpatent infections (i.e. missed by microscopy) increased from 43.8% to 73.1% as P. vivax transmission declined; (b) most (56.6%) P. vivax infections were asymptomatic and 32.8% of them were both subpatent and asymptomatic; (c) asymptomatic parasite carriers accounted for 54.4% of the total P. vivax biomass in the host population; (d) over 90% subpatent and asymptomatic P. vivax had PCR-detectable gametocytemias; and (e) few (17.0%) asymptomatic and subpatent P. vivax infections that were left untreated progressed to clinical disease over 6 weeks of follow-up and became detectable by routine malaria surveillance. Despite decades of control efforts, malaria remains a major public health concern in Brazil. A large proportion of the 243,000 cases diagnosed per year originate from areas of recent colonization in the densely forested Amazon Basin. This population-based longitudinal study addresses the epidemiology of malaria during the early stages of colonization of frontier settlements in Remansinho area, rural Amazonia. We documented a major decline in the prevalence of P. vivax infection, from 23.8% to 3.0%, between March–April 2010 and April–May 2013. Up to 73.1% of the P. vivax infections were missed by microscopy as malaria transmission declined and most (56.6%) of these infections caused no clinical signs or symptoms. Few (17.0%) asymptomatic P. vivax infections that were left untreated eventually progressed to clinical disease, becoming detectable by routine malaria surveillance, over 6 weeks of follow-up. Moreover, nearly all P. vivax infections that were undetected by microscopy had gametocytes, the parasite's blood stages responsible for malaria transmission to mosquito vectors, detected by molecular methods. These findings indicate that apparently healthy carriers of low-density parasitemias, who are often missed by conventional microscopy, contribute significantly to ongoing P. vivax transmission and may further complicate residual malaria elimination in Remansinho and similar endemic settings.

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.