Plasmodium vivax Reticulocyte Binding Proteins Are Key Targets of Naturally Acquired Immunity in Young Papua New Guinean Children

Background: Major gaps in our understanding of Plasmodium vivax biology and the acquisition of immunity to this parasite hinder vaccine development. P. vivax merozoites exclusively invade reticulocytes, making parasite proteins that mediate reticulocyte binding and/or invasion potential key vaccine or drug targets. While protein interactions that mediate invasion are still poorly understood, the P. vivax Reticulocyte-Binding Protein family (PvRBP) is thought to be involved in P. vivax restricted host-cell selectivity. Methodology/Principal findings: We assessed the binding specificity of five members of the PvRBP family (PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, PvRBP2-P2 and a non-binding fragment of PvRBP2c) to normocytes or reticulocytes. PvRBP2b was identified as the only reticulocyte-specific binder (P<0.001), whereas the others preferentially bound to normocytes (PvRBP1a/b P≤0.034), or showed comparable binding to both (PvRBP2a/2-P2, P = 0.38). Furthermore, we measured levels of total and IgG subclasses 1, 2, 3 and 4 to the six PvRBPs in a cohort of young Papua New Guinean children, and assessed their relationship with prospective risk of P. vivax malaria. Children had substantial, highly correlated (rho = 0.49–0.82, P<0.001) antibody levels to all six PvRBPs, with dominant IgG1 and IgG3 subclasses. Both total IgG (Incidence Rate Ratio [IRR] 0.63–0.73, P = 0.008–0.041) and IgG1 (IRR 0.56–0.69, P = 0.001–0.035) to PvRBP2b and PvRBP1a were strongly associated with reduced risk of vivax-malaria, independently of age and exposure. Conclusion/Significance: These results demonstrate a diversity of erythrocyte-binding phenotypes of PvRBPs, indicating binding to both reticulocyte-specific and normocyte-specific ligands. Our findings provide further insights into the naturally acquired immunity to P. vivax and highlight the importance of PvRBP proteins as targets of naturally acquired humoral immunity. In-depth studies of the role of PvRBPs in P. vivax invasion and functional validation of the role of anti-PvRBP antibodies in clinical immunity against P. vivax are now required to confirm the potential of the reticulocyte-binding PvRBP2b and PvRBP1a as vaccine candidate antigens

Pharmacokinetics of lamivudine and lamivudine-triphosphate after administration of 300 milligrams and 150 milligrams once daily to healthy volunteers: Results of the ENCORE 2 Study

There is interest in evaluating the efficacy of lower doses of certain antiretrovirals for clinical care. We determined here the bio-equivalence of plasma lamivudine (3TC) and intracellular 3TC-triphosphate (3TC-TP) concentrations after the administration of two different doses. ENCORE 2 was a randomized crossover study. Subjects received 3TC at 300 and 150 mg once daily for 10 days (arm 1; n = 13) or vice versa (arm 2; n = 11), separated by a 10-day washout. Pharmacokinetic (PK) profiles (0 to 24 h) were assessed on days 10 and 30. Plasma 3TC and 3TC-TP levels in peripheral blood mononuclear cells were quantified by high-performance liquid chromatography-tandem mass spectrometry. Within-subject changes in PK parameters (the area under the concentration-time curve from 0 to 24 h [AUC0-24], the trough concentration of drug in plasma at 24 h [C24], and the maximum concentration of drug in plasma [Cmax]) were evaluated by determining the geometric mean ratios (GMRs) adjusted for study arm, period, and intra-individual variation. Regimens were considered bioequivalent if the 90% confidence interval (90% CI) fell within the range of 0.8 to 1.25. A total of 24 subjects completed the study. The GM (90% CI) 3TC AUC0-24), expressed as ng•h/ml, for the 300- and 150-mg doses were 8,354 (7,609 to 9,172) and 4,773 (4,408 to 5,169), respectively. Bioequivalence in 3TC PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC0-24, C24, and Cmax were 0.57 (0.55 to 0.60), 0.63 (0.59 to 0.67), and 0.56 (0.53 to 0.60), respectively. The GM (90% CI) 3TC-TP AUC0-24 values (pmol•h/106 cells) for the 300- and 150-mg doses were 59.5 (51.8 to 68.3) and 44.0 (38.0 to 51.0), respectively. Bioequivalence in 3TC-TP PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC0-24, C24, and Cmax were 0.73 (0.64 to 0.83), 0.82 (0.68 to 0.99), and 0.70 (0.61 to 0.82), respectively. We found that 3TC at 150 mg is not bioequivalent to the standard regimen of 300 mg, indicating that saturation of cytosine phosphorylation pathways is not achieved at a dose of 150 mg

Differential Patterns of Infection and Disease with P. falciparum and P. vivax in Young Papua New Guinean Children

BACKGROUND: Where P. vivax and P. falciparum occur in the same population, the peak burden of P. vivax infection and illness is often concentrated in younger age groups. Experiences from malaria therapy patients indicate that immunity is acquired faster to P. vivax than to P. falciparum challenge. There is however little prospective data on the comparative risk of infection and disease from both species in young children living in co-endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: A cohort of 264 Papua New Guinean children aged 1-3 years (at enrolment) were actively followed-up for Plasmodium infection and febrile illness for 16 months. Infection status was determined by light microscopy and PCR every 8 weeks and at each febrile episode. A generalised estimating equation (GEE) approach was used to analyse both prevalence of infection and incidence of clinical episodes. A more pronounced rise in prevalence of P. falciparum compared to P. vivax infection was evident with increasing age. Although the overall incidence of clinical episodes was comparable (P. falciparum: 2.56, P. vivax 2.46 episodes / child / yr), P. falciparum and P. vivax infectious episodes showed strong but opposing age trends: P. falciparum incidence increased until the age of 30 months with little change thereafter, but incidence of P. vivax decreased significantly with age throughout the entire age range. For P. falciparum, both prevalence and incidence of P. falciparum showed marked seasonality, whereas only P. vivax incidence but not prevalence decreased in the dry season. CONCLUSIONS/SIGNIFICANCE: Under high, perennial exposure, children in PNG begin acquiring significant clinical immunity, characterized by an increasing ability to control parasite densities below the pyrogenic threshold to P. vivax, but not to P. falciparum, in the 2(nd) and 3(rd) year of life. The ability to relapse from long-lasting liver-stages restricts the seasonal variation in prevalence of P. vivax infections

Features and Prognosis of Severe Malaria Caused by Plasmodium falciparum, Plasmodium vivax and Mixed Plasmodium Species in Papua New Guinean Children

BACKGROUND: Mortality from severe pediatric falciparum malaria appears low in Oceania but Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking. METHODS AND FINDINGS: We followed 340 Papua New Guinean (PNG) children with PCR-confirmed severe malaria (77.1% P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax) hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8%) presented with hypoglycemia, seven (2.7%) were discharged with neurologic impairment, and one child died (0.4%). The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P=0.001); one child died (3.7%). The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality (8.0%; P=0.003 vs falciparum malaria). Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO). CONCLUSIONS: The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis

How Much Remains Undetected? Probability of Molecular Detection of Human Plasmodia in the Field

BACKGROUND: In malaria endemic areas, most people are simultaneously infected with different parasite clones. Detection of individual clones is hampered when their densities fluctuate around the detection limit and, in case of P. falciparum, by sequestration during part of their life cycle. This has important implications for measures of levels of infection or for the outcome of clinical trials. This study aimed at measuring the detectability of individual P. falciparum and P. vivax parasite clones in consecutive samples of the same patient and at investigating the impact of sampling strategies on basic epidemiological measures such as multiplicity of infection (MOI). METHODS: Samples were obtained in a repeated cross-sectional field survey in 1 to 4.5 years old children from Papua New Guinea, who were followed up in 2-monthly intervals over 16 months. At each follow-up visit, two consecutive blood samples were collected from each child at intervals of 24 hours. Samples were genotyped for the polymorphic markers msp2 for P. falciparum and msp1F3 and MS16 for P. vivax. Observed prevalence and mean MOI estimated from single samples per host were compared to combined data from sampling twice within 24 h. FINDINGS AND CONCLUSION: Estimated detectability was high in our data set (0.79 [95% CI 0.76-0.82] for P. falciparum and, depending on the marker, 0.61 [0.58-0.63] or 0.73 [0.71-0.75] for P. vivax). When genotyping data from sequential samples, collected 24 hours apart, were combined, the increase in measured prevalence was moderate, 6 to 9% of all infections were missed on a single day. The effect on observed MOI was more pronounced, 18 to 31% of all individual clones were not detected in a single bleed. Repeated sampling revealed little difference between detectability of P. falciparum and P. vivax

Comparison of Plasmodium falciparum allelic frequency distribution in different endemic settings by high-resolution genotyping

BACKGROUND: The diversity of genotyping markers of Plasmodium falciparum depends on transmission intensity. It has been reported that the diversity of the merozoite surface protein 2 (msp2) is greater in areas of high compared to low endemicity, however, results for msp1 were inconsistent. These previous reports relied on low resolution genotyping techniques. METHODS: In the present study, a high-resolution capillary electrophoresis-based technique was applied to genotype samples from areas of different endemicity in Papua New Guinea and Tanzania. For both endemic settings, the diversity of msp1 and msp2 was investigated; the mean multiplicity of infection (MOI) and the FST values were determined to investigate whether more accurate sizing generates different results. RESULTS AND CONCLUSION: The results of the present study confirmed previous reports of a higher mean MOI for both marker genes and increased genetic diversity in areas of higher endemicity as estimated by the total number of distinct alleles for msp2. For msp1 a minor increase in diversity was observed. Measures of between population variance in allele frequencies (FST) indicated little genetic differentiation for both marker genes between the two populations from different endemic settings. MOI adjusted for the probability of multiple infections sharing the same allele was estimated by using the msp2 allele frequency distribution and the distribution of observed numbers of concurrent infections. For the high-resolution typing technique applied in this study, this adjustment made little difference to the estimated mean MOI compared to the observed mean MO

Reduced risk of Plasmodium vivax malaria in Papua New Guinean children with Southeast Asian ovalocytosis in two cohorts and a case-control study

BACKGROUND: The erythrocyte polymorphism, Southeast Asian ovalocytosis (SAO) (which results from a 27-base pair deletion in the erythrocyte band 3 gene, SLC4A1Delta27) protects against cerebral malaria caused by Plasmodium falciparum; however, it is unknown whether this polymorphism also protects against P. vivax infection and disease. METHODS AND FINDINGS: The association between SAO and P. vivax infection was examined through genotyping of 1,975 children enrolled in three independent epidemiological studies conducted in the Madang area of Papua New Guinea. SAO was associated with a statistically significant 46% reduction in the incidence of clinical P. vivax episodes (adjusted incidence rate ratio [IRR] = 0.54, 95% CI 0.40-0.72, p>0.0001) in a cohort of infants aged 3-21 months and a significant 52% reduction in P. vivax (blood-stage) reinfection diagnosed by PCR (95% CI 22-71, p = 0.003) and 55% by light microscopy (95% CI 13-77, p = 0.014), respectively, in a cohort of children aged 5-14 years. SAO was also associated with a reduction in risk of P. vivax parasitaemia in children 3-21 months (1,111/microl versus 636/microl, p = 0.011) and prevalence of P. vivax infections in children 15-21 months (odds ratio [OR] = 0.39, 95% CI 0.23-0.67, p = 0.001). In a case-control study of children aged 0.5-10 years, no child with SAO was found among 27 cases with severe P. vivax or mixed P. falciparum/P. vivax malaria (OR = 0, 95% CI 0-1.56, p = 0.11). SAO was associated with protection against severe P. falciparum malaria (OR = 0.38, 95% CI 0.15-0.87, p = 0.014) but no effect was seen on either the risk of acquiring blood-stage infections or uncomplicated episodes with P. falciparum. Although Duffy antigen receptor expression and function were not affected on SAO erythrocytes compared to non-SAO children, high level (<90% binding inhibition) P. vivax Duffy binding protein-specific binding inhibitory antibodies were observed significantly more often in sera from SAO than non-SAO children (SAO, 22.2%; non-SAO, 6.7%; p = 0.008). CONCLUSIONS: In three independent studies, we observed strong associations between SAO and protection against P. vivax malaria by a mechanism that is independent of the Duffy antigen. P. vivax malaria may have contributed to shaping the unique host genetic adaptations to malaria in Asian and Oceanic populations. Please see later in the article for the Editors' Summar

Mapping of the Plasmodium chabaudi Resistance Locus char2

Animals congenic for the char2 host response locus to the murine malarial parasite Plasmodium chabaudi have been bred, and they demonstrated a phenotypic difference from the parental lines. These congenic lines have been crossed back to the parental line to generate recombinants across the congenic intervals. The recombinants were inbred, and the subcongenic intervals were fixed. These lines were then challenged with parasites and assessed as being either resistant or susceptible. From the analysis of many subcongenic lines, it has become obvious that there are at least two loci underlying the char2 locus and that both of these mediate resistance when the haplotype derives from the resistant C57BL/6 strain