Fadwa Al-Yaman

A Recombinant Blood-Stage Malaria Vaccine Reduces Plasmodium falciparum Density and Exerts Selective Pressure on Parasite Populations in a Phase 1-2b Trial in Papua New Guinea

The malaria vaccine Combination B comprises recombinant Plasmodium falciparum ring-infected erythrocyte surface antigen and 2 merozoite surface proteins (MSP1 and MSP2) formulated in oil-based adjuvant. A phase 1-2b double-blind, randomized, placebo-controlled trial in 120 children (5-9 years old) in Papua New Guinea demonstrated a 62% (95% confidence limits: 13%, 84%) reduction in parasite density in children not pretreated with sulfadoxine-pyrimethamine. Vaccinees had a lower prevalence of parasites carrying the MSP2-3D7 allelic form (corresponding to that in the vaccine) and a higher incidence of morbid episodes associated with FC27-type parasites. These results demonstrate functional activity of Combination B against P. falciparum in individuals with previous malaria exposure. The specific effects on parasites with particular msp2 genotypes suggest that the MSP2 component, at least in part, accounted for the activity. The vaccine-induced selection pressure exerted on the parasites and its consequences for morbidity strongly argue for developing vaccines comprising conserved antigens and/or multiple components covering all important allelic types.

The epidemiology of malaria in the Wosera area, East Sepik Province, Papua New Guinea, in preparation for vaccine trials. I. Malariometric indices and immunity

The epidemiological features of malaria were studied through seven community-based surveys in a population of 4000 in the Wosera area, East Sepik Province, Papua New Guinea. Prevalence of parasitaemia (all species, all ages) was 60%. Plasmodium falciparum was the predominant species in all surveys (55%), followed by P. vivax (25%) and P. malariae (20%). The highest prevalence for asexual forms of P. falciparum occurred in the 5-9-year age group, whereas P. falciparum gametocytaemia and P. vivax parasitaemia were observed most frequently in the 1-4-year age group and P. malariae in the 10-15-year age group. Mean densities of all species decreased with age except for that of P. malariae, which was lower in children aged < 1 year than in those aged 1-4 years. The prevalence of enlarged spleen was 57% in children and 10% in adults and closely matched the corresponding age-related parasite rate. Seroprevalence of antibody to the major merozoite surface antigen 2 rapidly increased with age, with > 90% of individuals older than 5 years being positive. Malariological indices showed irregular changes over time but there was no clear-cut seasonal pattern. The geographical distribution of these indices and immune responses was not uniform within the study area. Bednet use and drug consumption were negatively correlated with malariometric indices. Identification of significant temporal and local variations in malaria endemicity is important for the design and evaluation of intervention studies, including field trials of an antimalarial vaccine.

Safety and immunogenicity of a three-component blood-stage malaria vaccine (MSP1, MSP2, RESA) against Plasmodium falciparum in Papua New Guinean children

Combination B is a malaria vaccine that comprises recombinant Plasmodium falciparum (P. falciparum) blood-stage proteins MSP1, MSP2 and RESA, formulated with the adjuvant Montanide ISA 720. A phase I-IIb double-blind randomised placebo-controlled trial was undertaken in 120 children aged 5-9 years. Subjects were randomised in four groups: (i) No sulphadoxine-pyrimethamine (SP)+vaccine, (ii) No SP+placebo, (iii) SP+vaccine, (iv) SP+placebo. 15 microg of each protein were given in the thigh, at both first and second injection (4 weeks apart). The placebo was adjuvant emulsified with saline. No serious or severe AEs occurred. Moderate AEs were seen in 3% of the vaccine and 3% of the placebo recipients after first injection and in 12 and 10% after second injection. The vaccine induced significant antibody responses to all three antigens but triggered an IFN-gamma response to MSP1 only. At Week 12, the IFN-gamma response to MSP1 was substantially higher in the vaccine group where No SP had been given. Combination B proved to be safe and immunogenic in children aged 5-9 years. Vaccine immunogenicity was neither impaired by circulating parasites nor increased after pre-treatment with SP and pre-treatment is not advisable in future trials of malaria vaccines, at least for those including blood-stage antigens.

Safety and immunogenicity of a three-component blood-stage malaria vaccine in adults living in an endemic area of Papua New Guinea.

A Phase I safety and immunogenicity study with a three-component blood-stage malaria vaccine was conducted in adult male subjects living in an endemic area of Papua New Guinea. The preparations were recombinant proteins which corresponded to parts of the two merozoite surface proteins of Plasmodium falciparum (MSP1 and 2), and of the ring-infected erythrocyte surface antigen (RESA). The three proteins were emulsified with the adjuvant Montanide ISA720. Ten subjects were injected twice (four weeks apart) with the vaccine formulation and two with the adjuvant alone. Mild pain at the site of injection was reported by about half of the subjects but no systemic reaction related to the formulation occurred. There was a sharp rise in geometric mean stimulation index after the second dose compared to baseline for MSP1 and RESA, while the rise was small for MSP2. Geometric mean antibody titres increased for MSP1 during the study, whereas they hardly changed for MSP2 and RESA. The vaccine formulation was safe when used in an already immune population. The vaccine induced good cellular responses, especially for MSP1 and RESA. Boosting of humoral responses was weak, probably because of high baseline antibody levels.

Human cerebral malaria: lack of significant association between erythrocyte resetting and disease severity

The ability of Plasmodium falciparum isolates from 103 Papua New Guinea children with cerebral malaria and 158 children with uncomplicated malaria to form rosettes in vitro was studied. Of these, 81 isolates from cerebral malaria and 151 isolates from uncomplicated malaria grew to schizogony and were included in the rosetting analysis. Wide variation occurred in the level of rosette formation, with all isolates from both cerebral and uncomplicated malaria patients being able to form rosettes. No statistically significant difference existed between the geometric mean rosetting rate of isolates obtained from cerebral malaria and those from uncomplicated malaria (9% versus 8.6%, P = 0.27). The ability of acute sera to inhibit rosette formation was not significantly different between 18 cerebral malaria cases and 20 controls tested [mean reduction in rosetting rate 6.1% (SD 11.5) versus 8.4% (SD 12.3), P = 0.57]. The rosetting rate of cerebral malaria cases was not associated with the clinical outcome. Among the clinical and laboratory variables tested, only blood group and parasite density were significantly associated with rosetting. These data do not support the hypothesis that rosette formation is associated with cerebral malaria in Papua New Guinea, but indicate that rosetting is an intrinsic property of parasites occurring in all manifestations of the disease.

ASSESSMENT OF THE ROLE OF THE HUMORAL RESPONSE TO PLASMODIUM FALCIPARUM MSP2 COMPARED TO RESA AND SPF66 IN PROTECTING PAPUA NEW GUINEAN CHILDREN FROM CLINICAL MALARIA

The prevalence and concentration of naturally acquired humoral response (IgG) to merozoite surface protein 2 (MSP2), RESA, SPf66 and crude schizont extract were measured in a population living in a malaria highly endemic area of Papua New Guinea. A prospective longitudinal study in 0–5–15 year old children was conducted for one year in order to examine the relationship between the humoral response to these antigens and subsequent susceptibility to clinical malaria using a series of clinical definitions. The prevalence and concentration of antibodies to all antigens increased with age. Such correlation with age was most marked for MSP2 recombinant proteins. When age and previous exposure were controlled for, only antibody levels to MSP2 recombinant proteins (3D7 andd3D7) and to RESA predicted a reduction in incidence rate of episodes of clinical malaria. Our results support the inclusion of the recombinant proteins of the 3D7 allelic family of merozoite surface antigen 2 and RESA into a subunit vaccine against malaria.,

The epidemiology of malaria in the Wosera area, East Sepik Province, Papua New Guinea, in preparation for vaccine trials. II: Mortality and morbidity

Malaria mortality and morbidity were studied in a rural population of 4000 in the Wosera area, East Sepik Province, Papua New Guinea. Malaria accounted for 4.9% of the 162 deaths investigated by verbal autopsy and for 12.2% of the 49 deaths assessed through medical records. Malaria was the first cause of death in children aged 0.5-4 years. Of the 7795 subjects interviewed and bled during six cross-sectional community-based surveys, children of 1-4 years had the highest malaria-related morbidity. In this age group, point prevalences of fever, fever associated with parasitaemia, and fever plus Plasmodium falciparum (Pf) parasitaemia > or = 10,000 parasites/microliters blood were 5%, 4.1% and 1.5%, respectively. The corresponding figures for adults were 2%, 0.9% and 0.1%, respectively. The calculation of attributable fraction (AF) using a multiple logistic regression model showed that malaria accounted for 0.44 of all fevers in children of 1-4 years and 0.08 of the fevers in adults. Prevalence data derived from the AF estimate were compared with those calculated using different accepted density thresholds. The prevalences which best approximated the results from the logistic regression model were obtained using parasitaemia cut-offs of > or = 1000 Pf parasites/microliter in children aged 1-4 years and adults older than 19 years and of > or = 10,000 parasites/microliter in those aged 5-19 years. Prevalence of fever associated with parasitaemia was highly seasonal, with a peak at the beginning of the wet season. The geographical distribution of malaria morbidity was not uniform. The measurement of malaria-related morbidity, the identification of significant seasonal and local variation as well as the assessment of different methods of defining a clinical episode of Pf malaria are crucial for the design and evaluation of intervention studies, including field trials of antimalarial vaccines.

Malaria: how useful are clinical criteria for improving the diagnosis in a highly endemic area?

To assess the validity of clinical criteria, we investigated 2096 outpatients diagnosed as malaria cases by nurses at a rural health subcentre in a highly endemic area of Papua New Guinea. 73% of the children < 10 years old had a positive blood slide for any species of Plasmodium and 32% had > or = 10,000 P. falciparum parasites per microL. For adults the frequencies were 51% and 9%, respectively. Stepwise logistic regression identified spleen size, no cough, temperature, no chest indrawing, and normal stools as significant predictors for a positive blood slide in children; no cough and normal stools predicted a positive blood slide in adults. Fever, no cough, vomiting, and enlarged spleen were significant predictors for a P. falciparum parasitaemia > or = 10,000/microL in children; in adults the only predictor was vomiting. In children the association of no cough and enlarged spleen had the best predictive value for a positive blood slide, and a temperature > or = 38 degrees C had the best predictive value for a P. falciparum parasitaemia > or = 10,000 microL. In adults, no major symptom had a good predictive value for a positive blood slide but vomiting had the best predictive value for a P. falciparum parasitaemia > or = 10,000/microL. When microscopy is not available, these findings can help in areas of high endemicity to determine which patients with a history of fever are most likely to have malaria and, more importantly, for which patients another diagnosis should be strongly considered.

Relationships between Plasmodium falciparum infection and morbidity in a highly endemic area

A total of 736 outpatients diagnosed as having malaria using clinical criteria at a health centre in a highly endemic area of Papua New Guinea were investigated parasitologically. Plasmodium falciparum-attributable fractions were determined using a logistic regression model to compare parasite densities in cases with those of healthy individuals in community surveys. Thirty-seven percent of presumptive cases were found to have raised P. falciparum parasitaemia. This corresponds to an average reporting rate for the population of 0.53 attributable episodes per annum. Whilst the maximum prevalence of parasitaemia in the community was in children aged 5-9 years, the maximum age-specific incidence of attributable cases at the outpatient clinic was 2 cases per annum in the 2- to 4-year-old age group. The procedure for estimating attributable fractions makes it possible to compare morbidity rates between age groups, and to examine how the relationship between morbidity risk and parasite density changes with age, without diagnosing individual episodes. The average tolerance of parasites in an age group was measured by considering the level of parasitaemia associated with a given risk of malaria-attributable morbidity. In contrast to anti-parasite immunity, tolerance of parasites declines with age since at parasite isodensity the probability of being symptomatic increases with age.

The use of untreated bednets and malaria infection, morbidity and immunity

The effect of bednet use was investigated, without undertaking a specific intervention, in four cross-sectional community-based surveys in 10 villages of a highly endemic area of Papua New Guinea. Over half (55%) of the villagers interviewed reported that they had used a bednet on the previous night. In general and after adjustment for age, village and housing characteristics, bednet users, particularly children, had lower parasite prevalences and spleen rates and less enlarged spleens than non-users. However, users were similar to non-users in terms of fever reported for the previous week, axillary temperature, parasite density and haemoglobin level. The prevalence of antibody to the ring erythrocyte surface antigen and the major merozoite surface antigen 2 was lower in users than non-users. The association with malariometric indices and immune responses remained significant when bednet users were compared with non-users in houses without bednets. Thus, untreated bednets do not reduce malaria transmission sufficiently to decrease morbidity. They might paradoxically increase the risk of clinical malaria by lowering the development of humoral immunity.