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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.

Through a collaborative project led by the Papua New Guinea Institute of Medical Research (PNGIMR), Papua New Guinea has a significant role in the global effort to develop a malaria vaccine, ensuring that the malaria patterns in Asia and the Pacific region are considered in vaccine development strategies. Some of the major perspectives and achievements of the program are discussed here, one of the most successful being the trial of Combination B, a vaccine comprising three asexual blood-stage proteins [merozoite surface protein (MSP)1, MSP2 and ring-infected erythrocyte surface antigen (RESA)], which led to a considerable reduction of parasite density in the immunized children.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is a prime malaria vaccine candidate. Antigenic diversity within parasite populations is one of the main factors potentially limiting the efficacy of any asexual-stage vaccine, including one based on AMA1. The DNA coding for the most variable region of this antigen, domain I, was sequenced in 168 samples from the Wosera region of Papua New Guinea, including samples from symptomatic and asymptomatic infections. Neutrality tests applied to these sequences provided strong evidence of selective pressure operating on the sequence of ama1 domain I, consistent with AMA1 being a target of protective immunity. Similarly, a peculiar pattern of geographical diversity and the particular substitutions found were suggestive of strong constraints acting on the evolution of AMA1 at the population level, probably as a result of immune pressure. In addition, a strong imbalance between symptomatic and asymptomatic infections was detected in the frequency of particular residues at certain polymorphic positions, pointing to AMA1 as being one of the determinants of the morbidity associated with a particular strain. The information yielded by this study has implications for the design and assessment of AMA1-based vaccines and provides additional data supporting the importance of AMA1 as a malaria vaccine candidate.

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.

A prospective community study in a highly malaria endemic area of Papua New Guinea found that infection with multiple Plasmodium falciparum genotypes was an indicator of lowered risk of subsequent clinical attack. The results suggest that concurrent or very recent infections provide protection from superinfecting parasites. The finding of an association between reduced risk of clinical malaria and infection with parasites of merozoite surface protein 1 (MSP-1) type RO33 or MSP-2 type 3D7 further suggests that the concomitant immunity is, at least in part, a consequence of a response to these major merozoite surface proteins.

The prevalence and concentration of antibodies to ring-infected erythrocyte surface antigen (RESA) were measured in blood samples collected during a cross-sectional survey conducted in Papua New Guinea. Antibodies were measured by enzyme-linked immunosorbent assay to the recombinant RESA protein in 1,398 subjects and to RESA 8 and RESA 11 synthetic peptides in a subsample of 200 adults. Overall, the seropositivity rate to recombinant RESA was 66% and the geometric mean antibody concentration was 28 micrograms/ml. There was a slow increase in antibody prevalence and concentration with age that continued to occur even after 40 years of age. In children less than 10 years of age, there was a significant positive correlation between both RESA antibody prevalence and concentration and concurrent infection with Plasmodium falciparum. The opposite was true in adults more than 20 years of age, with those having a high antibody concentration to RESA being less likely to be parasitemic at the time of the survey. This observation was consistent with the finding of a weak but significant negative correlation between log antibody concentration and log P. falciparum density, which was mainly found in adults. No consistent correlation was found between humoral immune response to RESA and morbidity indicators.

The prevalence and concentration of antibodies to merozoite surface antigen-2 (MSA-2) were measured in blood samples collected during a cross-sectional survey. Antibodies were measured by enzyme-linked immunosorbent assay using two recombinant proteins that closely approximated the full-length mature MSA-2 polypeptides expressed by the Plasmodium falciparum isolate FC27 and the cloned line 3D7 and that were representative of the dimorphic forms of MSA-2. Antibodies were also measured to a form of the 3D7 MSA-2 lacking the central repetitive sequences (d3D7). High antibody prevalence was observed to all three antigens: the overall prevalence of IgG to FC27, 3D7, and d3D7 was 91%, 90%, and 90%, respectively. The majority of individuals > or = 5 years of age had antibodies to both forms of MSA-2. The geometric mean antibody units increased with age with a plateau being reached by 15-20 years of age. There was a significant positive association of antibody prevalence with both the presence of the parasite and an enlarged spleen in children. This study provides the first evidence that antibodies against nonrepeat regions of MSA-2 are associated with fewer fever episodes and less anemia, both known to be indicators of malaria morbidity.

The relationship between antigenic variation, cytoadherence, rosette formation, and the pathogenesis of malaria has led to great interest in the diversity of these properties in Plasmodium falciparum isolates from different communities. In this study, we extend previous investigations by delineating the spectrum of agglutinating phenotypes, adherence to C32 melanoma cells, human umbilical vein endothelial cells (HUVEC), CD36, and intracellular adhesion molecule-1 (ICAM-1), and rosette-forming ability of a group of 20 P. falciparum isolates from Papua New Guinean children. Agglutination phenotypes were determined by using both the children's convalescent serum and a panel of adult immune sera. The wide range of variant antigenic types in the community was demonstrated by the failure of the agglutination assays to identify any two isolates with the same agglutinating phenotype in this, the largest study of its kind. Comparison of agglutination profiles from fresh and cryopreserved isolates demonstrated the general acceptability of cryopreservation before testing, but cautioned that some isolates may undergo selection and phenotypic change during the process. Nineteen isolates were able to bind to at least one of the four ligands studied and showed marked variation in both avidity and specificity of binding. The purified proteins ICAM-1 and CD36 proved to be the most useful assay ligands for investigating field isolates, with 18 isolates binding to at least one protein and 14 to both. No correlation was found between the binding of isolates to any two ligands nor between the binding of a standardized inoculum and the level of the patient's presenting parasitemia. All isolates from the study group were found to form rosettes (at a mean rate of 14.6% of cultured trophozoites involved in rosettes). A lack of correlation between rosette formation and CD36 binding suggests that the previously reported role of CD36 as a rosette formation receptor may not be important for isolates from Papua New Guinea.

Immunoglobulin isolated from the sera of individuals living in a malarious area of Papua New Guinea was tested for an effect on the growth in vitro of four isolates of Plasmodium falciparum, three from Papua New Guinea and one from Thailand. The Papua New Guinea isolates were inhibited to the same degree by individual immunoglobulin preparations, and inhibition varied from 0 to 98% (assessed by a radioisotopic readout). Immunoglobulin preparations which inhibited the Papua New Guinea isolates caused less inhibition of the Thai parasites. Biosynthetically labeled parasite proteins were analyzed by two-dimensional gel electrophoresis, and differences were detected in the protein and antigenic composition of isolates which differed in their sensitivity to inhibitory immunoglobulin. Three acidic proteins (Mr 200,000, 150,000, and 65,000) were found only in the Papua New Guinea isolates. All Papua New Guinea isolates contained a high-molecular-weight basic protein with an Mr of 220,000 (Pf220), but the corresponding protein of the same molecular weight in the Thai isolate had a more acidic isoelectric point. Another isolate (from Africa) initially showed a degree of resistance to inhibition by Papua New Guinea immunoglobulin (although not to the same extent as the Thai parasite), but in later experiments, this isolate was susceptible to inhibition. During the course of this series of experiments, the antigenic composition of this (uncloned) isolate changed so that it became similar (but not identical) to the Papua New Guinea isolates.

Cross-sectional and longitudinal village-based studies of the transmission dynamics of an S-antigen serotype of the asexual erythrocyte stages of Plasmodium falciparum have been carried out in Madang, Papua New Guinea (PNG). Sera collected from village residents were screened for circulating S-antigen of the FC27 serotype by enzyme-linked immunosorbent assay (ELISA). The prevalence of the FC27 S-antigen was found to vary between villages at a given point in time, as well as within a village over time. Residents of villages 2-5 km apart were infected with P. falciparum of different S-antigen serotypes. This study documents the periodic nature of transmission of a sub-population of P. falciparum defined by the FC27 S-antigen. The variation in a small area in the prevalence of this serotype of P. falciparum in Madang illustrates the complexities of malaria transmission which must be considered in the design of malaria vaccine trials.