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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.

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 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.

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.

A prospective study in 207 children aged 0.5-15 years was carried out to examine the relationship between cellular responses to Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA) and malaria infection and morbidity. The prevalence of lymphoproliferative response to RESA was 13%, IFN-gamma prevalence was 40% and IL-4 prevalence was 22%. Only the IFN-gamma, response to RESA increased significantly with age. When proliferation or stimulation of either cytokine was used to assess T-cell activation the overall frequency of responders increased to 55%. The proliferative and IFN-gamma response to RESA were positively associated. Although there was no association between any of the CMI responses to RESA and concurrent morbidity the prevalence of IL-4 response to RESA was significantly lower in children who experienced clinical malaria in the following year. These results coupled with our earlier data showing a negative relationship between humoral responses to RESA and malaria morbidity support the inclusion of RESA in a subunit vaccine against malaria.

A prospective study in 207 children aged 0.5-15 years was carried out in a highly endemic area of Papua New Guinea to examine the relationship between cellular responses to Plasmodium falciparum merozoite surface protein 2 (MSP2) and malaria infection and morbidity. In vitro proliferation, IFN-gamma and IL-4 induction were measured against two recombinant proteins of MSP2, FC27 and 3D7 as well as against a form of the 3D7 MSP2 lacking the central repetitive sequences (d3D7). The prevalence of proliferative response was generally low, 6% for FC27, 9% for 3D7 and 11% for d3D7. A higher prevalence of IL-4 response was obtained being 27% for FC27, 34% for 3D7 and 30% for d3D7 while the prevalence of IFN-gamma response was 13%, 15% and 18%, respectively. There was no correlation between age and proliferative responses; in contrast cytokine production increased with age for all three antigens. When proliferation or stimulation of either cytokine was used to assess T-cell activation the frequency of responders increased to 39%, 47% and 46% for FC27, 3D7 and d3D7 respectively. Analysis of the relation of T cell responses to concurrent infection and morbidity showed that lymphoproliferative response only to d3D7 was significantly associated with parasitaemia; while lymphoproliferative responses to all 3 MSP2 antigens were highest in the group of clinical malaria cases. There was no significant correlation between proliferation or cytokine production to MSP2 and concurrent or subsequent malaria morbidity.

Isolates of Plasmodium falciparum obtained from 12 children attending different health facilities in the Madang Province, Papua New Guinea were typed for allelic variants of merozoite surface protein-1 and merozoite surface protein-2. Blood was obtained just before treatment with either amodiaquine or chloroquine and at intervals following treatment. All patients examined were found to be infected with genetically different parasites. Nine of the children were found to have single infections while three had mixed infections. In all patients, parasites reappearing in the blood following treatment had the same genotype as parasites in the primary infection. These results indicate that parasites reappearing in the blood following treatment were the result of true recrudescence and not new infections.

The prevalence and concentration of IgG antibodies to defined Plasmodium falciparum antigens were assessed in serum samples of 97 children with cerebral malaria and 146 children with uncomplicated malaria. The antigens used included the schizont extract, ring-infected erythrocyte surface antigen, the C-terminal region of merozoite surface antigen-1 (MSA-1) (BVp42), and three recombinant proteins of MSA-2 (FC27, 3D7, and d3D7). Parasite isolates from 24 children with cerebral malaria and 22 children with uncomplicated malaria were genotyped for MSA-1 and MSA-2. The distribution of parasite genotypes belonging to the different allelic families was similar in both the cerebral and uncomplicated malaria groups. There were higher antibody levels to antigens derived from the infecting parasite genotype than to heterologous genotypes, but this difference was only statistically significant for antibody against the d3D7 antigen among children infected with the 3D7 parasite genotype (mean log = 4.72 versus 3.45 antibody units [AU]; P = 0.029). Those who died were more likely to be infected with the FC27 genotype and had lower antibody levels to MSA-2 of the 3D7 type than had cerebral malaria patients who survived (mean log = 2.94 versus 3.79 AU; P = 0.049). Antibodies against parasites of the 3D7 genotype are associated with a better prognosis among children with cerebral malaria partly because these children are more likely to be infected with parasites of this genotype rather than the FC27 genotype, which appears to be more virulent.

We investigated the prevalence and magnitude of naturally acquired humoral immune response to the major merozoite surface protein (MSP-1) in a malaria-endemic population in Papua New Guinea. A prospective longitudinal study in 0.5-15-year-old children was conducted for one year to examine the relationship between acquired immune response to MSP-1 and subsequent susceptibility to clinical disease. The prevalence and concentration of antibodies to both N-(195A) and C-terminal (BVp42) regions of MSP-1 as well as to the parasite-derived MSP-1 increased with age, with the highest prevalence and concentration of antibodies being detected for the parasite-derived MSP-1 molecule and the C-terminal region of MSP-1. As malaria morbidity decreases with age, a significant negative correlation was observed between antibody levels to both 195A and BVp42 and the incidence rate of clinical malaria. When age and past exposure were corrected for, only antibody concentrations against BVp42 and to a lesser extent parasite-derived MSP-1 were significantly associated with protection from clinical malaria and severe parasitemia. The reduction in the incidence rate of clinical malaria observed in individuals with high antibody concentration to MSP-1 may be due to antibodies directed against epitopes within the C-terminal region of MSP-1.

The in vivo response of Plasmodium falciparum parasites to amodiaquine or chloroquine was assessed in children with symptomatic malaria attending different health facilities in the Madang area. Among the 27 subjects who were completely followed up, 4 (15%) were infected with parasites fully susceptible and 23 (85%) with parasites exhibiting some degree of resistance. Out of the latter group, 52% were of RI level, 26% RII and 22% RIII. 14 subjects out of 42 (33%) failed to clear their parasitaemia by day 7 and 92 out of 134 (69%) had persistent or recrudescent parasitaemia at day 21. The level of in vivo resistance was similar for amodiaquine and chloroquine. 86% of the isolates tested in vitro showed resistance to amodiaquine, 86% to chloroquine and 7% to quinine. In ten years the prevalence of resistant isolates in vivo has increased from 47% to 85%. Of more concern is the shift from RI level of resistance to RII and RIII: the proportion of resistant strains that were RI dropped from 90% to 52% over the ten-year period. To determine if the standard antimalarial regimens are still appropriate, there is a need not only to assess the level of parasite resistance but also the prevalence of treatment failure in different parts of Papua New Guinea.

People living in areas endemic for Plasmodium falciparum develop humoral responses which may contribute to protection against clinical disease but the specificity of such protective antibody responses remains to be defined. Antibodies disrupting erythrocyte rosettes have been associated with protection against cerebral malaria, and antibodies agglutinating infected erythrocytes with reduced episodes of clinical disease. We have studied the capacity of serum from Papua New Guinean adults and children with a spectrum of malaria exposure, including children and adults at the time of clinical disease, to disrupt erythrocyte rosettes and cause agglutination of infected erythrocytes. Using a single parasite isolate, almost all sera from adults from highly endemic areas agglutinated infected erythrocytes, and the majority disrupted rosettes, in some cases at greater titres than hitherto described. There was a correlation between rosette disruption and agglutination in highly exposed adults. Rosette disrupting antibodies were equally frequent in children with cerebral and uncomplicated malaria. Antibodies causing rosette disruption were frequent only in adults with a long history of malarial exposure. Rosette disrupting antibodies do not appear to protect Papua New Guinean children or adults against cerebral 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 and d3D7) 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 prevalence and concentration of antibodies to a yeast-expressed N-terminal region (195A) and a baculo-virus-expressed C-terminal region (BVp42) of merozoite surface antigen 1 (MSA-1) were measured during a cross-sectional survey in the Wosera area of East Sepik Province, Papua New Guinea, in order to obtain baseline data on naturally acquired antibody response to this antigen in preparation for a vaccine trial. Overall, the seropositivity rate was 78% for 195A and 91% for BVp42. Although antibody prevalence to both molecules increased with age, higher antibody prevalence rates were observed for BVp42 in all age groups studied. In children, significant positive associations were found between parasite prevalence and antibody prevalence for both regions of MSA-1 and between spleen rates and anti-BVp42 antibody prevalence. Concentration of antibody against both regions increased significantly with age, but was always higher for BVp42. In children, antibody levels to both regions of MSA-1 were significantly higher in those infected (symptomatic and asymptomatic), while in adults no significant difference in antibody concentration was observed between those infected and those uninfected. However, enlarged spleens were associated with higher antibody concentration to both regions of MSA-1 in both children and adults. The C-terminal of MSA-1 appeared to be more recognized than the N-terminal, in terms of both antibody prevalence and concentration.

A parasitological and immunological survey was carried out in an area in Papua New Guinea highly endemic for malaria. Two hundred fourteen adult individuals were selected for studies to assess their immune responses against the malaria vaccine candidate ring-infected erythrocyte surface antigen (RESA). Total immunoglobulin G (IgG) antibodies directed against RESA as well as specific IgG1, IgG2, and IgG3 antibodies were determined. Humoral responses directed against RESA were frequent in all IgG subclasses. Only IgG3 responses were found to be age dependent. Total anti-RESA IgG antibodies were not correlated with protection against malaria as measured by parasite prevalence, parasite density, or health center attendance. In contrast, cytophilic antibodies (IgG1 and IgG3) were associated with reduced Plasmodium falciparum prevalence and reduced health center attendance. T-cell proliferation in general was low and very infrequent. No correlation between humoral and cellular immune responses could be found. Parasite density, parasite prevalence, and health center visits tended to be reduced in individuals with good humoral and cell-mediated immune responses.

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 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.

Genes for two lethal diseases, thalassemia and sickle cell anemia, are favored by evolution because, in their heterozygous form, they protect against cerebral malaria. Rosette formation, the binding of uninfected red cells (RBCs) to Plasmodium falciparum-infected RBCs (PRBCs), has previously been found to be associated with cerebral malaria, the most important severe manifestation of P falciparum malaria. We show here that thalassemic RBCs and, under certain conditions, even hemoglobin S (HbS)-containing RBCs possess an impaired ability to bind to PRBCs, forming small and weak erythrocyte rosettes compared with rosettes formed by normal RBCs. This decreased rosetting ability is associated with the small size of the thalassemic RBCs and with distortion of the mechanical properties of HbS-containing RBCs. The impairment of rosette formation may hinder the development of cerebral malaria by abatement of sequestration.

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.

To assess natural immunity against the circumsporozoite (CS) protein and the synthetic vaccine SPf66, immunologic studies were carried out in a highly endemic malarious area of Papua New Guinea. Antibody prevalence, antibody titers, and T cell proliferation against both antigens were measured in 214 adults. Immunologic data were analyzed with respect to longitudinal malariologic and morbidity data. Evidence of genetic traits such as glucose-6-phosphate dehydrogenase deficiency and ovalocytosis was analyzed. Antibody prevalence was high, with 79% and 84% for CS protein and SPf66, respectively, while T cell proliferation was infrequent and low, with 14% and 12% responders, respectively. Anti-CS protein antibodies increased with age but showed no association to malaria indices or morbidity. No protective value was observed with T cell responses or with humoral response to SPf66. These results provide a first description of naturally developed immunity against SPf66 and suggest further studies in to fully understand the mechanism of immunity against this antigen.

Some strains of Plasmodium falciparum form erythrocyte rosettes that are believed to result from a lectin interaction between malaria-infected and uninfected erythrocytes. The sulfated glycoconjugate heparin and certain heparin derivatives have been observed to disrupt rosettes. To investigate this interaction further, we have studied the effects of four sulfated glycoconjugates on 15 fresh isolates of P. falciparum from Papua New Guinea. A broader range of sulfated glycoconjugates has been tested against a laboratory strain. A concentration of 1,000 micrograms/ml of dextran sulfate (molecular weight [MW] 500,000) was the most potent disrupter of rosettes. Fucoidan, heparin, and dextran sulfate (MW 5,000) were of decreasing effectiveness in 14 of 15 fresh isolates. The same relationship was true for the laboratory strain. Pentosan polysulfate and sulfatide also disrupted rosettes; chondroitin sulfates A, B, and C and keratan sulfate gave either minimal or no rosette disruption. Thus, some sulfated glycoconjugates are potent disrupters of P. falciparum erythrocyte rosettes. Sulfated glycoconjugates that are potent disrupters of P. falciparum rosettes may prove useful in identifying ligands involved in rosette formation.

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.