Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria

The merozoite surface protein 2 (MSP2) of Plasmodium falciparum is recognized by human antibodies elicited during natural infections, and may be a target of protective immunity. In this prospective study, serum IgG antibodies to MSP2 were determined in a cohort of 329 Gambian children immediately before the annual malaria transmission season, and the incidence of clinical malaria in the following 5 months was monitored. Three recombinant MSP2 antigens were used, representing each of the two major allelic serogroups and a conserved region. The prevalence of serum IgG to each antigen correlated positively with age and with the presence of parasitaemia at the time of sampling. These antibodies were associated with a reduced subsequent incidence of clinical malaria during the follow-up. This trend was seen for both IgG1 and IgG3, although the statistical significance was greater for IgG3, the most common subclass against MSP2. After adjusting for potentially confounding effects of age and pre-season parasitaemia, IgG3 reactivities against each of the major serogroups of MSP2 remained significantly associated with a lower prospective risk of clinical malaria. Individuals who had IgG3 reactivity to both of the MSP2 serogroup antigens had an even more significantly reduced risk. Importantly, this effect remained significant after adjusting for a simultaneous strong protective association of antibodies to another antigen (MSP1 block 2) which itself remained highly significant.     

Variation in the relationship between anti-MSP-1(19) antibody response and age in children infected with Plasmodium falciparum during the dry and rainy seasons

Malaria remains a major parasitic disease in Africa, with 300-500 million new infections each year. There is therefore an urgent need for the development of new effective measures, including vaccines. Plasmodium falciparum merozoite surface protein-1(19) (MSP-1(19)) is a prime candidate for a blood-stage malaria vaccine. Blood samples were collected from children aged 10 days to 15 years in the months of January-March (N = 351) and October-November (N = 369) corresponding to the dry and rainy seasons, respectively. P. falciparum infection was determined by microscopy and enzyme linked immunosorbent assay (ELISA) was used to determine the total IgG and IgG subclasses. There was a significant increase in the mean anti-MSP-1(19) antibody titre in the dry season (p < 0.05), compared to the rainy season. A significantly positive correlation between the anti-MSP-1(19) antibody titre and parasite density (p < 0.01, r = 0.138) was observed. In the rainy season, unlike in the dry season, P. falciparum positive children had higher anti-MSP-1(19) antibody titres than P. falciparum negative children and this difference was significant (p < 0.05). When all individuals were grouped together, the anti-MSP-1(19) antibody titre increased with age in both seasons (r = 0.186 and 0.002), this increase was more apparent in the dry season. However, when the study population was divided into P. falciparum positive and negative groups, it was observed that in the rainy season, there was a negative correlation between anti-MSP-1(19) titre and age in P. falciparum positive individuals, while those who were P. falciparum negative had a positive correlation between anti-MSP-1(19) titre and age. Analysis of anti-MSP-1(19) IgG subclass showed that IgG1 and IgG3 mean titres were highest in both the dry and rainy seasons with an increase in the mean antibody titres for IgG1, IgG2 and IgG3 in the rainy season. In the dry season there was a positive correlation between IgG1, IgG2, and IgG3 titres with age, while IgG4 was negative, whereas in the rainy season there was a positive correlation between IgG2 and IgG4 (non-cytophilic antibodies) with age and a negative correlation for IgG1 and IgG3 (cytophilic antibodies) with age. Seasonal differences in the level of MSP-1(19) IgG subclass titres were observed for P. falciparum negative and positive individuals. Only samples, which were positive for IgG2 and IgG4, showed positive correlation between parasitemia and total IgG. The incidence of P. falciparum infection, which increases during the rainy season, might be an important determinant of anti-MSP-1(19) antibody levels in children living in Igbo-Ora and the results point to the fact that non-cytophilic antibodies to MSP-1(19) in children might be associated with an increase in total IgG and parasitemia.     

Lymphocyte proliferation and antibody responses to Plasmodium falciparum liver-stage antigen-1 in a highland area of Kenya with seasonal variation in malaria transmission

Lymphocyte proliferation and antibody responses to five peptides corresponding to the N- and C-terminal non-repeat and central repeat regions of Plasmodium falciparum liver-stage antigen-1 (LSA-1) were examined in residents of a highland area of Kenya where malaria transmission is episodic and varies with rainfall. The frequency of lymphocyte proliferation responses (stimulation index > 2) by children (persons > or = 6 years old) and adults (persons > or = 18 years old) was similar and did not differ significantly across seasons. In contrast, the proportion of individuals with IgG antibodies to LSA-1 peptides was higher in the rainy than dry season, and the frequency of these responses was greater for adults than children (39.4% versus 18.7% during the period of high transmission; P = 0.009). Antibodies to LSA-1 were primarily of the IgG1 and IgG3 subclasses, and these also varied with season (30.1% and 32.5% of individuals had IgG1 and IgG3 in the rainy season versus none and 10.9% in the dry season). There was no significant difference in the time to re-infection between groups of persons with or without IgG antibody or lymphocyte proliferation responses to LSA-1 peptides. These data indicate that age and transmission intensity independently affect IgG antibody responses to LSA-1 but do not influence lymphocyte proliferation in this highland area where malaria transmission is highly variable.     

Serum antibody levels to glycosylphosphatidylinositols in specimens derived from matched Malian children with severe or uncomplicated Plasmodium falciparum malaria and healthy controls

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.     

Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2

There is longstanding evidence for a role of immunoglobulin (Ig)G in protection against malarial disease and infection. IgG1 and IgG3 have been shown to be particularly efficient at associating with monocytes in potentially protective mechanisms (i.e. antibody-dependent cellular inhibition, opsonization and phagocytosis). Conversely, there is some evidence that IgG2 (and possibly IgG4) antibodies may be antagonistic to this protection. The protective effect of IgG subclass antibody activity present before the beginning of a malaria transmission season (preseason antibody levels) against severe malaria has not been tested in longitudinal studies. We measured IgG class and subclass antibody levels specific to crude Plasmodium falciparum lysates by enzyme linked immunosorbent assay in a case-control study of 76 children on the coast of Kenya. The mean optical density values for both IgG class and subclass antibodies were not significantly different between the children who developed severe malaria and those who remained healthy during an observation period of two malaria transmission seasons. However, elevated levels of IgG1 in relation to levels of IgG2 and IgG4 antibodies were associated with protection from severe malaria (P = 0.02). Conversely, elevated levels of IgG2 in relation to IgG1 and IgG3 antibodies were associated with a higher risk of developing severe malaria (P = 0.006).     

Antibodies to the conserved C-terminal domain of the Plasmodium falciparum merozoite surface protein 1 and to the merozoite extract and their relationship with in vitro inhibitory antibodies and protection against clinical malaria in a Senegalese village

Antibodies to Plasmodium falciparum C-terminal merozoite surface protein 1 (PfMSP-1p19) have been correlated with protection against malaria, but this association may apply to many merozoite antigens. To address this question, we conducted a prospective serological study of 205 individuals in an active 5-month clinical survey in a Senegalese village where malaria is mesoendemic. Before the 2000 rainy season, antibody responses specific for recombinant baculovirus PfMSP-1p19 or merozoite extracts were compared with 2 in vitro functional antibody activities (inhibition of parasite grown and erythrocyte invasion) and with the number of clinical episodes during 5 months of follow-up. Antibody levels to PfMSP-1p19 and merozoite extract correlated, respectively, with erythrocyte invasion and parasite growth inhibition. Although antibody levels to both antigen preparations were associated with age, functional parameters were not. High levels of anti-PfMSP-1p19 immunoglobulin G were associated with reduced malaria in an age-adjusted multivariate analysis. These results support baculovirus PfMSP-1p19-based vaccine development.     

Season, fever prevalence and pyrogenic threshold for malaria disease definition in an endemic area of Mali

BACKGROUND: Modelling malaria parasitaemia as function of fever has been proposed as best alternative to estimate the attributable fraction of malaria fever and the sensitivity and specificity of different case definitions of malaria disease. OBJECTIVES: To determine the prevalence of fever and its relation to malaria parasitaemia and to establish a pyrogenic threshold for malaria disease in the area. METHODS: We conducted two cross-sectional surveys in children of 6 months to 9 years of age (2434 during the rainy season of 1993 and 2353 during the dry season of 1994) randomly selected from 21 areas of Bandiagara district, Mali. RESULTS: The relationship between fever and Plasmodium falciparum parasitaemia depends strongly on the season, thus affecting the malaria-attributable fraction of fever cases and the sensitivity and specificity of malaria case definitions. The overall proportion of fever attributable to malaria parasitaemia was 33.6% during the rainy season and 23.3% during the dry season, with the highest proportion occurring among the youngest children. The cut-off value, where the sensitivity curve crosses the specificity curve, was around 3200 pf/microl for all age categories during the rainy season and 200 pf/microl during the dry season. CONCLUSIONS: Malaria remains a main cause of fever in this area of Mali. The pyrogenic threshold of parasitaemia depends strongly on the season, and different cut-off levels of parasitaemia should be used during the two seasons to define malaria cases in this area.     

Prevalence of asymptomatic malaria parasitaemia amongst pregnant women

Two hundred and forty-six apparently healthy pregnant women aged 19-40 years, without symptoms were recruited (147 recruited during the dry season and 99 recruited during the rainy season) for the present study. Blood examinations for malaria parasites, Plasmodium falciparum specific-IgG concentration and serological reactivity with P. falciparum-histidine rich protein-2 (HRP-2) antigens were conducted on all the pregnant women during the dry and rainy seasons of the year. During the dry season, 109 (74%) of the recruited pregnant women without symptoms had P. falciparum parasitaemia, while 79 (80%) of the recruited pregnant women without symptoms had P. falciparum parasitaemia during the rainy season. However, the P. falciparum malaria parasites density was significantly raised during the dry season compared with that of in the rainy season (p < 0.05). Serological analysis with P. falciparum histidine rich protein-2 antigen (HRP-2) showed 108 (73%) and 71 (77%) of the pregnant women without symptoms as seropositive during the dry and rainy seasons respectively. The P. falciparum specific-IgG concentration was similar during both seasons in the HRP-2 seropositive pregnant women without symptoms (p > 0.05). The results showed no seasonal tide in the incidences of asymptomatic P. falciparum parasitaemia; however, the significantly raised parasitaemia during the dry season may suggest possible increased parasites tolerance. The P. falciparum specific-IgG concentration during both seasons may not be the primary effector mechanism offering tolerance in asymptomatic parasitaemia in pregnant women.     

Does radical cure of asymptomatic Plasmodium falciparum place adults in endemic areas at increased risk of recurrent symptomatic malaria?

A cohort of 197 adults in Kassena-Nankana District (northern Ghana) was radically cured of malaria parasites to study subsequent incidence of malaria infection. During the following 20 weeks of the malaria transmission season, 49% experienced clinical attacks associated with Plasmodium falciparum parasitaemia. In a group of 202 adults identically followed-up 1 year later without being treated, only 38% experienced such episodes (log-rank test for equality of survivor functions, P=0.035). Clinical attacks in radically cured individuals presented with lower parasite densities but more symptoms. Randomized studies are needed to test the hypothesis that radical cure of P. falciparum enhances the risk and severity of subsequent clinical malaria attacks.     

Boosting antibody responses to Plasmodium falciparum merozoite antigens in children with highly seasonal exposure to infection

Longitudinal cohort studies are important to describe the dynamics of naturally acquired antibody response profiles to defined Plasmodium falciparum malaria antigens relative to clinical malaria episodes. In children under 7 years of age in The Gambia, serum IgG responses were measured to P. falciparum merozoite antigens AMA1, EBA175, MSP1₁₉, MSP2 and crude schizont extract, over a 10‐month period. Persistence of antibody responses was measured in 152 children during the dry season when there was virtually no malaria transmission, and 103 children were monitored for new episodes of clinical malaria during the subsequent wet season when transmission occurred. Children who experienced clinical malaria had lower antibody levels at the start of the study than those who remained free from malaria. Associations between dry season antibody persistence and subsequent wet season antibody levels suggested robust immunological memory responses. Mean antibody levels to all antigens were elevated by the end of the wet season in children who experienced clinical malaria; each of these children had a boosted antibody response to at least one antigen. In all children, antibody avidities were lower against MSP2 than other antigens, a difference that did not change throughout the study period or in relation to clinical malaria episodes.     

Immunological evaluation of cell-mediated and humoral immunity in Thai patients with cerebral and non cerebral Plasmodium falciparum malaria: II. Evolution of serum levels of immunoglobulins, antimalarial antibodies, complement fractions and alpha interferon

In Thai patients with Plasmodium falciparum malaria, IgG and IgM values were elevated, whereas IgA levels were within normal ranges. No association of Ig values with parasitaemia was noted. IFA-IgM antibody levels were lower in cerebral malaria (CM) than in the non cerebral malaria (NCM) group. IFA-IgG antibodies were present in all patients. The mean C3 and C4 values were similar among patients from the CM and NCM groups. Interferon like activity was detected in all CM and NCM patients, and no correlation was found with either antimalarial antibodies, complement or parasitaemia.     

Revaccination with Bacillus Calmette-Guerin (BCG) vaccine does not reduce morbidity from malaria in African children

BACKGROUND: Studies in West Africa and elsewhere have suggested that Bacillus Calmette-Guérin (BCG) vaccine given at birth is beneficial for child survival. It is possible that this effect is mediated partly through an effect on malaria, a hypothesis supported by animal studies. We investigated whether revaccination with BCG at 19 months of age reduced morbidity from malaria. METHOD: In the capital of Guinea-Bissau, between January and November 2003, children who had previously received BCG vaccination and who did not have a strong reaction to tuberculin were individually randomised to either receive revaccination with BCG at the age of 19 months or to be a control. Episodes of malaria were recorded during the 2003 malaria transmission season through passive case detection at health centres in the study area and at the national hospital. Cross-sectional surveys were carried out at the beginning and at the end of the rainy season. RESULTS: Incidence rates of first episodes of malaria associated with any level of parasitaemia were 0.16 episodes per child-year among 713 revaccinated children and 0.12 among 720 control children [incidence rate ratio (IRR) = 1.37; 95% confidence intervals (CI): 0.84-2.25]. Results were similar when the diagnosis of malaria was based on the presence of parasitaemia >5000 parasites/microl (IRR = 1.30; 95% CI: 0.61-2.77). The incidence of all-cause hospitalisation was higher among BCG-revaccinated children than among controls (IRR = 2.13; 95% CI: 1.10-4.13). There were no significant differences in the prevalence of parasitaemia between the two groups of children at cross-sectional surveys. CONCLUSION: We found no evidence that BCG revaccination reduces morbidity from malaria.     

Ability of Anopheles gambiae mosquitoes to transmit malaria during the dry and wet seasons in an area of irrigated rice cultivation in The Gambia

The seasonality of malaria transmission was studied in a Gambian village situated in an area where rice was cultivated. Observations were made during two dry seasons, when pump-fed irrigation was used to grow rice, and in the intervening rainy season, when rice was cultivated using a combination of irrigated and rain-fed paddies. Clinical episodes of malaria were mainly confined to the months during and soon after the rainy season. In the wet season the prevalence of parasitaemia was higher in febrile subjects than in afebrile controls but the reverse applied during the dry seasons. However, the biting rates of Anopheles gambiae complex mosquitoes in the two dry seasons (2.5 and 0.8 bites/child/night respectively) were greater than or similar to that in the rainy season (0.6 bites/child/night). The proportion of human bloodmeals (0.53 vs 0.75) and the survival of mosquitoes (parity rates of 0.41 vs 0.58) were both lower in the dry seasons than in the rains. The low prevalence of morbidity due to malaria in the dry season and the observed fall in the sporozoite rate may therefore have been due to a reduction in the vectorial capacity of the An. gambiae population. However, reduced transmission in the dry season may also have been due to the direct effect of high temperatures on the parasite in the vector.     

Cellular mechanisms involved in recovery from acute malaria in Gambian children

This paper reports the results of in vitro experiments which attempt to elucidate the mechanisms whereby Gambian children control acute infections of Plasmodium falciparum. It was shown initially that mononuclear cells from children with acute malaria, in the presence of specific antibody, caused a marked reduction in in vitro parasite growth. IgM antibodies appeared to be considerably more effective than IgG. T or B lymphocytes were ineffective in the system; adherent cells alone had some effect, but much less than the unfractionated cell population. Adherent cells were however fully effective after exposure to supernatants from T cells activated either non-specifically by phytohaemagglutinin (PHA), or specifically by P. falciparum antigens. Depression of parasite growth was also observed, independent of anti-malarial antibody. This was achieved when adherent cells from healthy Europeans, as well as those from infected children, were exposed to the supernatants from previously stimulated T cells before adding to the culture. Furthermore, intra-erythrocytic parasite death occurred after a short exposure to the supernatants of 'activated' adherent cells from both infected children and Europeans.     

Seasonal fluctuation of antibody levels to Plasmodium falciparum parasitized red blood cell-associated antigens in two Senegalese villages with different transmission conditions

The recombinant R23, PfEB200, and GST-5 antigens derive from conserved antigens associated with the Plasmodium falciparum-infected erythrocyte membrane. They were identified as targets of protective antibodies in the Saimiri sciureus model. We have assessed here the humoral response to these antigens in humans. Cross-sectional surveys were conducted in two Senegalese villages with different levels of endemicity. The prevalence of specific IgG and IgM was similar and influenced by age in both localities. The anti-R23 antibodies decreased after the rainy season, particularly in the children less than ten years old. The anti-PfEB200 response did not show significant seasonal variation. The anti-GST-5 response increased in both the less-than 10-year-old and the greater-than 10-year-old groups after the rainy season in Dielmo, but only in the Ndiop villagers who were more than 10-years-old. Thus, antigen-specific seasonal variations of antibody levels were influenced differently by age in both villages. The isotype distribution was antigen-specific and differed for both seasons.     

Coincidence of malaria parasitaemia and abnormal chest X-ray findings in young Gambian children

Weekly surveillance of Gambian children aged less than 5 years for both acute lower respiratory infections (ALRI) and clinical malaria showed a high rate of coincidence between abnormal chest X-ray findings and high levels of malaria parasitaemia. Generalized interstitial X-ray changes were particularly associated with these cases of malaria parasitaemia. It is suggested that such ALRIs in these children may be attributable to malaria.     

The influence of placental malaria infection and maternal hypergammaglobulinemia on transplacental transfer of antibodies and IgG subclasses in a rural West African population

Two hundred thirteen mother-baby pairs in The Gambia were studied to determine the influence of placental malaria infection and maternal hypergammaglobulinemia on transplacental antibody transfer. Antibody transfer for herpes simplex virus 1 (HSV-1), respiratory syncytial virus (RSV), and varicella-zoster virus (VZV) was significantly reduced by placental malaria infection by 69%, 58%, and 55%, respectively. Maternal hypergammaglobulinemia was associated with a significant reduction in antibody transfer for HSV-1, RSV, VZV, and pneumococcus by 89%, 90%, 91%, and 88%, respectively. In addition, placental malaria infection was associated with a significant reduction in transfer of IgG1, IgG2, and IgG4 (P<.01, P=.01, and P=.03, respectively) but not of IgG3 (P=.59). Maternal hypergammaglobulinemia significantly impaired the transfer of IgG1 and IgG2 (P=.01) but not of IgG3 or IgG4 (P=.62 and P=.59, respectively). Placental malaria infection and maternal hypergammaglobulinemia were associated with reduction in the transplacental transfer of these specific antibodies, IgG1, and IgG2 in this Gambian population.     

Antibody responses to Plasmodium falciparum vaccine candidate antigens in three areas distinct with respect to altitude

Antibody levels against malaria antigens were measured among patients presenting with uncomplicated malaria at health centers from three locations in Zimbabwe (Bindura, Chiredzi and Kariba) that are distinct with regard to altitude and climatic conditions. Antibody levels were determined by ELISA using the antigens, apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), circumsporozoite surface protein (CSP), merozoite surface protein 1 (MSP-1) and Pfg27. For all the antigens tested, IgG and IgM levels were higher for Bindura (altitude 1100 m) compared to Kariba (<600 m, altitude) and Chiredzi (approximately 600 m, altitude) with the exception of IgG and IgM to AMA-1 and EBA-175 which were similar between Chiredzi and Bindura. Plasma samples were further analyzed for their functional activity by testing their ability to inhibit the growth of Plasmodium falciparum in culture. Our results, determined by microscopy and verified by the LDH assay revealed that plasma from the three locations had similar inhibitory activity against the growth of P. falciparum in vitro. Our data revealed that highest growth inhibition correlated with the highest levels of MSP-1 antibody values.     

The direct antiglobulin test in P. falciparum malaria

The direct antiglobulin test (DAT) was performed on 134 Gambian children with P. falciparum malaria. 52 children had a positive DAT and in 25 this was due to the adherence of C3 to their red cells whilst 13 had sensitization with IgG as well as C3. Sensitization with C4 alone or associated with IgG and/or C3 was only rarely found.