The pathologic manifestations of Onchocerca volvulus infection depend on the interplay between the host and the parasite. A genetic single nucleotide polymorphism in the FcγRIIa gene, resulting in arginine (R) or histidine (H) at position 131, affects the binding to the different IgG subclasses and may influence the clinical variations seen in onchocerciasis. This study investigated the relationship between this polymorphism and disease outcome. FcγRIIa genotyping was performed on clinically characterized onchocerciasis patients (N = 100) and healthy controls (N = 74). FcγRIIa genotype R/R131 frequencies were significantly higher among patients with severe dermatopathology (P < 0.001). Increased risk of developing this form was mostly associated with one tribe (Masalit) (OR = 3.2, 95% CI 1-9.9, P = 0.042). The H131 allele was found to be significantly associated with a reduced risk of having the severe form of the disease (adjusted OR = 0.26, 95% CI = 0.13-0.46, P < 0.001). Our findings suggest that the polymorphism influences the clinical outcome of onchocerciasis.

Previous studies have implicated reactive antibodies to the low molecular weight rhoptry-associated proteins (RAP-1, RAP-2/RSP-2 and RAP-3) in erythroid cell destruction during Plasmodium falciparum infection. In this pilot study, the frequency, specificity and functional capacity of naturally acquired anti-RAP-2/RSP-2 antibodies were investigated in the sera of anaemic and nonanaemic malaria-infected Cameroonian children. All sera recognized RAP-2/RSP-2 by FACS, irrespective of the clinical status of the subjects. However, the anaemic children showed higher levels of IgG antibodies than the nonanaemic group, while both groups showed similar levels of IgM antibodies. Only few individuals had detectable levels of RAP-2/RSP-2-specific IgG1 and IgG3 subclass antibodies, while no IgG2 and IgG4 subclass antibodies were detected in these subjects. By ELISA, the anaemic group tended to show higher levels of antibodies to RAP-2/RSP-2 regarding all antibody classes tested, except for IgG4 and IgE. Unexpectedly, sera from the nonanaemic group activated complement to a greater extent than those from the anaemic group. These results need to be confirmed in extended studies but indicate that the effector functions of the RAP-2/RSP-2-reactive antibodies may be more important than their amounts. Such antibodies could play a role in both immunity and pathogenesis during P. falciparum infection.

Plasmodium falciparum malaria is a major cause of morbidity and mortality throughout the tropics. Anaemia is a constant feature of the disease. Pregnant women mostly primigravidae and children below the age of 5 years are the most afflicted. Its pathogenesis is multifactorial and incompletely understood. Among several factors, the destruction of erythrocytes (RBCs) is the most frequently observed cause of severe malarial anaemia and the removal of non-parasitized RBCs (nEs) is thought to be the most important, accounting for approximately 90% of the reduction in haematocrit in acute malaria. Previous studies demonstrated that the tagging of nEs with the parasite antigen RAP-2 (rhoptry-associated protein-2; also designated RSP-2) due to either failed or aborted invasion by merozoites resulted in the destruction of these cells. In this study we further investigated the mechanisms mediating the destruction of nEs in the development of severe malarial anaemia and the possible involvement of RAP-2/RSP-2 and other members of the low molecular weight rhoptry complex (RAP-1: rhoptry-associated protein-1 and RAP-3: rhoptry-associated protein-3). Antibodies to the rhoptry-associated proteins were found to recognise the surface of nEs in a parasitaemia-dependent manner after merozoite release in P. falciparumin vitro cultures. These cells, as well as erythroblasts co-cultured with infected RBCs (IEs), could then be destroyed by either phagocytosis or lysis after complement activation. The ability of anti-rhoptry antibodies to mediate the destruction of RAP-2/RSP-2-tagged erythroblasts in the presence of effector cells was also investigated. Data obtained suggest that mouse monoclonal antibodies to the low molecular weight RAP proteins mediate the death of RAP-2/RSP-2-tagged erythroblasts on interaction with adherent monocytes. The mechanism of cell death is not yet fully known, but seems to involve primarily apoptosis. The above observations suggest that the antibody response against RAP-2/RSP-2 and other members of the complex could trigger the destruction of RAP-2/RSP-2-tagged host cells. Taken together it appears that during severe anaemia a defective bone marrow or dyserythropoiesis possibly due to erythroblast cell death, may overlap with the accelerated destruction of normal erythroid cells, either by opsonisation or complement activation further aggravating the anaemia which may become fatal. These observations could therefore have implications in the design, development and deployment of future therapeutic interventions against malaria.

Acquisition of antibodies against blood stage antigens is crucial in malaria immunity and the Plasmodium falciparum antigen Pf332, which is present in close association with the infected red blood cell membrane, is one such antigen. In this study, the antibody response to a Duffy binding like fragment of Pf332, termed Pf332-DBL was investigated in sera from naturally exposed individuals living in Dielmo village, Senegal, with regard to immunoglobulin classes (IgG, IgM, IgE) and IgG subclasses (IgG1-4). While the levels of IgM, IgG, IgG1 and IgG2 only displayed a moderate trend to increase with age, Pf332-DBL specific IgG3 levels increased significantly in the older villagers. In multivariate analysis, when controlling for confounding factors, and in a linear model with a Poisson distribution, anti-Pf332-DBL IgG3 as well as the ratio of cytophilic to non cytophilic anti-Pf332-DBL antibodies were found significantly associated with a reduced risk of malaria attack. This association was also present when the IgG3:IgG1 ratio was tested. Finally, two subgroups of villagers with the same mean age, were delineated by IgG3 concentrations either lower or higher than the median value. A total of 45.2% of the individuals with low anti-Pf332-DBL-IgG3 levels but only 21.4% of the villagers in the group with high levels of such antibodies had a clinical malaria attack during a period of 3 years of continuous follow-up after the blood sampling. In conclusion, Pf332-DBL induces naturally the acquisition of antibodies, and Pf332-DBL-specific IgG3 appears to be associated with protection against malaria in this endemic setting.

The Plasmodium falciparum antigen 332 (Pf332), is a megadalton parasite protein expressed at the surface of infected red cells during later stages of the parasite's developmental cycle. Antibodies to different parts of this antigen have been shown to inhibit parasite growth and adherence to host cells with or without ancillary cells. However, the mechanisms involved in these inhibitions remain largely unknown. We further analysed the activities of specific antibodies with regard to their specific mechanisms of action. For these analyses, affinity purified human antibodies against epitopes in the C-terminal fragment of Pf332 (Pf332-C231) were employed. All purified antibodies recognized Pf332-C231 both by immunofluorescence and ELISA. IgG was the main antibody isotype detected, although all sera investigated had varying proportions of IgG and IgM content. All the antibodies showed a capacity to inhibit parasite growth in P. falciparum cultures to different extents, mainly by acting on the more mature parasite stages. Morphological analysis revealed the antibody effects to be characterized by the presence of a high proportion of abnormal schizonts (15-30%) and pyknotic parasites. There was also an apparent antibody effect on the red cell integrity, as many developing parasites (up to 10% of trophozoites and schizonts) were extracellular. In some cases, the infected red cells appeared to be disintegrating/fading, staining paler than surrounding infected and uninfected cells. Antigen reversal of inhibition confirmed that these inhibitions were antigen specific. Furthermore, the growth of parasites after 22-42 h exposure to antibodies was investigated. Following the removal of antibody pressure, a decreased growth rate of these parasites was seen compared to that of control parasites. The present study confirms the potential of Pf332 as a target antigen for parasite neutralizing antibodies, and further indicates that epitopes within the C231 region of Pf332 should constitute important tools in the dissection of the role of Pf332 in the biology of the malaria parasite, as well as in the design of a malaria vaccine.

Antigen Pf332, a megadalton protein has been shown to be associated with the membrane of infected erythrocytes. Detailed functional studies on the antigen have remained hampered by the cross-reactive nature of antibodies generated to Pf332. Pf332-C231, identified in the C-terminal region of Pf332 was cloned and antibodies against the C231 fragment were shown to react with intact Pf332 antigen by both immunofluorescence and immunoblotting analyses. Antibodies to C231 inhibited in vitro Plasmodium falciparum growth efficiently. In addition, human sera from malaria-exposed individuals reacted with recombinant C231. We show that Pf332-C231 represents a functional domain and is expected to facilitate further studies on Pf332 as a potential target for protective immune responses and the function of the antigen.

In Burkina Faso, where malaria is hyper-endemic and transmission intensity is very high, the majority of malaria-related morbidity and mortality occurs in children less than 5 years of age. A control measure such as the use of insecticide-treated curtains (ITC) significantly reduces transmission of malaria infection. Concerns remain whether reduced transmission intensity may lead to a delay in the development of immunity in younger children and even to a partial loss of already acquired immunity. In this study, the levels of P. falciparum-specific IgG subclasses, the number of infecting parasite clones determined by PCR-based genotyping of the msp2 gene and the parasite density were analysed in 154 asymptomatic children (3–6 years) living in 16 villages (8 with and 8 without ITC) in the vicinity of Ouagadougou, the capital of Burkina Faso. In addition, the parasite inhibitory effects of Ig fractions, prepared from selected children, in co-operation with normal human monocytes were studied. Blood samples from asymptomatic ITC-users showed a significant decrease in P. falciparum prevalence as well as in parasite density. However, no significant difference was observed in P. falciparum-specific antibodies or in parasite multiplicity of infection between the two groups. Furthermore, Ig fractions from children of both groups showed similar levels of inhibitory activity against autologous parasite growth both on their own and in co-operation with monocytes.

The well-established relative resistance to malaria observed in the Fulani ascompared with other sympatric tribes in West Africa has been attributed totheir higher levels of serum immunoglobulin (Ig) G antibodies to malarialantigens. In this study, we confirm and extend the previous findings by analysesof the levels of IgM, IgG and IgG subclasses of anti-malarial antibodies inasymptomatic individuals of different sympatric tribes in Burkina Faso(Fulani/Mossi) and Mali (Fulani/Dogon). The Fulani showed significantlyhigher median concentrations of anti-malarial IgG and IgM antibodies thanthe sympatric tribes at both locations. Although the overall subclass pattern ofantibodies did not differ between the tribes, with IgG1 and IgG3 as dominant,the Fulani showed consistently significantly higher levels of these subclasses ascompared with those of the non-Fulani individuals. No significant differenceswere seen in the levels of total IgG between the tribes, but the Fulani showedsignificantly higher levels of total IgM than their neighbours in both countries.While the antibody levels to some nonmalarial antigens showed the same patternof differences seen for antibody levels to malaria antigens, no significant suchdifferences were seen with antibodies to other nonmalarial antigens. In conclusion,our results show that the Fulani in two different countries show higherlevels of anti-malarial antibodies than sympatric tribes, and this appears not tobe a reflection of a general hyper-reactivity in the Fulani.

A malaria vaccine targeting Plasmodium falciparum remains a strategic goal for malaria control. If a polyvalent vaccine is to be developed, its subunits would probably be chosen based on immunogenicity (concentration of elicited antibodies) and associations of selected antigens with protection. We propose an additional possible selection criterion for the inclusion of subunit antigens; that is, coordination between elicited antibodies. For the quantitative estimation of this coordination, we developed a malaria serological map (MSM). Construction of the MSM was based on three categories of variables: (i) malaria antigens, (ii) total IgG and IgG subclasses, (iii) different sources of plasma. To validate the MSM, in this study, we used four malaria antigens (AMA1, MSP2-3D7, MSP2-FC27 and Pf332-C231) and re-grouped the plasma samples into five pairs of subsets based on age, gender, residence, HbAS and malaria morbidity in 9 years. The plasma total IgG and IgG subclasses to the test antigens were measured, and the whole material was used for the MSM construction. Most of the variables in the MSM were previously tested and their associations with malaria morbidity are known. The coordination of response to each antigens pair in the MSM was quantified as the correlation rate (CR = overall number of significant correlations/total number of correlations x 100 %). Unexpectedly, the results showed that low CRs were mostly associated with variables linked with malaria protection and the antigen eliciting the least CRs was the one associated with protection. The MSM is, thus, of potential value for vaccine design and understanding of malaria natural immunity.

In malaria, drug resistance and treatment failure (TF) are not synonymous, although are escalating together. Over 9 years of surveillances for malaria morbidity and TF in Daraweesh village in eastern Sudan (1991-2004), 136 donors (15-78 years) from 43 households, treated for 278 malaria episodes and had experienced 46 incident of TF, were included in this study. Blood obtained from the donors in 2005, was used for measurement of IgG subclasses against Pf332-C231 antigen and GM/KM allotyping and for genotyping of the donors for; FcgammaRIIA 131 (HH, RH, RR), CRP 286 (C

The role of inflammation in malaria pathogenesis is not fully understood, although C-reactive protein (CRP) may have a negative influence on host immunity to infections. An upstream polymorphism, -286 (C > T > A), in the CRP gene is known to influence CRP levels. In this study, a cohort of 192 Sudanese donors, followed for malaria infection for 9 years, had their CRP -286 gene locus genotyped by pyrosequencing. The number of malaria episodes experienced by each individual over the study period was used as an index for malaria susceptibility. The prevalence of the CRP alleles A, C and T were 21%, 52% and 27%, respectively. Importantly, the A-allele, unlike the C- and T-alleles or CRP genotypes, was significantly associated with an increased number of malaria episodes, P = 0.007. The proportion of A-allele carriers among donors not known to have had malaria during the study period was 18%, whereas it was 43% and 63% among donors who had experienced 1-4 and > or =5 malaria episodes, respectively, over the same period (P = 0.002). Furthermore, the A-allele was associated with higher parasite counts. In conclusion, the CRP -286 A-allele was associated with an increased susceptibility to uncomplicated Plasmodium falciparum malaria.

ABSTRACT: BACKGROUND: Plasmodium falciparum malaria is a complex disease in which genetic and environmental factors influence susceptibility. IgG isotypes are in part genetically controlled, and GM/KM allotypes are believed to be involved in this control. METHODS: In this study, 216 individuals from Daraweesh, an area of seasonal malaria transmission in Sudan, were followed for nine years for malaria infection. Total IgG and IgG isotypes against four malaria antigens, MSP2-3D7, MSP2-FC27, AMA1, and Pf332-C231 were measured in plasma obtained from the cohort at the end of the study, during the dry malaria-free period. The GM/KM allotypes of the donors were determined. RESULTS: The GM 1,17 5,13,14,6 phenotype was associated with a higher incidence of malaria compared with the non-1,17 5,13,14,6 phenotypes (P = 0.037). Paradoxically, the carriers of the GM 1,17 5,13,14,6 phenotype had significantly higher baseline levels of total IgG and non-cytophilic IgG isotypes as compared to non-carriers. The KM allotypes influence on IgG isotypes level was limited. Finally, the differences in the baseline concentrations of total IgG and IgG isotypes between the different GK/KM phenotype carriers were antigen-dependent. DISCUSSION: The results show that GM but not KM allotypes appeared to influence host susceptibility to uncomplicated malaria as well as the antibody profile of the donors, and the carriers of the GM 1,17 5,13,14,6 phenotype were the most susceptible CONCLUSIONS: The GM allotypes have significant influence on susceptibility to uncomplicated P. falciparum malaria and antigen-dependent influence on total IgG and IgG subclasses.

The certainty of the protective role of acquired immunity in malaria is the major drive for malaria vaccine development. In this study, we measured the levels of total IgG and IgG subclasses to four candidate malaria vaccine antigens; MSP2-3D7, MSP2-FC27, AMA-1 and Pf332-C231, in plasma obtained from a cohort of 136 donors from Daraweesh in Sudan. The cohort was followed for malaria infection for 9 years. After an initial analysis, the immune response to Pf332-C231 antigen was the only one found associated with protection, thus taken for further analysis. The number of previous clinical malaria episodes experienced by the donors was used as an index for relative protection. The number of these episodes was found to be negatively correlated with the levels of pre-existing total IgG, IgG2 and IgG3 to Pf332-C231 (correlation coefficient, CC - 0.215, p=0.012; CC - 0.195, p=0.023 and CC - 0.211, p=0.014, respectively), and also with age (CC - 0.311, p<0.001). Unexpectedly, equal levels of Pf332-C231 antibodies were induced by both patent and sub-patent infections regardless of the number of previous malaria episodes (1-7). Combining the correlation analysis with a multi-linear regression, three variable markers for protection were emerged, two age-dependent, the antibody response to Pf332-C231 and an unidentified marker (likely immune response to other antigens), and the third was an age-independent unidentified marker (possibly gene polymorphisms). In conclusion, this report suggests a protective effect for IgG subclasses to Pf332-C231 antigen against malaria.

Malaria signature on human genome is marked by several gene polymorphisms. HemoglobinAS (HbAS) is known to protect against severe malaria, but barely proved to protect against uncomplicated malaria (UM). Similarly, the influence of Fc gamma RIIa-RH131 polymorphism on malaria is controversial. Polymorphisms in both genes were examined and levels of IgG subclasses against four malaria antigens were measured for 250 Fulani's from Daraweesh, eastern Sudan. Morbidity data for up to nine years was available for 214 donors. Number of malaria episodes experienced by each individual during the study period was used as indicator for susceptibility to UM. PCR and RFLP were used for donors DNA genotyping and ELISA for antibodies measurement. Results revealed that neither Fc gamma RIIa-RH131 alleles/genotypes nor HbAA/AS was significantly associated with malaria morbidity or with levels of IgG to test antigens. Both polymorphisms were in Hardy-Weinberg Equilibrium, interestingly, there was strong association between the two polymorphisms (linkage disequilibrium - LD) with D' = 0.89. The association between the two polymorphisms was confirmed by analysis of independent material from a neighboring village. In conclusion, in Daraweesh both Fc gamma RIIa-RH131 and HbAA/AS genotypes, independently or together, were not major markers for UM susceptibility, however, marked LD was observed between the two polymorphisms.

Susceptibility to uncomplicated malaria (UM), as to other forms of the disease, is genetically determined. Over 9-years of clinical and parasitological follow up of inhabitants of Daraweesh, in Eastern Sudan, the relative susceptibility to UM was estimated in terms of number of episodes experienced by each individual. Previously, we reported that the levels of IgG2 and IgG3 to Pf332-C231 malaria antigen are negatively correlated with number of malaria episodes. In addition, four molecular markers for malaria susceptibility (CRP -286, GM/KM haplotypes, FcγRIIa131 and HbAS) were tested. In this study, the above data were combined and reanalysed. The CRP -286A allele and GM 1,17 5,13,14,6 phenotype were previously found to be associated with increased susceptibility to malaria; however, individuals have both polymorphism together were not more susceptible to UM than the non-carriers of the same double polymorphism. The FcγRIIa-RR131 and HbAA genotypes taken individually or as double polymorphism were not associated with malaria susceptibility; however, their combination with any or both of the former polymorphisms was mostly associated with increased susceptibility to malaria. None of the four markers were associated with the levels of IgG2 and IgG3 against Pf332-C231. In conclusion, while our data support the polygenic nature of susceptibility to UM and highlighted the role of immune markers polymorphisms, the combinations of these markers were not predictable, i.e. the combination of the susceptibility markers will not necessarily render the carriers more susceptible to UM.

Despite many intervention programmes aimed at curtailing the scourge, malaria remains a formidable problem of human health. Immunity to asexual blood-stage of Plasmodium falciparum malaria is thought to be associated with protective antibodies of certain immunoglobulin classes and subclasses. We have analysed immunoglobulin G profiles to six leading blood-stage antigens in relation to clinical malaria outcome in a hospital-based study in Sudan. Our results revealed a linear association with anti-AMA-1-IgG1 antibodies in children <5 years and reduced risk of severe malaria, while the responses of the IgG3 antibodies against MSP-2, MSP-3, GLURP in individuals above 5 years were bi-modal. A dominance of IgG3 antibodies in >5 years was also observed. In the final combined model, the highest levels of IgG1 antibodies to AMA-1, GLURP-R0, and the highest levels of IgG3 antibodies to 3D7 MSP-2 were independently associated with protection from clinical malaria. The study provides further support for the potential importance of the studied merozoite vaccine candidate antigens as targets for parasite neutralizing antibody responses of the IgG1 and IgG3 subclasses.

BACKGROUND: The ability of the host immune system to efficiently clear Plasmodium falciparum parasites during a malaria infection depends on the type of immune response mounted by the host. STUDY DESIGN: In a cross-sectional study, we investigated the cellular-and antibody responses in individuals with P. falciparum infection, in an attempt to identify immunological signs indicative of the development of natural immunity against malaria in Ibadan, Nigeria. Levels of IL-10, IL-12(p70), IFN-gamma, and IgM, IgG and IgG1-4 subclasses in the serum of 36 symptomatic children with microscopically confirmed malaria parasitaemia and 54 asymptomatic controls were analysed by ELISA. RESULTS: IFN-gamma and IL-10 were significantly higher in the symptomatic children (p=0.009, p=0.025 respectively) than in the asymptomatic controls but no differences were seen for IL-12(p70). Estimated higher ratios of IFN-gamma/IL-10 and IFN-gamma/IL-12 were also observed in the symptomatic children while the asymptomatic controls had higher IL-12/IL-10 ratio. The mean concentration levels of anti-P. falciparum IgG1, IgG2, IgG3 antibodies were statistically significantly higher in the individuals >5 years of age than <5 years while anti-P. falciparum IgG3 antibodies were notably low in <5 years category. Children <5 years had higher IgM antibodies than IgG and the expression of IgG subclasses increased with age. CONCLUSION: Taken together, malaria infection is on a delicate balance of pro- and anti-inflammatory cytokines. The higher levels of IFN-gamma seen in the symptomatic children (<6 months) may be instrumental in immune-protection against malaria by limiting parasite replication. The observed variations in immunoglobulin subclass levels were age-dependent and exposure-related.