Naturally acquired immunity against malaria is highly prevalent in adults residing in malaria endemic regions. It is the strongest known resistance against severe clinical malaria, and it develops over a long period with repeated infections. Passive transfer of purified IgG from hyperimmune sera to malaria patients has demonstrated that IgG mediates anti-parasite activity and protection against malaria. However, the mechanisms involved in the acquisition of naturally acquired immunity and the targets of the protective IgG have not been completely elucidated. This report demonstrates a differential parasite antigen reactivity of IgG responses acquired against P. falciparum asexual blood stage antigens at different exposure levels with a particular focus on parasite antigens expressed in mature schizonts. Using parasite PEMS preparation as the source of antigens, antigenic profiling of the naturally acquired IgG has been carried out using ELISA and Western Blot analysis of parasite antigens separated by SDS PAGE. Conventionally, the approaches widely used to study host immune response against malaria antigens have relied on the use of recombinant proteins or synthetic peptides usually representing subdomains of parasite proteins which could differ from their corresponding native parasite counter-parts in terms of their overall structures and post-translational modifications both of which could potentially alter the antigenic property of the target protein. Since the selection of antigenic targets of interest has traditionally been the first step for studying naturally acquired immunity against malaria, very few studies have attempted an unbiased pan-profiling of antibody reactivity against a broad range of parasite antigens . Alternatively, other studies have attempted to profile antibody reactivity against the whole parasite extract primarily through ELISA [23, 26], an approach which does not yield information regarding the molecular characteristics of the specific antigens recognized by the sera samples. Recently, protein microarray approaches have been developed which enable antigenic analysis against wider range of antigens, but still this technique relies on use of recombinant proteins or synthetic peptides and may suffer from the above listed limitations [27–29]. The present approach of unbiased profiling of naturally acquired antibodies against parasite antigens using native parasite extract has obvious advantages over the use of recombinant proteins or synthetic peptides. As the Western blot method involves denaturing and reducing the antigens the responses reported here might preferentially be against denatured antigens, whereby reactivity against some antigens characterized by having three-dimensional epitopes could have been missed. However, a recent study on the antibody response against Schistosoma mansoni showed that the antigenic profiling was the same whether antigens had been denatured and reduced or not . Other advantages of using proteins from the parasite extract over recombinant proteins or peptides would include covering all linear epitopes over the full-length protein together with all the post-translational modifications which could affect antigenic properties .
The analysis of total anti-parasite IgG in individuals subject to different levels of exposure revealed unexpectedly high levels of anti-parasite IgG in the Danish travellers' plasma after a single episode of clinical malaria. Though polyclonal activation of host B-cell response has been suggested to be mediated by certain parasite antigens , this does not appear to explain the profile of the primary attack IgG subclass specific reactivity observed in the Western blot analysis. Primary attack plasma samples showed selective and restricted reactivity against high molecular weight parasite antigens with IgG1, IgG2 and IgG3 responses, but very little IgG4 and IgM reactivity, indicating activation of selected B-cell clones rather than polyclonal activation. It is interesting to note that the pattern of IgG2 reactivity developed against high molecular weight antigens as a result of primary attack is also observed in plasma samples collected from Ghanaian children and Liberian hyperimmune individuals with multiple attacks. Additionally, increasing exposure leads to a broader repertoire of P. falciparum reactive IgGs with the highest number of antigens being recognized by plasma from clinically immune Liberian adults. A degree of antigenic influence on subclass specificity is particularly evident for the IgG1 and IgG3 subclasses, with some bands being recognized predominantly by IgG1 or IgG3 whilst others are recognized by both classes. In contrast, the IgG2 antibody response seems relatively independent of host age and exposure to P. falciparum. It appears that the antigens recognized by IgG2 are few and in most cases of high molecular weight (> 70 kDa). One antigen of approximately 70 kDa was found to be particularly strongly recognized by majority of the plasma samples from all exposure groups. The apparently low prevalence of IgG2 antibodies to some malaria antigens is in agreement with many previous reports [6, 33–35]. However, the observation that the IgG2 response in general seems independent of age and exposure is inconsistent with the prevailing hypothesis that the initial antibody response against P. falciparum is dominated by non-cytophilic antibodies (IgG2/IgG4 and IgM), and that this response gradually develops into a more protective response dominated by cytophilic IgG1 and IgG3 antibodies . The data presented here suggests that the anti-P. falciparum IgG2 response is directed against a few high-molecular weight antigens and that the age-dependent IgG1 and/or IgG3 responses observed against several low-molecular weight proteins are not preceded by corresponding IgG2 responses.
This observation suggests that clonal B-cell activation is age and exposure dependent. It seems that upon subsequent malaria attacks, additional B-cell clones are activated, which primarily generate IgG1/IgG3 responses and which may contribute to the gradual development of immunity. The immune response to a wide range of antigens is weak upon the first few exposures to malaria infections. It is likely that the parasite may produce immune-suppressive factors [36–38] resulting in poor immunogenicity of at least some malaria antigens. However, upon repeated exposures to malaria infection a widened range of parasite antigens react with the developed antibodies. It is hypothesized that a first step towards developing IgG reactivity to wide range of parasite antigens and thus clinical protection would be to develop effective neutralizing factors towards such parasite encoded immune suppressive factors. This could happen in combination with age dependent physiological changes .
Results from this study suggest that the progressive development of anti-parasite immunity in older children is not mediated by a general switch of malaria-specific antibodies from the non-cytophilic to the cytophilic subclasses. Taken together, the data suggests that the long time required to acquire clinical protection against P. falciparum malaria is not only related to isotype switching towards ADCI-effective antigens but also to a gradual development of IgG1 and IgG3 antibodies against some previously non-targeted antigens like the distinct low-molecular weight P. falciparum proteins detected in this study.