Immunoglobulin G Subclass-Specific Responses against Plasmodium falciparum Merozoite Antigens Are Associated with Control of Parasitemia and Protection from Symptomatic Illness

Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.Effective immunity against Plasmodium falciparum malaria in humans develops slowly over time after repeated exposure and protects against the development of symptomatic and severe illness. Although the targets of protective immunity in humans remain ill-defined, substantial evidence suggests that antibodies against merozoite antigens play an important role, and several merozoite antigens are leading vaccine candidates (5, 15, 29, 35, 37, 45). Antibodies to merozoite antigens are thought to function in vivo by inhibition of merozoite invasion of erythrocytes, opsonization of merozoites for phagocytosis, and antibody-dependent cellular inhibition (3, 9, 13, 21, 24).The subclass of antibodies produced against antigens is likely to be important for protective activity, as immunoglobulin G (IgG) subclasses differ in their structures and mediate different immune effector functions (32). Knowledge of subclass responses associated with protection against malaria is important for understanding immunity and guiding vaccine development. IgG1 and IgG3 are the predominant subclasses produced in response to merozoite antigens (31, 37, 40, 43, 46, 48). IgG1 and IgG3 are cytophilic and T cell dependent, have high affinity for Fc receptors, and mediate phagocyte activation and complement fixation (7). It has been suggested that IgG3 is more efficient at mediating these processes (7). For reasons that are not well understood, different merozoite antigens induce different relative levels of IgG1 and IgG3 (14, 29, 31, 37, 40, 46, 48). It is unclear whether individuals have a bias toward producing a specific subclass regardless of the antigen or if instead the IgG subclass response is generated independently for each antigen and how this relates to protective immunity. While factors determining subclass responses to antigens are not clearly defined, antigen properties, host age, cumulative exposure, and genetic determinants have been linked with the nature of subclass responses (2, 4, 17, 33, 34, 41, 42, 47, 48). Some studies have suggested that increasing age (and therefore malaria exposure) leads to an increasing polarization of IgG subclass responses to merozoite antigens (41, 48).Antibodies to merozoite antigens have been linked with protection from malaria in humans in some longitudinal studies (6, 11, 15, 23, 25, 29, 31, 35, 37-39, 45). Results from these studies have been conflicting, which results partly from the use of different endpoints for evaluating the protective role of antibodies (i.e., different parasitemia thresholds versus symptomatic illness). It is thought that acquired immunity largely targets blood-stage antigens and acts by limiting parasite replication, thereby preventing the development of high-density parasitemia, but is less effective at protecting from parasitization per se (26). However, there are limited data that directly address this question and few studies have evaluated antibody associations with protection from symptomatic malaria, high-density parasitemia, and parasitization per se in the same cohort because of challenges in performing these studies in community-based settings. Additionally, the detection of parasitization has generally been performed using light microscopy, which is not sufficiently sensitive to detect parasitemias of very low density. The development of high-throughput molecular methods to detect parasitemia in cohort studies has provided new opportunities to better define these associations between immune responses and parasitization and symptomatic malaria. Furthermore, most studies of immunity have been conducted in sub-Saharan Africa, and there are little data from populations in Asia, where a large portion of the global malaria burden occurs (44).We addressed these important issues in a treatment-reinfection study of 206 children resident in an area of malaria endemicity in Papua New Guinea. We prospectively examined associations between subclass-specific responses to P. falciparum merozoite antigens (the 19-kDa C-terminal region of merozoite surface protein 1 [MSP1-19], apical membrane antigen 1 [AMA-1], and merozoite surface protein 2 [MSP2]) and the risks of high-density parasitemia, symptomatic malaria, and reinfection, as detected by sensitive molecular-based methods. Furthermore, we evaluated the influences of host age, exposure, and concurrent P. falciparum infection on the nature of responses and assessed whether individuals demonstrated a bias toward specific subclass responses and whether polymorphisms in antigens influenced the nature of subclass responses.