In this study the differences in antibody responses to MSP complex proteins among CM patients, MM patients and HC subjects in a malaria-endemic part of India was investigated. This is one of the first studies to address systematically the antibody responses to the two major allelic forms of all the four major subunits of MSP-1 antigens (MSP-130 MSP-138 MSP-183, MSP-142) together with their associated proteins, MSP-636 and MSP-722 in malaria patients. The HC group tended to have lower antibody levels than MM and CM patients consistent with previous studies, which demonstrated that antibodies to merozoite antigens were higher in parasitaemic compared to aparasitaemic subjects [38, 52–54]. An important finding is the observation that CM patients showed significantly lower antibody responses to some of the MSP family of antigens as compared to MM patients. These differences included both low prevalence and low mean IgG levels and IgG subclasses in the CM group. On the contrary, MM patients showed significantly elevated IgG responses to many of the antigens compared to the HC group.
The association of lower antibody titers to certain P. falciparum antigens with malaria severity (such as CM) has been demonstrated in previous studies. For example, significantly lower levels of P. falciparum anti-GPI IgGs were observed in CM patients as compared to MM patients in a study in Senegal . In the Senegal study, the differences in the responses to MSP-119 antigen were slightly lower in the CM patients compared to the MM patients, although significantly lower levels were only observed in the CM non-survivors sub-group. In the current study, antibody responses to the MSP-119 antigen and some other MSP complex antigens were lower in CM patients as compared to MM patients. On the contrary, other studies have reported higher levels of anti- GPI antibodies in CM patients compared to non-severe malaria patients or HC subjects . In another study, higher levels of IgG2 and IgG4 antibody responses to the variant surface glycoprotein RIF-29 were found exclusively in CM children but not in the non-cerebral malaria controls . Overall, findings from the current study are consistent with the hypothesis that CM patients may have some deficiency in mounting optimal antibody responses to some antigens essential for clinical protection. However, additional evidence to confirm this hypothesis is required and validation will depend on further studies.
The seropositivity of antibodies varied considerably depending on the MSP antigen as previously demonstrated . Antibody responses were relatively high for most of the antigens, which was not surprising given that this study was conducted in a malaria endemic region where the majority of people are exposed to malaria. Both MSP-142 and MSP-183 demonstrated high antibody prevalence compared to the other fragments, consistent with a previous study . Anti-MSP-636 antibodies were shown to be generated in individuals naturally infected with P. falciparum . Additionally, these antibodies were thought to play a role in the inhibition of erythrocyte invasion [55, 56] and parasite multiplication [23, 57]. This study demonstrates that naturally exposed residents in central India also generate both anti-MSP-636 and anti-MSP-722 antibodies. In contrast, the antibody prevalence to the MSP-1d30 antigen was low as had been observed in previous studies , suggesting that this may be a poorly immunogenic antigen.
Results from this study confirm that the entire MSP-1/MSP-6/MSP-7 complex contains B-cell epitopes capable of generating specific antibodies in naturally exposed individuals.
The F allelic form of MSP-130 and MSP-138 antigens showed higher seroprevalence than the corresponding D allelic forms, suggesting that individuals generate specific antibodies to the different allelic forms of these antigens. The higher prevalence of antibodies to the F allelic forms of MSP-130 and MSP-138 antigens may be due to a predominant presence of P. falciparum parasites with this allele in this population in India. However, there is no published information to verify the relative proportions of these alleles within this population. A few studies that have investigated the prevalence of the different MSP-1 alleles in India have either demonstrated the predominance of the MAD20 allele (D), as defined by sequence analysis of the 16th and 17th block , or no bias to any allele [59, 60].
However, the MSP-142 and MSP-183 antigens showed similar antibody prevalence to the two allelic forms of the antigens, suggesting the development of antibodies cross reactive to both allelic forms. Significant correlations in the antibody levels elicited by the two allelic forms of MSP-1 were observed except for the p30 fragment. The MSP-130 is located within the dimorphic region of the MSP-1 protein . Therefore, different epitopes in these dimorphic regions may be presented to B-cells generating heterogeneity in antibody responses between the two alleles. These results demonstrate that, at least for p38, p42 and p83, responses to one allelic form predict positive responses to the other allelic form. It is possible that conserved epitopes between the two allelic forms are presented to the immune system leading to the generation of cross-reactive antibodies. In fact, a previous study demonstrated a high degree of cross-inhibition between antibodies generated against the D and the F allelic forms of MSP-1 .
IgG subclass analysis showed that IgG1 and IgG3 were the predominant subclasses for most of the antigens studied, consistent with previous studies [34, 51, 61]. While mixed IgG1/IgG3 responses to all MSP antigens used was detected, the relative proportions of these two subclasses were different for the different MSP antigens, implying that IgG class switching may be greatly influenced by the characteristics of the antigen as previously suggested [40, 51, 62]. It has been suggested that conserved antigens, such as MSP-119, typically induce IgG1, while highly polymorphic antigens induce IgG3 . Interestingly in this study, such a correlation was not observed except for the MSP-119 antigen which elicited more IgG1 than IgG3 as previously observed [40, 51, 61, 64]. In contrast, a mixed response to the conserved MSP-636 was observed while a previous study in southern-central Vietnam demonstrated a skewing towards IgG1 . Similarly, predominant IgG3 responses were observed for the highly polymorphic block 2 region (found within the p83 subunit) of MSP-1 in an African population  and for polymorphic MSP-722 in Vietnam . However, in the current study, MSP-722 induced more IgG1 antibodies than IgG3 and MSP-1d83 showed a mixed response, while MSP-1f83 elicited more IgG1 antibodies than IgG3. These differences may be due to differences in innate characteristics of the host population or epidemiologic differences.