Antibody responses to the merozoite surface protein-1 complex in cerebral malaria patients in India

One life-threatening complication of Plasmodium falciparum infection is cerebral malaria (CM). This complex syndrome affects mainly young children (two to six years old) in sub-Saharan Africa with an estimated incidence of 1.12 cases per 1,000 children per year and an estimated mortality of 18.6% [1]. In addition, a subset of CM survivors have an increased risk of developing persistent neurocognitive sequelae post-recovery [2‐4] and reviewed in [5]. In Asia and South America, where the intensity of P. falciparum is much lower than in Africa, all age groups are at risk for CM [1, 6‐9]. The pathogenesis of CM is complex and it is still poorly understood as to why only a subset of patients develop CM. Various factors, such as sequestration of infected erythrocytes, and inflammatory cytokines and chemokines, have been postulated to play major roles in CM pathogenesis [10‐17]. The role of antibodies in CM pathogenesis or protection is not well understood.

The merozoite surface protein (MSP)-1, a large multiprotein complex exposed on the surface of merozoites, is one of the well characterized antigens of P. falciparum. During late schizogony, MSP-1 is proteolytically processed from its ~190 kDa precursor into four major cleavage products: p83, p30, p38, and p42 [18] designated as MSP-1₈₃, MSP-1₃₀, MSP-1₃₈ and MSP-1₄₂ respectively. During erythrocyte invasion, the MSP-1₄₂ fragment is further cleaved into MSP-1₃₃ and MSP-1₁₉ which is essential for invasion (Figure 1A) [19]. The proteolytically processed MSP-1 appears to exist in association with the processed products of MSP-6 and MSP-7 (Figure 1B) [20‐22]. Major biochemical and immunological parameters of this multipartite have been described recently [23].

Humoral immune responses to MSP-1 protein subunits, especially, MSP-1₄₂ and MSP-1₁₉ fragments, are known to be protective against P. falciparum infection and clinical malaria [24‐33]. Antibodies specific for these antigens have been shown to inhibit both erythrocyte invasion and parasite growth in vitro [26, 27]. In some studies, antibody responses to MSP-1₁₉ were correlated with clinical immunity to P. falciparum [29, 30, 34] and with reduced parasitaemia and fever [31]. In addition, presence of several T-cell epitopes within the MSP-1₄₂ fragment were identified [35] and these epitopes may provide T- helper function needed for the production of anti-MSP-1 antibodies.

Studies of the msp-1 gene sequence obtained from different P. falciparum isolates demonstrate significant antigenic diversity comprising highly conserved, dimorphic and variable regions. There are two major allelic forms of MSP-1 belonging to either the K1, (as in the FCB-1 strain, here referred to as F allelic form) or the MAD20 (as in the 3D7 strain, here referred to as D allelic form) allelic families [36, 37]. Therefore, one would postulate that naturally exposed individuals would mount immune responses to different fragments and allelic forms of MSP-1. However, only a few field studies have investigated humoral responses to both of these allelic forms of the four major subunits of MSP-1 and their associated proteins, MSP-6₃₆ and MSP-7₂₂[25, 38‐40] in humans naturally exposed to malaria. A complete characterization of humoral immune responses to this complex protein is therefore required to determine their role in protective immunity and pathogenesis.

The potential adverse effect of malarial antibody responses, including antibodies to the C-terminal region of MSP-1, in the manifestation of CM have been implicated in some studies [41‐45]. For example, higher levels of IgM and IgG antibodies to glycosylphosphatidylinositols (GPI) were associated with CM and death in young children in a study conducted in Mali [45]. On the contrary, in another study, reduced anti-GPI antibodies were found in CM patients compared to mild malaria patients [41]. In a Ghanaian study, IgG2 and IgG4 antibody responses to a recombinant P. falciparum RIFIN antigen, RIF-29, were exclusively shown to be associated with CM, suggesting that these antibodies might be involved in CM pathogenesis [44].

In the current study, the antibody response to the two major allelic forms (F and D) of MSP-1 antigens (MSP-1₈₃, MSP-1₃₀, MSP-1₃₈ and MSP-1₄₂) and their associated proteins, MSP-6₃₆ and MSP-7₂₂, was systematically characterized in an Indian cohort and the responses compared between two different group of malaria patients and healthy controls.

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 [41]. 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 [45]. 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 [44]. 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 [39]. 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 [39]. Anti-MSP-6₃₆ antibodies were shown to be generated in individuals naturally infected with P. falciparum [55]. 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-1_d30 antigen was low as had been observed in previous studies [39], 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 16^th and 17^th block [58], 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 [36]. 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 [39].

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 [63]. 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 [63]. 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 [40] and for polymorphic MSP-7₂₂ in Vietnam [63]. However, in the current study, MSP-7₂₂ induced more IgG1 antibodies than IgG3 and MSP-1_d83 showed a mixed response, while MSP-1_f83 elicited more IgG1 antibodies than IgG3. These differences may be due to differences in innate characteristics of the host population or epidemiologic differences.