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Cytophilic Antibodies Against Key Plasmodium falciparum Blood Stage Antigens Contribute to Protection Against Clinical Malaria in a High Transmission Region of Eastern India

Cytophilic Antibodies Against Key Plasmodium falciparum Blood Stage Antigens Contribute to... Abstract Background The collection of clinical data from a tribal population in a malaria-endemic area of India suggests the occurrence of naturally acquired immunity (NAI) against Plasmodium falciparum malaria. Methods Quantity and functionality of immunoglobulin G (IgG) antibodies against intact merozoites and recombinant proteins were assessed in a 13-month longitudinal cohort study of 121 individuals, 3–60 years of age. Results Opsonic phagocytosis of merozoites activity was strongly associated (hazard ratio [HR] = 0.34; 95% confidence interval [CI] = .18–.66; P = .0013) with protection against febrile malaria. Of the different IgG subclasses, only IgG3 antibodies against intact whole merozoites was significantly associated with protection against febrile malaria (HR = 0.47; 95% CI = .26–.86; P = .01). Furthermore, a combination of IgG3 antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 was strongly associated with protection against febrile malaria (HR = 0.15; 95% CI, .06–.37; P = .0001). Conclusions These data suggest that NAI may, at least in part, be explained by opsonic phagocytosis of merozoites and IgG3 responses against whole merozoites, and in particular to a combination of 4 antigens is critical in this population. These results may have implications in the development of a subunit malaria vaccine. Opsonic phagocytosis of Plasmodium falciparum merozoites was associated with protection against clinical malaria in an India population. Antibody profiling identified four merozoite antigens (Pf12, MSP3.7, MSP3.3, and MSP2) as targets of protective Immunoglobuline G3 antibodies. merozoites antigens, naturally acquired immunity, opsonic phagocytosis Malaria remains a worldwide health problem with 216 million cases and 445000 deaths in 2016 [1]. A significant amount of effort currently aimed at finding an efficacious vaccine against blood stage malaria has focused mainly on seroepidemiological and functional studies in sub-Saharan African populations where the highest burden of the disease occurs. Although such an approach is clearly justified, malaria studies in other endemic areas such as India [2] may also contribute to deepening the current understanding of malaria immunity. Among a total of 15 countries, India is the only country outside of sub-Saharan Africa that carried 80% of the global malaria burden [1], although the exact number of deaths in the Indian population remains unclear [3, 4]. Of the 6 Plasmodium species known to cause malaria in humans [5], India is endemic to Plasmodium falciparum and Plasmodium vivax. Of these, P falciparum is responsible for the most severe forms of malaria and more likely to cause death. Immunity against P falciparum malaria develops naturally after repeated exposure to the parasite, and the passive transfer of immunoglobulin G (IgG) from immune African adults has demonstrated that antibodies can control parasite multiplication and clinical symptoms [6, 7]. The exact targets of these malaria protective antibodies and the mechanisms by which they exert their antimalarial action have not been conclusively ascertained and have remained the subject of intense studies for decades. Because merozoites are exposed to the human immune system, merozoite-associated proteins are likely targets of naturally acquired immunity (NAI) [8–10]. Numerous studies performed in human populations have shown positive associations between cytophilic (IgG1 and IgG3) antibodies against merozoite surface proteins and protection against clinical malaria [11–17]. Some recent studies have further demonstrated that combinations of specific antibodies are stronger predictors of protection than the individual responses [13, 14, 17, 18]. How antibodies against the merozoite control parasite multiplication in the infected individuals remains unclear, and several mechanisms including growth inhibition [19], complement-mediated lysis [20, 21], antibody-dependent cellular inhibition (ADCI) [22], opsonic phagocytosis [23–25], and antibody-dependent respiratory burst (ADRB) activity by polymorphonuclear neutrophils [26–28] have been proposed. Of these, monocyte-mediated mechanisms have proven to be particularly strong predictors of protective immunity in different endemic populations [9, 23, 29, 30]. So far, the merozoite surface proteins MSP2 [23], MSP3 [23], GLURP [9, 31], MSPDBL1, and MSPDBL2 [32] have been identified as targets of opsonizing antibodies and may be crucial to NAI against malaria. To improve our understanding of the mechanism(s) underlying NAI and to identify new malaria vaccine candidate antigens, this study characterized antibody responses against whole intact merozoites and recombinant P falciparum proteins in whole blood samples from individuals living in tribal communities of Eastern India endemic for malaria. MATERIAL AND METHODS Ethics Statement The study was approved by the Institutional Ethics Committee of the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi. Villagers were informed about the purpose of the study, and informed consent was obtained from study participants or their guardians before enrollment in the study. Samples used for this study were obtained in accordance with the Indian Council of Medical Research Material Transfer Agreement and the Health Ministry’s Screening Committee. Study Area, Population, and Baseline Sampling The data presented here were generated from a longitudinal malaria cohort study conducted in Dumargarhi village, described in detail elsewhere [33]. In brief, 386 of the 945 villagers were sampled at cross-sectional survey-1 ([CSS1] conducted in October–December 2014) and followed up actively and passively for malaria case detection in a 13-month longitudinal cohort study (LCS) [33]. Febrile malaria was defined as fever (axillary temperature ≥36.5°C, measured or reported) in conjunction with microscopically confirmed P falciparum infection plus any other symptom of malaria such as vomiting, diarrhoea, or malaise. At the end of the study, villagers in whom parasitemia was associated with febrile malaria were considered susceptible, whereas those who did not experience any febrile malaria despite parasitemia were considered protected. Most of the febrile malaria cases (95%) were due to P falciparum during the longitudinal follow-up [33]. Therefore, we focused on the individuals (n = 121; aged 3–60 years) who were definitively exposed to P falciparum malaria as determined by microscopy and excluded those with P vivax or mixed infections. Parasite Culture and Merozoites Isolation A previously published method was used to culture the laboratory-adapted P falciparum line NF54 [34]. Isolation of merozoites was performed as previously described [35]. In brief, mature trophozoite-early schizoint stage parasites were harvested by using a magnetic separation unit and then cultured in 10 μM E64 (protease inhibitor)-supplemented parasite medium for up to 10 hours. Free merozoites were obtained by filtering the mature schizionts through a 1.2-μm syringe filter. After the hemozoin removal, merozoites were stained with ethidium bromide (EtBr) at 10 μg/mL final concentration. After a couple of washes with THP-1 medium (Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum [FCS]), merozoites were counted using a Beckman Coulter cytometer and kept in THP-1 medium until use. Samples and Controls Blood samples from CSS1 were stored as dried blood spot samples (DBSS) [36]. Antibodies were extracted from DBSS as described in detail [36]. Filter paper debris and residuals blood cells were removed by centrifugation at 2000× g for 30 minutes. Complement was eliminated by heat-inactivating samples, thereby allowing us to focus functional studies on Fc-receptor-mediated phagocytosis. Hyperimmune sera (HS) and normal sera obtained from Liberians and Danes were used as positive and negative controls, respectively. Flow Cytometry-Based Immunofluorescence Assay The flow cytometry-based immunofluorescence assay (FC-IFA) was performed as described in detail [9] with some modifications. In brief, each well of a 96-well U-bottom plate was seeded with 4 × 105 merozoites in 100 μL wash buffer (0.5% bovine serum albumin [BSA] in phosphate-buffered saline [PBS]). Test samples were added at a dilution of 1:100, and the mixture was incubated for 1 hour on a vibrating shaker at room temperature (RT). Plates were centrifuged, washed twice, and then incubated for 1 hour with 100 μL fluorescein isothiocyanate-conjugated sheep anti-human IgG1, IgG2, or IgG3 at 1:4000, 1:1000, or 1:3000, respectively. After thorough washing, merozoites were resuspended in 200 μL wash buffer and analyzed in a Beckman Coulter cytometer. Centrifugations were performed at 2000× g for 5 minutes, and washes were performed with wash buffer. Kaluza Analysis Software was used to obtain median fluorescence intensities (MFIs). Opsonic Phagocytosis Assay The opsonic phagocytosis of merozoite assay was performed as previously described [9], with minor modifications. In brief, 1 μL extracted DBSS was mixed with 4 × 105 EtBr-stained merozoites resuspended in 100 μL THP-1 medium and incubated for 30 minutes. After a centrifugation step at 2000× g for 5 minutes, merozoite pellets were resuspended in fresh 100 μL THP-1 medium before being transferred to FCS-coated, 96-well U-bottom plates containing 1 × 105 THP-1 cells/100 μL in each well. After half-hour incubation at 37°C, phagocytosis was stopped by washing the plates twice with chilled fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% BSA + 2 mM ethylenediaminetetraacetic acid). Before analysis with flow cytometry, samples were fixed in ice cold FACS fixative (2% paraformaldehyde in FACS buffer). Phagocytosis index (PI) refers to the percentage of EtBr-positive THP-1 cells (level of phagocytosis). Expression of Recombinant Merozoite Antigens We expressed a panel of 25 merozoite antigens including antigens that are exclusively expressed on the merozoite surface and those released by rhoptries and micronemes during or before erythrocyte invasion. All antigens were expressed as C-terminally His-tagged proteins (see Supplementary Table 1) using the Lactococcus lactis expression system [37]. Both conserved and variable subdomains of certain antigens were expressed as separate recombinant proteins to assess antibody responses to allele-specific as well as conserved regions. Multiplex Assay Conjugation of antigens to beads and antibody quantification were done according to a previously published method [38], with minor adjustments. In brief, 50 μL bead suspension containing 1250 beads of each antigen-coupled bead region was transferred to each well of a 96-well filter microtiter plate. Test samples were added at a dilution of 1:100 and incubated for 2 hours on vibrating shaker at RT. After 3 washes, plates were incubated for 1 hour with mouse antihuman IgG3 (diluted 1:5000), followed by 3 more washes before 1 hour incubation with phycoerythrin-labeled goat antimouse IgG (diluted 1:200). Finally, beads were resuspended in 100 μL assay buffer E (0.05% sodium azide, 0.05% Tween 20, 0.1% BSA in PBS), which was also used during washing steps, and analyzed on a Luminex. Mean fluorescence intensities represented the antibody levels, and positive control (HS) was used as calibrator to correct for interplate variability. Statistics Multiple linear regression determined the association between PI and age. The relationship among the covariates (see Supplementary Table 2), IgG3 antibody responses to individual antigens, and febrile malaria was assessed by multivariate logistic regression analyses. Antibody responses to each antigen were stratified into quartiles and assigned 0, 1, 2, or 3 for the lowest, 2nd, 3rd, or highest quartile, respectively, and summed across antigens to generate a breadth score. The PI, FC-IFA-derived MFI values, and breadth scores were stratified into 2 equal groups (low or high) based on the median. Survival analyses were performed to investigate associations between time-to-first malaria episode and these categorical variables. Mann-Whitney and Kruskal-Wallis tests were used to determine differences in breadth score between categorical variables. Depending upon the analysis used, age alone (categorized at 3 levels: 1–10, 11–15 and 16–60 years) or categorized age plus IgG3 reactivity (continuous scale) to whole merozoites were included as potential confounder(s). P < .05 was considered significant. RESULTS Study Design, Demographics, and Malaria Incidence Detailed demographics, baseline characteristics of the study participants, malaria prevalence, and parasite density-related information has been published previously [33]. The study area consisted of 5 hamlets inhabited by 945 individuals living in 164 households. Blood samples were collected from 386 individuals who were monitored in a LCS to assess antibody-mediated protection against malaria. Of these, 121 individuals were parasitaemic at least once during the study. Fifty individuals experienced 1 or more episodes of febrile malaria (susceptible group) and 71 individuals were asymptomatic throughout the follow-up and were considered protected against febrile malaria. Individuals with no evidence of malaria parasitemia by microscopy were excluded from further analysis. Of the different covariates studied, only age (16–60 years group) was significantly (odds ratio [OR] = .30; 95% confidence interval [CI] = .11–.77; P = .01) associated with protection against febrile malaria. Average age of individuals (n = 121) considered for this study was 21.2 years. Baseline parasite density, gender, bed net use, type of housing, and ethnicity were not identified as confounders (Supplementary Table 2). Association Between Opsonic Phagocytosis Activity and Protection Against Febrile Malaria A significant increase in opsonic phagocytosis was observed with increasing age (OR = 1.36; 95% CI = 1.17–1.59; P = .0001), with protected individuals particularly displaying higher opsonic phagocytosis activity than the susceptible (Figure 1A). There was a significant difference in the time to first malaria episode between individuals in the high and low opsonic phagocytosis groups (log rank, P < .0001) (Figure 1B). This association remained significant (hazard ratio [HR] = .34; 95% CI, .18–.66; P = .0013) after adjusting for age in a Cox regression analysis. Figure 1. View largeDownload slide Opsonic phagocytosis (OP) is positively correlated with age and a reduced risk of febrile malaria. (A) Opsonic phagocytosis activity in the whole cohort (n = 121) was positively correlated with age (Pearson’s r [overall] = 0.34; P = .0001). Red and blue (dots and lines) represent protected and susceptible individuals, respectively. (B) The probability of remaining free of malaria was significantly higher for individuals in the high OP group (blue curve) compared with those in the low OP group (red curve). Figure 1. View largeDownload slide Opsonic phagocytosis (OP) is positively correlated with age and a reduced risk of febrile malaria. (A) Opsonic phagocytosis activity in the whole cohort (n = 121) was positively correlated with age (Pearson’s r [overall] = 0.34; P = .0001). Red and blue (dots and lines) represent protected and susceptible individuals, respectively. (B) The probability of remaining free of malaria was significantly higher for individuals in the high OP group (blue curve) compared with those in the low OP group (red curve). Association Between Levels of Antibodies Against Intact Merozoites and Febrile Malaria To investigate the role of IgG subclasses in mediating protective immunity in this population, levels of antimerozoite antibodies were determined by FC-IFA, because this assay proved useful for detecting protective antibodies in Ghana [9]. The antibody responses were categorized as low or high based on respective MFIs. Of the 3 IgG subclasses evaluated, high levels of cytophilic IgG1 and IgG3 were significantly associated with a higher probability of remaining malaria-free during follow-up by Cox regression analysis (unadjusted HR [uHR] = .54; 95% CI, .31–.96; P = .037 for IgG1; uHR = .41; 95% CI, .23–.74; P = .003 for IgG3). However, only IgG3 subclass remained significant (HR = .47; 95% CI, .26–.86; P = .01) after adjusting for age (Figure 2). Figure 2. View largeDownload slide Cytophillic antibodies against intact merozoites predict protection against febrile malaria. Median fluorescence intensities (MFIs) for each immunoglobulin G (IgG) subclass (IgG1, IgG2, and IgG3), determined by the flow cytometry-based immunofluorescence assay, were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios (HRs), 95% confidence intervals (CIs), and P values for each IgG subclass in turn, comparing those with high versus low (reference group) MFIs, for the probability of febrile malaria over a 13-month follow-up. Only first febrile malaria episode was considered in each analysis. Values represent age-adjusted (filled circles) and unadjusted (filled squares) HRs, with 95% CI. Figure 2. View largeDownload slide Cytophillic antibodies against intact merozoites predict protection against febrile malaria. Median fluorescence intensities (MFIs) for each immunoglobulin G (IgG) subclass (IgG1, IgG2, and IgG3), determined by the flow cytometry-based immunofluorescence assay, were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios (HRs), 95% confidence intervals (CIs), and P values for each IgG subclass in turn, comparing those with high versus low (reference group) MFIs, for the probability of febrile malaria over a 13-month follow-up. Only first febrile malaria episode was considered in each analysis. Values represent age-adjusted (filled circles) and unadjusted (filled squares) HRs, with 95% CI. Association Between Antigen-Specific Antibodies and Protection Against Febrile Malaria To delineate individual targets of protective antibodies, DBSS were evaluated against a panel of merozoite antigens. Antigen-specific IgG3 levels were higher in older individuals than the younger age groups (Supplementary Table 3), suggesting that merozoite-specific antibodies results from cumulative exposure to P falciparum infections in this population. Therefore, levels of antigen-specific antibodies were assessed in multivariable logistic regression adjusting for age and IgG3 reactivity to whole merozoites because these variables were individually associated with protection against febrile malaria. We found that antibodies against some but not all antigens were associated with protection against febrile malaria (Supplementary Table 4). The proteins most strongly associated with protection from febrile malaria (Figure 3) were either integral membrane proteins or peripherally associated proteins. Figure 3. View largeDownload slide Antimerozoite antibody responses are associated with a reduced risk of febrile malaria. Immunoglobulin G3 antibody levels against a panel of merozoite antigens were assessed for their protective association in a longitudinal cohort of 121 individuals (71 protected and 50 susceptible). Multivariate logistic regression model was used to determine odds ratios (ORs), 95% confidence intervals (CIs), and P values. Each analysis was adjusted for age and immunoglobulin G3 reactivity to whole merozoites. Antibody titers were log to base 2 transformed; therefore, ORs and 95% CIs represent effect associated with 2-fold increase in antibody titers. Antigens are ranked based on increasing ORs (top to bottom), with circles indicating the ORs and error bars showing the 95% CI. The vertical dotted line indicates an OR of 1 (ie, no reduced risk of febrile malaria). Figure 3. View largeDownload slide Antimerozoite antibody responses are associated with a reduced risk of febrile malaria. Immunoglobulin G3 antibody levels against a panel of merozoite antigens were assessed for their protective association in a longitudinal cohort of 121 individuals (71 protected and 50 susceptible). Multivariate logistic regression model was used to determine odds ratios (ORs), 95% confidence intervals (CIs), and P values. Each analysis was adjusted for age and immunoglobulin G3 reactivity to whole merozoites. Antibody titers were log to base 2 transformed; therefore, ORs and 95% CIs represent effect associated with 2-fold increase in antibody titers. Antigens are ranked based on increasing ORs (top to bottom), with circles indicating the ORs and error bars showing the 95% CI. The vertical dotted line indicates an OR of 1 (ie, no reduced risk of febrile malaria). Association of Breadth of Antibody Specificities and Protection Against Febrile Malaria Several studies have shown that increasing breadth of antibody specificities is a predictor of protection [39, 40]. Accordingly, each DBSS was assigned a specific score depending on the magnitude of the antibody response (Figure 4). Protected individuals had significantly higher breadth score compared with susceptible (Figure 5A). The overall breadth score based on all 25 antigens increased with age in both protected and susceptible villagers (Figure 5B). In general, the breadth score was higher in protected than in susceptible individuals when comparing the same age groups (Figure 5B). However, susceptible individuals in the 16–60 year age group had a higher median breadth scores than that of protected children in the 1–10 year age group, suggesting that some of the antigens included in this analysis are markers of repeated infection and/or exposure. Figure 4. View largeDownload slide Heat map showing immunoglobulin G3 antibody responses against a panel of antigens that were tested in a prospective longitudinal cohort study in India (n = 121). Antibody levels against each antigen were divided into quartiles, and a score was assigned to each quartile; 0, 1, 2, and 3 for first, second, third, and fourth quartile, respectively. Each score is represented by a color going from white (0) to dark color (3). For each individual, these designated scores (0 to 3) were added for all 25 antigens giving a breadth score between 0 and 75. Each column represents an individual, and each row represents an antigen. Individuals are divided into those who did not have a case of febrile malaria (protected) and those who did (susceptible). Individuals are also sorted in increasing age. Figure 4. View largeDownload slide Heat map showing immunoglobulin G3 antibody responses against a panel of antigens that were tested in a prospective longitudinal cohort study in India (n = 121). Antibody levels against each antigen were divided into quartiles, and a score was assigned to each quartile; 0, 1, 2, and 3 for first, second, third, and fourth quartile, respectively. Each score is represented by a color going from white (0) to dark color (3). For each individual, these designated scores (0 to 3) were added for all 25 antigens giving a breadth score between 0 and 75. Each column represents an individual, and each row represents an antigen. Individuals are divided into those who did not have a case of febrile malaria (protected) and those who did (susceptible). Individuals are also sorted in increasing age. Figure 5. View largeDownload slide Protective efficacy is associated with breadth of immunoglobulin G3 (IgG3) responses. (A) A significant difference was observed in the overall breadth scores of protected (n = 71) and susceptible (n = 50) individuals. (B) Overall, the breadth scores (for 25 antigens) significantly increased with age in both protected and susceptible individuals. (C) Breadth scores obtained for combinations of 25, 7 significantly associated, or 4 most protective antigens were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios, 95% confidence intervals (CIs), and P values for each antigen combination in turn, comparing those with high versus low (reference group) breadth score for the probability of febrile malaria over a 13-month follow up. Only first febrile malaria episode was considered in each analysis. Values represent age and IgG3 reactivity to whole merozoites-adjusted (filled circles) and unadjusted (filled squares) hazard ratios, with 95% CI. (D) Breadth scores were strongly associated with age in the protected but not the susceptible individuals when scores were considered only for a combination of the 4 most protective antigens (Pf12, MSP3.3, MSP3.7, and MSP2FC27). For A, B, and D, data are plotted as box-and-whisker plots showing medians and interquartile ranges. Statistical significance was determined by Mann-Whitney test for A and by Kruskal-Wallis test for B and D. **, P < .01; ***, P < .001. Abbreviation: ns, not significant. Figure 5. View largeDownload slide Protective efficacy is associated with breadth of immunoglobulin G3 (IgG3) responses. (A) A significant difference was observed in the overall breadth scores of protected (n = 71) and susceptible (n = 50) individuals. (B) Overall, the breadth scores (for 25 antigens) significantly increased with age in both protected and susceptible individuals. (C) Breadth scores obtained for combinations of 25, 7 significantly associated, or 4 most protective antigens were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios, 95% confidence intervals (CIs), and P values for each antigen combination in turn, comparing those with high versus low (reference group) breadth score for the probability of febrile malaria over a 13-month follow up. Only first febrile malaria episode was considered in each analysis. Values represent age and IgG3 reactivity to whole merozoites-adjusted (filled circles) and unadjusted (filled squares) hazard ratios, with 95% CI. (D) Breadth scores were strongly associated with age in the protected but not the susceptible individuals when scores were considered only for a combination of the 4 most protective antigens (Pf12, MSP3.3, MSP3.7, and MSP2FC27). For A, B, and D, data are plotted as box-and-whisker plots showing medians and interquartile ranges. Statistical significance was determined by Mann-Whitney test for A and by Kruskal-Wallis test for B and D. **, P < .01; ***, P < .001. Abbreviation: ns, not significant. Individuals with high breadth score (based on all 25 antigens) had a significantly (HR = 0.34; 95% CI, .15–.79; P = .01) reduced risk of febrile malaria compared with those with low breadth score in a Cox regression adjusting for age and merozoites IgG3 responses (Figure 5C). Next, the analysis was restricted to include only the 7 antigens (Figure 5C) whose antibody responses were significantly associated with protection against febrile malaria (Supplementary Table 4). Finally, when highly correlated antibodies with higher odds ratios were excluded, Pf12, MSP3.7, MSP3.3, and MSP2FC27 were the strongest predictors of protection (HR = .15; 95% CI, .06–.37; P = .0001) (Figure 5C). More importantly, the median breadth score of this combination was always higher in protected than susceptible individuals across all age groups (Figure 5D). Relationship Among Opsonic Phagocytosis, Merozoite Immunoglobulin G3 Responses, Breadth Score, and Malaria Status The interrelationships among the malaria protective variables opsonic phagocytosis, merozoite IgG3 responses in the IFA, breadth score based on the final 4 antigens (Pf12, MSP3.7, MSP3.3, and MSP2FC27), and malaria status of the study participants were visualized using a bubble plot (Figure 6). Both opsonic phagocytosis and breadth score increased simultaneously with increasing merozoite IgG3 responses. Susceptible individuals had lower opsonic phagocytosis, merozoite IgG3 responses, and smaller breadth score values (Figure 6, blue circles) compared with protected individuals (Figure 6, red circles). Figure 6. View largeDownload slide Bubble plot of the interrelationship among opsonic phagocytosis, merozoite immunoglobulin G3 (IgG3) responses, breadth score, and malaria status. Opsonic phagocytosis was plotted against merozoite-specific IgG3 responses together with breadth score for the final 4 antigens (Pf12, MSP3.7, MSP3.3, and MSP2FC27) for susceptible (blue circle) and protected (red circles) individuals. Each circle represents an individual, and the size of the circle is proportional to the breadth score (range, 0–12). Protected individuals have higher breadth score compared with susceptible individuals. Abbreviations: IFA, immunofluorescence assay; MFI, mean fluorescence intensity. Figure 6. View largeDownload slide Bubble plot of the interrelationship among opsonic phagocytosis, merozoite immunoglobulin G3 (IgG3) responses, breadth score, and malaria status. Opsonic phagocytosis was plotted against merozoite-specific IgG3 responses together with breadth score for the final 4 antigens (Pf12, MSP3.7, MSP3.3, and MSP2FC27) for susceptible (blue circle) and protected (red circles) individuals. Each circle represents an individual, and the size of the circle is proportional to the breadth score (range, 0–12). Protected individuals have higher breadth score compared with susceptible individuals. Abbreviations: IFA, immunofluorescence assay; MFI, mean fluorescence intensity. DISCUSSION Increasing evidence from studies performed mainly in malaria-endemic sub-Saharan African populations indicate that opsonic phagocytosis of merozoites plays a critical role in NAI against malaria [23, 30, 41]. However, data on the relevance of merozoite-specific antibodies for NAI in other malaria-endemic populations such as India are presently lacking. Using samples and data from an LCS performed in a tribal population in India, we have quantified functional antibodies against whole merozoites and assessed the importance of specific merozoite antigens as targets of NAI. We found that opsonic phagocytosis of merozoites is a strong predictor of protection against febrile malaria and that antibodies of the IgG3 subclass play a key role. More importantly, levels of opsonic phagocytosis as well as levels of merozoite IgG3 increased with age, which is consistent with acquisition of NAI in this population [33]. Thus, our data reinforce previously published data that opsonic phagocytosis of merozoites is a strong correlate of NAI worldwide [23, 30, 41]. Notwithstanding, other immune mechanisms such as antibody-mediated complement-dependent inhibition [20], direct growth inhibition [19], ADRB [26], and ADCI [29] are likely to contribute to NAI. The relative importance of these mechanisms in this population remains to be investigated. The proposed importance of merozoites in immunity is further underscored by a detailed profiling of protective immunity against a large panel of merozoite antigens. Several of these merozoite antigens are disulfide-bonded, and, because the correct cysteine connectivity is essential for antibody recognition [42], we have used antigens produced in the L lactis expression system, which is highly suited for the production of cysteine-rich malaria proteins [37]. The concept of screening large numbers of antigens with the aim of prioritizing antigenic targets of malaria protective immunity is relatively new, and methods for down-selection are still developing. The theoretical outcome of combining the 25 antigens in our study taking any 2, 3, or 4 at a time would have resulted in a total of 15250 possible combinations. Thus, there is a need for specialized algorithms that will allow a more intuitive way of creating such combinations conditioned on some functional biological parameters. Others have studied protective roles of combined antigen responses by using a summation of graded quartile responses and categorizing the sums into different levels of magnitude to obtain different combinations [10]. In this study, we favored different regression approaches for arriving at combinations of potentially protective antigens because these have been used extensively in malaria studies and the interpretations are more straightforward. In this study, as in most malaria immune-epidemiological studies, age was associated with protection against symptomatic infection. It has been hypothesized (1) that cumulative exposure to malaria infection alone does not sufficiently explain the susceptibility of children and resistance of adults to symptomatic infection and (2) that intrinsic age-dependent immune factors may be key to NAI [43]. Thus, it is plausible that the humoral and cellular components of NAI differ qualitatively and/or quantitatively with age, an idea that warrants further investigations. Multivariate logistic regression models correcting for the confounding effects of age and merozoite IgG3 identified levels of IgG3 antibodies against 7 antigens: Pf12, MSP3.7, MSP3.3, GLURP-R0, cMSP3, MSP119k, and MSP2FC27, as associated with protection against febrile malaria. It is interesting to note that these antigens were characterized as weakly associated with protection in Papua New Guinean children [10], whereas the top ranking antigens in that previous study showed no association with protection in the Indian population studied here. The exact reason for this discrepancy is unknown, but differences in study design, ethnicity, and statistical modeling may provide an explanation. Some of the top ranking antigens including GLURP and MSP3 (reviewed in [44]), MSP2 [45, 46], and MSP1 [47] have been studied extensively in several longitudinal cohorts, whereas there is little published information available on the relevance of Pf12, MSP3.7, and MSP3.3 [10] for malaria immunity. Although levels of antibodies against these antigens were highly associated with protection against febrile malaria, the combined antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 were more strongly associated with protective immunity than the individual antigens, suggesting an additive effect on protective associations. The combined effect of these 4 antigens was associated with an 85% reduction in the probability of developing febrile malaria during follow up. It is generally agreed that antibodies against no single antigen can explain NAI against febrile malaria [8, 13, 40]. It seems that protective immunity relies on a robust antibody response against multiple antigens [13, 17, 18, 40], and it has been hypothesized that both the breadth and the magnitude of specific responses are critical for protective immunity [40]. Some studies have already investigated this issue in different populations, and it appears that multiple antigen combinations may provide protective immunity in the different populations [10, 14, 40, 48, 49]. In some populations EBA, PfRh2, and PfRh4 proteins are important [10], whereas in other populations combinations of AMA1, MSP2, and MSP3 were strong predictors of protection [40]. Thus, neither a single antigen nor a specific combination of antigens is required for the development of protective immunity. Most likely, protective immunity results from antibody responses against multiple antigens and depends on a certain threshold concentration [13]. In this study, we have identified a combination of both integral membrane proteins and peripherally associated proteins as targets of protective immunity in a tribal population. Thus, reinforcing the perception that the breadth of antibody responses to merozoite antigens is a better predictor of protective immunity than the responses to the individual antigens [10, 14, 40, 50]. The observation in this study that the breadth score correlates with opsonic phagocytosis, IgG3 antibody responses against merozoites, and protection against malaria further corroborates this assertion. CONCLUSIONS In conclusion, this study shows evidence of the opsonic phagocytosis of merozoites as an important player in NAI against febrile malaria in a high transmission region of Eastern India and has further identified Pf12, MSP3.7, MSP3.3, and MSP2FC27 as potentially important antigenic targets of protective immunity. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Acknowledgments. We are grateful to the Indian Council of Medical Research and the director of National Institute of Malaria Research for support in conducting this study. Financial support. This work was funded by the Danish Council for Strategic Research (grant 13127], the Department of Biotechnology, Government of India (BT/IN/Denmark/13/SS/2013), and Danish Ministry of Foreign Affairs (DFC file no. 14-P01-GHA). Potential conflicts of interest. All authors: No reported conflicts of Interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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Cytophilic Antibodies Against Key Plasmodium falciparum Blood Stage Antigens Contribute to Protection Against Clinical Malaria in a High Transmission Region of Eastern India

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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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Abstract

Abstract Background The collection of clinical data from a tribal population in a malaria-endemic area of India suggests the occurrence of naturally acquired immunity (NAI) against Plasmodium falciparum malaria. Methods Quantity and functionality of immunoglobulin G (IgG) antibodies against intact merozoites and recombinant proteins were assessed in a 13-month longitudinal cohort study of 121 individuals, 3–60 years of age. Results Opsonic phagocytosis of merozoites activity was strongly associated (hazard ratio [HR] = 0.34; 95% confidence interval [CI] = .18–.66; P = .0013) with protection against febrile malaria. Of the different IgG subclasses, only IgG3 antibodies against intact whole merozoites was significantly associated with protection against febrile malaria (HR = 0.47; 95% CI = .26–.86; P = .01). Furthermore, a combination of IgG3 antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 was strongly associated with protection against febrile malaria (HR = 0.15; 95% CI, .06–.37; P = .0001). Conclusions These data suggest that NAI may, at least in part, be explained by opsonic phagocytosis of merozoites and IgG3 responses against whole merozoites, and in particular to a combination of 4 antigens is critical in this population. These results may have implications in the development of a subunit malaria vaccine. Opsonic phagocytosis of Plasmodium falciparum merozoites was associated with protection against clinical malaria in an India population. Antibody profiling identified four merozoite antigens (Pf12, MSP3.7, MSP3.3, and MSP2) as targets of protective Immunoglobuline G3 antibodies. merozoites antigens, naturally acquired immunity, opsonic phagocytosis Malaria remains a worldwide health problem with 216 million cases and 445000 deaths in 2016 [1]. A significant amount of effort currently aimed at finding an efficacious vaccine against blood stage malaria has focused mainly on seroepidemiological and functional studies in sub-Saharan African populations where the highest burden of the disease occurs. Although such an approach is clearly justified, malaria studies in other endemic areas such as India [2] may also contribute to deepening the current understanding of malaria immunity. Among a total of 15 countries, India is the only country outside of sub-Saharan Africa that carried 80% of the global malaria burden [1], although the exact number of deaths in the Indian population remains unclear [3, 4]. Of the 6 Plasmodium species known to cause malaria in humans [5], India is endemic to Plasmodium falciparum and Plasmodium vivax. Of these, P falciparum is responsible for the most severe forms of malaria and more likely to cause death. Immunity against P falciparum malaria develops naturally after repeated exposure to the parasite, and the passive transfer of immunoglobulin G (IgG) from immune African adults has demonstrated that antibodies can control parasite multiplication and clinical symptoms [6, 7]. The exact targets of these malaria protective antibodies and the mechanisms by which they exert their antimalarial action have not been conclusively ascertained and have remained the subject of intense studies for decades. Because merozoites are exposed to the human immune system, merozoite-associated proteins are likely targets of naturally acquired immunity (NAI) [8–10]. Numerous studies performed in human populations have shown positive associations between cytophilic (IgG1 and IgG3) antibodies against merozoite surface proteins and protection against clinical malaria [11–17]. Some recent studies have further demonstrated that combinations of specific antibodies are stronger predictors of protection than the individual responses [13, 14, 17, 18]. How antibodies against the merozoite control parasite multiplication in the infected individuals remains unclear, and several mechanisms including growth inhibition [19], complement-mediated lysis [20, 21], antibody-dependent cellular inhibition (ADCI) [22], opsonic phagocytosis [23–25], and antibody-dependent respiratory burst (ADRB) activity by polymorphonuclear neutrophils [26–28] have been proposed. Of these, monocyte-mediated mechanisms have proven to be particularly strong predictors of protective immunity in different endemic populations [9, 23, 29, 30]. So far, the merozoite surface proteins MSP2 [23], MSP3 [23], GLURP [9, 31], MSPDBL1, and MSPDBL2 [32] have been identified as targets of opsonizing antibodies and may be crucial to NAI against malaria. To improve our understanding of the mechanism(s) underlying NAI and to identify new malaria vaccine candidate antigens, this study characterized antibody responses against whole intact merozoites and recombinant P falciparum proteins in whole blood samples from individuals living in tribal communities of Eastern India endemic for malaria. MATERIAL AND METHODS Ethics Statement The study was approved by the Institutional Ethics Committee of the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi. Villagers were informed about the purpose of the study, and informed consent was obtained from study participants or their guardians before enrollment in the study. Samples used for this study were obtained in accordance with the Indian Council of Medical Research Material Transfer Agreement and the Health Ministry’s Screening Committee. Study Area, Population, and Baseline Sampling The data presented here were generated from a longitudinal malaria cohort study conducted in Dumargarhi village, described in detail elsewhere [33]. In brief, 386 of the 945 villagers were sampled at cross-sectional survey-1 ([CSS1] conducted in October–December 2014) and followed up actively and passively for malaria case detection in a 13-month longitudinal cohort study (LCS) [33]. Febrile malaria was defined as fever (axillary temperature ≥36.5°C, measured or reported) in conjunction with microscopically confirmed P falciparum infection plus any other symptom of malaria such as vomiting, diarrhoea, or malaise. At the end of the study, villagers in whom parasitemia was associated with febrile malaria were considered susceptible, whereas those who did not experience any febrile malaria despite parasitemia were considered protected. Most of the febrile malaria cases (95%) were due to P falciparum during the longitudinal follow-up [33]. Therefore, we focused on the individuals (n = 121; aged 3–60 years) who were definitively exposed to P falciparum malaria as determined by microscopy and excluded those with P vivax or mixed infections. Parasite Culture and Merozoites Isolation A previously published method was used to culture the laboratory-adapted P falciparum line NF54 [34]. Isolation of merozoites was performed as previously described [35]. In brief, mature trophozoite-early schizoint stage parasites were harvested by using a magnetic separation unit and then cultured in 10 μM E64 (protease inhibitor)-supplemented parasite medium for up to 10 hours. Free merozoites were obtained by filtering the mature schizionts through a 1.2-μm syringe filter. After the hemozoin removal, merozoites were stained with ethidium bromide (EtBr) at 10 μg/mL final concentration. After a couple of washes with THP-1 medium (Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum [FCS]), merozoites were counted using a Beckman Coulter cytometer and kept in THP-1 medium until use. Samples and Controls Blood samples from CSS1 were stored as dried blood spot samples (DBSS) [36]. Antibodies were extracted from DBSS as described in detail [36]. Filter paper debris and residuals blood cells were removed by centrifugation at 2000× g for 30 minutes. Complement was eliminated by heat-inactivating samples, thereby allowing us to focus functional studies on Fc-receptor-mediated phagocytosis. Hyperimmune sera (HS) and normal sera obtained from Liberians and Danes were used as positive and negative controls, respectively. Flow Cytometry-Based Immunofluorescence Assay The flow cytometry-based immunofluorescence assay (FC-IFA) was performed as described in detail [9] with some modifications. In brief, each well of a 96-well U-bottom plate was seeded with 4 × 105 merozoites in 100 μL wash buffer (0.5% bovine serum albumin [BSA] in phosphate-buffered saline [PBS]). Test samples were added at a dilution of 1:100, and the mixture was incubated for 1 hour on a vibrating shaker at room temperature (RT). Plates were centrifuged, washed twice, and then incubated for 1 hour with 100 μL fluorescein isothiocyanate-conjugated sheep anti-human IgG1, IgG2, or IgG3 at 1:4000, 1:1000, or 1:3000, respectively. After thorough washing, merozoites were resuspended in 200 μL wash buffer and analyzed in a Beckman Coulter cytometer. Centrifugations were performed at 2000× g for 5 minutes, and washes were performed with wash buffer. Kaluza Analysis Software was used to obtain median fluorescence intensities (MFIs). Opsonic Phagocytosis Assay The opsonic phagocytosis of merozoite assay was performed as previously described [9], with minor modifications. In brief, 1 μL extracted DBSS was mixed with 4 × 105 EtBr-stained merozoites resuspended in 100 μL THP-1 medium and incubated for 30 minutes. After a centrifugation step at 2000× g for 5 minutes, merozoite pellets were resuspended in fresh 100 μL THP-1 medium before being transferred to FCS-coated, 96-well U-bottom plates containing 1 × 105 THP-1 cells/100 μL in each well. After half-hour incubation at 37°C, phagocytosis was stopped by washing the plates twice with chilled fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% BSA + 2 mM ethylenediaminetetraacetic acid). Before analysis with flow cytometry, samples were fixed in ice cold FACS fixative (2% paraformaldehyde in FACS buffer). Phagocytosis index (PI) refers to the percentage of EtBr-positive THP-1 cells (level of phagocytosis). Expression of Recombinant Merozoite Antigens We expressed a panel of 25 merozoite antigens including antigens that are exclusively expressed on the merozoite surface and those released by rhoptries and micronemes during or before erythrocyte invasion. All antigens were expressed as C-terminally His-tagged proteins (see Supplementary Table 1) using the Lactococcus lactis expression system [37]. Both conserved and variable subdomains of certain antigens were expressed as separate recombinant proteins to assess antibody responses to allele-specific as well as conserved regions. Multiplex Assay Conjugation of antigens to beads and antibody quantification were done according to a previously published method [38], with minor adjustments. In brief, 50 μL bead suspension containing 1250 beads of each antigen-coupled bead region was transferred to each well of a 96-well filter microtiter plate. Test samples were added at a dilution of 1:100 and incubated for 2 hours on vibrating shaker at RT. After 3 washes, plates were incubated for 1 hour with mouse antihuman IgG3 (diluted 1:5000), followed by 3 more washes before 1 hour incubation with phycoerythrin-labeled goat antimouse IgG (diluted 1:200). Finally, beads were resuspended in 100 μL assay buffer E (0.05% sodium azide, 0.05% Tween 20, 0.1% BSA in PBS), which was also used during washing steps, and analyzed on a Luminex. Mean fluorescence intensities represented the antibody levels, and positive control (HS) was used as calibrator to correct for interplate variability. Statistics Multiple linear regression determined the association between PI and age. The relationship among the covariates (see Supplementary Table 2), IgG3 antibody responses to individual antigens, and febrile malaria was assessed by multivariate logistic regression analyses. Antibody responses to each antigen were stratified into quartiles and assigned 0, 1, 2, or 3 for the lowest, 2nd, 3rd, or highest quartile, respectively, and summed across antigens to generate a breadth score. The PI, FC-IFA-derived MFI values, and breadth scores were stratified into 2 equal groups (low or high) based on the median. Survival analyses were performed to investigate associations between time-to-first malaria episode and these categorical variables. Mann-Whitney and Kruskal-Wallis tests were used to determine differences in breadth score between categorical variables. Depending upon the analysis used, age alone (categorized at 3 levels: 1–10, 11–15 and 16–60 years) or categorized age plus IgG3 reactivity (continuous scale) to whole merozoites were included as potential confounder(s). P < .05 was considered significant. RESULTS Study Design, Demographics, and Malaria Incidence Detailed demographics, baseline characteristics of the study participants, malaria prevalence, and parasite density-related information has been published previously [33]. The study area consisted of 5 hamlets inhabited by 945 individuals living in 164 households. Blood samples were collected from 386 individuals who were monitored in a LCS to assess antibody-mediated protection against malaria. Of these, 121 individuals were parasitaemic at least once during the study. Fifty individuals experienced 1 or more episodes of febrile malaria (susceptible group) and 71 individuals were asymptomatic throughout the follow-up and were considered protected against febrile malaria. Individuals with no evidence of malaria parasitemia by microscopy were excluded from further analysis. Of the different covariates studied, only age (16–60 years group) was significantly (odds ratio [OR] = .30; 95% confidence interval [CI] = .11–.77; P = .01) associated with protection against febrile malaria. Average age of individuals (n = 121) considered for this study was 21.2 years. Baseline parasite density, gender, bed net use, type of housing, and ethnicity were not identified as confounders (Supplementary Table 2). Association Between Opsonic Phagocytosis Activity and Protection Against Febrile Malaria A significant increase in opsonic phagocytosis was observed with increasing age (OR = 1.36; 95% CI = 1.17–1.59; P = .0001), with protected individuals particularly displaying higher opsonic phagocytosis activity than the susceptible (Figure 1A). There was a significant difference in the time to first malaria episode between individuals in the high and low opsonic phagocytosis groups (log rank, P < .0001) (Figure 1B). This association remained significant (hazard ratio [HR] = .34; 95% CI, .18–.66; P = .0013) after adjusting for age in a Cox regression analysis. Figure 1. View largeDownload slide Opsonic phagocytosis (OP) is positively correlated with age and a reduced risk of febrile malaria. (A) Opsonic phagocytosis activity in the whole cohort (n = 121) was positively correlated with age (Pearson’s r [overall] = 0.34; P = .0001). Red and blue (dots and lines) represent protected and susceptible individuals, respectively. (B) The probability of remaining free of malaria was significantly higher for individuals in the high OP group (blue curve) compared with those in the low OP group (red curve). Figure 1. View largeDownload slide Opsonic phagocytosis (OP) is positively correlated with age and a reduced risk of febrile malaria. (A) Opsonic phagocytosis activity in the whole cohort (n = 121) was positively correlated with age (Pearson’s r [overall] = 0.34; P = .0001). Red and blue (dots and lines) represent protected and susceptible individuals, respectively. (B) The probability of remaining free of malaria was significantly higher for individuals in the high OP group (blue curve) compared with those in the low OP group (red curve). Association Between Levels of Antibodies Against Intact Merozoites and Febrile Malaria To investigate the role of IgG subclasses in mediating protective immunity in this population, levels of antimerozoite antibodies were determined by FC-IFA, because this assay proved useful for detecting protective antibodies in Ghana [9]. The antibody responses were categorized as low or high based on respective MFIs. Of the 3 IgG subclasses evaluated, high levels of cytophilic IgG1 and IgG3 were significantly associated with a higher probability of remaining malaria-free during follow-up by Cox regression analysis (unadjusted HR [uHR] = .54; 95% CI, .31–.96; P = .037 for IgG1; uHR = .41; 95% CI, .23–.74; P = .003 for IgG3). However, only IgG3 subclass remained significant (HR = .47; 95% CI, .26–.86; P = .01) after adjusting for age (Figure 2). Figure 2. View largeDownload slide Cytophillic antibodies against intact merozoites predict protection against febrile malaria. Median fluorescence intensities (MFIs) for each immunoglobulin G (IgG) subclass (IgG1, IgG2, and IgG3), determined by the flow cytometry-based immunofluorescence assay, were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios (HRs), 95% confidence intervals (CIs), and P values for each IgG subclass in turn, comparing those with high versus low (reference group) MFIs, for the probability of febrile malaria over a 13-month follow-up. Only first febrile malaria episode was considered in each analysis. Values represent age-adjusted (filled circles) and unadjusted (filled squares) HRs, with 95% CI. Figure 2. View largeDownload slide Cytophillic antibodies against intact merozoites predict protection against febrile malaria. Median fluorescence intensities (MFIs) for each immunoglobulin G (IgG) subclass (IgG1, IgG2, and IgG3), determined by the flow cytometry-based immunofluorescence assay, were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios (HRs), 95% confidence intervals (CIs), and P values for each IgG subclass in turn, comparing those with high versus low (reference group) MFIs, for the probability of febrile malaria over a 13-month follow-up. Only first febrile malaria episode was considered in each analysis. Values represent age-adjusted (filled circles) and unadjusted (filled squares) HRs, with 95% CI. Association Between Antigen-Specific Antibodies and Protection Against Febrile Malaria To delineate individual targets of protective antibodies, DBSS were evaluated against a panel of merozoite antigens. Antigen-specific IgG3 levels were higher in older individuals than the younger age groups (Supplementary Table 3), suggesting that merozoite-specific antibodies results from cumulative exposure to P falciparum infections in this population. Therefore, levels of antigen-specific antibodies were assessed in multivariable logistic regression adjusting for age and IgG3 reactivity to whole merozoites because these variables were individually associated with protection against febrile malaria. We found that antibodies against some but not all antigens were associated with protection against febrile malaria (Supplementary Table 4). The proteins most strongly associated with protection from febrile malaria (Figure 3) were either integral membrane proteins or peripherally associated proteins. Figure 3. View largeDownload slide Antimerozoite antibody responses are associated with a reduced risk of febrile malaria. Immunoglobulin G3 antibody levels against a panel of merozoite antigens were assessed for their protective association in a longitudinal cohort of 121 individuals (71 protected and 50 susceptible). Multivariate logistic regression model was used to determine odds ratios (ORs), 95% confidence intervals (CIs), and P values. Each analysis was adjusted for age and immunoglobulin G3 reactivity to whole merozoites. Antibody titers were log to base 2 transformed; therefore, ORs and 95% CIs represent effect associated with 2-fold increase in antibody titers. Antigens are ranked based on increasing ORs (top to bottom), with circles indicating the ORs and error bars showing the 95% CI. The vertical dotted line indicates an OR of 1 (ie, no reduced risk of febrile malaria). Figure 3. View largeDownload slide Antimerozoite antibody responses are associated with a reduced risk of febrile malaria. Immunoglobulin G3 antibody levels against a panel of merozoite antigens were assessed for their protective association in a longitudinal cohort of 121 individuals (71 protected and 50 susceptible). Multivariate logistic regression model was used to determine odds ratios (ORs), 95% confidence intervals (CIs), and P values. Each analysis was adjusted for age and immunoglobulin G3 reactivity to whole merozoites. Antibody titers were log to base 2 transformed; therefore, ORs and 95% CIs represent effect associated with 2-fold increase in antibody titers. Antigens are ranked based on increasing ORs (top to bottom), with circles indicating the ORs and error bars showing the 95% CI. The vertical dotted line indicates an OR of 1 (ie, no reduced risk of febrile malaria). Association of Breadth of Antibody Specificities and Protection Against Febrile Malaria Several studies have shown that increasing breadth of antibody specificities is a predictor of protection [39, 40]. Accordingly, each DBSS was assigned a specific score depending on the magnitude of the antibody response (Figure 4). Protected individuals had significantly higher breadth score compared with susceptible (Figure 5A). The overall breadth score based on all 25 antigens increased with age in both protected and susceptible villagers (Figure 5B). In general, the breadth score was higher in protected than in susceptible individuals when comparing the same age groups (Figure 5B). However, susceptible individuals in the 16–60 year age group had a higher median breadth scores than that of protected children in the 1–10 year age group, suggesting that some of the antigens included in this analysis are markers of repeated infection and/or exposure. Figure 4. View largeDownload slide Heat map showing immunoglobulin G3 antibody responses against a panel of antigens that were tested in a prospective longitudinal cohort study in India (n = 121). Antibody levels against each antigen were divided into quartiles, and a score was assigned to each quartile; 0, 1, 2, and 3 for first, second, third, and fourth quartile, respectively. Each score is represented by a color going from white (0) to dark color (3). For each individual, these designated scores (0 to 3) were added for all 25 antigens giving a breadth score between 0 and 75. Each column represents an individual, and each row represents an antigen. Individuals are divided into those who did not have a case of febrile malaria (protected) and those who did (susceptible). Individuals are also sorted in increasing age. Figure 4. View largeDownload slide Heat map showing immunoglobulin G3 antibody responses against a panel of antigens that were tested in a prospective longitudinal cohort study in India (n = 121). Antibody levels against each antigen were divided into quartiles, and a score was assigned to each quartile; 0, 1, 2, and 3 for first, second, third, and fourth quartile, respectively. Each score is represented by a color going from white (0) to dark color (3). For each individual, these designated scores (0 to 3) were added for all 25 antigens giving a breadth score between 0 and 75. Each column represents an individual, and each row represents an antigen. Individuals are divided into those who did not have a case of febrile malaria (protected) and those who did (susceptible). Individuals are also sorted in increasing age. Figure 5. View largeDownload slide Protective efficacy is associated with breadth of immunoglobulin G3 (IgG3) responses. (A) A significant difference was observed in the overall breadth scores of protected (n = 71) and susceptible (n = 50) individuals. (B) Overall, the breadth scores (for 25 antigens) significantly increased with age in both protected and susceptible individuals. (C) Breadth scores obtained for combinations of 25, 7 significantly associated, or 4 most protective antigens were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios, 95% confidence intervals (CIs), and P values for each antigen combination in turn, comparing those with high versus low (reference group) breadth score for the probability of febrile malaria over a 13-month follow up. Only first febrile malaria episode was considered in each analysis. Values represent age and IgG3 reactivity to whole merozoites-adjusted (filled circles) and unadjusted (filled squares) hazard ratios, with 95% CI. (D) Breadth scores were strongly associated with age in the protected but not the susceptible individuals when scores were considered only for a combination of the 4 most protective antigens (Pf12, MSP3.3, MSP3.7, and MSP2FC27). For A, B, and D, data are plotted as box-and-whisker plots showing medians and interquartile ranges. Statistical significance was determined by Mann-Whitney test for A and by Kruskal-Wallis test for B and D. **, P < .01; ***, P < .001. Abbreviation: ns, not significant. Figure 5. View largeDownload slide Protective efficacy is associated with breadth of immunoglobulin G3 (IgG3) responses. (A) A significant difference was observed in the overall breadth scores of protected (n = 71) and susceptible (n = 50) individuals. (B) Overall, the breadth scores (for 25 antigens) significantly increased with age in both protected and susceptible individuals. (C) Breadth scores obtained for combinations of 25, 7 significantly associated, or 4 most protective antigens were stratified into 2 equal groups (low or high) based on the median. Cox proportional-hazards model was used to calculate hazard ratios, 95% confidence intervals (CIs), and P values for each antigen combination in turn, comparing those with high versus low (reference group) breadth score for the probability of febrile malaria over a 13-month follow up. Only first febrile malaria episode was considered in each analysis. Values represent age and IgG3 reactivity to whole merozoites-adjusted (filled circles) and unadjusted (filled squares) hazard ratios, with 95% CI. (D) Breadth scores were strongly associated with age in the protected but not the susceptible individuals when scores were considered only for a combination of the 4 most protective antigens (Pf12, MSP3.3, MSP3.7, and MSP2FC27). For A, B, and D, data are plotted as box-and-whisker plots showing medians and interquartile ranges. Statistical significance was determined by Mann-Whitney test for A and by Kruskal-Wallis test for B and D. **, P < .01; ***, P < .001. Abbreviation: ns, not significant. Individuals with high breadth score (based on all 25 antigens) had a significantly (HR = 0.34; 95% CI, .15–.79; P = .01) reduced risk of febrile malaria compared with those with low breadth score in a Cox regression adjusting for age and merozoites IgG3 responses (Figure 5C). Next, the analysis was restricted to include only the 7 antigens (Figure 5C) whose antibody responses were significantly associated with protection against febrile malaria (Supplementary Table 4). Finally, when highly correlated antibodies with higher odds ratios were excluded, Pf12, MSP3.7, MSP3.3, and MSP2FC27 were the strongest predictors of protection (HR = .15; 95% CI, .06–.37; P = .0001) (Figure 5C). More importantly, the median breadth score of this combination was always higher in protected than susceptible individuals across all age groups (Figure 5D). Relationship Among Opsonic Phagocytosis, Merozoite Immunoglobulin G3 Responses, Breadth Score, and Malaria Status The interrelationships among the malaria protective variables opsonic phagocytosis, merozoite IgG3 responses in the IFA, breadth score based on the final 4 antigens (Pf12, MSP3.7, MSP3.3, and MSP2FC27), and malaria status of the study participants were visualized using a bubble plot (Figure 6). Both opsonic phagocytosis and breadth score increased simultaneously with increasing merozoite IgG3 responses. Susceptible individuals had lower opsonic phagocytosis, merozoite IgG3 responses, and smaller breadth score values (Figure 6, blue circles) compared with protected individuals (Figure 6, red circles). Figure 6. View largeDownload slide Bubble plot of the interrelationship among opsonic phagocytosis, merozoite immunoglobulin G3 (IgG3) responses, breadth score, and malaria status. Opsonic phagocytosis was plotted against merozoite-specific IgG3 responses together with breadth score for the final 4 antigens (Pf12, MSP3.7, MSP3.3, and MSP2FC27) for susceptible (blue circle) and protected (red circles) individuals. Each circle represents an individual, and the size of the circle is proportional to the breadth score (range, 0–12). Protected individuals have higher breadth score compared with susceptible individuals. Abbreviations: IFA, immunofluorescence assay; MFI, mean fluorescence intensity. Figure 6. View largeDownload slide Bubble plot of the interrelationship among opsonic phagocytosis, merozoite immunoglobulin G3 (IgG3) responses, breadth score, and malaria status. Opsonic phagocytosis was plotted against merozoite-specific IgG3 responses together with breadth score for the final 4 antigens (Pf12, MSP3.7, MSP3.3, and MSP2FC27) for susceptible (blue circle) and protected (red circles) individuals. Each circle represents an individual, and the size of the circle is proportional to the breadth score (range, 0–12). Protected individuals have higher breadth score compared with susceptible individuals. Abbreviations: IFA, immunofluorescence assay; MFI, mean fluorescence intensity. DISCUSSION Increasing evidence from studies performed mainly in malaria-endemic sub-Saharan African populations indicate that opsonic phagocytosis of merozoites plays a critical role in NAI against malaria [23, 30, 41]. However, data on the relevance of merozoite-specific antibodies for NAI in other malaria-endemic populations such as India are presently lacking. Using samples and data from an LCS performed in a tribal population in India, we have quantified functional antibodies against whole merozoites and assessed the importance of specific merozoite antigens as targets of NAI. We found that opsonic phagocytosis of merozoites is a strong predictor of protection against febrile malaria and that antibodies of the IgG3 subclass play a key role. More importantly, levels of opsonic phagocytosis as well as levels of merozoite IgG3 increased with age, which is consistent with acquisition of NAI in this population [33]. Thus, our data reinforce previously published data that opsonic phagocytosis of merozoites is a strong correlate of NAI worldwide [23, 30, 41]. Notwithstanding, other immune mechanisms such as antibody-mediated complement-dependent inhibition [20], direct growth inhibition [19], ADRB [26], and ADCI [29] are likely to contribute to NAI. The relative importance of these mechanisms in this population remains to be investigated. The proposed importance of merozoites in immunity is further underscored by a detailed profiling of protective immunity against a large panel of merozoite antigens. Several of these merozoite antigens are disulfide-bonded, and, because the correct cysteine connectivity is essential for antibody recognition [42], we have used antigens produced in the L lactis expression system, which is highly suited for the production of cysteine-rich malaria proteins [37]. The concept of screening large numbers of antigens with the aim of prioritizing antigenic targets of malaria protective immunity is relatively new, and methods for down-selection are still developing. The theoretical outcome of combining the 25 antigens in our study taking any 2, 3, or 4 at a time would have resulted in a total of 15250 possible combinations. Thus, there is a need for specialized algorithms that will allow a more intuitive way of creating such combinations conditioned on some functional biological parameters. Others have studied protective roles of combined antigen responses by using a summation of graded quartile responses and categorizing the sums into different levels of magnitude to obtain different combinations [10]. In this study, we favored different regression approaches for arriving at combinations of potentially protective antigens because these have been used extensively in malaria studies and the interpretations are more straightforward. In this study, as in most malaria immune-epidemiological studies, age was associated with protection against symptomatic infection. It has been hypothesized (1) that cumulative exposure to malaria infection alone does not sufficiently explain the susceptibility of children and resistance of adults to symptomatic infection and (2) that intrinsic age-dependent immune factors may be key to NAI [43]. Thus, it is plausible that the humoral and cellular components of NAI differ qualitatively and/or quantitatively with age, an idea that warrants further investigations. Multivariate logistic regression models correcting for the confounding effects of age and merozoite IgG3 identified levels of IgG3 antibodies against 7 antigens: Pf12, MSP3.7, MSP3.3, GLURP-R0, cMSP3, MSP119k, and MSP2FC27, as associated with protection against febrile malaria. It is interesting to note that these antigens were characterized as weakly associated with protection in Papua New Guinean children [10], whereas the top ranking antigens in that previous study showed no association with protection in the Indian population studied here. The exact reason for this discrepancy is unknown, but differences in study design, ethnicity, and statistical modeling may provide an explanation. Some of the top ranking antigens including GLURP and MSP3 (reviewed in [44]), MSP2 [45, 46], and MSP1 [47] have been studied extensively in several longitudinal cohorts, whereas there is little published information available on the relevance of Pf12, MSP3.7, and MSP3.3 [10] for malaria immunity. Although levels of antibodies against these antigens were highly associated with protection against febrile malaria, the combined antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 were more strongly associated with protective immunity than the individual antigens, suggesting an additive effect on protective associations. The combined effect of these 4 antigens was associated with an 85% reduction in the probability of developing febrile malaria during follow up. It is generally agreed that antibodies against no single antigen can explain NAI against febrile malaria [8, 13, 40]. It seems that protective immunity relies on a robust antibody response against multiple antigens [13, 17, 18, 40], and it has been hypothesized that both the breadth and the magnitude of specific responses are critical for protective immunity [40]. Some studies have already investigated this issue in different populations, and it appears that multiple antigen combinations may provide protective immunity in the different populations [10, 14, 40, 48, 49]. In some populations EBA, PfRh2, and PfRh4 proteins are important [10], whereas in other populations combinations of AMA1, MSP2, and MSP3 were strong predictors of protection [40]. Thus, neither a single antigen nor a specific combination of antigens is required for the development of protective immunity. Most likely, protective immunity results from antibody responses against multiple antigens and depends on a certain threshold concentration [13]. In this study, we have identified a combination of both integral membrane proteins and peripherally associated proteins as targets of protective immunity in a tribal population. Thus, reinforcing the perception that the breadth of antibody responses to merozoite antigens is a better predictor of protective immunity than the responses to the individual antigens [10, 14, 40, 50]. The observation in this study that the breadth score correlates with opsonic phagocytosis, IgG3 antibody responses against merozoites, and protection against malaria further corroborates this assertion. CONCLUSIONS In conclusion, this study shows evidence of the opsonic phagocytosis of merozoites as an important player in NAI against febrile malaria in a high transmission region of Eastern India and has further identified Pf12, MSP3.7, MSP3.3, and MSP2FC27 as potentially important antigenic targets of protective immunity. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Acknowledgments. We are grateful to the Indian Council of Medical Research and the director of National Institute of Malaria Research for support in conducting this study. Financial support. This work was funded by the Danish Council for Strategic Research (grant 13127], the Department of Biotechnology, Government of India (BT/IN/Denmark/13/SS/2013), and Danish Ministry of Foreign Affairs (DFC file no. 14-P01-GHA). Potential conflicts of interest. All authors: No reported conflicts of Interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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Journal

The Journal of Infectious Diseases – Oxford University Press

Published: May 4, 2018

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