Allele‐specific antibodies to Plasmodium falciparum merozoite surface protein‐2 and protection against clinical malaria

IgG and IgG3 antibodies to merozoite surface protein‐2 (MSP‐2) of Plasmodium falciparum have been associated with protection from clinical malaria in independent studies. We determined whether this protection was allele‐specific by testing whether children who developed clinical malaria lacked IgG/IgG3 antibodies specific to the dominant msp2 parasite genotypes detected during clinical episodes. We analysed pre‐existing IgG and IgG1/IgG3 antibodies to antigens representing the major dimorphic types of MSP‐2 by ELISA. We used quantitative real‐time PCR to determine the dominant msp2 alleles in parasites detected in clinical episodes. Over half (55%, 80/146) of infections contained both allelic types. Single or dominant IC1‐ and FC27‐like alleles were detected in 46% and 42% of infections respectively, and both types were equally dominant in 12%. High levels of IgG/IgG3 antibodies to the FC27‐like antigen were not significantly associated with a lower likelihood of clinical episodes caused by parasites bearing FC27‐like compared to IC1‐like alleles, and vice versa for IgG/IgG3 antibodies to the IC1‐like antigen. These findings were supported by competition ELISAs which demonstrated the presence of IgG antibodies to allele‐specific epitopes within both antigens. Thus, even for this well‐studied antigen, the importance of an allele‐specific component of naturally acquired protective immunity to malaria remains to be confirmed.     

Variant-specific antibodies to merozoite surface protein 2 and clinical expression of Plasmodium falciparum malaria in rural Amazonians

Naturally acquired antibodies to five variants of the merozoite surface protein 2 (MSP-2), a target of clinical immunity to Plasmodium falciparum malaria, were measured in a cohort of rural Amazonians. Local MSP-2 variants comprised both highly divergent families of alleles (FC27 and 3D7). Total IgG antibodies to two FC27-type antigens were found in 22-28% of subjects at baseline, with substantial cross-reactivity between variants and stable concentrations and specificities over time. The IgG antibodies to three 3D7-type antigens were less prevalent (6-7%), less cross-reactive, and short-lived; subsequent exposure to 3D7-type parasites rarely elicited homologous response. The clinical spectrum of 109 incident P. falciparum infections in our cohort ranged between asymptomatic infection and fully symptomatic but uncomplicated disease. Parasitemia at the time of diagnosis, rather than cumulative malaria exposure or acquired immunity (presence of variant-specific antibodies matching the MSP-2 type in infecting parasites), was a major predictor of perceived symptom severity.     

Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3

Many studies on the role of merozoite surface protein 3 (MSP3) in immunity against malaria have focused on a conserved section of MSP3. New evidence suggests that polymorphic sequences within MSP3 are under immune selection. We report a detailed analysis of naturally-acquired antibodies to allele-specific and conserved parts of MSP3 in a Kenyan cohort. Indirect and competition ELISA to heterologous recombinant MSP3 proteins were used for antibody assays, and parasites were genotyped for msp3 alleles. Antibody reactivity to allele-specific and conserved epitopes of MSP3 was heterogeneous between individuals. Overall, the prevalence of allele-specific antibody reactivity was significantly higher (3D7-specific 54%, K1-specific 41%) than that to a recombinant protein representing a conserved portion of C-terminal MSP3 (24%, P < 0.01). The most abundant IgG subclass was IgG3, followed by IgG1. Allele-specific reactivity to the K1-type of MSP3 was associated with a lower risk of clinical malaria episodes during a 6-month follow-up in individuals who were parasitized at the start of the malaria transmission season (Relative risk 0.41 with 95% confidence interval 0.20-0.81, P = 0.011). The potential importance of allele-specific immunity to MSP3 should be considered in addition to immunity to conserved epitopes, in the development of an MSP3 malaria vaccine.     

Human antibodies to the 19kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro

The 19kDa, C-terminal fragment of the major surface protein of Plasmodium falciparum (PfMSP1(19)) is a candidate for inclusion in a subunit malaria vaccine. In this study, we show that PfMSP1(19)-specific antibodies, affinity purified from malaria-immune human serum, can: (i) compete with invasion-inhibitory monoclonal antibodies for binding to PfMSP1(19) and (ii) mediate inhibition of parasite growth in vitro, in the absence of complement and mononuclear cells, at physiological antibody concentrations (100 micrograms/ml). Parasites expressing either the Kl or 3D7 allele of PfMSP1(19) were equally susceptible to inhibition of merozoite invasion, indicating that the target epitopes of inhibitory antibodies are conserved or cross-reactive. These studies suggest that vaccines designed to induce antibodies to PfMSP1(19) may protect against the high levels of malaria parasitaemia which are associated with clinical disease.     

Naturally acquired antibodies to the glutamate-rich protein are associated with protection against Plasmodium falciparum malaria

The development of effective malaria vaccines depends on the identification of targets of well-defined protective immune responses. Data and samples from a longitudinal study of a cohort of children from coastal Ghana were used to investigate the role of antibody responses to 3 regions of the Plasmodium falciparum glutamate-rich protein (GLURP). The data show that levels of the GLURP-specific IgG that occurs in the nonrepeat region of the antigen are significantly correlated with clinical protection from P. falciparum malaria, after correction for the confounding effect of age. Furthermore, levels of cytophilic antibodies were found to be of particular importance for protection, lending support to the hypothesis that antibody-dependent cellular inhibition is the important element in GLURP-specific protective immunity.     

Antibodies to Plasmodium falciparum and Plasmodium vivax merozoite surface protein 5 in Indonesia: species-specific and cross-reactive responses

BACKGROUND: Merozoite surface protein (MSP) 5 is a candidate antigen for a malaria vaccine. In cross-sectional and longitudinal studies, we measured MSP5 antibody responses in Papuans with acute Plasmodium falciparum malaria, Plasmodium vivax malaria, and mixed P. falciparum and P. vivax malaria and in those with past exposure. METHODS: Enzyme-linked immunosorbant assay (ELISA) was used to quantitate antibody responses to P. falciparum MSP5 (PfMSP5) and P. vivax MSP5 (PvMSP5) in 82 subjects with P. falciparum infection, 86 subjects with P. vivax infection, 85 subjects with mixed infection, and 87 asymptomatic individuals. Longitudinal responses through day 28 were tested in 20 persons. Cross-reactivity was tested by competition ELISA. RESULTS: PfMSP5 or PvMSP5 immunoglobulin (Ig)Gwas detected in 39%-52% of subjects, and IgM was detected in 44%-72%. IgG responses were distributed equally between IgG3 and IgG1 for PfMSP5 but were predominantly IgG3 for PvMSP5. Although IgG responses were generally specific for PfMSP5 or PvMSP5, cross-species reactivity was found in 7 of 107 dual-positive responders. No significant difference was seen in the magnitude, frequency, or subclass of PfMSP5 or PvMSP5 IgG antibodies between groups. There was no significant association between antibody responses and therapeutic response. CONCLUSION: PfMSP5 and PvMSP5 were frequently recognized by short-lived, species-specific antibodies. Although infrequent, the cross-reactive MSP5 antibodies indicate that an appropriately formulated vaccine may elicit and/or enhance cross-species recognition, which may be very useful in areas where both parasites are endemic.     

Distinct surrogate markers for protection against Plasmodium falciparum infection and clinical malaria identified in a Senegalese community after radical drug cure

Plasmodium falciparum expresses many antigens, which elicit various immune responses in exposed individuals, but no simple surrogate marker for protection has yet been developed. In this prospective survey, we looked for immune responses predictive of protection at various stages of progression from parasite inoculation to onset of disease. We studied 110 Senegalese volunteers from an area in which malaria is mesoendemic after they had received eradication therapy. We evaluated 4 protection-related outcomes (reappearance of parasitemia, duration of asymptomatic carriage, time to first clinical episode, and incidence of clinical episodes) in terms of levels of immunoglobulin G (IgG) against 3 crude parasite extracts and 5 conserved antigens during a 5-month period. Kaplan-Meier estimates and age-adjusted regression models showed these 4 outcomes to be associated with different patterns of IgG response to PfEMP3-cl5 (derived from P. falciparum erythrocyte membrane protein 3), PfEB200, MSP-1(19) (derived from merozoite surface protein-1), [NANP]10, infected red blood cell membrane, and merozoite and schizont extracts. It should, therefore, be possible to develop surrogate markers for each end point on the basis of IgG response to a limited number of conserved antigens.     

Association of the IgG response to Plasmodium falciparum merozoite protein (C-terminal 19 kD) with clinical immunity to malaria in the Brazilian Amazon region

The antibody response to the C-terminal 19-kD fragment of Plasmodium falciparum merozoite surface protein-1 (PfMSP1-19) was investigated in groups of subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PfMSP1-19 increased with the time of exposure and were positively correlated with the absence of clinical symptoms in parasitemic patients. The frequency of positive response and the mean level of IgG were higher in areas where malaria prevalence was more intense, especially among asymptomatic patients. The serum absorbance values of the IgG1 isotype were significantly higher among subjects with long-term exposure and in asymptomatic infections. These data suggest a protective role of IgG1 in naturally acquired immunity in spite of the unstable transmission levels in the Brazilian Amazon.     

恶性疟原虫主要裂殖子表面蛋白1C末端片段在毕赤酵母表达系统的高效表达与纯化(英文)

目的利用毕赤酵母表达系统高效表达并获得纯度较为理想的恶性疟原虫主要裂殖子表面蛋白1 C末端片段(MSP-119)重组蛋白。方法将带有6-his基因的MSP-119基因序列插入毕赤酵母分泌型表达载体pPIC9k中,用高压电穿孔转化法将目的基因转化入酵母感受态细胞GS115,筛选出高拷贝转化子,优化表达条件,利用甲醇进行诱导表达。表达产物用SDS-PAGE和免疫印迹进行检测。结果毕赤酵母分泌表达MSP-119蛋白,免疫印迹结果表明MSP-119基因表达蛋白能被抗MSP-119的单抗所识别,出现特异条带,将培养上清利用Ni-NTA柱纯化后,推算MSP-119蛋白的表达量为1.0g/L。结论酵母细胞表达系统可高效表达可免疫识别的MSP-119重组蛋白。     

Antibodies to pre-erythrocytic Plasmodium falciparum antigens and risk of clinical malaria in Kenyan children

BACKGROUND: IgG antibodies to pre-erythrocytic antigens are involved in prevention of infection and disease in animal models of malaria but have not been associated with protection against disease in human malaria. METHODS: Levels of IgG antibodies to circumsporozoite protein (CSP), liver-stage antigen type 1 (LSA-1), and thrombospondin-related adhesive protein (TRAP) were measured in 86 children in a malaria-holoendemic area of Kenya. The children were then monitored for episodes of clinical malaria for 52 weeks. RESULTS: Children with high levels of IgG antibodies to CSP, LSA-1, and TRAP had a decreased risk of clinical malaria (adjusted hazard ratio, 0.29; 95% confidence interval 0.10-0.81; P = .02), a lower incidence of clinical malaria (P=.006), protection from clinical malaria with a parasite level of > or =4000 parasites/microL (P= .03), and a higher hemoglobin level at enrollment (P= .009), compared with children with lower antibody levels. Protection against malaria morbidity was associated primarily with antibodies to CSP and LSA-1. CONCLUSIONS: Kenyan children with high levels of IgG antibodies to the pre-erythrocytic antigens CSP, LSA-1, and TRAP have a lower risk of developing clinical malaria than children without high levels of these antibodies. The decreased risk of clinical malaria may be mediated in part by prevention of high-density parasitemia.     

Antibodies directed against merozoite surface protein-6 are induced by natural exposure to Plasmodium falciparum in a low transmission environment

Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and there is currently no licensed vaccine that provides protection against infection or disease. Several P. falciparum vaccine targets have undergone early testing, but many more candidates remain with little data to support their development. Plasmodium falciparum Merozoite Surface Protein 6 (PfMSP6) is a candidate of particular interest because it is a member of the PfMSP3 multi‐gene family, raising the possibility that vaccine‐induced immune responses could cross‐react across multiple family members. However, few immunoepidemiological studies of PfMSP6 have been carried out to measure domain‐specific anti‐PfMSP6 responses. This study investigated anti‐PfMSP6 responses in P. falciparum‐infected individuals from the Peruvian Amazon, using two different PfMSP6 N‐terminal allele antigens and a single C‐terminal domain antigen, and compared the responses with both PfMSP6 genotyping data and anti‐PfMSP3 response data that had been previously generated for the same samples. Anti‐PfMSP6 responses were detected despite the low transmission setting, but were less frequent and of considerably lower intensity than anti‐PfMSP3 responses. There was a positive correlation between anti‐PfMSP3 and PfMSP6 responses, suggesting that the possibility that PfMSP3 family antigens could induce cross‐reactive responses requires further detailed investigation.     

恶性疟原虫主要裂殖子表面蛋白1C末端19ku片段(MSP1-19)的表达及免疫效应检测

目的 探讨恶性疟原虫主要裂殖子表面蛋白1C末端19ku片段（MSP1-19）重组蛋白的免疫活性。方法 利用毕赤酵母高效表达系统分泌表达MSP1-19，表达产物纯化后免疫新西兰兔，3次免疫后ELISA检测其血清中IgG滴度的变化，观测重组MSP“。免疫效应。结果 MSP1-19在毕赤酵母高效表达；重组MSP1-19免疫后，兔血清中IgG滴度的变化与对照组有显著差异。结论 MSP1-19重组蛋白具有较好的免疫原性。     

Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria

The merozoite surface protein 2 (MSP2) of Plasmodium falciparum is recognized by human antibodies elicited during natural infections, and may be a target of protective immunity. In this prospective study, serum IgG antibodies to MSP2 were determined in a cohort of 329 Gambian children immediately before the annual malaria transmission season, and the incidence of clinical malaria in the following 5 months was monitored. Three recombinant MSP2 antigens were used, representing each of the two major allelic serogroups and a conserved region. The prevalence of serum IgG to each antigen correlated positively with age and with the presence of parasitaemia at the time of sampling. These antibodies were associated with a reduced subsequent incidence of clinical malaria during the follow-up. This trend was seen for both IgG1 and IgG3, although the statistical significance was greater for IgG3, the most common subclass against MSP2. After adjusting for potentially confounding effects of age and pre-season parasitaemia, IgG3 reactivities against each of the major serogroups of MSP2 remained significantly associated with a lower prospective risk of clinical malaria. Individuals who had IgG3 reactivity to both of the MSP2 serogroup antigens had an even more significantly reduced risk. Importantly, this effect remained significant after adjusting for a simultaneous strong protective association of antibodies to another antigen (MSP1 block 2) which itself remained highly significant.     

Sequence diversity of the merozoite surface protein 1 of Plasmodium falciparum in clinical isolates from the Kilombero District, Tanzania

Merozoite surface protein 1 of Plasmodium falciparum (PfMSP-1) is regarded as a key candidate antigen for malaria vaccine development. It exhibits significant antigenic polymorphism and has been divided into 17 building blocks based on the analysis of sequence diversity. Differences in the antigenic composition of PfMSP-1 in local P. falciparum populations may result in differences in the efficacy of vaccines, which contain sequences of particular allelic variant(s) of PfMSP-1. To contribute to the required knowledge of genetic diversity of malaria parasites in geographically diverse regions, we have used the polymerase chain reaction (PCR) to analyze the sequence diversity of blocks 1-4 of PfMSP-1 in disease isolates from the Kilombero District in Tanzania. In the semi-conserved block 1, in which dimorphic amino acid variances have been described at three positions, we found three of the five previously described combinations of these three pairs of amino acids. In addition one combination was found, which has not been reported before in parasite isolates from different locations worldwide. Of the two sequence variants, which were dominating, one (S44-Q47-V52) corresponded to the 83.1 sequence incorporated into the SPf66 malaria peptide vaccine, while the other one (G44-H47-I52) differed from the previous in all three dimorphic amino acids. The partial protection observed in a phase III SPf66 trial conducted in the Kilombero District in children aged 1-5, thus does not seem to be associated with a clear dominance of favourable variants of block 1 of PfMSP-1 in this area. All three different principle types of block 2, the major polymorphic region of PfMSP-1, were found in the Tanzanian isolates. Most of the sequences contained K1-type tripeptide repeats, but clones with MAD20-type repeats or no repetitive sequence (RO33-type block 2) were also present. K1- and MAD20-type tripeptide repeat motifs were never mixed within one parasite clone. In one sequence a hexapeptide repeat was found at the end of block 2, which has not been reported before. Dimorphism in 13 of the 17 previously described variable positions of the semi-conserved block 3 and three of four recombination types of block 4 (K/K, M/K and M/M) were found among the Tanzanian isolates. Apart from previously described dimorphic amino acid positions, polymorphism was rare in the non-repeated building blocks. Selection and spreading of parasite variants, which contain amino acid exchanges at other than the dimorphic positions thus, is not a common event. Parasite isolates frequently harboured more than one PfMSP-1 allele. Three of the four heterogeneous isolates analysed contained two different general types of sequences. One isolate contained at least four distinct clones, demonstrating the high endemicity of malaria in the Kilombero District, which is a well-established site for malaria vaccine field trials.     

Plasmodium falciparum merozoite surface protein 1 (MSP1): genotyping and humoral responses to allele-specific variants.

The present study is the first to investigate Plasmodium falciparum merozoite surface protein 1 (MSP1) allele-specific humoral responses in residents of central Africa. In endemic areas, acquired immune responses to malaria are assumed to reflect the need to be infected with a large number of antigenically diverse parasite populations. In the work presented here, the relationship between antibody specificity and the infecting parasite genotype was investigated in asymptomatic subjects and patients with uncomplicated malaria in order to possibly clarify the relationship between anti-MSP1 block2 antibodies and clinical malaria. Overall isolates were typed by nested PCR using allele-specific primers of the P. falciparum MSP1 gene to identify the infecting parasite genotype. The K1 type was the predominant allelic family in both clinical groups. Polyinfection (number of isolates with more than one parasite genotype) and the complexity of infections (mean number of parasite genotype per infected subject) were higher in isolates from asymptomatic individuals. Total immunoglobulins G (IgG) responses to schizont crude extract antigens and to MSP1 variant-specific peptides were assessed by ELISA test. More than 90% of the sera reacted against schizont extract, whatever the clinical group and the K1 seroprevalence was the highest in both clinical groups. Our results showed an age-dependence in the number of different variants of MSP1 block2 recognised by serum. Indeed, isolates from older (>14 years) subjects showed lower multiplicity of infection and higher was the mean number of different MSP1 variants recognised by their serum. This corresponded to the age reported for the acquisition of anti-parasite immunity under high malaria endemicity. The contribution of variant-specific immunity in asymptomatic malaria infections is discussed.     

Antibody responses to Plasmodium falciparum merozoite surface protein-1 and efficacy of amodiaquine in Gabonese children with P. falciparum malaria

The relationship between the efficacy of amodiaquine for the treatment of uncomplicated Plasmodium falciparum malaria and preexisting antibodies against merozoite surface protein (MSP)-1, a blood-stage P. falciparum antigen, was investigated. The immunoglobulin G antibody response to different MSP-1 recombinant proteins was evaluated in plasma samples from Gabonese children with uncomplicated malaria who were treated with amodiaquine. The prevalence of anti-MSP-1 antibodies was similar among patients with either parasitological and clinical cure after treatment (n=102) or treatment failure (n=51) by day 28 (83% in both groups). However, associations between antibody responses to K1 and MAD20 allelic families and therapeutic success were found (P< .001 and P= .034, respectively). A high proportion of plasma samples recognizing several antigens was found in the cured group. This association was significant even when data were stratified by age, particularly for the K1 family antigens (P= .029). These results suggest that humoral immune responses play a supportive role in the efficacy of amodiaquine treatment.     

Genetic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum in different malaria-endemic localities

A number of stage-specific antigens have been characterized for vaccine development in Plasmodium falciparum malaria. The polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a major asexual blood stage malaria vaccine candidate antigen. In the present study, we analyzed the impact of hyperendemic malaria transmission, mesoendemic malaria transmission, and multiple infection on allelic diversity. We have used a simple strategy of polymerase chain reaction amplification and slot-blot hybridization to analyze variable regions of block-2, block-4 and blocks 6-10 of the MSP-1 gene. The allelic types of isolates collected from regions of hyperendemic malaria transmission (RHEMT) and mesoendemic malaria transmission (RMEMT) were compared. In RHEMT, 20 of 24 possible gene types were found among 163 isolates and more than one allelic type was found in 82 (50.3%) of the isolates. Thirteen of 24 possible gene types were found among 125 isolates in RMEMT and 27 (21.6%) of them contained more than one allele type. Our results suggest for the first time that the allelic distribution or allelic diversity and chances of finding multi-strain parasites in isolates in an area vary with the rate of transmission. Analyses of isolates containing more than one strain of parasite suggest that allelic types are randomly distributed, no specific type of alleles predominately show multi-strain infection, and neither strain of the parasite affect the process of infection and development of another.     

Naturally acquired antibody responses to the components of the Plasmodium falciparum merozoite surface protein 1 complex

Merozoite surface protein 6 (MSP6) and 7 (MSP7) of Plasmodium falciparum are peripheral membrane proteins whose cleaved products, MSP636, MSP722 and MSP719, are found on the merozoite surface as components of a non-covalently bound complex which also contains four polypeptides derived from merozoite surface protein 1 (MSP1). We have expressed both the precursor regions and the processed mature products of MSP6 and MSP7 in Escherichia coli and showed that these recombinant proteins react with human immune sera. In a set of sera collected from individuals living in malaria-endemic areas of Southern-central Vietnam, antibodies to the mature polypeptides of MSP636 and MSP722 were detected in 50.6 and 85.6% of the serum samples, whereas antibodies to the precursor regions of MSP6 and MSP7 were detected in only 12.1 and 42.5% of the serum samples, respectively. The predominant subclass of anti-MSP6 antibodies was IgG1, whereas the predominant subclass of anti-MSP7 antibodies was IgG3. In the same set of serum samples, the antibody responses to MSP119 are predominantly IgGI, whereas antibodies to merozoite surface protein 4 (MSP4) are mainly IgG3. This data is consistent with the proposition that, during malaria infection, variable proteins induce responses that are predominantly of the IgG3 isotype, and conserved proteins induce responses that are predominantly IgG1. The antibodies to MSP6, MSP7 and MSP119 all decreased at the time of infection, but increased during the convalescent period. No correlation was observed between the antibodies at the commencement of the study and absence of parasitaemia during surveillance in this population.     

Longevity of naturally acquired antibody responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1.

In an earlier study, we found that individuals with patent infection had significantly higher IgG antibody titers to the 19-kD C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP1) than individuals treated for malaria 1-4 months earlier. These results suggested that the antibody levels decreased rapidly following treatment. The present study was designed to determine the persistence of antibody response to the N- and C-terminal regions of PvMSP1 after infection with P. vivax in individuals from the city of Bélem in northern Brazil. Our results demonstrated that the vast majority of individuals had a significant decrease in antibody titers to the C-terminal region of PvMSP1 in a period of two months following treatment. Among responders to the C-terminal region, 44.4% became serologically negative and 44.4% had their antibody titers reduced by an average of 13-fold. Only 11.2% of the individuals had their antibody titers maintained or slightly increased during that period. A decrease in the antibody response to the recombinant protein representing the N-terminal region of PvMSP1 was also noted; however, it was not as dramatic. The rapid decrease in the antibody levels to the C-terminal region of PvMSP1 might contribute to the high risk of reinfection in these individuals.