HLA class II and antibody responses to circumsporozoite protein repeats of P. vivax (VK210, VK247 and P. vivax-like) in individuals naturally exposed to malaria

We studied the seroreactivity against the circumsporozoite protein (CSP) repeats of Plasmodium vivax variants in individuals living in malaria-endemic area of the Brazilian Amazon region (Candeias do Jamari - RO). The prevalence of IgG antibodies for at least one of the P. vivax CSP repeats was 49%. Among these positive individuals, 34.2% were positive for the standard repeat sequence VK210, 24% for the VK247 and 31.5% for the P. vivax-like sequence. HLA typing showed an association between antibody responses to the CS repeats of VK247 and the presence of HLA-DR16 and between HLA-DR7 and the absence of antibody responses to the CS repeats of VK210. We also investigated the potential relationship between HLA-DQB1 allele profile and antibody response to the CSP repeats of P. vivax but no segregation with responding profile was evidenced. The observed findings indicate that antibody responses to the CSP repeats of P. vivax variants appear to be modulated by HLA class II molecules in malaria naturally exposed individuals.     

Reactivity of sera from cases of Plasmodium vivax malaria towards three recombinant antigens based on the surface proteins of the parasite

Sera collected in South Korea, from 61 cases of Plasmodium vivax malaria and, as controls, 40 healthy volunteers, were tested in ELISA for IgG or IgM reacting with any of three recombinant P. vivax proteins. The antigens used, representing the parasite's major merozoite surface protein (MSP), circumsporozoite surface protein (CSP) and Duffy-binding protein (DBP), had all been expressed in an Escherichia coli system and purified. The ELISA results were recorded as optical densities (OD). The highest ratio observed between the mean OD for a malaria serum and that for a control serum was that for IgG against MSP, although CSP gave a higher ratio than MSP or DBP in the IgM ELISA. In the ELISA for IgG, the OD for MSP were found to be correlated with those for DBP (r = 0.53; P < 0.5) but the OD for CSP were not correlated with those for MSP or DBP. As the most intense reactions observed were those between the IgG from the malaria sera and the recombinant MSP, the latter antigen may be useful in diagnostic tests and as a component of any vaccine used to protect against P. vivax malaria.     

Humoral immune responses against Plasmodium vivax MSP1 in humans living in a malaria endemic area in Flores, Indonesia

The aim of this study was to evaluate the relationship among age, parasitemia status, spleen size, hematocrit, and antibody levels to Plasmodium vivax merozoite surface protein 1 (MSP1) in individuals chronically exposed to P. vivax. Subjects were recruited from the population of three adjacent villages on the Island of Flores in Indonesia where malaria transmission is hyperendemic and tropical splenomegaly syndrome is highly prevalent. Subjects were evaluated for spleen size, hematocrit, presence of parasitemia, and presence of antibodies to a recombinant peptide consisting of 90 amino acids from the carboxy terminus of MSP1. Fifty-seven percent of 2-4 year olds, 45% of 5-9 years old, and 7% of > or = 15 years old were parasitemic; 99% of the > or = 15 years old had splenomegaly, and 31% of them had Hackett 4 or 5 spleens. The frequency of antibody positivity to MSP1 antigen in ELISA increased with age reaching a maximum of 89% in > or = 20 years old. The frequency of antibody positivity to MSPI also increased with spleen size, and with a decline in the prevalence of parasitemia.     

Anti-Plasmodium vivax duffy binding protein antibodies measure exposure to malaria in the Brazilian Amazon

Plasmodium vivax Duffy binding protein (DBP) is functionally important in the erythrocyte invasion process and provides a logical target for vaccine-mediated immunity. In the current study, we demonstrated that DBP is naturally immunogenic in different populations of the Brazilian Amazon, and the proportions of DBP IgG positive subjects increased with exposure to malaria, reaching a peak in those subjects with long-term exposure (> 15 years) in the Amazon area. This profile of antibody response was significantly different from the one observed for the P. vivax merozoite surface protein 1 (MSP1(19)), which was relatively uniform in areas with markedly different levels of malaria transmission. In a small sample of adults with symptomless P. vivax infection, we could not detect any significant correlation between antibodies against these P. vivax proteins and asymptomatic infection. Our study provided an additional insight by demonstrating cumulative exposure as a determinant that acts independently of host age in generation of anti-DBP IgG response.     

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.     

Plasmodium falciparum merozoite surface protein 3 is a target of allele-specific immunity and alleles are maintained by natural selection

BACKGROUND: Plasmodium falciparum merozoite surface protein (MSP) 3 is an asexual blood-stage malaria vaccine candidate antigen. Sequence polymorphisms divide alleles into 2 major types, but the adaptive and immunological significance of the types has not been defined. METHODS: One hundred one msp3 allele sequences were sampled from 2 populations living in areas where malaria is endemic and were analyzed for evidence of natural selection. Recombinant antigens representing full-length sequences of different allelic types and a relatively conserved C-terminal region were produced, to evaluate immunization-induced antibody responses in mice and protective associations for naturally acquired antibodies in a cohort of 319 Gambian children under surveillance for malaria. RESULTS: Frequency-based statistical analyses indicated that polymorphisms are maintained by balancing selection in each of the 2 populations studied. Immunization of mice with full-length MSP3 antigens induced predominantly type-specific antibodies, and a large proportion of naturally acquired antibodies to MSP3 in humans also discriminated between the alleles. Among Gambian children, antibodies to allele-specific and conserved epitopes in MSP3 were associated prospectively with protection from clinical malaria, even after adjustment for age and for the presence of antibodies to other merozoite antigens. CONCLUSIONS: A vaccine incorporating both major allelic types of this promising candidate antigen could be particularly useful for induction of protective immunity in infants and young children.     

Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand

Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.     

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.     

Assessment of the role of the humoral response to Plasmodium falciparum MSP2 compared to RESA and SPf66 in protecting Papua New Guinean children from clinical malaria

The prevalence and concentration of naturally acquired humoral response (IgG) to merozoite surface protein 2 (MSP2), RESA, SPf66 and crude schizont extract were measured in a population living in a malaria highly endemic area of Papua New Guinea. A prospective longitudinal study in 0–5–15 year old children was conducted for one year in order to examine the relationship between the humoral response to these antigens and subsequent susceptibility to clinical malaria using a series of clinical definitions. The prevalence and concentration of antibodies to all antigens increased with age. Such correlation with age was most marked for MSP2 recombinant proteins. When age and previous exposure were controlled for, only antibody levels to MSP2 recombinant proteins (3D7 andd3D7) and to RESA predicted a reduction in incidence rate of episodes of clinical malaria. Our results support the inclusion of the recombinant proteins of the 3D7 allelic family of merozoite surface antigen 2 and RESA into a subunit vaccine against malaria.,     

Polyspecific malaria antibodies present at the time of infection inhibit the development of immunity to malaria but antibodies specific for the malaria merozoite surface protein,MSP1, facilitate immunity

Serum taken from mice immune to malaria as a result of infection and drug cure, or from mice immunized with a recombinant form of the merozoite surface protein, MSP1, can provide passive protection of recipient mice against the lethal parasite, Plasmodium yoelii YM. However, recipients of MSP1-immune serum go on to develop long-term immunity, whereas recipients of serum from mice naturally immune to malaria rapidly lose their resistance to infection. We demonstrate that 'infection/cure' serum suppresses the development of both antibody and cell-mediated parasite-specific responses in recipients, whereas these develop in recipients of MSP1-specific antibodies. These data have profound implications for our understanding of the development of malaria immunity in babies who passively acquire antibodies from their mothers.     

Acquisition of invasion-inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein 1 in a transmigrant population requires multiple infections

Antibodies against the 19 kDa C-terminal fragment of merozoite surface protein 1 (MSP1(19)) are a major component of the invasion-inhibitory response in individuals immune to malaria. We report here the acquisition of MSP1(19)-specific invasion-inhibitory antibodies in a group of transmigrants who experienced their sequential malaria infections during settlement in an area of Indonesia where malaria is highly endemic. We used 2 transgenic Plasmodium falciparum parasite lines that expressed either endogenous MSP1(19) or the homologous region from P. chabaudi to measure the MSP1(19)-specific invasion-inhibitory antibodies. The results revealed that the acquisition of MSP1(19)-specific invasion-inhibitory antibodies required 2 or more P. falciparum infections. In contrast, enzyme-linked immunosorbent assays on the same serum samples showed that MSP1(19)-specific antibodies are present after the first malaria infection. This delay in the acquisition of functional antibodies by residents of areas where malaria is endemic is consistent with the observation that multiple malaria infections are required before clinical immunity is acquired.     

我国华南间日疟原虫裂殖子表面蛋白1等位基因分型和序列分析

目的　了解我国间日疟原虫MSP1(PvMSP1)的等位基因类型和序列的多态性。方法　用特异的引物通过套式PCR方法体外扩增包含PvMSP1的ICB5与ICB6之间的基因片段 ,并对部分扩增产物进行序列测定和分析。结果　 2 7份间日疟原虫患者血样中 ,有 16份 (5 9 9% )扩增得到Belem型基因产物 ,2 5 (92 6 % )份扩增出Sal- 1型基因产物 ;两种不同等位基因型虫株的混合感染率为 5 1 9%。序列分析结果发现一个以前未见报告的基因内重组类型。结论　我国间日疟原虫虫株存在两种PvMSP1等位基因型 ,Sal- 1型可能是主导型 ;不同等位基因型的混合感染率较高     

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.     

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.     

ITN protection, MSP1 antibody levels and malaria episodes in young children of rural Burkina Faso

Malaria blood-stage vaccines are in an early phase of clinical development with MSP1 being a major antigen candidate. There are limited data on the protective efficacy of antibodies against subunits of MSP1 in the malaria endemic areas of sub-Saharan Africa. This prospective cohort study was nested into a large insecticide-treated mosquito net (ITN) trial during which neonates were individually randomised to ITN protection from birth vs. protection from month six onwards in rural Burkina Faso. A sub sample of 120 children from three villages was followed for 10 months with six measurements of MSP1(42) antibodies (ELISA based on recombinant 42kDa fragment) and daily assessment of malaria episodes. Time to the next malaria episode was determined in relation to MSP1(42) antibody titres. MSP1(42) antibody titres were dependent on age, season, ITN-group, number of previous malaria episodes and parasitaemia. There were no significant differences in time until the next malaria episode in children with low compared to children with high MSP1(42) antibody titres at any point in time (101 vs. 97 days in May, p=0.6; 58 vs. 84 days in September, p=0.3; 144 vs. 161 days in March, p=0.5). The findings of this study support the short-lived nature of the humoral immune response in infants of malaria endemic areas. The study provides no evidence for antibodies against a subunit of MSP1 being protective against new malaria episodes in infants.     

Genetic diversity of Plasmodium vivax Pvcsp and Pvmsp1 in Guyana, South America

Approximately 55% of malaria infections in the Guyana Amazon region are attributed to Plasmodium falciparum while the other 45% are attributed to non-falciparum, mostly Plasmodium vivax. However, little is known about the P. vivax strain types circulating in the region. Using PCR for Plasmodium detection and two genetic markers specific to P. vivax to detect the polymorphic circumsporozoite protein (CSP) and the conserved 19-kDa region of the merozoite surface protein-1 (MSP-1), we investigated the overall Plasmodium strain distribution and population diversity within P. vivax in isolates collected from the blood of infected individuals in the interior Amazon region of Guyana, South America. Out of a total of 250 samples positive for Plasmodium, P. vivax was detected in 30% (76/250) and P. falciparum was detected in 76% (189/250). Mixed infections containing both P. falciparum and P. vivax constituted 6% (15/250) of the total positive samples. Further analysis of P. vivax strains showed that 92% (56/61) of the P. vivax samples hybridized with a probe specific to type VK210, 39% (24/61) hybridized with a probe specific for type VK247, and 25% (15/61) hybridized with a probe specific for the P. vivax-like CS genotype. DNA sequencing of the 19-kDa C-terminal domain in block 13 of MSP-1 amplified from 61 samples from patients infected with P. vivax demonstrated that this region is highly conserved, and all samples were identical at the nucleotide level to the Belem and Salvador-1 types. No synonymous or nonsynonymous mutations were observed in this region of the gene, indicating that current vaccine-development efforts based on the MSP-1(19) fragment would be applicable in Guyana.     

Acquisition of antibody isotypes against Plasmodium falciparum blood stage antigens in a birth cohort

Information on the period during which infants lose their maternally derived antibodies to malaria and begin to acquire naturally their own immune responses against parasite antigens is crucial for understanding when malaria vaccines may be best administered. This study investigated the rates of decline and acquisition of serum antibody isotypes IgG1, IgG2, IgG3, IgG4, IgM and IgA to Plasmodium falciparum antigens apical membrane antigen (AMA1), merozoite surface proteins (MSP1‐19, MSP2 and MSP3) in a birth cohort of 53 children living in an urban area in the Gambia, followed over the first 3 years of life (sampled at birth, 4, 9, 18 and 36 months). Antigen‐specific maternally transferred antibody isotypes of all IgG subclasses were detected at birth and were almost totally depleted by 4 months of age. Acquisition of specific antibody isotypes to the antigens began with IgM, followed by IgG1 and IgA. Against the MSP2 antigen, IgG1 but not IgG3 responses were observed in the children, in contrast with the maternally derived antibodies to this antigen that were mostly IgG3. This confirms that IgG subclass responses to MSP2 are strongly dependent on age or previous malaria experience, polarized towards IgG1 early in life and to IgG3 in older exposed individuals.     

Antibody responses to repetitive epitopes of the circumsporozoite protein,liver stage antigen-1, and merozoite surface protein-2 in infants residing in a Plasmodium falciparum-hyperendemic area of western Kenya. XIII. Asembo Bay Cohort Project

The present study was initiated to characterize antibody responses to repetitive epitopes of the circumsporozoite protein (CSP), liver stage antigen-1 (LSA-1), and merozoite surface protein-2 (MSP-2) of Plasmodium falciparum in infants residing in a P. falciparum-hyperendemic area of western Kenya. In this study, development and maintenance of these antibody responses in 28 infants were studied longitudinally by use of monthly serum samples collected from birth to age 1 year. Mother plasma and infant umbilical cord plasma were also tested to assess the transplacental transfer of maternal antibodies. Results showed that antibodies passively transferred from mothers were detectable for CSP, LSA-1, and MSP-2 repeat epitopes. Infants were able to mount and maintain a strong antibody response against LSA-1 in their first year of life. Infants often responded to CSP repeats, but with a much lower antibody titer. Antibody responses in infants against Fc27 and 3D7 repeats of MSP-2 were low throughout their first year. In addition, 51 infants whose first detected infection occurred at > 4 months of age were selected to determine antibody responses to the antigens tested upon their first and second detected infections. Antibody responses to LSA-1 and, to a lesser degree, CSP increased in positivity rates and titer upon second infection. Antibody responses to Fc27-type and 3D7-type repeats of MSP-2 were low upon both infections. There was no association between maternally transferred anti-LSA-1, anti-CSP, or anti-MSP-2 antibodies and an infant's first detected infection. No significant correlation was found between an infant's antibody responses to the 4 antigen repetitive epitopes and protection against malarial parasitemia during the first year of life.     

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.     

IgG antibody profile to c-terminal region of Plasmodium vivax merozoite surface protein-1 in Thai individuals exposed to malaria

Naturally acquired immune response to C-terminal region of Plasmodium vivax merozoite surface protein1 (PvMSP1) in 200 individuals with recent clinical episodes of malaria from malaria endemic areas along Thai-Myanmar border in the west and Thai-Cambodia border in the east of Thailand was evaluated by enzyme-linked immunosorbent assay (ELISA). The anti-PvMSP1-IgG antibody was observed in 110 individuals (55%). Among IgG responders, IgG1 coexpressed with IgG3 were the predominant subclasses. The levels of anti-PvMSP1 total IgG, IgG1 and IgG3 antibody response seem to be increased with age although no detectable significant correlation was found (r = 0.004, p = 0.484 for total IgG; r = 0.035, p = 0.386 for IgG1; r = -0.600, p = 0.142 for IgG2; r = 0.077, p = 0.227 for IgG3; r = 0.664, p = 0.051 for IgG4). However, the mean level of specific total IgG was highest in the age group of >40 years. These levels of either specific total IgG or each IgG isotype did not vary among individuals with different malaria episodes. A higher level of specific total IgG, IgG1 and IgG3 antibody response related with the lower of parasitemia density was observed although no significant correlation was found. Our data indicate that individuals exposed to vivax malaria in Thailand developed antibodies to the potential candidate vaccine antigen, PvMSP1 (C-terminal).