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.     

Characteristics of natural antibody responses to the circumsporozoite protein of Plasmodium vivax

The antibody response to the prototype circumsporozoite (CS) protein of Plasmodium vivax (CSPV) was studied in Thai soldiers experiencing occupational malaria. Seventy-four (65%) of 114 men followed during assignment to a malaria transmission area developed blood-stage infection with P. vivax. IgG antibodies against the central repeat region of the CSPV protein were quantitated by ELISA using the recombinant protein, NS181V20, as the capture antigen. One quarter of the subjects had detectable anti-CSPV antibodies at the beginning of the study. CSPV antibody seroconversion was documented in 16 of 26 subjects assessed during their first observed episodes of vivax malaria. This antibody response was of moderate magnitude, fell off after the first week post-diagnosis and appeared, at the low levels observed, to be unassociated with protection. Continued assessment of anti-CSPV antibody after subjects left the transmission area found no increase associated with release of P. vivax. These findings indicate that CS antibody responses to P. vivax during occupational malaria share many characteristics with responses to P. falciparum.     

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.     

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax

We evaluated the influence of allelic frequency of the human leukocyte antigen (HLA) -DRB1 on the acquisition of antibody response against malaria sporozoite and merozoite peptides in patients with Plasmodium vivax malaria acquired in endemic areas of Brazil. IgG antibodies were detected by enzyme-linked immunosorbent assay against four peptides of circumsporozoite protein (CSP) (amino, carboxyl, and VK210 and VK247 repeats) and peptides of merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and Duffy-binding protein (DBP). We found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to CSP amino terminal, as well as an association between HLA-DR3 and the highest antibody response to MSP1 (Pv200L). In conclusion, we suggest a potential regulatory role of the HLA-DRB1 alleles in the production of antibodies to a conserved region of P. vivax CSP and MSP1 in Brazilian population exposed to malaria.     

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role in Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey in the Brazilian Amazon Basin. Despite continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the population was reexamined 12 months later. Antibodies from 16 of 50 (36.0%) subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.     

Antibodies to Plasmodium vivax apical membrane antigen 1: persistence and correlation with malaria transmission intensity.

The antibody responses to the apical membrane antigen 1 of the Plasmodium vivax (PvAMA-1) were investigated in subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PvAMA-1 increased with the time of exposure. The frequency of a positive response and the mean IgG level were higher in areas where malaria prevalence was more intense, especially among non-infected subjects exposed to moderate transmission over a period of 20 years. The proportions and levels of IgG1and IgG3 isotypes were significantly higher among those subjects with long-term exposure. Antibodies, mainly IgG1, to PvAMA-1 persisted for seven years among subjects briefly exposed to malaria in an outbreak outside the Brazilian malaria-endemic area. These data show the highly immunogenic properties of PvAMA-1 and emphasize its possible use as a malaria vaccine candidate.     

Analysis of naturally acquired antibody responses to the 19-kd C-terminal region of merozoite surface protein-1 of Plasmodium vivax from individuals in Sanliurfa, Turkey

Plasmodium vivax is the second most prevalent global Plasmodium species causing malaria after P. falciparum. These two Plasmodium spp. co-exist in most endemic areas, apart from west and central Africa, which has only P. falciparum. However, southeastern Turkey is one of the exceptional regions with the sole presence of P. vivax infection, where a thorough epidemiologic survey has not been performed. Here, we report for the first time the identification of naturally acquired antibodies against the 19-kd C-terminal region of the merozoite surface protein-1 of P. vivax (PvMSP1(19)), using ELISA, from residents in the Sanliurfa region of southeastern Turkey. Among the 82 samples from patients with patent P. vivax malaria, 85% of the individuals were sero-reactive to PvMSP1(19). Particularly, 69.5% of the subjects were positive for IgM, 53.6% were positive for IgG (predominantly IgG1 and IgG3), and 7.3% were positive for IgA.     

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.     

Cross-reactive cellular immune response to circumsporozoite proteins of Plasmodium vivax and P. falciparum in malaria-exposed individuals

Acquired immunity against the recombinant circumsporozoite protein of P. falciparum (rPfCS) or P. vivax (rPvCS) was studied in two malarious areas of the Brazilian Amazon. Cellular responsiveness, evaluated by proliferative assays, was detected in about 45% of individuals who had recovered from recent acute malaria infections. Peripheral blood mononuclear cells of individuals whose last malaria infection was by P. vivax responded more to the rCS proteins than those who had P. falciparum . Since in P. vivax infections hypnozoites in the liver retain CS antigen, this stage may have contributed to the increased cellular response. The unexpected result was that in primoinfections by P. falciparum or P. vivax the proliferative response did not correspond to the rPfCS and rPvCS, respectively. Furthermore, among the malaria-exposed individuals, there was a positive correlation between the intensity of the responses to the two rCS proteins. Our results suggest that cross-reactive epitopes exist in the CS protein of P. falciparum and P. vivax . In the areas studied, the frequency of antibodies against rPvCS and/or rPfCS ranged from 43% to 11%. Species-specific antibodies against the CS protein were detected in the primoinfected individuals. Some individuals living in the endemic area but with no clinical history of malaria were positive by serology (8%) or by in vitro proliferation (21%). However, antibodies and cellular responses against rCS were detected only in malaria-exposed individuals, since those living outside the endemic area were all negative.     

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.     

Natural malaria infections in anophelines in Rondonia State, Brazilian Amazon

The use of an Immunoassay for the detection of Plasmodium falciparum and P. vivax circumsporozoite (CS) antigens in anophelines has recently incriminated other malaria vectors besides Anopheles darlingi in the Brazilian Amazon. In this study we analyzed 12,336 field-collected anophelines from endemic areas in Rondonia for plasmodial infection. Sixty-one specimens from 6 species were positive: 47 An. darlingi, 5 An. triannulatus, 4 An. albitarsis, 2 An. braziliensis, 2 An. strodei, and 1 An. oswaldoi. As concerns the species, 41 anopheles harbored P. falciparum and 20 were infected with P. vivax. An. darlingi was the most important local vector, as it was the one most frequently found infected and the only one clearly related to areas where malaria transmission was being recorded.     

Polymerase chain reaction and molecular genotyping to monitor parasitological response to anti-malarial chemotherapy in the Peruvian Amazon

Over the past decade, anti-malarial drug resistance has rapidly become a major public health problem in the Peruvian Amazon. This study compared polymerase chain reaction (PCR) to light microscopy for diagnosing and monitoring the parasitological response of malaria patients to anti-malarial chemotherapy in the Peruvian Amazon region of Iquitos. Typing of P. falciparum using MSP1, MSP2, and glutamine-rich protein distinguished among infecting parasites. Most (73%) P. falciparum patients were parasitologically resistant to sulfadoxine-pyrimethamine (RI = 10, RII = 1). Sensitivity of microscopy was lower than PCR (69% for P. vivax and 78% for P. falciparum), but parasite clearance times were comparable between microscopy and PCR. PCR sensitively and specifically detected mixed infections and low-level parasitemia indicative of drug resistance, making this approach of practical use for the control of malaria at the public health level. Genotyping malaria parasites will be useful to distinguish drug failure from new infections in clinical trials of anti-malarial drugs in the Peruvian Amazon region.     

Ahaptoglobinemia (HpO) and malaria in India

Haptoglobin (Hp) polymorphism analysed among P. vivax and P. falciparum patients and malaria negative subjects from areas with different epidemiological situations had shown high incidence of ahaptoglobinemia (HpO) among malaria patients. A definite association of HpO with P. vivax as well as P. falciparum malaria in Indian subjects had been observed. However, low sensitivity and reliability of HpO index indicates that it can not be a good indicator for determination of malaria endemicity. About 75 per cent of HpO subjects with P. vivax infection when treated with chloroquine showed typable Hp polymorphs by 8-9 days of post-treatment.     

Hidden Plasmodium falciparum infections

Mixed infection of P. vivax and P. falciparum malaria frequently recorded in field survey. However mixed infection was frequently misdiagnosed as single infection due to low parasite density, difficult species identification and procedure error of microscopic examination. Our previous report showed high rates (32-33%) of P vivax infection following treatment of previously assumed to be only P. falciparum infection. In a study of 992 patients with initial presentation with P. falciparum, we found that 104 (10.5%) of patients had P. falciparum appearing during 28 days in the hospital (ranged 1-28 days) following chloroquine treatment for P. vivax. The potential for P. falciparum appearing following elimination of P. vivax must be considered in malaria treatment.     

Apical membrane protein 1‐specific antibody profile and temporal changes in peripheral blood B‐cell populations in Plasmodium vivax malaria

Plasmodium falciparum‐specific antibodies tend to be short‐lived, but their cognate memory B cells (MBCs) circulate in the peripheral blood of exposed subjects for several months or years after the last infection. However, the time course of antigen‐specific antibodies and B‐cell responses to the relatively neglected parasite Plasmodium vivax remains largely unexplored. Here, we showed that uncomplicated vivax malaria elicits short‐lived antibodies but long‐lived MBC responses to a major blood‐stage P vivax antigen, apical membrane protein 1 (PvAMA‐1), in subjects exposed to declining malaria transmission in the Amazon Basin of Brazil. We found that atypical (CD19⁺CD10^?CD21^?CD27^?) MBCs, which appear to share a common precursor with classical MBCs but are unable to differentiate into antibody‐secreting cells, significantly outnumbered classical MBCs by 5:1 in the peripheral blood of adult subjects currently or recently infected with P vivax and by 3:1 in healthy residents in the same endemic communities. We concluded that malaria can drive classical MBCs to differentiate into functionally impaired MBCs not only in subjects repeatedly exposed to P falciparum, but also in subjects living in areas with low levels of P vivax transmission in the Amazon, leading to an impaired B‐cell memory that may affect both naturally acquired and vaccine‐induced immunity.     

Comparison with the Pan Malaria IgG assays for malaria diagnosis and direct microscopy among suspected malaria patients in Sanliurfa

In this study, we evaluated the usage of the Pan Malaria IgG CELISA test in the diagnosis of malarial infections in Siverek-Sanliurfa, Turkey, where malarial infection is endemic and there are minimal health services available. The Pan Malaria IgG CELISA (Cellabs) test, which uses recombinant antigens and detects exposure to all four forms of malaria (P. falciparum, P. vivax, P. ovale and P. malariae) was used as individuals. Using the consensus microscopy results as the standard, sensitivity of ELISA for detection of any malarial infection in the rural populations was 83%, specificity was 85%. These results show that the performance of ELISA for the detection of any malarial infection is adequate for acute- and post-emergency situations and rural populations when the alternative is just clinical diagnosis.     

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.     

Comparison of IgG reactivities to Plasmodium vivax merozoite invasion antigens in a Brazilian Amazon population

Naturally acquired antibody reactivity to two major Plasmodium vivax vaccine candidates was investigated in 294 donors from three malaria-endemic communities of Rond?nia state, Brazil. Antibody recognition of recombinantly expressed antigens covering five different regions of P. vivax reticulocyte binding protein 1 (PvRBP1) and region II of P. vivax Duffy binding protein (PvDBP-RII) were compared. Positive IgG responses to these antigens were significantly related to the level of malaria exposure in terms of past infections and years of residence in the endemic area when corrected for age. The highest prevalence of anti-PvRBP1 total IgG antibodies corresponded to the amino acid regions denoted PvRBP1(431-748) (41%) and PvRBP1(733-1407) (47%). Approximately one-fifth of positively responding sera had titers of at least 1:1,600. Total IgG responses to PvDBP-RII were more prevalent (67%), of greater magnitude, and acquired more rapidly than those to individual PvRBP1 antigens. Responses to both PvRBP1 and PvDBP-RII were biased toward the cytophilic subclasses IgG1 and IgG3. These data provide the first insights on acquired antibody responses to PvRBP1 and a comparative view with PvDBP-RII that may prove valuable for understanding protective immune responses to these two vaccine candidates as they are evaluated as components of multitarget blood-stage vaccines.