RESUMEN
Background: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.
Asunto(s)
Anticuerpos Antiprotozoarios , Biomarcadores , Malaria Vivax , Proteína 1 de Superficie de Merozoito , Plasmodium vivax , Humanos , Malaria Vivax/inmunología , Malaria Vivax/sangre , Malaria Vivax/parasitología , Malaria Vivax/transmisión , Malaria Vivax/diagnóstico , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium vivax/inmunología , Biomarcadores/sangre , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Adulto , Femenino , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Adulto Joven , Adolescente , Secuencia de AminoácidosRESUMEN
Plasmodium vivax serological exposure markers (SEMs) have emerged as promising tools for the actionable surveillance and implementation of targeted interventions to accelerate malaria elimination. To determine the dynamic profiles of SEMs in current and past P. vivax infections, we screened and selected 11 P. vivax proteins from 210 putative proteins using protein arrays, with a set of serum samples obtained from patients with acute P. vivax and documented past P. vivax infections. Then we used a murine protein immune model to initially investigate the humoral and memory B cell response involved in the generation of long-lived antibodies. We show that of the 11 proteins, especially C-terminal 42-kDa region of P. vivax merozoite surface protein 1 (PvMSP1-42) induced longer-lasting long-lived antibodies, as these antibodies were detected in individuals infected with P. vivax in the 1960-1970s who were not re-infected until 2012. In addition, we provide a potential mechanism for the maintenance of long-lived antibodies after the induction of PvMSP1-42. The results indicate that PvMSP1-42 induces more CD73+CD80+ memory B cells (MBCs) compared to P. vivax GPI-anchored micronemal antigen (PvGAMA), allowing IgG anti-PvMSP1-42 antibodies to be maintained for a long time.
Asunto(s)
Anticuerpos Antiprotozoarios , Malaria Vivax , Células B de Memoria , Proteína 1 de Superficie de Merozoito , Plasmodium vivax , Plasmodium vivax/inmunología , Humanos , Malaria Vivax/inmunología , Anticuerpos Antiprotozoarios/inmunología , Animales , Proteína 1 de Superficie de Merozoito/inmunología , Ratones , Células B de Memoria/inmunología , Inmunidad Humoral/inmunología , Biomarcadores/sangre , Femenino , Memoria Inmunológica/inmunología , Linfocitos B/inmunología , Antígenos de Protozoos/inmunologíaRESUMEN
The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface of the invasive merozoite stages of Plasmodium falciparum and has long been considered a key target of protective immunity. We used samples from a single controlled human malaria challenge study to test whether the full-length version of MSP1 (MSP1FL) induced antibodies that mediated Fc-IgG functional activity in five independent assays. We found that anti-MSP1FL antibodies induced complement fixation via C1q, monocyte-mediated phagocytosis, neutrophil respiratory burst, and natural killer cell degranulation as well as IFNγ production. Activity in each of these assays was strongly associated with protection. The breadth of MSP1-specific Fc-mediated effector functions was more strongly associated with protection than the individual measures and closely mirrored what we have previously reported using the same assays against merozoites. Our findings suggest that MSP1FL is an important target of functional antibodies that contribute to a protective immune response against malaria.
Asunto(s)
Anticuerpos Antiprotozoarios , Malaria Falciparum , Proteína 1 de Superficie de Merozoito , Fagocitosis , Plasmodium falciparum , Humanos , Proteína 1 de Superficie de Merozoito/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/inmunología , Anticuerpos Antiprotozoarios/inmunología , Fagocitosis/inmunología , Inmunoglobulina G/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Interferón gamma/metabolismo , Interferón gamma/inmunología , Femenino , Merozoítos/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
BACKGROUND: Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119. METHODS: A total of 37 clinical Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012-2016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp119 (pocmsp119) and P. ovale wallikeri (pow) msp119 (powmsp119) genes by polymerase chain reaction. The genetic diversity of pomsp119 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (GST) proteins were expressed in an Escherichia coli expression system and analyzed by western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays. RESULTS: Sequences of pomsp119 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as ~ 37-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively. CONCLUSIONS: This study revealed highly conserved gene sequences of pomsp119. In addition, naturally acquired humoral immune responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design.
Asunto(s)
Proteína 1 de Superficie de Merozoito , Plasmodium ovale/genética , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Humanos , Inmunogenicidad Vacunal , Vacunas contra la Malaria , Proteína 1 de Superficie de Merozoito/genética , Proteína 1 de Superficie de Merozoito/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmodium ovale/inmunología , Polimorfismo Genético , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Desarrollo de VacunasRESUMEN
The polymorphic nature of merozoite surface protein 1(MSP1) raises doubts whether it may serve as a vaccine target against Plasmodium vivax malaria. This study analyses the impact of genetic variability on the epitope organization of different Pvmsp1 blocks. Ten blood samples collected from P. vivax infected malaria patients from West Bengal, India were used to analyze sequence and antigenic diversities of block 2 region of Pvmsp1. An additional 48 block 2 sequences from other countries were also analyzed. Global genetic framework of Pvmsp1 block 2 was represented by 12 indel clusters & 33 haplotypes (haplotype diversiy = 0.965 ± 0.024). Parasite sequences pertaining to other Pvmsp1 modules, namely block 6 and 10 displayed 14 & 29 (haplotype diversiy = 0.975 ± 0.003) and 22 & 30 indel clusters and haplotypes (haplotype diversiy = 0.947 ± 0.004), respectively. In spite of this remarkable genetic diversity, a small number of conserved epitopes were detected in all three PvMSP1 blocks. This novel finding substantiates that MSP1 could serve as a promising vaccine candidate against vivax malaria.
Asunto(s)
Epítopos de Linfocito B/inmunología , Variación Genética/inmunología , Vacunas contra la Malaria/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium vivax/inmunología , Plasmodium vivax/genéticaRESUMEN
Research on erythrocytic Plasmodium vivax merozoite antigens is critical for identifying potential vaccine candidates in reducing P. vivax disease. However, many P. vivax studies are constrained by its inability to undergo long-term culture in vitro Conserved across all Plasmodium spp., merozoite surface proteins are essential for invasion into erythrocytes and highly expressed on erythrocytic merozoites, thus making it an ideal vaccine candidate. In clinical trials, the P. vivax merozoite surface protein 1 (PvMSP1-19) vaccine candidate alone has shown to have limited immunogenicity in patients; hence, we incorporate the highly conserved and immunogenic C terminus of both P. vivax merozoite surface protein 8 (PvMSP8) and PvMSP1-19 to develop a multicomponent chimeric protein rPvMSP8+1 for immunization of mice. The resulted chimeric rPvMSP8+1 antibody was shown to recognize native protein MSP8 and MSP1-19 of mature P. vivax schizonts. In the immunized mice, an elevated antibody response was observed in the rPvMSP8+1-immunized group compared to that immunized with single-antigen components. In addition, we examined the growth inhibition of these antibodies against Plasmodium cynomolgi (Berok strain) parasites, which is phylogenetically close to P. vivax and sustains long-term culture in vitro Similarly, the chimeric anti-rPvMSP8+1 antibodies recognize P. cynomolgi MSP8 and MSP1-19 on mature schizonts and showed strong inhibition in vitro via growth inhibition assay. This study provides support for a new multiantigen-based paradigm rPvMSP8+1 to explore potential chimeric vaccine candidates against P. vivax malaria using sister species P. cynomolgi.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Malaria Vivax/genética , Malaria Vivax/inmunología , Proteína 1 de Superficie de Merozoito/genética , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium vivax/genética , Plasmodium vivax/inmunología , Virulencia/inmunología , Animales , Anticuerpos Antiprotozoarios/genética , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Eritrocitos/inmunología , Regulación de la Expresión Génica , Humanos , Ratones , Modelos Animales , Virulencia/genéticaRESUMEN
BACKGROUND: Suriname has accomplished a steep decline in malaria burden, even reaching elimination levels. Plasmodium serology data are not available for Suriname and even extremely scarce within the region, therefore malaria serology testing was introduced, country customized cut-off values were determined and a study was performed to explore the antibody status for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. METHODS: A cross-sectional survey was conducted between July 2017 and March 2018 in two areas of the interior with different malaria settings: Stoelmanseiland, representing Maroon villages and Benzdorp, a gold mining area, with mostly Brazilian miners. Dried blood spots (DBS) were collected (n = 197) and antibody presence against seven Plasmodium antigens was detected using a multiplex bead-based, IgG antibody assay. Demographic information was gathered through a questionnaire. Country customized cut-off values were generated from a Surinamese malaria-naïve reference population (n = 50). RESULTS: Serological analysis for the reference population revealed cut-off values ranging from 14 MFI for LSA-1 to 177 MFI for PmMSP-119. Seroprevalence against any of the three MSP-119 antibodies was similar in both regions and surpassed 75%. Single seropositivity against PfMSP-119 antibodies was higher in Stoelmanseiland (27.0%) than Benzdorp (9.3%), in line with the historical malaria burden of Stoelmanseiland, while the reverse was observed for PvMSP-119 antibodies. Despite sporadic reports of P. malariae infections, PmMSP-119 antibody presence was 39.6%. A more detailed examination of P. falciparum serology data displayed a higher seroprevalence in villagers (90.7%) than in Brazilians (64.6%) and a highly diverse antigenic response with 22 distinct antibody combinations. CONCLUSIONS: The results on the malaria antibody signature of Maroon villagers and Brazilian miners living in Suriname displayed a high Plasmodium seroprevalence, especially for P. falciparum in villagers, still reflecting the historical malaria burden. The seroprevalence data for both regions and the observed combinations of P. falciparum antibodies provided a valuable dataset from a historically important region to the international malaria serology knowledge. First insight in malaria serology data for Suriname indicated that the use of other target groups and assessment of age-dependent seroprevalence are required to successfully use malaria serology as tool in the national elimination strategy.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/sangre , Inmunoglobulina G/sangre , Malaria Falciparum/epidemiología , Malaria Vivax/epidemiología , Malaria/epidemiología , Adulto , Anciano , Brasil/etnología , Estudios Transversales , Femenino , Humanos , Masculino , Proteína 1 de Superficie de Merozoito/inmunología , Persona de Mediana Edad , Minería , Plasmodium falciparum/fisiología , Plasmodium malariae/fisiología , Plasmodium vivax/fisiología , Prevalencia , Estudios Seroepidemiológicos , Suriname/epidemiología , Adulto JovenRESUMEN
Morbidity and mortality related to malaria in Indonesia are attributed to both Plasmodium falciparum and P. vivax parasites. In addition to vaccines for P. falciparum, vaccines against P. vivax are urgently needed for the prevention of the disease. An extensively studied antigen is the carboxyl-terminus of the 42 kDa region of P. vivax merozoite surface protein-1 (PvMSP1-42). The design of a vaccine based on this antigen requires an understanding of the extent of polymorphism. However, there is no information on the genetic diversity of the antigen in Indonesia. This study aimed to profile the diversity of PvMSP1-42 and its two subdomains (PvMSP1-33 and PvMSP1-19) among Indonesian P. vivax isolates. A total of 52 P. vivax-infected blood samples were collected from patients in two different endemic areas in Indonesia: Banjarmasin (Kalimantan) and Sumba Timur (Nusa Tenggara Timur). The polymorphic characteristics and natural selection of PvMSP1-42 were analyzed using the DnaSP, MEGA, and Structure software. Thirty distinct haplotypes of PvMSP1-42 were identified. They displayed amino acid changes compared to the reference PVP01 sequence. Most of the mutations were concentrated in the 33 kDa fragment. PvMSP1-42 of the Indonesian isolates appeared to be under positive selection. Recombination may also play a role in the resulting genetic diversity of PvMSP1. In conclusion, PvMSP1-42 of Indonesian isolates displayed allelic polymorphisms caused by mutation, recombination, and positive selection. These results will aid the understanding of the P. vivax population in Indonesia and to develop a PvMSP1 based vaccine against P. vivax.
Asunto(s)
Antígenos de Protozoos/genética , Variación Genética , Malaria Vivax/parasitología , Proteína 1 de Superficie de Merozoito/genética , Plasmodium vivax/genética , Alelos , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Humanos , Indonesia/epidemiología , Vacunas contra la Malaria/inmunología , Malaria Vivax/epidemiología , Malaria Vivax/prevención & control , Proteína 1 de Superficie de Merozoito/química , Proteína 1 de Superficie de Merozoito/inmunología , Modelos Moleculares , Plasmodium vivax/inmunología , Plasmodium vivax/aislamiento & purificación , Polimorfismo Genético , Selección Genética , Relación Estructura-ActividadRESUMEN
Rapid diagnostic tests are first-line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population-screening assays, high-quality reagents and well-characterized antigens and antibodies are needed. An important property of antigen-antibody binding is recognition specificity, which best can be estimated by mapping an antibody's epitope on the respective antigen. We have cloned a malarial antigen-containing fusion protein, MBP-pfMSP119, in Escherichia coli, which then was structurally and functionally characterized before and after high pressure-assisted enzymatic digestion. We then used our previously developed method, intact transition epitope mapping-targeted high-energy rupture of extracted epitopes (ITEM-THREE), to map the area on the MBP-pfMSP119 antigen surface that is recognized by the anti-pfMSP119 antibody G17.12. We identified three epitope-carrying peptides, 386GRNISQHQCVKKQCPQNSGCFRHLDE411, 386GRNISQHQCVKKQCPQNSGCFRHLDEREE414, and 415CKCLLNYKQE424, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP-pfMSP119 antigen whose identification was substantiated by positive and negative control experiments. In conclusion, our data help to establish a workflow to obtain high-quality control data for diagnostic assays, including the use of ITEM-THREE as a powerful analytical tool. Data are available via ProteomeXchange: PXD019717.
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Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Epítopos/inmunología , Malaria Falciparum/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Secuencia de Aminoácidos , Animales , Complejo Antígeno-Anticuerpo/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida/métodos , Mapeo Epitopo/métodos , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Plasmodium vivax remains an important cause of malaria in South America and Asia, and analyses of the antibody immune response are being used to identify biomarker of parasite exposure. The IgG antibody naturally acquired predominantly occurs against targets on blood-stage parasites, including C-terminal of the merozoite surface protein 1 (MSP1-19). Epidemiological and immunological evidence has been showed that antibodies to malaria parasite antigens are lost in the absence of ongoing exposure. We describe the IgG antibody response in individuals living in an unstable malaria transmission area in Pará state, Amazon region, Brazil, where an epidemic of P. vivax malaria was recorded and monitored over time. As indicated by epidemiological data, the number of P. vivax-caused malaria cases decreased by approximately 90% after three years and the prevalence of IgG positive to PvMSP1-19 decreased significantly over time, in 2010 (93.4%), 2012 (78.3%), and 2013 (85.1%). Acquisition and decay of the IgG antibody against P. vivax MSP1-19 showed variability among individuals living in areas with recent circulating parasites, where the malaria epidemic was being monitored until transmission had been completely controlled. We also found that previous malaria episodes were associated with an increased in the IgG positivity . Our results showed epidemiological, spatial, temporal and individual variability. The understanding on dynamics of antibodies may have implications for the design of serosurveillance tools for monitoring parasite circulation, especially in a context with spatial and temporal changes in P. vivax malaria transmission.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Inmunoglobulina G/sangre , Malaria Vivax/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium vivax/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Antiprotozoarios/inmunología , Brasil/epidemiología , Niño , Preescolar , Femenino , Humanos , Malaria Vivax/transmisión , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The lack of continuous in vitro cultures has been an obstacle delaying pre-clinical testing of Plasmodium vivax vaccine formulations based on known antigens. In this study, we generated a model to test available formulations based on the P. vivax MSP119 antigen. The Plasmodium berghei strains ANKA and NK65 were modified to express PvMSP119 instead of the endogenous PbMSP119. The hybrid parasites were used to challenge C57BL/6 or BALB/c mice immunized with PvMSP119-based vaccine formulations. The PvMSP119 was correctly expressed in the P. berghei hybrid mutant lines as confirmed by immunofluorescence using anti-PvMSP119 monoclonal antibodies and by Western blot. Replacement of the PbMSP119 by the PvMSP119 had no impact on asexual growth in vivo. High titers of specific antibodies to PvMSP119 were not sufficient to control initial parasitemia in the immunized mice, but late parasitemia control and a balanced inflammatory process protected these mice from dying, suggesting that an established immune response to PvMSP119 in this model can help immunity mounted later during infection.
Asunto(s)
Antígenos de Protozoos/inmunología , Inmunogenicidad Vacunal , Vacunas contra la Malaria/inmunología , Malaria Vivax/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Proteína 1 de Superficie de Merozoito/metabolismo , Plasmodium berghei/metabolismo , Plasmodium vivax/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Femenino , Malaria Vivax/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Parasitemia/inmunología , Plásmidos/genética , Plasmodium berghei/genética , Proteínas Protozoarias/inmunología , Transfección , Resultado del Tratamiento , VacunaciónRESUMEN
Plasmodium vivax reemerged in 1993. It has been sustained for more than 25 years and become one of the important indigenous parasitic diseases in northern and western parts of the Republic of Korea near the demilitarized zone. In particular, relapse is a significant concern for the control of malaria, as short- and long-term incubation periods vary among those infected in Korea. In this study, the prevalence of asymptomatic carriers was examined among residents of high endemic areas of vivax malaria during nonseasonal transmission of mosquitoes. Blood samples from 3 endemic regions in northwestern Korea were evaluated by microscopic examination, rapid diagnostic testing, and nested PCR to identify asymptomatic patients carrying malaria parasites in the community. However, no positive malaria case among residents of endemic areas was detected. Additionally, serological analysis was carried out to measure antibodies against 3 antigenic recombinant proteins of P. vivax, merozoite surface protein 1-19, circumsporozoite surface protein-VK210, and liver-stage antigen (PvLSA-N), by the protein array method. Interestingly, seropositivity of sera between previous exposure and samples without exposure to malaria was significantly higher using the PvLSA-N antigen than the other antigens, suggesting that PvLSA-N can be used as a serological marker to analyze the degree of exposure for malaria transmission in endemic areas. This indicates a very low asymptomatic carrier prevalence during the nonmalaria season in the endemic areas of Korea.
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Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Portador Sano/diagnóstico , Portador Sano/epidemiología , Enfermedades Endémicas , Malaria Vivax/diagnóstico , Malaria Vivax/epidemiología , Plasmodium vivax/inmunología , Pruebas Serológicas/métodos , Biomarcadores/sangre , Enfermedades Endémicas/prevención & control , Enfermedades Endémicas/estadística & datos numéricos , Femenino , Humanos , Malaria Vivax/prevención & control , Masculino , Proteína 1 de Superficie de Merozoito/inmunología , Prevalencia , Proteínas Protozoarias/inmunología , República de Corea/epidemiologíaRESUMEN
BACKGROUND: Identifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-119) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-119 antigens in individuals living in three geographically diverse malaria endemic regions of India. METHODS: A total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-119 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed. RESULTS: The overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-119. Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-119, seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-119 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-119. CONCLUSION: These data suggest that natural acquired antibody response is higher for PvMSP-119 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-119 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Malaria Vivax/inmunología , Proteínas de la Membrana/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Proteínas Protozoarias/inmunología , Adolescente , Adulto , Formación de Anticuerpos , Antígenos de Protozoos/sangre , Niño , Enfermedades Endémicas , Ensayo de Inmunoadsorción Enzimática , Femenino , Geografía , Humanos , Inmunoglobulina G/sangre , India , Malaria Vivax/prevención & control , Masculino , Proteínas de la Membrana/sangre , Proteína 1 de Superficie de Merozoito/sangre , Persona de Mediana Edad , Plasmodium vivax , Proteínas Protozoarias/sangre , Estudios Seroepidemiológicos , Adulto JovenRESUMEN
BACKGROUND: Plasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1. METHODS: Five regions of PmMSP1 were expressed in Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry. RESULTS: We found that N-terminal, central regions, and PmMSP119 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns (predominantly IgG1, IgG2a, and IgG2b), characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins. CONCLUSION: Recombinant PmMSP1 proteins are very useful diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae.
Asunto(s)
Malaria/diagnóstico , Malaria/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium malariae/inmunología , Proteínas Recombinantes/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Proliferación Celular , Citocinas/metabolismo , Humanos , Inmunización , Inmunoglobulina G/inmunología , Interleucina-4/metabolismo , Malaria/sangre , Malaria/parasitología , Proteína 1 de Superficie de Merozoito/química , Ratones Endogámicos BALB C , Bazo/metabolismoRESUMEN
Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4+ T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Formación de Anticuerpos , Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Transmisión de Enfermedad Infecciosa/prevención & control , Vacunas contra la Malaria/inmunología , Malaria Vivax/prevención & control , Plasmodium vivax/inmunología , Animales , Homólogo de la Proteína Chromobox 5 , Vacunas contra la Malaria/administración & dosificación , Malaria Vivax/transmisión , Proteína 1 de Superficie de Merozoito/inmunología , Ratones , Proteínas Recombinantes de Fusión/inmunología , Factores de Tiempo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: Plasmodium vivax contains approximately 5400 coding genes, more than 40% of which code for hypothetical proteins that have not been functionally characterized. In a previous preliminary screening using pooled serum samples, numerous hypothetical proteins were selected from among those that were highly transcribed in the schizont-stage of parasites, and highly antigenic P. vivax candidates including hypothetical proteins were identified. However, their immunological and functional activities in P. vivax remain unclear. From these candidates, we investigated a P. vivax 50-kDa protein (Pv50, PVX_087140) containing a highly conserved signal peptide that shows high transcription levels in blood-stage parasites. RESULTS: Recombinant Pv50 was expressed in a cell-free expression system and used for IgG prevalence analysis of patients with vivax malaria and healthy individuals. Immune responses were analyzed in immunized mice and mouse antibodies were used to detect the subcellular localization of the protein in blood-stage parasites by immunofluorescence assay. A protein array method was used to evaluate protein-protein interactions to predict protein functional activities during the invasion of parasites into erythrocytes. Recombinant Pv50 showed IgG prevalence in patient samples with a sensitivity of 42.9% and specificity of 93.8% compared to that in healthy individuals. The non-cytophilic antibodies IgG1 and IgG3 were the major components involved in the antibody response in Pv50-immunized mice. Pv50 localized on the surface of merozoites and a specific interaction between Pv50 and PvMSP1 was detected, suggesting that Pv50-PvMSP1 forms a heterodimeric complex in P. vivax. CONCLUSIONS: Increased immune responses caused by native P. vivax parasites were detected, confirming its immunogenic effects. This study provides a method for detecting new malaria antigens, and Pv50 may be a vivax malaria vaccine candidate with PvMSP1.
Asunto(s)
Plasmodium vivax/química , Proteínas Protozoarias/aislamiento & purificación , Adolescente , Adulto , Animales , Anticuerpos Antiprotozoarios/sangre , Western Blotting , Citocinas/sangre , Femenino , Humanos , Inmunidad Humoral , Recuento de Linfocitos , Malaria Vivax/inmunología , Malaria Vivax/parasitología , Proteína 1 de Superficie de Merozoito/inmunología , Proteína 1 de Superficie de Merozoito/metabolismo , Merozoítos/química , Merozoítos/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmodium/química , Plasmodium vivax/genética , Plasmodium vivax/inmunología , Unión Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/genética , Linfocitos T/inmunología , Adulto JovenRESUMEN
Merozoite surface proteins (MSPs) are critical for parasite invasion; they represent attractive targets for antibody-based protection against clinical malaria. To identify protection-associated target MSPs, the present study analysed antibody responses to whole merozoite extract (ME) and to defined MSP recombinant antigens in hospitalized patients from a low endemic urban area as a function of disease severity (mild versus cerebral malaria). Sera from 110 patients with confirmed severe cerebral malaria (CM) and 91 patients with mild malaria (MM) were analysed (mean age = 29 years) for total and subclass immunoglobulin (Ig)G to ME and total IgG to MSP1p19, MSP2, MSP3, MSP4 and MSP5 by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were evaluated using the antibody-dependent respiratory burst (ADRB) assay in a subset of sera. There was a trend towards higher IgG1 and IgG4 levels to ME in CM compared to MM; only ME IgM responses differed significantly between fatal and surviving CM patients. Increased prevalence of IgG to individual MSPs was found in the CM compared to the MM group, including significantly higher levels of IgG to MSP4 and MSP5 in the former. Sera from fatal (24·5%) versus surviving cases showed significantly lower IgG to MSP1p19 and MSP3 (P < 0·05). ADRB assay readouts correlated with high levels of anti-MSP IgG, and trended higher in sera from patients with surviving compared to fatal CM outcome (P = 0·07). These results document strong differential antibody responses to MSP antigens as targets of protective immunity against CM and in particular MSP1p19 and MSP3 as prognostic indicators.
Asunto(s)
Antígenos de Protozoos/inmunología , Extractos Celulares/inmunología , Malaria Cerebral/inmunología , Malaria Falciparum/inmunología , Merozoítos/inmunología , Plasmodium falciparum/inmunología , Población Urbana , Adolescente , Adulto , Anciano , Anticuerpos Antiprotozoarios/sangre , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Hospitalización , Humanos , Inmunoglobulina M/sangre , Lactante , Malaria Cerebral/mortalidad , Malaria Falciparum/mortalidad , Masculino , Proteína 1 de Superficie de Merozoito/inmunología , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Antibodies against the merozoite surface protein 1-19 (MSP1-19) and the apical membrane antigen 1 (AMA1) of the malaria parasite (Plasmodium vivax) are proven to be important in protection against clinical disease. Differences in the production/maintenance of antibodies may be due to many factors including host genetics. This paper discusses the association of 4 anti-malarial antibodies with selected host genetic markers. METHODS: Blood was collected from individuals (n = 242) with a history of malaria within past 15 years for DNA and serum. ELISA was carried out for serum to determine the concentration of anti-malarial antibodies MSP1-19 and AMA1 for both vivax and falciparum malaria. 170 SNPs related to malaria were genotyped. Associations between seropositivity, antibody levels and genetic, non-genetic factors were determined. RESULTS: Age ranged 13-74 years (mean age = 40.21 years). Majority were females. Over 90% individuals possessed either one or more type(s) of anti-malarial antibodies. Five SNPs were significantly associated with seropositivity. One SNP was associated with MSP1-19_Pv(rs739718); 4 SNPs with MSP1-19_Pf (rs6874639, rs2706379, rs2706381 and rs2075820) and1 with AMA1_Pv (rs2075820). Eleven and 7 genotypes (out of 15) were significantly associated with either presence or absence of antibodies. Three SNPs were found to be significantly associated with the antibody levels viz. rs17411697 with MSP1-19_Pv, rs2227491 with AMA1_Pv and rs229587 with AMA1_Pf. Linkage of the markers in the two groups was similar, but lower LOD scores were observed in seropositives compared to seronegatives. DISCUSSION AND CONCLUSIONS: The study suggests that several SNPs in the human genome that exist in Sri Lankan populations are significantly associated with anti-malarial antibodies, either with generation and/or maintenance of antibodies for longer periods, which can be due to either individual polymorphisms or most probably a combined effect of the markers.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Inmunidad Humoral , Malaria Falciparum/inmunología , Malaria Vivax/inmunología , Proteínas de la Membrana/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Polimorfismo Genético , Proteínas Protozoarias/inmunología , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Plasmodium falciparum/fisiología , Plasmodium vivax/fisiología , Sri Lanka , Adulto JovenRESUMEN
A subset of atypical memory B cells accumulates in malaria and several infections, autoimmune disorders and aging in both humans and mice. It has been suggested these cells are exhausted long-lived memory B cells, and their accumulation may contribute to poor acquisition of long-lasting immunity to certain chronic infections, such as malaria and HIV. Here, we generated an immunoglobulin heavy chain knock-in mouse with a BCR that recognizes MSP1 of the rodent malaria parasite, Plasmodium chabaudi. In combination with a mosquito-initiated P. chabaudi infection, we show that Plasmodium-specific atypical memory B cells are short-lived and disappear upon natural resolution of chronic infection. These cells show features of activation, proliferation, DNA replication, and plasmablasts. Our data demonstrate that Plasmodium-specific atypical memory B cells are not a subset of long-lived memory B cells, but rather short-lived activated cells, and part of a physiologic ongoing B-cell response.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Memoria Inmunológica , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium chabaudi/inmunología , Animales , Subgrupos de Linfocitos B/química , Linfocitos B/química , Citometría de Flujo , Técnicas de Sustitución del Gen , Inmunoglobulina G/genética , Malaria/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Enfermedades de los Roedores/inmunologíaRESUMEN
Malaria is a significant public health problem in several tropical countries including Thailand. The prevalence of Plasmodium vivax infection has been increasing in the past decades. Plasmodium vivax merozoite surface protein (PvMSP) gene encodes a malaria vaccine candidate antigen. Its polymorphic nature leads to antigenic variation, the barrier for vaccine development, drug resistance, and potential for multiple-clone infections within the malaria patients. The objective of this study was to investigate the genetic diversity of PvMSP1 and PvMSP3 gene in P. vivax populations in Thailand. A total of 100 P. vivax isolates collected from the western (Kanchanaburi and Tak Provinces) and southern (Ranong Provinces) regions along the Thai-Myanmar border were analyzed using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Analysis of the F1, F2, and F3 regions of PvMSP1 revealed 5, 2, and 3 allelic variants, respectively. Three major types of PvMSP3-α and two major types of PvMSP3-ß were identified based on the PCR product sizes. After digestion with restriction enzymes, 29, 25, 26, and 18 patterns were distinguished by RFLP for PvMSP1 (F2, Alu I), PvMSP1 (F2, Mnl I), PvMSP3-α, and PvMSP3-ß, respectively. Combination of each family variant (PvMSP1 and PvMSP3) resulted in high genetic polymorphism of P. vivax population. Additionally, using PvMSP1 polymorphic marker revealed a significant association between multiple-genotype infections and P. vivax parasitemia. The results strongly supported that P. vivax populations in the endemic areas along the Thai-Myanmar border are highly diverse.