O Antígeno 1 de Membrana Apical (AMA-1) tem sido sugerido como candidato a compor uma vacina contra estágios assexuados sanguíneos de Plasmodium. Recentemente nosso grupo identificou o domínio II (DII) de AMA-1 de Plasmodium vivax (PvAMA-1) como uma região altamente reconhecida por anticorpos IgG de indivíduos brasileiros infectados por P. vivax. No presente estudo avaliamos as propriedades imunogênicas da proteína recombinante DII, produzida a partir de Escherichia coli. Grupos de 6 camundongos fêmeas BALB/c foram imunizados quatro vezes com 10 µg dessa proteína na presença de diferentes formulações de adjuvantes [Adjuvante Completo/Incompleto de Freund (ACF/AIF), MPL-TDM, TiterMax, Hidróxido de Alumínio (Alum), Quil A, QS-21 e CpG-ODN 1826], individualmente, ou em combinação (Alum + QS-21 ou Alum + CpG-ODN 1826)). Nosso objetivo foi avaliar comparativamente a resposta de anticorpos (IgM, IgG e isotipos de IgG), induzida pelos diferentes esquemas de imunizações, visando futuros estudos pré-clínicos em primatas não humanos. Os títulos de anticorpos IgG contra (o ectodomínio) PvAMA-1 foram determinados por ELISA, duas semanas após cada imunização. A presença de IgM e dos isotipos de IgG também foi avaliada após o final do esquema de imunizações. Nossos resultados demonstraram que a proteína recombinante DII foi altamente imunogênica em camundongos BALB/c quando administrada na presença dos adjuvantes testados. Altos títulos de IgG1, IgG2a e IgG2b foram observados na maioria dos grupos (com exceção do adjuvante Alum), sugerindo uma resposta mista Th1/Th2. Finalmente, demonstramos que anticorpos monoclonais e policlonais anti-DII reconheceram a proteína nativa expressa na superfície de merozoítas de P. vivax, por imunofluorescência. Em conclusão, nossos resultados mostraram que a proteína recombinante o domínio II de PvAMA-1 (DII) foi imunogênico em camundongos BALB/c quando administrado na presença das diferentes formulações de adjuvantes testadas, sugerindo que esse antígeno possa ser utilizado como uma vacina de subunidade contra a malária vivax. / The Apical Membrane Antigen 1 (AMA-1) has been considered a malaria vaccine candidate against asexual blood stages of Plasmodium. Recently, we identified the domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a region highly recognized by IgG antibodies from Brazilian individuals infected by P. vivax. In the present study, we evaluated the immunogenic properties of a bacterial recombinant PvAMA-1 DII. Groups of 6 female BALB/c were immunized four times with 10 µg of recombinant protein in the presence of different adjuvant formulations [Complete/Incomplete Freunds Adjuvant (CFA/IFA), MPL-TDM, TiterMax, Aluminum hydroxide (Alum), Quil A, QS-21, CpG-ODN 1826] separately or in combination (Alum + QS-21 or Alum + CpG-ODN 1826). Our goal was to compare the antibody response (IgM, IgG and IgG subclass) induced by different protocols of immunization aiming at future pre-clinical studies in non-human primates. The IgG antibody titers against PvAMA-1 were determined by ELISA two weeks after each immunizing dose. The presence of IgM and IgG subclass were evaluated after the end of immunizations schedule. We found that the recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of all adjuvant tested. High titers of IgG1, IgG2a and IgG2b were observed in all groups (except for Alum adjuvant), suggesting a mixed Th1/Th2 response. Finally, we demonstrated that monoclonal and polyclonal antibodies against DII recognized the native protein expressed on the P. vivax merozoite surface parasites by immunofluorescence. Together, our data demonstrated that the recombinant PvAMA-1(DII) was immunogenic in mice when administered in different adjuvant formulations, suggesting that this protein can be used as part of a sub-unit vaccine against malaria vivax.

O Antígeno 1 de Membrana Apical (AMA-1) tem sido sugerido como candidato a compor uma vacina contra estágios assexuados sanguíneos de Plasmodium. Recentemente nosso grupo identificou o domínio II (DII) de AMA-1 de Plasmodium vivax (PvAMA-1) como uma região altamente reconhecida por anticorpos IgG de indivíduos brasileiros infectados por P. vivax. No presente estudo avaliamos as propriedades imunogênicas da proteína recombinante DII, produzida a partir de Escherichia coli. Grupos de 6 camundongos fêmeas BALB/c foram imunizados quatro vezes com 10 µg dessa proteína na presença de diferentes formulações de adjuvantes [Adjuvante Completo/Incompleto de Freund (ACF/AIF), MPL-TDM, TiterMax, Hidróxido de Alumínio (Alum), Quil A, QS-21 e CpG-ODN 1826], individualmente, ou em combinação (Alum + QS-21 ou Alum + CpG-ODN 1826)). Nosso objetivo foi avaliar comparativamente a resposta de anticorpos (IgM, IgG e isotipos de IgG), induzida pelos diferentes esquemas de imunizações, visando futuros estudos pré-clínicos em primatas não humanos. Os títulos de anticorpos IgG contra (o ectodomínio) PvAMA-1 foram determinados por ELISA, duas semanas após cada imunização. A presença de IgM e dos isotipos de IgG também foi avaliada após o final do esquema de imunizações. Nossos resultados demonstraram que a proteína recombinante DII foi altamente imunogênica em camundongos BALB/c quando administrada na presença dos adjuvantes testados. Altos títulos de IgG1, IgG2a e IgG2b foram observados na maioria dos grupos (com exceção do adjuvante Alum), sugerindo uma resposta mista Th1/Th2. Finalmente, demonstramos que anticorpos monoclonais e policlonais anti-DII reconheceram a proteína nativa expressa na superfície de merozoítas de P. vivax, por imunofluorescência. Em conclusão, nossos resultados mostraram que a proteína recombinante o domínio II de PvAMA-1 (DII) foi imunogênico em camundongos BALB/c quando administrado na presença das diferentes formulações de adjuvantes testadas, sugerindo que esse antígeno possa ser utilizado como uma vacina de subunidade contra a malária vivax. / The Apical Membrane Antigen 1 (AMA-1) has been considered a malaria vaccine candidate against asexual blood stages of Plasmodium. Recently, we identified the domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a region highly recognized by IgG antibodies from Brazilian individuals infected by P. vivax. In the present study, we evaluated the immunogenic properties of a bacterial recombinant PvAMA-1 DII. Groups of 6 female BALB/c were immunized four times with 10 µg of recombinant protein in the presence of different adjuvant formulations [Complete/Incomplete Freunds Adjuvant (CFA/IFA), MPL-TDM, TiterMax, Aluminum hydroxide (Alum), Quil A, QS-21, CpG-ODN 1826] separately or in combination (Alum + QS-21 or Alum + CpG-ODN 1826). Our goal was to compare the antibody response (IgM, IgG and IgG subclass) induced by different protocols of immunization aiming at future pre-clinical studies in non-human primates. The IgG antibody titers against PvAMA-1 were determined by ELISA two weeks after each immunizing dose. The presence of IgM and IgG subclass were evaluated after the end of immunizations schedule. We found that the recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of all adjuvant tested. High titers of IgG1, IgG2a and IgG2b were observed in all groups (except for Alum adjuvant), suggesting a mixed Th1/Th2 response. Finally, we demonstrated that monoclonal and polyclonal antibodies against DII recognized the native protein expressed on the P. vivax merozoite surface parasites by immunofluorescence. Together, our data demonstrated that the recombinant PvAMA-1(DII) was immunogenic in mice when administered in different adjuvant formulations, suggesting that this protein can be used as part of a sub-unit vaccine against malaria vivax.

Malaria is a global pandemic that affects millions of people each year. It is a parasitic infection caused by the Plasmodium family, with Plasmodium falciparum being the most virulent strain. Malaria is transmitted to humans by the female Anopheles mosquito. The parasite undergoes two different cycles of its life cycle within the human host: the liver and intraerythrocytic life cycle. The latter consists of an asexual and sexual cycle. The intraerythrocytic cycle is perhaps the most important stage of the parasite's life cycle as it promotes the spread of the disease within and between hosts. The focus of this investigation was aimed at the invasion process of the merozoites into the erythrocytes. The Plasmodium merozoite utilises a cascade of proteins during the erythrocyte invasion process, which is a swift action that takes place in approximately 30 seconds. A number of surface proteins are expressed during merozoite development and are distributed along the merozoite surfaces to assist with attachment and invasion, the most crucial being MSP-1, AMA-1 and RON-2. MSP-1 and AMA-1 are vital targets for the development of malaria vaccines. AMA-1 is the central target protein of this investigation as it plays an essential role in the invasion process. AMA-1 commits the merozoite to invade the erythrocyte, as it assists the RON proteins in the formation of an irreversible tight-junction with the membrane of the erythrocyte. Antibodies, specific to AMA-1, bind to the protein, which prevents the formation of the tight junction and inhibits the invasion of the merozoite into the erythrocyte, therefore preventing the spread of the disease. However, before invasion, AMA-1 undergoes a number of proteolytic processes. It is synthesized as an 83 kDa (AMA-183) precursor protein in the apical organelle of the merozoite. This is then cleaved at the N-terminus to give rise to a 66 kDa (AMA-166) fragment, which is secreted onto the surface of the merozoite. The AMA-166 fragment is then cleaved into either a 48 kDa (AMA-148) or 44 kDa (AMA-144) fragment. One of these three fragments is then used by the merozoite for erythrocyte invasion. The aim of this investigation was to isolate and characterise each of the fragments of the Plasmodium falciparum AMA-1 (PfAMA-1) protein using the 3D7 lab strain of P. falciparum and to visualise the merozoite-erythrocyte invasion process, to possibly identify which of the AMA-1 fragments are involved in the invasion process. In order to achieve this large clusters of merozoites from sorbitol-synchronised cultures were isolated. Schizonts were isolated from culture by magnetic separation and incubated with E64 to prevent the release of merozoites. Merozoites that were required for the isolation of PfAMA-1 were harvested from the schizonts by saponin lysis, then homogenised, separated by SDS-PAGE and digested for LC-MS/MS analysis. Merozoites that were required for the visualisation procedures were not incubated with E64, to allow natural egression from the erythrocyte. The transmission electron microscopy results produced clear images of the merozoiteerythrocyte invasion process and the positioning of PfAMA-1 on the merozoite, before and after schizont rupture, was visualised from results obtained from confocal microscopy. Then PfAMA-1 was identified in isolated merozoite samples by LC-MS/MS analysis. However, due to its low abundance, isolation of high enough concentrations of PfAMA-1 to characterise its different fragments was not achieved. Further investigation into the development of the culturing and isolating methods could help in future projects aimed at isolating higher concentrations of merozoite proteins from synchronised cultures with a lower merozoite egression window period, in order to accomplish detailed analysis on invading proteins for the future development of treatments against malaria. / Dissertation (MSc)--University of Pretoria, 2016. / Pharmacology / MSc / Unrestricted

O Antígeno 1 de Membrana Apical (AMA-1) de Plasmodium sp tem sido sugerido como candidato a compor uma vacina contra a malária. No presente estudo geramos cinco proteínas recombinantes baseadas em diferentes regiões do ectodomínio de AMA-1 de Plasmodium vivax, o qual compreende os domínios I a III, com intuito de mapear regiões particularmente imunogênicas da proteína. Cada uma das cinco proteínas recombinantes foi expressa em Eschericha coli a partir do vetor pET-28a em fusão com a cauda de histidina e purificadas por cromatografia de afinidade. As diferentes proteínas recombinantes foram comparadas, por ELISA, quanto ao reconhecimento por anticorpos IgM, IgG e subclasses de IgG de 100 indivíduos infectados por P. vivax procedentes de áreas endêmicas do Estado do Pará e 32 indivíduos não infectados que relataram terem sido acometidos de mais de 10 episódios prévios de malária procedentes do município de Terra Nova do Norte (MT). As freqüências de indivíduos que apresentaram anticorpos IgM foram mais baixas e variaram de 4% (DIII) a 36% (DII-III). Por outro lado, as freqüências de indivíduos que apresentaram anticorpos IgG para DI, DII, DIII, DI-II e DII-III foram 13%, 65%, 12%, 59% e 58%, respectivamente. Podemos observar que as proteínas recombinantes contendo o DII foram particularmente imunogênicas durante a infecção natural. Com o objetivo de avaliar se os epítopos reconhecidos nas cinco proteínas baseadas nos diferentes domínios estão expostos na proteína recombinante correspondente ao ectodomínio (DI-III) gerada previamente, realizamos ensaios de inibição por ELISA utilizando placas sensibilizadas com a proteína DI-III. Nossos resultados sugerem a presença de um maior número de epítopos comuns entre as proteínas recombinantes baseadas nos domínios I-II e ectodomínio de AMA-1. Além disso, observamos que a proporção de indivíduos que apresentaram anticorpos contra DII, DI-II e DII-III aumentou de acordo com o maior número de exposições prévias ao P. vivax. As subclasses de IgG que predominaram contra todas as proteínas foram IgG1, IgG3 e IgG4. Em conjunto, nossos resultados sugerem que as proteínas recombinantes contendo o DII podem ser exploradas em futuros estudos de indução de imunidade protetora contra malária vivax em primatas não-humanos. / The Apical Membrane Antigen 1 (AMA-1) of Plasmodium sp has been suggested as a vaccine candidate against malaria. Herein, to identify novel antigenic epitopes on the Plasmodium vivax AMA-1 ectodomain, we have generated five recombinant proteins, comprising domains I to III. All recombinant proteins were expressed in Escherichia coli using the pET-28a vector system fused to hexahistidine tag for purification by affinity chromatography. Recognition of recombinant proteins by antibodies was evaluated using a panel of sera collected from onehundred P. vivax -infected patients resident in the State of Pará and from thirty-two non-infected individuals, living in the State of Mato Grosso and who have faced a minimum of ten malaria episodes. ELISA analyses demonstrated that protein recognition was highly dependent on IgG antibodies, raging from 13%, 65%, 12%, 59% up to 58%, respectively for DI, DII, DIII, DI-II and DII-III domains. Indeed, we have noticed a lower frequency of recognition, ranging from 4% (DIII) to 36% (DII-III), by sera from those individuals that presented IgM antibodies. Collectively, these data suggest that the DII domain is particularly immunogenic during natural infections. Next, to verify whether the epitopes recognized in these five different recombinant proteins were also expressed in a recombinant protein spanning domains I through III (DI-III), we carried out ELISA inhibition assays using plates coated with the DI-III recombinant protein. Our findings revealed the presence of a higher number of common epitopes among recombinant proteins based on domains I-II and the AMA-1 ectodomain. Moreover, we observed that the proportion of individuals who had presented antibodies against DII, DI-II and DII-III domains increased according to the previous number of P. vivax episodes. Overall, IgG1, IgG3 and IgG4 antibodies were prevalent to all proteins. Taken together, our results demonstrated that DII domain is highly recognized, mainly by IgG antibodies; and open promising perspectives to use this region as an experimental vaccine in non-human primates capable to induce protective immunity against vivax malaria.

O Antígeno-1 de Membrana Apical (AMA-1) de merozoítas de Plasmodium é um dos principais candidatos a compor uma vacina contra a malária. A função biológica de AMA-1 não é totalmente conhecida, entretanto, existem evidências que sugerem a participação dessa proteína na ligação a eritrócitos de diferentes espécies de Plasmodium. O objetivo deste estudo foi investigar a participação de AMA-1 de P. vivax (PvAMA-1) na ligação a eritrócitos humanos utilizando células COS-7 transfectadas com plasmídios recombinantes codificando diferentes regiões do ectodomínio de PvAMA-1. Para isso, os genes que codificam os domínios I-II ou III de PvAMA-1 foram inseridos no vetor pDisplay-EGFP, que permite a expressão das proteínas recombinantes em fusão com a porção N-terminal da Proteína Fluorescente Verde (GFP). Em paralelo, utilizamos construções contendo os genes que codificam a região C-terminal de 19 kDa da Proteína 1 de Superfície do Merozoíta (PvMSP119) e a região II da Duffy Binding Protein (PvDBP-RII). As quatro construções foram utilizadas para transfectar células COS-7 na presença de lipofectamina. A eficiência das transfecções transientes foi confirmada por imunofluorescência utilizando anticorpos específicos. Em seguida, estudamos a participação dos diferentes domínios de PvAMA-1 na ligação aos eritrócitos. Nossos resultados mostraram que os domínios contíguos I-II, ao contrário do domínio III, foram capazes de se ligar a eritrócitos in vitro. Essa ligação foi específica, pois soros de indivíduos infectados por P. vivax e soros policlonais de camundongos contendo anticorpos anti-PvAMA-1 foram capazes de inibir essa ligação em 82,0% e 79,8%, respectivamente. Além disso, anticorpos monoclonais dirigidos contra o domínio II dessa proteína foram capazes de inibir parcialmente essa ligação. Após o tratamento de eritrócitos com tripsina ou quimiotripsina, estas células perderam grande parte de sua capacidade de ligação, sugerindo que o receptor para PvAMA-1 tenha constituição predominantemente protéica. Em conjunto, nossos resultados podem servir de base para futuros estudos visando um melhor entendimento da função de anticorpos gerados durante a infecção natural ou induzidos após vacinação com PvAMA-1. / The Apical Membrane Antigen (AMA-1) of Plasmodium merozoites is one of the main candidates to be part of a vaccine against malaria. The biological function of AMA-1 is unknown. However, there are evidences that suggest the participation of this protein in the interaction with erythrocytes (RBC) of different Plasmodium species. Using transfected COS-7 cells with recombinant plasmids encoding different portions of the PvAMA-1 ectodomain, our aim was to identify possible domains of PvAMA-1 able to interact with human RBC. The genes that encoded domains I and II in combination or domain III of PvAMA-1 were cloned into the pDisplay-EGFP vector. This vector allows expression of the protein fused to the N-terminus of enhanced green fluorescent protein (GFP). In parallel, we also used constructions containing the genes that encoded the 19 kDa C-terminal region of Merozoite Surface Protein 1 (PvMSP119) and region II of the Duffy Binding Protein (PvDBP-RII). Constructions were used to transiently transfect COS-7 cells. The efficiency of expression of all constructs was confirmed by immunofluorescence assay using specific antibodies. After that, we studied the participation of the different domains of PvAMA-1 in the binding to human RBC. We found that COS-7 cells expressing domains I-II, but not domain III, bound to human RBC in vitro. This binding was specific, because sera from malaria-infected patients and mouse polyclonal sera containing antibodies to PvAMA-1 were able to block the adhesion by 82.0% and 79.8%, respectively. Moreover, monoclonal antibodies directed against domain II were partially inhibitory in the cytoadherence assays. The receptor recognized on the surface of COS-7 cells expressing the domains I and II was partially removed after human RBC were treated with trypsin or chymotrypsin, suggesting that its composition is predominantly protein. In conclusion, our results can be used as basis for future studies aimed at better understating the function of the antibodies generated during the natural infection or after vaccination with PVAMA-1.

O Antígeno 1 de Membrana Apical (AMA-1) de Plasmodium sp tem sido sugerido como candidato a compor uma vacina contra a malária. No presente estudo geramos cinco proteínas recombinantes baseadas em diferentes regiões do ectodomínio de AMA-1 de Plasmodium vivax, o qual compreende os domínios I a III, com intuito de mapear regiões particularmente imunogênicas da proteína. Cada uma das cinco proteínas recombinantes foi expressa em Eschericha coli a partir do vetor pET-28a em fusão com a cauda de histidina e purificadas por cromatografia de afinidade. As diferentes proteínas recombinantes foram comparadas, por ELISA, quanto ao reconhecimento por anticorpos IgM, IgG e subclasses de IgG de 100 indivíduos infectados por P. vivax procedentes de áreas endêmicas do Estado do Pará e 32 indivíduos não infectados que relataram terem sido acometidos de mais de 10 episódios prévios de malária procedentes do município de Terra Nova do Norte (MT). As freqüências de indivíduos que apresentaram anticorpos IgM foram mais baixas e variaram de 4% (DIII) a 36% (DII-III). Por outro lado, as freqüências de indivíduos que apresentaram anticorpos IgG para DI, DII, DIII, DI-II e DII-III foram 13%, 65%, 12%, 59% e 58%, respectivamente. Podemos observar que as proteínas recombinantes contendo o DII foram particularmente imunogênicas durante a infecção natural. Com o objetivo de avaliar se os epítopos reconhecidos nas cinco proteínas baseadas nos diferentes domínios estão expostos na proteína recombinante correspondente ao ectodomínio (DI-III) gerada previamente, realizamos ensaios de inibição por ELISA utilizando placas sensibilizadas com a proteína DI-III. Nossos resultados sugerem a presença de um maior número de epítopos comuns entre as proteínas recombinantes baseadas nos domínios I-II e ectodomínio de AMA-1. Além disso, observamos que a proporção de indivíduos que apresentaram anticorpos contra DII, DI-II e DII-III aumentou de acordo com o maior número de exposições prévias ao P. vivax. As subclasses de IgG que predominaram contra todas as proteínas foram IgG1, IgG3 e IgG4. Em conjunto, nossos resultados sugerem que as proteínas recombinantes contendo o DII podem ser exploradas em futuros estudos de indução de imunidade protetora contra malária vivax em primatas não-humanos. / The Apical Membrane Antigen 1 (AMA-1) of Plasmodium sp has been suggested as a vaccine candidate against malaria. Herein, to identify novel antigenic epitopes on the Plasmodium vivax AMA-1 ectodomain, we have generated five recombinant proteins, comprising domains I to III. All recombinant proteins were expressed in Escherichia coli using the pET-28a vector system fused to hexahistidine tag for purification by affinity chromatography. Recognition of recombinant proteins by antibodies was evaluated using a panel of sera collected from onehundred P. vivax -infected patients resident in the State of Pará and from thirty-two non-infected individuals, living in the State of Mato Grosso and who have faced a minimum of ten malaria episodes. ELISA analyses demonstrated that protein recognition was highly dependent on IgG antibodies, raging from 13%, 65%, 12%, 59% up to 58%, respectively for DI, DII, DIII, DI-II and DII-III domains. Indeed, we have noticed a lower frequency of recognition, ranging from 4% (DIII) to 36% (DII-III), by sera from those individuals that presented IgM antibodies. Collectively, these data suggest that the DII domain is particularly immunogenic during natural infections. Next, to verify whether the epitopes recognized in these five different recombinant proteins were also expressed in a recombinant protein spanning domains I through III (DI-III), we carried out ELISA inhibition assays using plates coated with the DI-III recombinant protein. Our findings revealed the presence of a higher number of common epitopes among recombinant proteins based on domains I-II and the AMA-1 ectodomain. Moreover, we observed that the proportion of individuals who had presented antibodies against DII, DI-II and DII-III domains increased according to the previous number of P. vivax episodes. Overall, IgG1, IgG3 and IgG4 antibodies were prevalent to all proteins. Taken together, our results demonstrated that DII domain is highly recognized, mainly by IgG antibodies; and open promising perspectives to use this region as an experimental vaccine in non-human primates capable to induce protective immunity against vivax malaria.

O Antígeno-1 de Membrana Apical (AMA-1) de merozoítas de Plasmodium é um dos principais candidatos a compor uma vacina contra a malária. A função biológica de AMA-1 não é totalmente conhecida, entretanto, existem evidências que sugerem a participação dessa proteína na ligação a eritrócitos de diferentes espécies de Plasmodium. O objetivo deste estudo foi investigar a participação de AMA-1 de P. vivax (PvAMA-1) na ligação a eritrócitos humanos utilizando células COS-7 transfectadas com plasmídios recombinantes codificando diferentes regiões do ectodomínio de PvAMA-1. Para isso, os genes que codificam os domínios I-II ou III de PvAMA-1 foram inseridos no vetor pDisplay-EGFP, que permite a expressão das proteínas recombinantes em fusão com a porção N-terminal da Proteína Fluorescente Verde (GFP). Em paralelo, utilizamos construções contendo os genes que codificam a região C-terminal de 19 kDa da Proteína 1 de Superfície do Merozoíta (PvMSP119) e a região II da Duffy Binding Protein (PvDBP-RII). As quatro construções foram utilizadas para transfectar células COS-7 na presença de lipofectamina. A eficiência das transfecções transientes foi confirmada por imunofluorescência utilizando anticorpos específicos. Em seguida, estudamos a participação dos diferentes domínios de PvAMA-1 na ligação aos eritrócitos. Nossos resultados mostraram que os domínios contíguos I-II, ao contrário do domínio III, foram capazes de se ligar a eritrócitos in vitro. Essa ligação foi específica, pois soros de indivíduos infectados por P. vivax e soros policlonais de camundongos contendo anticorpos anti-PvAMA-1 foram capazes de inibir essa ligação em 82,0% e 79,8%, respectivamente. Além disso, anticorpos monoclonais dirigidos contra o domínio II dessa proteína foram capazes de inibir parcialmente essa ligação. Após o tratamento de eritrócitos com tripsina ou quimiotripsina, estas células perderam grande parte de sua capacidade de ligação, sugerindo que o receptor para PvAMA-1 tenha constituição predominantemente protéica. Em conjunto, nossos resultados podem servir de base para futuros estudos visando um melhor entendimento da função de anticorpos gerados durante a infecção natural ou induzidos após vacinação com PvAMA-1. / The Apical Membrane Antigen (AMA-1) of Plasmodium merozoites is one of the main candidates to be part of a vaccine against malaria. The biological function of AMA-1 is unknown. However, there are evidences that suggest the participation of this protein in the interaction with erythrocytes (RBC) of different Plasmodium species. Using transfected COS-7 cells with recombinant plasmids encoding different portions of the PvAMA-1 ectodomain, our aim was to identify possible domains of PvAMA-1 able to interact with human RBC. The genes that encoded domains I and II in combination or domain III of PvAMA-1 were cloned into the pDisplay-EGFP vector. This vector allows expression of the protein fused to the N-terminus of enhanced green fluorescent protein (GFP). In parallel, we also used constructions containing the genes that encoded the 19 kDa C-terminal region of Merozoite Surface Protein 1 (PvMSP119) and region II of the Duffy Binding Protein (PvDBP-RII). Constructions were used to transiently transfect COS-7 cells. The efficiency of expression of all constructs was confirmed by immunofluorescence assay using specific antibodies. After that, we studied the participation of the different domains of PvAMA-1 in the binding to human RBC. We found that COS-7 cells expressing domains I-II, but not domain III, bound to human RBC in vitro. This binding was specific, because sera from malaria-infected patients and mouse polyclonal sera containing antibodies to PvAMA-1 were able to block the adhesion by 82.0% and 79.8%, respectively. Moreover, monoclonal antibodies directed against domain II were partially inhibitory in the cytoadherence assays. The receptor recognized on the surface of COS-7 cells expressing the domains I and II was partially removed after human RBC were treated with trypsin or chymotrypsin, suggesting that its composition is predominantly protein. In conclusion, our results can be used as basis for future studies aimed at better understating the function of the antibodies generated during the natural infection or after vaccination with PVAMA-1.

Babesia microti is a tickborne hemoprotozoan parasite that causes the disease babesiosis in humans. Babesia microti Apical Membrane Antigen-1 (AMA-1) is a micronemal protein suspected to play a role in erythrocyte invasion. To investigate interaction between AMA-1 and the host cell, the ectodomain region of the B. microti ama-1 gene was cloned into an expression vector, expressed as a histidine-tagged fusion protein, and used to probe red blood cell membrane proteins in far Western blot assays. The B. microti ama-1 ectodomain, which excludes the signal peptide and the transmembrane region of the open reading frame, was amplified from a cloned gene sequence. The AMA-1 ectodomain is a membrane bound polypeptide that extends into the extracellular space and is most likely to interact or initiate interaction with the host red blood cell surface receptor(s). The amplicon was ligated into a protein expression vector to produce a 58.1 kDa recombinant His-tagged fusion protein, which was confirmed by Western blot analysis. The recombinant B. microti AMA-1 fusion protein was enriched on nickel affinity columns and then used to probe mouse, human and horse red blood cell membrane proteins in far Western blot assays. Babesia microti AMA-1 consistently reacted strongly with a protein migrating at 49 kDa. A similar reaction occurred between the B. microti AMA-1 and horse red blood cell membrane proteins, suggesting that similar interacting proteins of this size are shared by red blood cells from the three species. The B. microti AMA-1 may bind to red blood cell membrane sialic-acid groups, as shown for other Babesia spp. This may explain the signal at the 49 kDa position observed between B. microti AMA-1 and red blood cell membrane proteins from three different species. Further studies may determine if the binding epitopes of the red blood cell binding partner at this position vary and contribute to the specificity of each parasite AMA-1 for their respective host cells.