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Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Malaria parasites are responsible for an increase in the morbidity and mortality in several tropical regions in Southern Africa. In this thesis, research was undertaken on Plasmodium, which is responsible for more than 95% of these cases. Although pharmacological prophylaxis is available, a worldwide resistance against existing drugs have been encountered. A study on chloroquineresistance in North-Eastern Transvaal in 1988, indicated that 11% of the strains in this area were resistant against chloroquine. Only 78% of these parasites were sensitive to the new drug, Mefloquine, which could serve as a substitute in chloroquine-resistant infections. Furthermore, no strain was found to be resistant or insensitive to both anti-malarial drugs. Malaria parasites can be obtained for research purposes from long term in vitro cultures. The initiation of cultures of some wild isolates of P.falciparum appears to be problematic at certain levels above sea-level. The gas composition of the medium was identified as a probable cause due to its dependence on the partial pressures of gasses at different heights above sealevel. The toxic effect of a too high oxygen concentration on the parasite, caused by the lower atmospheric pressure of the Highveld area, was prevented after the concentration of dissolved carbon dioxide in the medium was increased through equilibration with the special gas mixture. Possible shortcomings in the long-term culture method for malaria parasites that could retard optimum growth, were also investigated. Local parasite isolates could be supported on medium enriched with bovine serum although the growth rate was lower than when human serum was used. By increasing the frequency of medium replacement with progressing parasitemia, less stress were placed on the system since parasites was exposed for shorter periods and to lower concentrations of byproducts such as lactic acid. In addition, the ATP-concentration in infected cultures decreased by nearly 50 % over a growth period of 4 days. The stress in the host cell was reflected by the decrease in the total adenyl-nucleotide pool and the increase in AMP-concentration. An increase in the intracellular IMP-concentration indicated that the purine salvage pathway was inhibited which may explain the decrease in the ATP-concentration. It therefore appears that the regular replacement of medium and replenishment of erythrocytes only partially contribute to the successful establishment of malaria cultures. More research is necessary to identify the factors that are responsible for the inhibition of the purine salvage pathway. The stress placed on the human body by the parasite is complicated by the thrombocytopenia observed in some infections. Increases in the concentrations of thrombocyte-associated immunoglobulin G and M which are a characteristic for this condition, can be determined by a modified microo-method developed in our laboratory. By monitoring the parasite-infected patient over a period of time with the micro-method, the increase in thrombocytes and decrease in thrombocyte-associated antibodies were correlated with the recuperation of the patient. This method does not destroy the thrombocytes, thereby allowing displacement studies to be undertaken with purified parasite antigens of synthetic peptides. An investigation of parasite-infected erythrocytes by means of light microscopy, transmission and scanning electronmicroscopy, indicated that the local isolate could be composed of a mixture of strains, of which some have the ability to induce knobs on the erythrocyte. Furthermore, the investigation illustrated that fast fixation with gluteraldehyde is superior to slow fixation when transmission electronmicroscopy is performed. However, no difference could be observed when scanning electron microscopy was performed on infected erythrocytes that had been fixed by either of these methods. AFRIKAANS : Malaria parasiete is verantwoordelik vir 'n toenemende morbiditeit sowel as mortaliteit in verskeie tropiese streke in Suider-Afrika waarvan meer as 95 % van die gevalle deur Plasmodium falciparum veroorsaak word. Navorsing in die tesis is gevolglik op hierdie parasiet toegespits. Ten spyte daarvan dat far'makologiese profilakse toegepas word, kan malaria nog steeds opgedoen word as gevolg van n wereldwye weerstandigheid teen bestaande geneesmiddels. 'n Ondersoek van die klorokien-weerstandigheidstatus in die Noord-Oostelike Transvaal het in 1988 getoon dat 11 % van die stamme in die area weerstandig is teenoor klorokien. Slegs 78% van die parasiete was sensitief vir die nuwe middel, Meflokien, wat as moontlike plaasvervanger in klorokien-weerstandige parasietinfeksies gebruik kan word. Geen parasietstam was egter weerstandig of onsensitief teen beide middels nie. Langtermyn kultuurkweking van malaria parasiete is essensieël vir die verkryging van uitgangsmateriaal vir navorsing op die parasiet. Dit blyk egter dat inisiasie van kulture van wilde stamme van P. falciparum problematies by sekere hoogtes bo seespieël is. Die gassamestelling van die medium is as 'n moontlike oorsaak gëidentifiseer aangesien dit afhanklik is van die parsiële drukke van gasse by verskillende hoogtes bo seespieël. Die toksiese effek van te hoë suurstofkonsentrasies op die parasiet a.g.v. die laer atmosferiese druk op die Hoëveldstreek, is oorkom deur die konsentrasie van opgeloste koolsuurgas in die medium te verhoog deur vooraf 'n spesiale gasmengsel daardeur te borrel. Moontlike tekortkominge in die langtermyn kultuur metode vir malaria parasiete wat optimale groei kan belemmer, is ook ondersoek. Plaaslike parasietstamme kon op medium wat verryk is met beesserum onderhou word, alhoewel die groeitempo laer is as wanneer mensserum gebruik word. 'n Toenemende tempo van mediumvervanging soos die parasitemia in kulture toeneem, het getoon dat minder stres op die sisteem geplaas word deurdat die parasiete vir 'n korter periode en aan laer konsentrasies van byprodukte soos melksuur blootgestel word. Die ATP-konsentrasies in gëinfekteerde kulture daal egter met ongeveer die helfte oor 'n groeiperiode van 4 dae. Tesame hiermee, is daar ook 'n daling in die totale adeniel-nukleotied poel van die gëinfekteerde rooibloedsel en 'n styging in die AMP-konsentrasie wat die stres in die gasheersel weerspieël. 'n Verhoging in die intrasellulêre IMP-konsentrasie dui op 'n moontlike inhibisie van die purienherwinningspadweg wat die verlaagde ATP-konsentrasie mag verklaar. Uit die studie blyk dit dus of meer gereelde vervanging van medium en toevoeging van rooibloedselle slegs 'n gedeeltelike bydrae maak tot die suksesvolle vestiging van malariakulture. Die identifikasie van die faktore wat lei tot die inhibisie van die purienherwinningspadweg, verg nog verdere ondersoeke. Die stres wat die parasiet in die menslike liggaam veroorsaak, word ook weerspieël deur onderandere die trombositopenie wat in sommige infeksies waargeneem word. Verhogings in die konsentrasie van trombosiet-geassosieërde immunoglobuliene G en M wat kenmerkend is van die toestand, kan gemeet word deur 'n gemodifiseerde mikrometode wat in ons laboratoriums ontwikkel is. Deur die parasiet-gëinfekteerde pasiët oor 'n paar dae met die mikrometode te monitor, kon die verhoging in trombosiete en afname in trombosiet-geassosieërde antiliggame met herstel van die pasiënt gekorreleer word. Die metode veroorsaak geen skade aan die trombosiete nie sodat antigeenverplasingstudies ook na die tyd gedoen kan word. In 'n ondersoek van die parasiet met behulp van ligmikroskopie, deurstraal- sowel as skandeer-elektronmikroskopie, is gevind dat die plaaslike isolaat moontlik uit 'n mengsel van stamme bestaan, waarvan sommige die vermoë het om knoppe op die rooibloedsel te induseer. Verder het die ondersoek getoon dat indien deurstraal-elektronmikroskopie gebruik word, 'n vinnige fikseringsmetode met gluteraldehied beter is as 'n stadige fikseringsmetode. In vergelyking hiermee kon geen verskil waargeneem word met skandeer-elektronmikroskopie van geparasiteerde rooibloedselle wat met enige van hierdie metodes gefikseer is nie. Copyright / Dissertation (MSc)--University of Pretoria, 1992. / Biochemistry / unrestricted

O Antígeno 1 de Membrana Apical (AMA-1) é um dos mais promissores candidatos a vacina contra a fase sanguínea da malária. No presente estudo, geramos uma nova proteína recombinante baseada no ectodomínio de AMA-1 de P. vivax (PvAMA-1) a partir do gene sintético com códon otimizado para expressão secretada na levedura Pichia pastoris. Por ELISA, a proteína PvAMA-1 foi reconhecida por 62,5% dos soros de pacientes da Amazônia brasileira infectados por P. vivax. A imunogenicidade de PvAMA-1 foi avaliada em camundongos BALB/c, utilizando protocolos homólogos e heterólogos de indução e reforço com DNA plasmidial e/ou proteína recombinante, emulsificada em Adjuvante de Freund. Os resultados mostraram que a eficiência da imunização é dependente da presença da proteína emulsificada em adjuvante forte. As imunizações subseqüentes foram realizadas com a proteína recombinante emulsificada em diferentes adjuvantes: sais de alumínio (Alum), Monophosphoryl Lipid A (MPLA) de Bordetella pertussis, flagelina FliC de Salmonella Typhimurium, saponina Quil A e Adjuvante Incompleto de Freund (AIF). Os resultados demonstraram que as imunizações na presença de Quil A e AIF foram capazes de induzir altos títulos de anticorpos IgG e uma resposta de isotipos de IgG mais balanceada, caracterizando um padrão do tipo Th1/Th2. Títulos de anticorpos mais baixos, mas também expressivos, foram obtidos na presença de Alum, MPLA, Alum + MPLA e Alum + FliC. A análise da resposta proliferativa, por citometria de fluxo utilizando marcação com CFSE, mostrou que células esplênicas CD3+CD4+, assim como CD3+CD8+, de animais imunizados com PvAMA-1 na presença dos adjuvantes Alum, Alum + MPLA, Quil A e AIF foram capazes de proliferar especificamente in vitro. Por imunofluorescência, soros policlonais de camundongos imunizados com o antígeno na presença de MPLA e Quil A foram capazes de reconhecer a proteína nativa expressa em isolados de P. Vivax da Ásia. Visando futuros testes pré clínicos em primatas não humanos, a proteína PvAMA-1 foi produzida em boas práticas de laboratório (BPL) por uma companhia especializada e o potencial imunogênico da proteína foi confirmado em imunizações posteriores. Em conjunto, nossos resultados demonstram que PvAMA-1 é um antígeno promissor para ser explorado em protocolos de imunização contra malária vivax, individualmente, ou em combinação com outros antígenos. / The Apical Membrane Antigen 1 (AMA-1) is one of the most promissing vaccine candidates against the erythrocytic stage of malaria. In the present study we generated a new recombinant protein based on AMA-1 ectodomain of P. vivax (PvAMA-1) using a synthetic codon optimized gene for yeast secreted expression. By ELISA, the protein PvAMA-1 was recognized by 62,5% of the sera from Brazilian Amazonia patients infected by P. vivax. The immunogenicity of PvAMA-1 was evaluated in BALB/c mice using homologous and heterologous protocols of prime and boost with plasmidial DNA and/or recombinant protein emulsified in Freund adjuvant. The results showed that the efficiency of immunization is dependent on the presence of a strong adjuvant. The following immunizations were conducted using the protein emulsified in different adjuvants: aluminium salts (Alum), Monophosphoryl Lipid A (MPLA) of Bordetella pertussis, flagelin FliC of Salmonella Typhimurium, saponin Quil A and Incomplete Freund\'s Adjuvant (IFA). The results showed that immunizations in the presence of Quil A e IFA were able to induce high IgG titers and a more balanced IgG isotypes response, featuring a standard type Th1/Th2. Lower antibody titers, but also significant, were obtained in the presence of Alum, MPLA, Alum + MPLA and Alum + FliC. The proliferative cellular analysis, by flow cytometry using CFSE staining, showed that splenic cells CD3+CD4+, as well as CD3+CD8+ from immunized mice that received PvAMA-1 with Alum, Alum + MPLA, Quil A and IFA were specifically able to proliferate in vitro. By immunofluorescence, the polyclonal sera from mice immunized with the antigen in the presence of MPLA and Quil A were able to recognize native protein expressed in Asian P. vivax isolates. Aiming future pre-clinical assays in non-human primates, the protein PvAMA-1 was produced in good laboratory practices (GLP) conditions by a specialized company and its immunogenic efficiency was confirmed in later immunizations. Altogether, our results showed that PvAMA-1 is a promissor antigen to be explored in immunization protocols against vivax malaria, individually, or combined with other antigens.

Nos últimos anos, estudamos vários aspectos da resposta imune naturalmente adquirida em indivíduos de diferentes áreas endêmicas da Região Amazônica expostos à malária. Para isso, utilizamos proteínas recombinantes baseadas em antígenos de formas sanguíneas de P. vivax, os quais têm sido considerados candidatos à vacina contra a malária vivax. No presente trabalho, nossos estudos imunoepidemiológicos concentraram-se em 396 indivíduos de uma comunidade rural da Amazônia ocidental brasileira, localizada no Estado do Acre, com o objetivo de realizar um estudo transversal e longitudinal da resposta de anticorpos contra um painel de proteínas recombinantes derivadas de merozoítos de P. vivax (MSP119, AMA-1, MSP3α e MSP3β). Para isso, os soros desses indivíduos foram testados por ELISA quanto ao reconhecimento das quatro proteínas recombinantes. As proporções de indivíduos na linha de base com anticorpos IgG contra MSP119, AMA- 1, MSP3α e MSP3β foram de 59,8%, 50,0%, 23,5% e 26,5%, respectivamente. Dentre esses indivíduos, apenas 10,9% tinham infecções por P. vivax, P. falciparum ou malária mista. No grupo de infectados por P. vivax, a proporção de respondedores foi maior para as proteínas recombinantes MSP119 e AMA-1, atingindo 78,6%, em ambos os casos. A proporção de indivíduos com anticorpos para cada uma das proteínas associou-se com o maior tempo de exposição à malária, exceto para a MSP3β. Além disso, a positividade foi mais alta nas áreas de maior risco de transmissão. Não observamos associações relevantes entre o genótipo do antígeno Duffy dos indivíduos e presença de anticorpos para as proteínas estudadas. No estudo longitudinal, observamos um aumento da prevalência de respondedores durante a parasitemia patente, sendo de 81,9%, 80,9%, 31,9% e 48,9% para a MSP119, AMA-1, MSP3α e MSP3β, respectivamente. Em conclusão, nossos resultados confirmam a alta antigenicidade dessas proteínas, o que pode ser de grande importância para futuros ensaios clínicos na região. / In recent years we studied various aspects of the naturally acquired immune response in individuals from different endemic areas of the Amazon region exposed to malaria. For this purpose we used recombinant proteins based on P. vivax blood stage antigens considered candidates for a vaccine against vivax malaria. In the present study, we focused on 396 individuals from a rural community in western Brazilian Amazon located at the state of Acre. We conducted a transversal and longitudinal study of the antibody response to a panel of recombinant proteins representing P. vivax merozoites surface antigens (MSP119, AMA-1, MSP3α and MSP3β). The sera of these individuals were tested by ELISA for the recognition of the four antigens mentioned above. The proportions of individuals at the baseline with IgG antibodies to MSP119, AMA-1, MSP3α and MSP3β were 59.8%, 50.0%, 23.5% and 26.5%, respectively. Among these individuals, 10.9% had patent malaria infections with either P. vivax or P. falciparum or both. Among individuals with patent P. vivax infection, the frequency of responders was high for MSP119 and AMA-1, (78.6% in both cases). Except in the case of MSP3β, the proportion of individuals with antibodies to each protein correlated with the time of malaria exposure. Also, the positivity was higher in areas of higher transmission levels. No relevant association was found between the Duffy genotypes and presence of antibodies to the different antigen. In longitudinal study, we observed an increased prevalence of responders during patent parasitemia, 81.9% 80.9% 31.9% and 48.9% to MSP119, AMA-1, MSP3α and MSP3β, respectively. In conclusion, our results confirm the high antigenicity of these proteins, which can be of great importance for future clinical trials in the region.

Orientador: Rosangela Zacarias Machado / Coorientador: Silvia Maria Fátima Di Santi / Banca: Solange Maria Gennari / Banca: Karin Werther / Resumo: Os Estados que compõem a Amazônia Legal respondem por 99% dos casos de malária humana registrados no Brasil. O Estado do Maranhão, atualmente, é responsável por 1% dos casos dessa região. Acredita-se que, na Região Amazônica ocorra a transmissão de malária de primatas para humanos, devido a relatos atuais de resultados soroepidemiológicos de humanos e primatas. O presente estudo objetivou realizar o diagnóstico morfológico, sorológico e molecular de Plasmodium spp. em primatas neotropicais na Ilha de São Luís, Estado do Maranhão, Brasil. Para tal, foram utilizadas como técnicas de diagnóstico: a microscopia de luz, a reação em cadeia pela polimerase (PCR) e a imunofluorescência indireta (RIFI). No período de junho de 2009 a abril de 2010, foram amostrados 70 primatas, sendo 50 provenientes do Centro de Triagem Animais Silvestres (CETAS), localizado no município de São Luís, e 20 de vida livre, capturados em reserva particular localizada no município de São José de Ribamar, Estado do Maranhão. Foram avaliadas pela microscopia de luz 140 lâminas (duas de cada animal), das quais cinco (7,1%) foram positivas. Pela RIFI não se detectou anticorpos anti-Plasmodium spp. Pela PCR, dos 70 animais amostrados, foi possível observar produtos amplificados para Plasmodium spp. em 13 amostras, das quais oito (61,54%) eram de animais de vida livre e cinco (38,46%) de CETAS. Pelo pequeno tamanho dos fragmentos dos produtos amplificados, foi necessário cloná-los em vetor pGEM-T Easy para sequenciamento, no entanto, não foram obtidos resultados confirmatórios / Abstract: The states that comprise the Amazon region are incriminated for 99% of registered cases of human malaria in Brazil. Currently, the state of Maranhão is responsible for 1% of cases in Amazonic region. According to recent seroepidemiological studies, the transmission of malaria from primates to humans occurs in Amazon region. Our study aimed to detect Plasmodium spp. in neotropical primate from island of São Luis, Maranhão State, Brazil, using morphological (light microscopy), molecular (Polymerase Chain Reaction [PCR]) and serological (Indirect immunofluorescence Assay [IFA]) techniques. Between June/2009 and April/2010, we sampled 70 primates, 50 from the Wildlife Screening Center (CETA), located in the city of São Luís, and 20 captured in a private reserve located in São José de Ribamar, state of Maranhão. Based on blood smear examinations, five (7.1%) monkeys were positive to Plasmodium spp. All animals showed seronegative to Plasmodium spp. by IFA. Plasmodium spp.-amplicons were detected in 13 animals; eight out of positive monkeys (61.54%) were free-ranging animals and five (38.46%) maintained in capitivity in CETAS. Regarding the small size of amplified fragments, we cloned the amplicons into pGEM-T Easy vector aiming at obtaining a higher sequencing quality; however, we did not reached satisfactory results / Mestre

Made available in DSpace on 2014-06-11T19:27:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-09Bitstream added on 2014-06-13T18:56:57Z : No. of bitstreams: 1 figueiredo_map_me_jabo.pdf: 822824 bytes, checksum: 5e062bcbd66ebcff34c0479b4b807b5d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os Estados que compõem a Amazônia Legal respondem por 99% dos casos de malária humana registrados no Brasil. O Estado do Maranhão, atualmente, é responsável por 1% dos casos dessa região. Acredita-se que, na Região Amazônica ocorra a transmissão de malária de primatas para humanos, devido a relatos atuais de resultados soroepidemiológicos de humanos e primatas. O presente estudo objetivou realizar o diagnóstico morfológico, sorológico e molecular de Plasmodium spp. em primatas neotropicais na Ilha de São Luís, Estado do Maranhão, Brasil. Para tal, foram utilizadas como técnicas de diagnóstico: a microscopia de luz, a reação em cadeia pela polimerase (PCR) e a imunofluorescência indireta (RIFI). No período de junho de 2009 a abril de 2010, foram amostrados 70 primatas, sendo 50 provenientes do Centro de Triagem Animais Silvestres (CETAS), localizado no município de São Luís, e 20 de vida livre, capturados em reserva particular localizada no município de São José de Ribamar, Estado do Maranhão. Foram avaliadas pela microscopia de luz 140 lâminas (duas de cada animal), das quais cinco (7,1%) foram positivas. Pela RIFI não se detectou anticorpos anti-Plasmodium spp. Pela PCR, dos 70 animais amostrados, foi possível observar produtos amplificados para Plasmodium spp. em 13 amostras, das quais oito (61,54%) eram de animais de vida livre e cinco (38,46%) de CETAS. Pelo pequeno tamanho dos fragmentos dos produtos amplificados, foi necessário cloná-los em vetor pGEM-T Easy para sequenciamento, no entanto, não foram obtidos resultados confirmatórios / The states that comprise the Amazon region are incriminated for 99% of registered cases of human malaria in Brazil. Currently, the state of Maranhão is responsible for 1% of cases in Amazonic region. According to recent seroepidemiological studies, the transmission of malaria from primates to humans occurs in Amazon region. Our study aimed to detect Plasmodium spp. in neotropical primate from island of São Luis, Maranhão State, Brazil, using morphological (light microscopy), molecular (Polymerase Chain Reaction [PCR]) and serological (Indirect immunofluorescence Assay [IFA]) techniques. Between June/2009 and April/2010, we sampled 70 primates, 50 from the Wildlife Screening Center (CETA), located in the city of São Luís, and 20 captured in a private reserve located in São José de Ribamar, state of Maranhão. Based on blood smear examinations, five (7.1%) monkeys were positive to Plasmodium spp. All animals showed seronegative to Plasmodium spp. by IFA. Plasmodium spp.-amplicons were detected in 13 animals; eight out of positive monkeys (61.54%) were free-ranging animals and five (38.46%) maintained in capitivity in CETAS. Regarding the small size of amplified fragments, we cloned the amplicons into pGEM-T Easy vector aiming at obtaining a higher sequencing quality; however, we did not reached satisfactory results

O Antígeno 1 de Membrana Apical (AMA-1) é um dos mais promissores candidatos a vacina contra a fase sanguínea da malária. No presente estudo, geramos uma nova proteína recombinante baseada no ectodomínio de AMA-1 de P. vivax (PvAMA-1) a partir do gene sintético com códon otimizado para expressão secretada na levedura Pichia pastoris. Por ELISA, a proteína PvAMA-1 foi reconhecida por 62,5% dos soros de pacientes da Amazônia brasileira infectados por P. vivax. A imunogenicidade de PvAMA-1 foi avaliada em camundongos BALB/c, utilizando protocolos homólogos e heterólogos de indução e reforço com DNA plasmidial e/ou proteína recombinante, emulsificada em Adjuvante de Freund. Os resultados mostraram que a eficiência da imunização é dependente da presença da proteína emulsificada em adjuvante forte. As imunizações subseqüentes foram realizadas com a proteína recombinante emulsificada em diferentes adjuvantes: sais de alumínio (Alum), Monophosphoryl Lipid A (MPLA) de Bordetella pertussis, flagelina FliC de Salmonella Typhimurium, saponina Quil A e Adjuvante Incompleto de Freund (AIF). Os resultados demonstraram que as imunizações na presença de Quil A e AIF foram capazes de induzir altos títulos de anticorpos IgG e uma resposta de isotipos de IgG mais balanceada, caracterizando um padrão do tipo Th1/Th2. Títulos de anticorpos mais baixos, mas também expressivos, foram obtidos na presença de Alum, MPLA, Alum + MPLA e Alum + FliC. A análise da resposta proliferativa, por citometria de fluxo utilizando marcação com CFSE, mostrou que células esplênicas CD3+CD4+, assim como CD3+CD8+, de animais imunizados com PvAMA-1 na presença dos adjuvantes Alum, Alum + MPLA, Quil A e AIF foram capazes de proliferar especificamente in vitro. Por imunofluorescência, soros policlonais de camundongos imunizados com o antígeno na presença de MPLA e Quil A foram capazes de reconhecer a proteína nativa expressa em isolados de P. Vivax da Ásia. Visando futuros testes pré clínicos em primatas não humanos, a proteína PvAMA-1 foi produzida em boas práticas de laboratório (BPL) por uma companhia especializada e o potencial imunogênico da proteína foi confirmado em imunizações posteriores. Em conjunto, nossos resultados demonstram que PvAMA-1 é um antígeno promissor para ser explorado em protocolos de imunização contra malária vivax, individualmente, ou em combinação com outros antígenos. / The Apical Membrane Antigen 1 (AMA-1) is one of the most promissing vaccine candidates against the erythrocytic stage of malaria. In the present study we generated a new recombinant protein based on AMA-1 ectodomain of P. vivax (PvAMA-1) using a synthetic codon optimized gene for yeast secreted expression. By ELISA, the protein PvAMA-1 was recognized by 62,5% of the sera from Brazilian Amazonia patients infected by P. vivax. The immunogenicity of PvAMA-1 was evaluated in BALB/c mice using homologous and heterologous protocols of prime and boost with plasmidial DNA and/or recombinant protein emulsified in Freund adjuvant. The results showed that the efficiency of immunization is dependent on the presence of a strong adjuvant. The following immunizations were conducted using the protein emulsified in different adjuvants: aluminium salts (Alum), Monophosphoryl Lipid A (MPLA) of Bordetella pertussis, flagelin FliC of Salmonella Typhimurium, saponin Quil A and Incomplete Freund\'s Adjuvant (IFA). The results showed that immunizations in the presence of Quil A e IFA were able to induce high IgG titers and a more balanced IgG isotypes response, featuring a standard type Th1/Th2. Lower antibody titers, but also significant, were obtained in the presence of Alum, MPLA, Alum + MPLA and Alum + FliC. The proliferative cellular analysis, by flow cytometry using CFSE staining, showed that splenic cells CD3+CD4+, as well as CD3+CD8+ from immunized mice that received PvAMA-1 with Alum, Alum + MPLA, Quil A and IFA were specifically able to proliferate in vitro. By immunofluorescence, the polyclonal sera from mice immunized with the antigen in the presence of MPLA and Quil A were able to recognize native protein expressed in Asian P. vivax isolates. Aiming future pre-clinical assays in non-human primates, the protein PvAMA-1 was produced in good laboratory practices (GLP) conditions by a specialized company and its immunogenic efficiency was confirmed in later immunizations. Altogether, our results showed that PvAMA-1 is a promissor antigen to be explored in immunization protocols against vivax malaria, individually, or combined with other antigens.

Nos últimos anos, estudamos vários aspectos da resposta imune naturalmente adquirida em indivíduos de diferentes áreas endêmicas da Região Amazônica expostos à malária. Para isso, utilizamos proteínas recombinantes baseadas em antígenos de formas sanguíneas de P. vivax, os quais têm sido considerados candidatos à vacina contra a malária vivax. No presente trabalho, nossos estudos imunoepidemiológicos concentraram-se em 396 indivíduos de uma comunidade rural da Amazônia ocidental brasileira, localizada no Estado do Acre, com o objetivo de realizar um estudo transversal e longitudinal da resposta de anticorpos contra um painel de proteínas recombinantes derivadas de merozoítos de P. vivax (MSP119, AMA-1, MSP3α e MSP3β). Para isso, os soros desses indivíduos foram testados por ELISA quanto ao reconhecimento das quatro proteínas recombinantes. As proporções de indivíduos na linha de base com anticorpos IgG contra MSP119, AMA- 1, MSP3α e MSP3β foram de 59,8%, 50,0%, 23,5% e 26,5%, respectivamente. Dentre esses indivíduos, apenas 10,9% tinham infecções por P. vivax, P. falciparum ou malária mista. No grupo de infectados por P. vivax, a proporção de respondedores foi maior para as proteínas recombinantes MSP119 e AMA-1, atingindo 78,6%, em ambos os casos. A proporção de indivíduos com anticorpos para cada uma das proteínas associou-se com o maior tempo de exposição à malária, exceto para a MSP3β. Além disso, a positividade foi mais alta nas áreas de maior risco de transmissão. Não observamos associações relevantes entre o genótipo do antígeno Duffy dos indivíduos e presença de anticorpos para as proteínas estudadas. No estudo longitudinal, observamos um aumento da prevalência de respondedores durante a parasitemia patente, sendo de 81,9%, 80,9%, 31,9% e 48,9% para a MSP119, AMA-1, MSP3α e MSP3β, respectivamente. Em conclusão, nossos resultados confirmam a alta antigenicidade dessas proteínas, o que pode ser de grande importância para futuros ensaios clínicos na região. / In recent years we studied various aspects of the naturally acquired immune response in individuals from different endemic areas of the Amazon region exposed to malaria. For this purpose we used recombinant proteins based on P. vivax blood stage antigens considered candidates for a vaccine against vivax malaria. In the present study, we focused on 396 individuals from a rural community in western Brazilian Amazon located at the state of Acre. We conducted a transversal and longitudinal study of the antibody response to a panel of recombinant proteins representing P. vivax merozoites surface antigens (MSP119, AMA-1, MSP3α and MSP3β). The sera of these individuals were tested by ELISA for the recognition of the four antigens mentioned above. The proportions of individuals at the baseline with IgG antibodies to MSP119, AMA-1, MSP3α and MSP3β were 59.8%, 50.0%, 23.5% and 26.5%, respectively. Among these individuals, 10.9% had patent malaria infections with either P. vivax or P. falciparum or both. Among individuals with patent P. vivax infection, the frequency of responders was high for MSP119 and AMA-1, (78.6% in both cases). Except in the case of MSP3β, the proportion of individuals with antibodies to each protein correlated with the time of malaria exposure. Also, the positivity was higher in areas of higher transmission levels. No relevant association was found between the Duffy genotypes and presence of antibodies to the different antigen. In longitudinal study, we observed an increased prevalence of responders during patent parasitemia, 81.9% 80.9% 31.9% and 48.9% to MSP119, AMA-1, MSP3α and MSP3β, respectively. In conclusion, our results confirm the high antigenicity of these proteins, which can be of great importance for future clinical trials in the region.

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted