Host cell invasion by apicomplexan parasites: the junction conundrum

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Selection and development of adjuvants for immunizations with recombinant proteins of Plasmodium

The C-terminal region of Plasmodium merozoite surface protein 1 (MSP119) is being studied as one of the main targets for the development of a vaccine against malaria. Several studies have shown high imunogenicity of this region in experimental immunizations when injected in the presence of strong adjuvant formulations. In the present study we evaluate the possibility of using recombinant proteins based on the sequence of MSP119 to immunize mice by a mucosal route. Also, we generate new recombinant proteins consisting in the genetic fusion of the MSP119 to the flagellin FliC (flagellar protein of Salmonella enterica Typhimurium), to increase the immunogenicity of the antigen. Initially, we evaluated the capacity of the molecules cholera toxin (CT), heat-labile E. coli enterotixin (LT) or CpG ODN 1826, to act as adjuvants in mucosal intranasal immunization of mice with the recombinant proteins His6PvMSP119 or His6PvMSP119-PADRE (containing the universal T helper epitope PADRE). When administered in the presence of CT or LT, both recombinant proteins were highly immunogenic by the intranasal route. CpG ODN 1826 was less efficient as adjuvant by this route. The addition of CpG ODN 1826 in CT adjuvanted immunizations increased the specific IgG2c titers. These results showed that CT and LT are potent mucosal adjuvants in immunizations with recombinant malaria antigens and that CpG ODN 1826 can, in this case, be used as a tool to modulate the pattern of the immune response. Subsequently, we expressed a recombinant protein consisting of the sequence of PvMSP119-PADRE genetically fused to FliC (His6FliC-PvMSP119- PADRE). We showed that this fusion protein preserved the antigenic properties of PvMSP119 and the ability of flagellin to activate TLR5. The immunization of mice using this recombinant fusion protein induced high titers of specific antibodies and the presence of antigen-specific IFN-g producing cells in the spleen. The addition of CpG ODN 1826 in the vaccine formulations modulated the immune response by augmenting the specific titers of IgG2c. In addition, sera from immunized mice recognized the parasite in indirect immunofluorescence assay (IFA). Our results provided a new class of malaria vaccine formulation with intrinsic adjuvant property capable of stimulating specific humoral and cellular immune responses when administered alone or in the presence of other adjuvants. Finally, we expressed a recombinant protein containing the sequence of Plamodium falciparum MSP119 (PfMSP119) fused to FliC (His6FliC-PfMSP119). This fusion protein retained the capacity of flagellin to activate TLR5. Immunization of mice with the His6FliC-PfMSP119 alone induced high titers of specific antibodies and IFN-g producing cells. The addition of adjuvants such as CpG ODN 1826 or Quil-A (saponin of Quillaja saponaria) increased the levels of IgG2c and the cellular immune response, measured by the IFN-g secretion by immune spleen cells in culture. In addition, sera from immunized rabbits recognized the parasites in IFA and inhibited parasite growth in vitro. These results provide evidences that the fusion of malaria antigens to flagellin is an inexpensive and viable alternative for the development of a malaria vaccine.A região C-terminal da proteína 1 de superfície de merozoítas de Plasmodium (MSP119) vem sendo estudada como um dos principais alvos para desenvolvimento de uma vacina contra malária. Estudos têm demonstrado a imunogenicidade desta região em vacinações experimentais utilizando proteínas recombinantes na presença de adjuvantes fortes. No presente estudo, consideramos a possibilidade da utilização de proteínas recombinantes baseadas na sequência da MSP119 para imunização de camundongos por uma via de mucosa. Também geramos novas proteínas recombinantes de fusão da MSP119 com a flagelina (proteína do flagelo de Salmonella enterica Typhimurium), com o intuito de aumentar a imunogenicidade deste antígeno. Avaliamos inicialmente a capacidade de atuação das moléculas toxina colérica (CT), toxina termo-lábil de E. coli (LT) e do oligodeoxinucleotídeo CpG ODN 1826 como adjuvantes em imunizações de camundongos pela via de mucosa intranasal, utilizando como antígenos as proteínas recombinantes baseadas na MSP119 de P. vivax His6PvMSP119 ou His6PvMSP119-PADRE (contendo o epítopo “helper” universal PADRE). Quando administradas na presença dos adjuvantes CT ou LT, ambas foram altamente imunogênicas pela via intranasal, induzindo altos títulos de anticorpos, maiores do que quando utilizamos o CpG ODN 1826 como adjuvante. A adição do CpG ODN 1826 em imunizações na presença de CT foi capaz de aumentar a resposta específica de IgG2c. Nossos resultados demonstraram que CT e LT são adjuvantes potentes de mucosa em imunizações com antígenos recombinantes de malária, e que o CpG ODN 1826 pode ser utilizado como ferramenta de modulação da resposta imune nestas imunizações. Subsequentemente, expressamos em bactérias E. coli uma proteína recombinante contendo a sequência da PvMSP119-PADRE fusionada à flagelina FliC de S. enterica Typhimurium (His6FliC-PvMSP119-PADRE). Demonstramos que essa proteína de fusão retém as propriedades antigênicas da PvMSP119 e a capacidade da flagelina de ativar o receptor TLR5 da imunidade inata. A imunização de camundongos utilizando somente esta proteína recombinante de fusão induziu altos títulos de anticorpos, além de células específicas produtoras de IFN-g medidas no baço dos animais imunes. A adição de CpG ODN 1826 nas imunizações foi capaz de imunomodular a resposta de anticorpos, aumentando os títulos de IgG2c. Além disso, os soros dos camundongos imunizados foram capazes de reconhecer os parasitas por imunofluorescência indireta (IFA). Estes resultados demonstraram uma nova classe de antígenos candidatos à vacina contra malária, com atividade adjuvante intrínseca e capaz de estimular respostas imunológicas humoral e celular específicas, quando administrada sozinha ou na presença de outros adjuvantes. Por fim, expressamos em bactérias E. coli uma proteína recombinante contendo a sequência da MSP119 de P. falciparum (PfMSP119) fusionada à flagelina FliC de S. enterica Typhimurium (His6FliC-PfMSP119). Demonstramos que esta proteína de fusão retém a capacidade da flagelina de ativar o receptor TLR5 da imunidade inata. A imunização de camundongos com somente esta proteína recombinante de fusão induziu altos títulos de anticorpos, além de células produtoras de IFN-g específicas contra a PfMSP119. A adição de adjuvantes como CpG ODN 1826 ou Quil-A (saponina de Quillaja saponaria) nas imunizações com a proteína recombinante de fusão foi capaz de imunomodular a resposta de anticorpos, aumentando os títulos de IgG2c, bem como de potencializar a resposta celular medida pela produção de IFN-g contra a PfMSP119. Os soros de coelhos imunizados com a His6FliC-PfMSP119 sem a adição de nenhum adjuvante apresentaram altos títulos de anticorpos específicos contra a PfMSP119, que foram capazes de inibir o crescimento do parasita in vitro. Esses resultados sugerem a estratégia de fusão de antígenos de plasmódios como uma alternativa viável de desenvolvimento de uma vacina barata e eficaz contra a malária.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)TEDEBV UNIFESP: Teses e dissertaçõe

Diagnostic Methods for Non-Falciparum Malaria

Malaria is a serious public health problem that affects mostly the poorest countries in the world, killing more than 400,000 people per year, mainly children under 5 years old. Among the control and prevention strategies, the differential diagnosis of the Plasmodium–infecting species is an important factor for selecting a treatment and, consequently, for preventing the spread of the disease. One of the main difficulties for the detection of a specific Plasmodium sp is that most of the existing methods for malaria diagnosis focus on detecting P. falciparum. Thus, in many cases, the diagnostic methods neglect the other non-falciparum species and underestimate their prevalence and severity. Traditional methods for diagnosing malaria may present low specificity or sensitivity to non-falciparum spp. Therefore, there is high demand for new alternative methods able to differentiate Plasmodium species in a faster, cheaper and easier manner to execute. This review details the classical procedures and new perspectives of diagnostic methods for malaria non-falciparum differential detection and the possibilities of their application in different circumstances.Fil: Gimenez, Alba Marina. Universidade de Sao Paulo; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferreira Marqués, Rodolfo. Universidade de Sao Paulo; BrasilFil: Regiart, Daniel Matias Gaston. Universidade de Sao Paulo; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; ArgentinaFil: Bargieri, Daniel Youssef. Universidade de Sao Paulo; Brasi

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His(6)MSP1(19)-PADRE). in the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin ( LT) developed high and long lasting titers of specific serum antibodies. the induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. in contrast, mice immunized by intranasal route with His(6)MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.Universidade Federal de São Paulo, Dept Microbiol Imunol Parasitol, Escola Paulista Med, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Interdisciplinar Terapia Genica, Escola Paulista Med, BR-04044010 São Paulo, BrazilUniv São Paulo, Dept Microbiol, Inst Ciencias Biomed, São Paulo, BrazilUniv São Paulo, Dept Anal Clin & Toxicol, Fac Ciencias Farmaceut, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol Parasitol, Escola Paulista Med, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Interdisciplinar Terapia Genica, Escola Paulista Med, BR-04044010 São Paulo, BrazilWeb of Scienc

Malaria Vaccine Development: Are Bacterial Flagellin Fusion Proteins the Bridge between Mouse and Humans?

In the past 25 years, the development of an effective malaria vaccine has become one of the biggest riddles in the biomedical sciences. Experimental data using animal infection models demonstrated that it is possible to induce protective immunity against different stages of malaria parasites. Nonetheless, the vast body of knowledge has generated disappointments when submitted to clinical conditions and presently a single antigen formulation has progressed to the point where it may be translated into a human vaccine. In parallel, new means to increase the protective effects of antigens in general have been pursued and depicted, such as the use of bacterial flagellins as carriers/adjuvants. Flagellins activate pathways in the innate immune system of both mice and humans. The recent report of the first Phase I clinical trial of a vaccine containing a Salmonella flagellin as carrier/adjuvant may fuel the use of these proteins in vaccine formulations. Herein, we review the studies on the use of recombinant flagellins as vaccine adjuvants with malarial antigens in the light of the current state of the art of malaria vaccine development. The available information indicates that bacterial flagellins should be seriously considered for malaria vaccine formulations to the development of effective human vaccines

Primaquine and chloroquine nano-sized solid dispersion-loaded dissolving microarray patches for the improved treatment of malaria caused by Plasmodium vivax

Malaria is a global parasitic infection that leads to substantial illness and death. The most commonly-used drugs for treatment of malaria vivax are primaquine and chloroquine, but they have limitations, such as poor adherence due to frequent oral administration and gastrointestinal side effects. To overcome these limitations, we have developed nano-sized solid dispersion-based dissolving microarray patches (MAPs) for the intradermal delivery of these drugs. In vitro testing showed that these systems can deliver to skin and receiver compartment up to ≈60% of the payload for CQ-based dissolving MAPs and a total of ≈42% of drug loading for PQ-based dissolving MAPs. MAPs also displayed acceptable biocompatibility in cell tests. Pharmacokinetic studies in rats showed that dissolving MAPs could deliver sustained plasma levels of both PQ and CQ for over 7 days. Efficacy studies in a murine model for malaria showed that mice treated with PQ-MAPs and CQ-MAPs had reduced parasitaemia by up to 99.2%. This pharmaceutical approach may revolutionise malaria vivax treatment, especially in developing countries where the disease is endemic. The development of these dissolving MAPs may overcome issues associated with current pharmacotherapy and improve patient outcomes

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.FAPESPCNPq - INCTVCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Immunization with the MAEBL M2 domain protects against lethal Plasmodium yoelii infection

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOMalaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythr831037813792FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãosem informaçã

Integrative multi-kinase approach for the identification of potent antiplasmodial hits

Malaria is a tropical infectious disease that affects over 219 million people worldwide. Due to the constant emergence of parasitic resistance to the current antimalarial drugs, the discovery of new antimalarial drugs is a global health priority. Multi-target drug discovery is a promising and innovative strategy for drug discovery and it is currently regarded as one of the best strategies to face drug resistance. Aiming to identify new multi-target antimalarial drug candidates, we developed an integrative computational approach to select multi-kinase inhibitors for Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4) and protein kinase 6 (PK6). For this purpose, we developed and validated shape-based and machine learning models to prioritize compounds for experimental evaluation. Then, we applied the best models for virtual screening of a large commercial database of drug-like molecules. Ten computational hits were experimentally evaluated against asexual blood stages of both sensitive and multi-drug resistant P. falciparum strains. Among them, LabMol-171, LabMol-172, and LabMol-181 showed potent antiplasmodial activity at nanomolar concentrations (EC50 15 folds. In addition, LabMol-171 and LabMol-181 showed good in vitro inhibition of P. berghei ookinete formation and therefore represent promising transmission-blocking scaffolds. Finally, docking studies with protein kinases CDPK1, CDPK4, and PK6 showed structural insights for further hit-to-lead optimization studies.7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP405996/2016-0; 400760/2014-2Sem informação2018/05926-2; 2017/02353-9; 2012/16525-2; 2017/18611-7; 2018/07007-4; 2013/13119-6; 2018/24878-9; 2015/20774-