RESUMO
This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; â¼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides' 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.
Assuntos
Eritrócitos/parasitologia , Vacinas Antimaláricas/farmacologia , Plasmodium falciparum/patogenicidade , Animais , Antígenos de Protozoários/química , Aotidae , Proteínas de Transporte/química , Epitopos , Eritrócitos/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Peptídeos/imunologia , Peptídeos/metabolismo , Proteínas de Protozoários/química , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/farmacologiaRESUMO
In August 2017, the National Institute of Allergy and Infectious Diseases convened a meeting, entitled "Understanding the Liver-Stage Biology of Malaria Parasites to Enable and Accelerate the Development of a Highly Efficacious Vaccine," to discuss the needs and strategies to develop a highly efficacious, whole organism-based vaccine targeting the liver stage of malaria parasites. It was concluded that attenuated sporozoite platforms have proven to be promising approaches, and that late-arresting sporozoites could potentially offer greater vaccine performance than early-arresting sporozoites against malaria. New knowledge and emerging technologies have made the development of late-arresting sporozoites feasible. Highly integrated approaches involving liver-stage research, "omics" studies, and cutting-edge genetic editing technologies, combined with in vitro culture systems or unique animal models, are needed to accelerate the discovery of candidates for a late-arresting, genetically attenuated parasite vaccine.
Assuntos
Fígado/imunologia , Vacinas Antimaláricas/genética , Malária Falciparum/prevenção & controle , Malária Vivax/prevenção & controle , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Esporozoítos/imunologia , Animais , Modelos Animais de Doenças , Raios gama , Engenharia Genética/métodos , Humanos , Fígado/parasitologia , Malária/imunologia , Malária/parasitologia , Malária/prevenção & controle , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/metabolismo , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Vivax/imunologia , Malária Vivax/parasitologia , Camundongos , Plasmodium berghei/química , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Plasmodium berghei/efeitos da radiação , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/efeitos da radiação , Plasmodium vivax/química , Plasmodium vivax/genética , Plasmodium vivax/efeitos da radiação , Plasmodium yoelii/química , Plasmodium yoelii/genética , Plasmodium yoelii/imunologia , Plasmodium yoelii/efeitos da radiação , Esporozoítos/química , Esporozoítos/genética , Esporozoítos/efeitos da radiação , Vacinas AtenuadasRESUMO
Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed ß-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.
Assuntos
Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Vacinas Antimaláricas/química , Vacinas Antimaláricas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.
Assuntos
Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Fígado/metabolismo , Vacinas Antimaláricas/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Animais , Anopheles , Biomarcadores/metabolismo , Linhagem Celular , Feminino , Humanos , Fígado/parasitologia , Malária Falciparum/metabolismo , Malária Falciparum/prevenção & controle , Vacinas Atenuadas/metabolismoRESUMO
Malaria is a vector-borne disease affecting more than two million people and accounting for more than 600,000 deaths each year, especially in developing countries. The most serious form of malaria is caused by Plasmodium falciparum. The complex life cycle of this parasite, involving pre-erythrocytic, asexual and sexual stages, makes vaccine development cumbersome but also offers a broad spectrum of vaccine candidates targeting exactly those stages. Vaccines targeting the sexual stage of P. falciparum are called transmission-blocking vaccines (TBVs). They do not confer protection for the vaccinated individual but aim to reduce or prevent the transmission of the parasite within a population and are therefore regarded as an essential tool in the fight against the disease. Malaria predominantly affects large populations in developing countries, so TBVs need to be produced in large quantities at low cost. Combining the advantages of eukaryotic expression with a virtually unlimited upscaling potential and a good product safety profile, plant-based expression systems represent a suitable alternative for the production of TBVs. We report here the high level (300 µg/g fresh leaf weight (FLW)) transient expression in Nicotiana benthamiana leaves of an effective TBV candidate based on a fusion protein F0 comprising Pfs25 and the C0-domain of Pfs230, and the implementation of a simple and cost-effective heat treatment step for purification that yields intact recombinant protein at >90% purity with a recovery rate of >70%. The immunization of mice clearly showed that antibodies raised against plant-derived F0 completely blocked the formation of oocysts in a malaria transmission-blocking assay (TBA) making F0 an interesting TBV candidate or a component of a multi-stage malaria vaccine cocktail.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Precipitação Fracionada , Vacinas Antimaláricas/isolamento & purificação , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Temperatura Alta , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/genética , Vacinação/métodosRESUMO
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.
Assuntos
Flagelina/imunologia , Epitopos Imunodominantes/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Proteínas de Escherichia coli/imunologia , Flagelina/metabolismo , Epitopos Imunodominantes/metabolismo , Vacinas Antimaláricas/metabolismo , Malária Vivax/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/metabolismo , Receptor 5 Toll-Like/imunologiaRESUMO
The requirement for CD4 T cells in priming and maintaining cytotoxic T-lymphocyte responses presents a long-standing paradox in cellular immunology. In this study, we used sequential coadministration of a DNA vaccine encoding an invariant (Ii) chain in which the class II-associated Ii-peptide region is replaced with CD4 T-helper epitope, PADRE [Pan human leukocyte antigen-DR reactive epitope (Ii-PADRE)] or Bcl-xL with a DNA vaccine encoding Sig/E7/LAMP-1 to verify the role of CD4 T cells for the generation of effectors and memory E7-specific CD8 T-cell immune responses. Sequential vaccination, with Ii-PADRE+Sig/E7/LAMP-1 priming followed by Bcl-xL+Sig/E7/LAMP-1 boosting led to generation of E7-specific CD8 T cells, and was nearly equivalent in effect to coadministration with Ii-PADRE+Sig/E7/LAMP-1 or Bcl-xL+Sig/E7/LAMP-1 at both prime and boost. The mice vaccinated with the Ii-PADRE+Sig/E7/LAMP-1 prime-Bcl-xL+Sig/E7/LAMP-1 boost regimen exhibited better long-term E7-specific immune responses and tumor prevention effects in vivo than the mice vaccinated with the reverse sequential coadministration. After CD4 T-cell depletion, mice primed with Ii-PADRE+Sig/E7/LAMP-1 generated low numbers of E7-specific CD8 T cells and suppressed long-term memory CD8 T-cell response regardless of the sequence or combination of DNA vaccines administered. Mice primed with Bcl-xL+Sig/E7/LAMP-1 only suppressed long-term memory CD8 T-cell response after depletion of CD4 T cells before priming. Our findings suggest that activated CD4 T cells at prime phase are important to generate the antigen-specific CD8 T-cell immune responses and CD4 T cells, which are naive or activated, play a role to maintain the long-term memory responses.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer , Células Th1/metabolismo , Vacinas de DNA/administração & dosagem , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Comunicação Celular , Citotoxicidade Imunológica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunização Secundária , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Depleção Linfocítica , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Células Th1/imunologia , Células Th1/patologia , Vacinas de DNA/imunologia , Proteína bcl-X/genética , Proteína bcl-X/imunologia , Proteína bcl-X/metabolismoRESUMO
Developing a process to generate dendritic cells (DCs) applicable for multicenter trials would facilitate cancer vaccine development. Moreover, targeting multiple antigens with such a vaccine strategy could enhance the efficacy of such a treatment approach. We performed a phase 1/2 clinical trial administering a DC-based vaccine targeting multiple tumor-associated antigens to patients with advanced colorectal cancer (CRC). A qualified manufacturing process was used to generate DC from blood monocytes using granulocyte macrophage colony-stimulating factor and IL-13, and matured for 6 hours with Klebsiella-derived cell wall fraction and interferon-gamma (IFN-gamma). DCs were also loaded with 6 HLA-A*0201 binding peptides derived from carcinoembryonic antigen (CEA), MAGE, and HER2/neu, as well as keyhole limpet hemocyanin protein and pan-DR epitope peptide. Four planned doses of 35x10(6) cells were administered intradermally every 3 weeks. Immune response was assessed by IFN-gamma enzyme-linked immunosorbent spot (ELISPOT). Matured DC possessed an activated phenotype and could prime T cells in vitro. In the trial, 21 HLA-A2+ patients were apheresed, 13 were treated with the vaccine, and 11 patients were evaluable. No significant treatment-related toxicity was reported. T-cell responses to a CEA-derived peptide were detected by ELISPOT in 3 patients. T cells induced to CEA possessed high avidity T-cell receptors. ELISPOT after in vitro restimulation detected responses to multiple peptides in 2 patients. All patients showed progressive disease. This pilot study in advanced CRC patients demonstrates DC-generated granulocyte macrophage colony-stimulating factor and IL-13 matured with Klebsiella-derived cell wall fraction and IFN-gamma can induce immune responses to multiple tumor-associated antigens in patients with advanced CRC.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Neoplasias Colorretais/terapia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia Adotiva , Adjuvantes Imunológicos , Adolescente , Adulto , Idoso , Apresentação de Antígeno , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/efeitos adversos , Neoplasias Colorretais/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Feminino , Hemocianinas/imunologia , Hemocianinas/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Peptídeos/imunologia , Peptídeos/metabolismoRESUMO
Binding of P. falciparum-infected erythrocytes to vascular endothelium and to uninfected erythrocytes is mediated by the parasite-derived variant erythrocyte membrane protein PfEMP-1 and various receptors, both on the vascular endothelium and on the erythrocyte surface. Consecutive, non-overlapping peptides spanning the N-terminal segment (NTS) and Duffy-binding-like PfEMP1 sequence alpha-domain (DBLalpha) of this protein were tested in erythrocyte and C32 cell binding assays. Eight peptides specifically bound to C32 cells, and were named high-activity binding peptides (HABPs). No erythrocyte binding HABPs were found in this region. Strikingly, three HABPs [6504 ((1)MVELA KMGPK EAAGG DDIED(20)), 6505 ((21)ESAKH MFDRI GKDVY DKVKE(40)) and 6506 ((41)YRAKE RGKGL QGRLS EAKFEK(60))] are located within the NTS, for which no specific function has yet been described. HABP 6505 is neither immunogenic nor protection-inducing; therefore, based on our previous reports, critical amino acids (shown in bold) in HABP-C32 cell binding were identified and replaced to modify HABP immunogenicity and protectivity. Analogue peptide 12722 (ESAKH KFDRI GKDVY DMVKE) produced high antibody titres and completely protected three out of 12 vaccinated Aotus monkeys and 23410 (KHKFD FIGKI VYDMV KER) also produced high protection-inducing titres and completely protected one out of eight monkeys. (1)H NMR studies showed that all peptides were helical. Binding of these peptides to isolated HLADRbeta1 molecules did not reveal any preference, suggesting that they could bind to molecules not studied here.
Assuntos
Malária Falciparum/imunologia , Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Aotidae , Sítios de Ligação , Western Blotting , Eritrócitos/imunologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Espectroscopia de Ressonância Magnética , Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Células Tumorais CultivadasRESUMO
An effective malarial vaccine must contain multiple immunogenic, protection-inducing epitopes able to block and destroy the P. falciparum malaria parasite, the most lethal form of this disease in the world. Our strategy has consisted in using conserved peptides blocking parasite binding to red blood cells; however, these peptides are non-immunogenic and non-protection-inducing. Modifying their critical residues can make them immunogenic. Such peptides induced antibody titers (determined by immunofluorescence antibody test, IFA) and made the latter reactive (determined by Western blot) and protection inducing against experimental challenge with a highly infective Aotus monkey adapted P. falciparum strain. Modified peptides also induce highly non-protective long-lasting antibody levels. Modifications performed might allow them to bind specifically to different HLA-DRbeta purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by (1)H-NMR. It was found that modified, high non-protective long-lasting antibody level peptides bound to HLA-DR molecules from a different haplotype (to which immunogenic, protection-inducers bind) and had 4.6 +/- 1.4 A shorter distances between residues fitting into these molecules' Pocket 1 to Pocket 9, suggesting fitting into an inappropriate HLA-DR molecule. A multi-component, subunit-based, malarial vaccine is therefore feasible if modified peptides are suitably modified for an appropriate fit into the correct HLA-DRbeta1* molecule in order to form a proper MHC-II-peptide-TCR complex.
Assuntos
Anticorpos Antiprotozoários/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Malária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Aotidae , Western Blotting , Simulação por Computador , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Homologia de Sequência de AminoácidosRESUMO
With the aim of developing transmission-blocking vaccines based on the sexual stage-specific surface antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum, the gene encoding Pfs48/45 was incorporated into the genome of a recombinant vaccinia virus. In virus-infected mammalian tissue culture cells, recombinant Pfs48/45 antigen (rPfs48/45) is posttranslational modified to produce a highly N-glycosylated polypeptide. The rPfs48/45 protein was radiolabeled with ethanolamine, consisting of a further posttranslational modification in the form of a glycosylphosphatidylinositol anchor at its carboxy-terminal end. The rPfs48/45 was not detected on the surface of the infected cells; instead, it remained within the secretion pathway of mammalian cells irrespective of the duration of infection or culture temperature. Studies with monoclonal antibodies specific for disulfide band-dependent epitopes of Pfs48/45 revealed that recombinant Pfs48/45 is not folded in its authentic conformation even if N-glycosylation was chemically inhibited. Infection of mice and rabbits with recombinant virus elicited Pfs48/45-specific antibodies; however, the antisera failed to block parasite transmission in a standard mosquito membrane-feeding assay.