Immunization with mutant plasmodium parasites : how is that achieved?

Tese de doutoramento em Ciências Biomédicas (Ciências Biopatológicas), apresentada à Universidade de Lisboa através da Faculdade de Medicina, 200

Carbon nanotubes as a novel tool for vaccination against infectious diseases and cancer

Due to their unusual properties, carbon nanotubes have been extensively employed in electronics, nanotechnology and optics, amongst other. More recently, they have also been used as vehicles for drug and antigen delivery, the latter being a novel immunization strategy against infectious diseases and cancer. Here we discuss the potential of carbon nanotubes as an antigen delivery tool and suggest further directions in the field of vaccination. © 2013 Gottardi and Douradinha; licensee BioMed Central Ltd

Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

Background: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants,.pcrmp3 and.pcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Delta pcrmp3 and Delta pcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Delta crmp3 and Delta crmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Delta pcrmp3 and Delta pcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.Host-parasite interactio

Immunization with genetically attenuated P52-deficient Plasmodium berghei sporozoites induces a long-lasting effector memory CD8+ T cell response in the liver

Item does not contain fulltextABSTRACT: BACKGROUND: The induction of sterile immunity and long lasting protection against malaria has been effectively achieved by immunization with sporozoites attenuated by gamma-irradiation or through deletion of genes. For mice immunized with radiation attenuated sporozoites (RAS) it has been shown that intrahepatic effector memory CD8+ T cells are critical for protection. Recent studies have shown that immunization with genetically attenuated parasites (GAP) in mice is also conferred by liver effector memory CD8+ T cells. FINDINGS: In this study we analysed effector memory cell responses after immunization of GAP that lack the P52 protein. We demonstrate that immunization with p52-GAP sporozoites also results in a strong increase of effector memory CD8+ T cells, even 6 months after immunization, whereas no specific CD4+ effector T cells response could be detected. In addition, we show that the increase of effector memory CD8+ T cells is specific for the liver and not for the spleen or lymph nodes. CONCLUSIONS: These results indicate that immunization of mice with P. berghei p52-GAP results in immune responses that are comparable to those induced by RAS or GAP lacking expression of UIS3 or UIS4, with an important role implicated for intrahepatic effector memory CD8+ T cells. The knowledge of the mediators of protective immunity after immunization with different GAP is important for the further development of vaccines consisting of genetically attenuated sporozoites

Klebsiella pneumoniae Lipopolysaccharides Serotype O2afg Induce Poor Inflammatory Immune Responses Ex Vivo

Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium

Lipid droplet levels vary heterogeneously in response to simulated gastrointestinal stresses in different probiotic Saccharomyces cerevisiae strains

AbstractTo exert their therapeutic action, probiotic Saccharomyces cerevisiae strains must survive harsh digestive environments. Lipid droplets accumulate in cells which undergo stress-inducing situations, supposedly having a protective role. We assessed lipid droplet levels, either naturally accumulated or induced in response to digestive challenges, of probiotic strains S. boulardii, S. cerevisiae A-905, S. cerevisiae Sc47 and S. cerevisiae L11, and of non-probiotic strains S. cerevisiae BY4741 and S. cerevisiae BY4743. Strains 905 and Sc47 had lower and higher lipid droplet levels, respectively, when compared to the remaining strains, showing that higher accumulationof these neutral lipids is not a feature shared by all probiotic Saccharomyces strains. When submitted to simulated gastric or bile salts environments, lipid droplet levels increase in all tested probiotic strains, at least for one to the induced stresses, suggesting that lipid droplets participate in the protective mechanisms against gastrointestinal stresses in probiotic Saccharomyces yeasts

Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients

Proteomic Profiling of Plasmodium Sporozoite Maturation Identifies New Proteins Essential for Parasite Development and Infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito—early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans

Inactivation of a Plasmodium apicoplast protein attenuates formation of liver merozoites

Malaria parasites undergo a population expansion inside the host liver before disease onset. Developmental arrest inside host hepatocytes elicits protective immune responses. Therefore, elucidation of the molecular mechanisms leading to mature hepatic merozoites, which initiate the pathogenic blood phase, also informs anti-malaria vaccine strategies. Using targeted gene deletion in the rodent model malaria parasite Plasmodium berghei, we show that a Plasmodium-specific Apicoplast protein plays an important role for Liver Merozoite formation (PALM). While the resulting knockout mutants develop normally for most of the life cycle, merozoite release into the blood stream and the ability to establish an infection are severely impaired. Presence of a signature blood-stage antigen, merozoite surface protein 1 and normal apicoplast morphology indicate that the inability to finalize merozoite segregation is a direct consequence of loss of PALM function. Experimental immunization of mice with as few as two doses of palm- sporozoites can elicit sterile protection up to 110 days after final immunization. Our data establish that a tailor-made arrest in the final steps of hepatic merozoite formation can induce strong protective immune responses and that malaria parasites employ a distinct apicoplast protein for efficient formation of pre-erythrocytic merozoites

Hypoxia promotes liver stage malaria infection in primary human hepatocytes in vitro

Homeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that may influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells (MPCC) to model liver stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver stage malaria infection efficiencies may improve under hypoxia. Using the infection of MPCCs with P. berghei or P. yoelii as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes, and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters like hepatocyte density and media height, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments reveal that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt (II) chloride, as well as a HIF-1α activator, dimethyloxalylglycine, also enhance P. berghei infection, suggesting that the effect of hypoxia on infection is mediated in part by host-dependent HIF-1α mechanisms.Bill & Melinda Gates Foundation (Award 51066)Howard Hughes Medical Institut