Verwendung von Phagen-Display-Technologien zur Target-Erkennung, Antikörpergenerierung und Antigen-Antikörper-Interaktionsstudien zur Entwicklung eines Listeria spp. Nachweises

The genus Listeria comprises bacteria that are ubiquitous and commonly present in food production facilities. Even though Listeria monocytogenes is the only Listeria species with relevance in causing listeriosis, many new Listeria species have been described in the recent years increasing the importance of studies over the genus. In this study, phage display technologies were employed to discover, identify, and characterize novel biomarkers and monoclonal antibodies. In brief, antibody phage display was applied to select antibodies against subcellular fractions of L. monocytogenes. Then, the target of the antibodies was identified by ORFeome phage display and confirmed with immunomagnetic separation-mass spectrometry (IMS-MS). Dihydrolipoamide acetyltransferase (pyruvate dehydrogenase complex - enzyme 2, PDC-E2) was identified as the target of the initial antibodies against Listeria spp.. Immunoblot and microscopy showed that this protein is present on the bacterial cell surface and is detectable in fluorescent microscopy. In addition, recombinant antibodies were generated against already known targets (internalins A and B, and fructose bisphosphate aldolase). In total, a set of five scFv-Fc against L. monocytogenes and 22 against Listeria spp. were tested in indirect ELISA against 17 Listeria and 15 non-Listeria species. All the five scFv-Fc against the pathogenic species showed 100 % sensitivity (CI 88.78-100.0 %) and specificity (CI 88.78-100.0 %), while the two anti-fructose bisphosphate aldolase showed non-optimal diagnostic performance. On the other hand, two antibodies against PDC-E2 (anti-Listeria spp.) showed 100 % sensitivity (CI 82.35-100.0 %) and specificity (CI 78.20-100.0 %), confirming PDC-E2 as a suitable detection target for Listeria spp.. Furthermore, the binding regions of two hibridoma-derived antibodies and the scFv-Fc against PDC-E2 were defined via single gene phage display, showing that this technique can provide information over the recognized region of a target in a considerable resolution. In addition, it revealed that the two best anti-PDC-E2 antibodies are the only ones to bind a synthetic region composed of two parts of PDC-E2. Hence, through a particular combination of phage display techniques, the biomarker PDC-E2 and two corresponding scFv-Fc against it, as well as scFv-Fc against internalins A and B, are hereby reported as novel promising tools for Listeria spp. detection.Die Gattung Listeria umfasst Bakterien, die allgegenwärtig sind und in Lebensmittelproduktionsanlagen häufig vorkommen. Obwohl Listeria monocytogenes die einzige Listeria-Art ist, die für die Entstehung von Listeriosis relevant ist, wurden in den letzten Jahren viele neue Listeria-Arten beschrieben, die die Bedeutung von Studien über die Gattung erhöhen. In dieser Studie wurden Phagen-Display-Technologien eingesetzt, um neuartige Biomarker und monoklonale Antikörper zu entdecken, zu identifizieren und zu charakterisieren. Kurz gesagt, wurde die Antikörper-Phagenanzeige verwendet, um Antikörper gegen subzelluläre Fraktionen von L. monocytogenes auszuwählen. Anschließend wurde das Ziel der Antikörper mittels ORFeome Phagen-Display identifiziert und mit der immunomagnetischen Separationsmassenspektrometrie (IMS-MS) bestätigt. Dihydrolipoamid-Acetyltransferase (Pyruvat-Dehydrogenase-Komplex - Enzym 2, PDC-E2) wurde als Ziel der ersten Antikörper gegen Listeria spp.. Immunoblot und Mikroskopie zeigten, dass dieses Protein auf der Oberfläche der Bakterienzellen vorhanden ist und in der Fluoreszenzmikroskopie nachweisbar ist. Darüber hinaus wurden rekombinante Antikörper gegen bereits bekannte Targets (Internaline A und B sowie Fructosebisphosphat-Aldolase) gebildet. Insgesamt wurden fünf scFv-Fc gegen L. monocytogenes und 22 gegen Listeria spp. im indirekten ELISA gegen 17 Listerien und 15 Nicht-Listerienarten getestet. Alle fünf scFv-Fc gegen die pathogene Spezies zeigten 100 % Sensitivität (CI 88,78-100,0 %) und Spezifität (CI 88,78-100,0 %), während die beiden Anti-Fruktose-Bisphosphat-Aldolasen eine nicht optimale diagnostische Leistung zeigten. Andererseits zeigten zwei Antikörper gegen PDC-E2 (anti-Listeria spp.) 100 % Sensitivität (CI 82,35-100,0 %) und Spezifität (CI 78,20-100,0 %), was PDC-E2 als geeignetes Nachweisziel für Listeria spp. bestätigt. Darüber hinaus wurden die Bindungsbereiche von zwei aus dem Hibridom stammenden Antikörpern und dem scFv-Fc gegen PDC-E2 über ein einzelnes Genphagen-Display definiert, was zeigt, dass diese Technik Informationen über die erkannte Region eines Ziels in einer beträchtlichen Auflösung liefern kann. Darüber hinaus zeigte sich, dass die beiden besten Anti-PDC-E2-Antikörper die einzigen sind, die eine synthetische Region binden, die aus zwei Teilen von PDC-E2 besteht. Daher werden durch eine besondere Kombination von Phagen-Display Deep Ls, der Biomarker PDC-E2 und zwei entsprechende scFv-Fc dagegen, sowie scFv-Fc gegen Internaline A und B, hiermit als neuartige vielversprechende Werkzeuge für den Listeria spp. Nachweis berichtet

EPIGENÉTICA E CÉLULAS-TRONCO: UM NOVO CAMINHO PARA A COMPREENSÃO DOS MECANISMOS DE PLASTICIDADE, DIFERENCIAÇÃO E IMPRINTING

The characteristic of plasticity, especially of embryonic stem cells (ESC), confers them capacity to differentiate in any cellular type. The differentiation processes occur through loss of plasticity, specializing cells by blocking the expression of non-related genes. The epigenetics suggests explanations for this capacity, by regulatory processes that are not contained in the genome. The elucidation of the processes involved in the cellular differentiation phenomena is fundamental towards the development of cellular therapy techniques and can be extrapolated to studies about other phenomena, like the cellular malignant transformation. This review summarizes and discusses key-points on the recent topic of the stem-cells epigenetics.La característica de la plasticidad, sobretodo de las células madre embrionarias (ESC: Embrionyc Stem Cells), les confiere la capacidad de diferenciarse en cualquier tipo celular. Los procesos de diferenciación se producen por la pérdida de la plasticidad, especializando células mediante el bloqueo de la expresión de genes no relacionados. La epigenética sugiere explicaciones para esta capacidad, por parte de los procesos regulatorios que no están contenidos en el genoma. La elucidación de los procesos involucrados en los fenómenos de diferenciación celular es fundamental para el desarrollo de técnicas en terapia celular y puede ser extrapolada a estudios sobre otros fenómenos, como la transformación de células malignas. Esta revisión resume y analiza los puntos clave sobre el tema reciente de las células madre en epigenética.A característica de plasticidade, especialmente das células-tronco embrionárias (ESC: EmbrionycStemCells), confere a elas capacidade de diferenciação em qualquer tipo celular. Processos de diferenciação ocorrem através da perda desta plasticidade, especializando células pelo bloqueio da expressão de genes não-relacionados. A epigenética sugere explicações para esta capacidade, através de processos regulatórios que não estão contidos no genoma. A elucidação dos processos envolvidos nos fenômenos de diferenciação celular é fundamental em relação ao desenvolvimento de técnicas de terapia celular e pode ser extrapolada para estudos sobre outros fenômenos, como transformação celular maligna. Esta revisão sumariza e discute pontos chave do recente tópico da epigenética das células-tronco

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications

Targeting Aspergillus fumigatus Crf Transglycosylases With Neutralizing Antibody Is Relevant but Not Sufficient to Erase Fungal Burden in a Neutropenic Rat Model

Aspergillus fumigatus is an airborne opportunistic fungal pathogen responsible for severe infections. Among them, invasive pulmonary aspergillosis has become a major concern as mortality rates exceed 50% in immunocompromised hosts. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. Current treatments suffer from high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of Chitin Ring Formation cell wall transglycosylases of A. fumigatus to be therapeutic targets for therapeutic antibodies. We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the CRF1 gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for CRF1 gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% in vitro when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival. Overall, our study highlighted the potential relevance of targeting Crf cell wall protein (CWP) with therapeutic antibodies

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016

Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24-100.0) and specificity (CI 88.78-100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes

Pyruvate dehydrogenase complex-enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies

The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria

Targeting Aspergillus fumigatus Crf Transglycosylases With Neutralizing Antibody Is Relevant but Not Sufficient to Erase Fungal Burden in a Neutropenic Rat Model

International audienceAspergillus fumigatus is an airborne opportunistic fungal pathogen responsible for severe infections. Among them, invasive pulmonary aspergillosis has become a major concern as mortality rates exceed 50% in immunocompromised hosts. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. Current treatments suffer from high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of Chitin Ring Formation cell wall transglycosylases of A. fumigatus to be therapeutic targets for therapeutic antibodies. We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the CRF1 gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for CRF1 gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% in vitro when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival. Overall, our study highlighted the potential relevance of targeting Crf cell wall protein (CWP) with therapeutic antibodies