Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention

Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy—the use of specific viruses that infect bacteria—is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections

Papillomavirus pseudovirions packaged with the L2 gene induce cross-neutralizing antibodies

<p>Abstract</p> <p>Background</p> <p>Current vaccines against HPVs are constituted of L1 protein self-assembled into virus-like particles (VLPs) and they have been shown to protect against natural HPV16 and HPV18 infections and associated lesions. In addition, limited cross-protection has been observed against closely related types. Immunization with L2 protein in animal models has been shown to provide cross-protection against distant papillomavirus types, suggesting that the L2 protein contains cross-neutralizing epitopes. However, vaccination with L2 protein or L2 peptides does not induce high titers of anti-L2 antibodies. In order to develop a vaccine with the potential to protect against other high-risk HPV types, we have produced HPV58 pseudovirions encoding the HPV31 L2 protein and compared their capacity to induce cross-neutralizing antibodies with that of HPV L1 and HPV L1/L2 VLPs.</p> <p>Methods</p> <p>The titers of neutralizing antibodies against HPV16, HPV18, HPV31 and HPV58 induced in Balb/c mice were compared after immunization with L2-containing vaccines.</p> <p>Results</p> <p>Low titers of cross-neutralizing antibodies were detected in mice when immunized with L1/L2 VLPs, and the highest levels of cross-neutralizing antibodies were observed in mice immunized with HPV 58 L1/L2 pseudovirions encoding the HPV 31 L2 protein.</p> <p>Conclusions</p> <p>The results obtained indicate that high levels of cross-neutralizing antibodies are only observed after immunization with pseudovirions encoding the L2 protein. HPV pseudovirions thus represent a possible new strategy for the generation of a broad-spectrum vaccine to protect against high-risk HPVs and associated neoplasia.</p

Utilisation des bactériophages comme thérapie lors d'une infection à Pseudomonas aeruginosa dans le cadre de la mucoviscidose : efficacité et innocuité

Repetitive antibiotic treatments against Pseudomonas aeruginosa in cystic fibrosis patients lead to the emergence of multi-resistant strains calling for new therapeutic strategies. One of the most promising approach seems to be the use of bacteriophages. In Eastern Europe successful human treatments with bacteriophages are ongoing since decades, while in Western Europe the interest for phage therapy is still weak. Notably, no data are available on the potential application of bacteriophages to treat pulmonary infections in CF patients. In this study, we first demonstrated in mice model that bacteriophages were able to treat a lethal pulmonary infection caused by a clinical strain of P. aeruginosa. We also studied the efficacy of different bacteriophages to reduce in vitro a P. aeruginosa biofilm.In a second time, we evaluate inflammation induced by bacteriophages which was found to be negligible in different models in vitro and in vivo. The transepithelial ion transport, a crucial function in cystic fibrosis, was also evaluated by measuring the nasal potential difference (npd) in mice. When mice were treated with bacteriophages, npd values did not differ from the reference ones, demonstrating an aspect of bacteriophages safety. Finally, in order to develop this therapeutic approach towards clinical trials, we successfully developed a protocol for the evaluation of bacteriophages ability to infect bacteria within sputum of cystic fibrosis patients. Our work focusing on both safety and efficacy of bacteriophages treatments in a CF context should contribute to the future developments of clinical trialsFace au phénomène de multi-résistance aux antibiotiques des souches de Pseudomonas aeruginosa chez les patients atteints de mucoviscidose, de nouvelles approches doivent être envisagées. L'utilisation des bactériophages pour cibler les bactéries semble être l'une des plus prometteuses. L'efficacité de la phagothérapie semble démontrée par son utilisation en Europe de l'Est depuis des décennies et par les récents résultats obtenus sur des modèles expérimentaux. Cependant, la possibilité de son utilisation chez des patients atteints de mucoviscidose n'a pas encore fait l'objet d'études approfondies. Nous avons démontré l'efficacité des bactériophages in vivo, lors d'une infection pulmonaire létale provoquée par une souche clinique de P. aeruginosa, mais aussi in vitro, pour réduire un biofilm formé par P. aeruginosa. Nous avons aussi étudié la réponse inflammatoire induite par les bactériophages, dans différents modèles in vitro et in vivo, qui s'est révélée quasiment négligeable. Nous avons également mis au point la technique de mesure de différence de potentiel nasale chez la souris pour étudier le transport ionique transépithélial, paramètre fondamental de la mucoviscidose. Les mesures obtenues en présence de bactériophages ne diffèrent pas significativement par rapport aux normes préalablement définies. Enfin, nous avons mis au point une méthode permettant d'évaluer la capacité des bactériophages à infecter des bactéries au sein d'expectorations de patients atteints de mucoviscidose. Nous apportons ainsi des résultats scientifiques concrets qui permettront de mieux appréhender les conditions nécessaires au développement de futurs essais cliniques chez ces patients

Utilisation des bactériophages comme thérapie lors d'une infection à Pseudomonas aeruginosa dans le cadre de la mucoviscidose (efficacité et innocuité)

Face au phénomène de multi-résistance aux antibiotiques des souches de Pseudomonas aeruginosa chez les patients atteints de mucoviscidose, de nouvelles approches doivent être envisagées. L utilisation des bactériophages pour cibler les bactéries semble être l une des plus prometteuses. L efficacité de la phagothérapie semble démontrée par son utilisation en Europe de l Est depuis des décennies et par les récents résultats obtenus sur des modèles expérimentaux. Cependant, la possibilité de son utilisation chez des patients atteints de mucoviscidose n a pas encore fait l objet d études approfondies. Nous avons démontré l efficacité des bactériophages in vivo, lors d une infection pulmonaire létale provoquée par une souche clinique de P. aeruginosa, mais aussi in vitro, pour réduire un biofilm formé par P. aeruginosa. Nous avons aussi étudié la réponse inflammatoire induite par les bactériophages, dans différents modèles in vitro et in vivo, qui s est révélée quasiment négligeable. Nous avons également mis au point la technique de mesure de différence de potentiel nasale chez la souris pour étudier le transport ionique transépithélial, paramètre fondamental de la mucoviscidose. Les mesures obtenues en présence de bactériophages ne diffèrent pas significativement par rapport aux normes préalablement définies. Enfin, nous avons mis au point une méthode permettant d évaluer la capacité des bactériophages à infecter des bactéries au sein d expectorations de patients atteints de mucoviscidose. Nous apportons ainsi des résultats scientifiques concrets qui permettront de mieux appréhender les conditions nécessaires au développement de futurs essais cliniques chez ces patients.Repetitive antibiotic treatments against Pseudomonas aeruginosa in cystic fibrosis patients lead to the emergence of multi-resistant strains calling for new therapeutic strategies. One of the most promising approach seems to be the use of bacteriophages. In Eastern Europe successful human treatments with bacteriophages are ongoing since decades, while in Western Europe the interest for phage therapy is still weak. Notably, no data are available on the potential application of bacteriophages to treat pulmonary infections in CF patients. In this study, we first demonstrated in mice model that bacteriophages were able to treat a lethal pulmonary infection caused by a clinical strain of P. aeruginosa. We also studied the efficacy of different bacteriophages to reduce in vitro a P. aeruginosa biofilm.In a second time, we evaluate inflammation induced by bacteriophages which was found to be negligible in different models in vitro and in vivo. The transepithelial ion transport, a crucial function in cystic fibrosis, was also evaluated by measuring the nasal potential difference (npd) in mice. When mice were treated with bacteriophages, npd values did not differ from the reference ones, demonstrating an aspect of bacteriophages safety. Finally, in order to develop this therapeutic approach towards clinical trials, we successfully developed a protocol for the evaluation of bacteriophages ability to infect bacteria within sputum of cystic fibrosis patients. Our work focusing on both safety and efficacy of bacteriophages treatments in a CF context should contribute to the future developments of clinical trialsPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

La phagothérapie : cauchemar pour la bactérie et rêve pour le médecin ?

International audienceBacteriophages were discovered in the early 20th century and rapidly used to treat bacterial infections in humans. As the first specific antibacterial agents, they were used worldwide until antibiotics ramped up. Thereafter, rapidly forgotten, they be- came the favorite toolbox for researchers that used them to elucidate some of the most fundamental aspects of the cellular life at the molecular level. Today, facing the threat of antibiotic resistant bacteria, bacteriophages are being reconsidered for their use in medicine. During the past century, knowledge on bacteriophages has improved considerably, nevertheless phage therapy is still in its infancy. Taking two examples of recently published experimental phage therapy results, this article summarizes the hopes but also the challenges that surround the future development of human phage therapy.-Découverts au début du XX e siècle, les bactériophages ont rapidementété utilisés pour traiter des infections bactériennes, devenant ainsi les premiers antibactériens spécifiquesàêtre employés en médecine. Cependant, l'arrivée des antibiotiques a brutalement réduit leur utilisation, les confinant au rayon ((boîteà outils)) du chercheur. Celui-ci en fît bon usage permettant de dévoiler une très grande partie des secrets moléculaires de la cellule. Aujourd'hui, faceà la menace que représentent les pathogènes bactériens résistants aux antibiotiques, les bactériophages tentent de faire leur retour dans le domaine médical. Bien que la connaissance de ces virus bactériens ait considérablement progressé après presque un siècle d'études, leur utilisation thérapeutique est loin d'être maîtrisée. Cet article relate,à l'aide de deux exemples récents de l'utilisation expérimentale des bactériophages, les espoirs mais aussi les challenges qui entourent la mise en place de la phagothérapie appliquéeà l'Homme. Mots clés : Bactériophages thérapeutiques / infections bactériennes / résistance aux antibiotiques / pneumonie / diarrhée Abstract-Phagotherapy: a nightmare for bacteria, a dream for physicians? Bacteriophages were discovered in the early 20th century and rapidly used to treat bacterial infections in humans. As the first specific antibacterial agents, they were used worldwide until antibiotics ramped up. Thereafter, rapidly forgotten, they became the favorite toolbox for researchers that used them to elucidate some of the most fundamental aspects of the cellular life at the molecular level. Today, facing the threat of antibiotic resistant bacteria, bacteriophages are being reconsidered for their use in medicine. During the past century, knowledge on bacteriophages has improved considerably, nevertheless phage therapy is still in its infancy. Taking two examples of recently published experimental phage therapy results, this article summarizes the hopes but also the challenges that surround the future development of human phage therapy

Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

BACKGROUND: Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE)) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE) in CF mice must be well characterized for correct interpretation. METHODS: We performed V(TE) measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. RESULTS: We determined the typical V(TE) values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(-) solution was considered to indicate a normal response. CONCLUSIONS: These data will make it possible to interpret changes in nasal V(TE) in mouse models of CF, in future preclinical studies

Localized lipidomics in cystic fibrosis: TOF-SIMS imaging of lungs from Pseudomonas aeruginosa-infected mice.

International audience: A consistent body of research has linked cystic fibrosis (CF) with variations in the tissue and fluid content in a number of lipid molecules. However, little is known about the spatial localization of those variations. We have recently applied TOF-SIMS mass spectrometry imaging to detect differential lipid signatures at the colon epithelium between normal and cftr-/- mice. In the present work we have used this technology to investigate potential differences in the spatial distribution of lipids due to Pseudomonas aeruginosa (P.a.) infection in mouse lung expressing or not cftr. Wild-type and exon 10 cftr knockout mice were subjected to intranasal infection with a clinical strain of P.a. Lung cryosections from infected and non-infected mice were subjected to cluster TOF-SIMS analysis in the negative ion mode. We observed a highly specific localization of a phosphoinositol fragment ion at m/z 299.1 in bronchial epithelium. Using this ion to delineate a region of interest, we studied the relative abundance of ions below m/z 1500. We found a significant increase in m/z 465.4 (identified as cholesteryl sulfate) in cftr-/- epithelium and in response to bacterial infection, as well as a decrease in most carboxylic ions. In conclusion, the m/z 299.1 ion can be used as a marker of bronchial epithelium, where P.a. infection leads to increased presence of cholesteryl sulfate in this tissue. TOF-SIMS imaging reveals as a valuable tool for the study of respiratory epithelium. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances

Survival curve of infected mice treated with bacteriophage PAK-P3 compared to P3-CHA.

<p>Mice were infected intranasally with 3×10⁶ cfu (CHA strain) and 2 h later were treated with either PBS (♦) or 3×10⁷ pfu of bacteriophage PAK-P3 (•), or 3×10⁷ pfu of bacteriophage P3-CHA (□) also administered intranasally (P<0.005 for both P3-CHA and PAK-P3 bacteriophage doses compared to PBS and P<0.05 between P3-CHA and PAK-P3 bacteriophage doses).</p

Characterization of PAK-P3 and P3-CHA bacteriophages.

<p>(<b>A</b>, <b>C</b>) Electron micrographs of PAK-P3 (<b>A</b>) and P3-CHA (<b>C</b>). Scale bar: 100 nm. (<b>B</b>) SDS-PAGE of PAK-P3 and P3-CHA proteins; only the most abundant proteins give visible signals (MW: molecular weight markers, the arrow points to the major capsid proteins). (<b>D</b>) Clustal alignment of the three major capsid proteins of PAK-P1, PAK-P3 and P3-CHA bacteriophages with their closest homologs in the database with known function (the major capsid protein of Felix 01 bacteriophage; NP_944891). Major capsid proteins of PAK-P3 and P3-CHA are 100% identical.</p