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The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). ispartof: MOLECULAR THERAPY vol:30 issue:9 pages:2952-2967 ispartof: location:United States status: published

International audience; Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

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Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. To inform on the origin of SARS-CoV-2, we evaluated the clinical, epidemiological and evolutionary consequences of a potential BANAL-236 spillover into humans using animal models. The virus replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a more virulent strain like Wuhan SARS-CoV-2. Yet we found no evidence of antibodies recognizing bat sarbecoviruses in populations highly exposed to bats, indicating that such infections, if they occur, are rare. Six passages in mice or in human intestinal cells, mimicking putative early spillover events, selected adaptive mutations without appearance of a furin cleavage site and not change in virulence. We thus conclude that the hypothesis of the SARS-CoV-2 pandemic being preceded by silent circulation in humans of BANAL-236-like strains leading to the acquisition of a furin cleavage site is unlikely. Our studies suggest that a specific search for a furin cleavage site in sarbecoviruses in the wild should be pursued to understand the origin of the SARS-CoV-2 pandemics.

Summary The impact of treatment on the risk of lymphoma in patients with rheumatoid arthritis (RA) is unclear. Here, we aimed to assess if the risk of lymphoma differs according to the type of tumor necrosis factor inhibitor (TNFi), comparing monoclonal anti-TNF antibodies to the soluble TNF receptor. We used B cell activating factor belonging to the TNF family (BAFF)-transgenic (Tg) mice as a model of autoimmunity-associated lymphoma. Six-month-old BAFF-Tg mice were treated with TNFi for 12 months. Histological examination of the spleen, assessment of the cellular composition of the spleen by flow cytometry and assessment of B cell clonality were performed at euthanasia. Crude mortality and incidence of lymphoma were significantly higher in mice treated with monoclonal anti-TNF antibodies compared to both controls and mice treated with the soluble TNF receptor, even at a high dose. Flow cytometry analysis revealed decreased splenic macrophage infiltration in mice treated with monoclonal anti-TNF antibodies. Overall, this study demonstrates, for the first time, that a very prolonged treatment with monoclonal anti-TNF antibodies increase the risk of lymphoma in B cell-driven autoimmunity. These data suggest a closer monitoring for lymphoma development in patients suffering from B cell-driven autoimmune disease with long-term exposure to monoclonal anti-TNF antibodies.

PURPOSE: 12b80 combines doxorubicin bound to a bone targeting hydroxybisphosphonate vector using a pH-sensitive linker, designed to specifically trigger doxorubicin release in an acidic bone tumor microenvironment. This phase I study aimed to determine the safety and toxicity profiles of 12b80 in dogs with naturally occurring osteosarcoma, with the objective to translate findings from dogs to humans.EXPERIMENTAL DESIGN: Ten client-owned dogs with osteosarcoma were enrolled in an accelerated dose-titration design followed by 3 + 3 design. Dogs received three cycles of 12b80 intravenous injection at 4 mg/kg (n = 1), 6 mg/kg (n = 2), 8 mg/kg (n = 3), and 10 mg/kg (n = 4). Endpoints included safety, tolerability, maximum tolerated dose (MTD), and dose-limiting toxicity (DLT).RESULTS: The MTD of 12b80 was 8 mg/kg (i.e., equivalent dose of doxorubicin of 110 mg/m2, range: 93-126). Most adverse events included grade ≤ 2 gastrointestinal disorders and hypersensitivity reactions. No hematological or cardiac DLT were observed at any dose tested.CONCLUSIONS: In dogs, 12b80 is overall well tolerated and expends the MTD of doxorubicin up to four times the standard dose of 30 mg/m2. These results demonstrate the potential therapeutic benefit of 12b80 in canine and human osteosarcoma.

Non-human primates (NHPs) are particularly relevant as preclinical models for SARS-CoV-2 infection and nuclear imaging may represent a valuable tool for monitoring infection in this species. We investigated the benefit of computed X-ray tomography (CT) and [

A 4 mo old female Staffordshire bull terrier puppy was presented with chronic Escherichia coli cystitis. Ultrasound and cystoscopic examination revealed innumerable, intraluminal, finger-like proliferations arising from the dorsal urinary bladder (UB) wall. Histological examination of mucosal biopsies obtained by cystoscopy was suggestive of granulomatous cystitis. The proliferative lesions were removed surgically and submitted for histological examination. The UB submucosa was heavily infiltrated by macrophages with periodic acid-Schiff–positive cytoplasm exhibiting rare Michaelis-Gutmann bodies, leading to the diagnosis of malakoplakia. The puppy was prescribed with sulfamethoxazole-trimethoprim. The urinary signs disappeared despite the persistent UB wall thickening revealed by abdominal ultrasound. Urine culture performed during the ninth week of treatment showed a persistent infection by E coli resistant to sulfamethoxazole-trimethoprim. The dog was switched to doxycycline but was then lost to follow-up. Malakoplakia is a chronic granulomatous inflammation well documented in humans. Its pathophysiology is not fully understood, but bacterial infection, immunodepression, and a defective lysosomal function may lead to the intracytoplasmic accumulation of partially degraded bacteria that can subsequently mineralize to form the Michaelis-Gutmann bodies. Malakoplakia should be suspected when UB mass lesions are identified in a young dog with bacterial cystitis.

Accumulating data highlight the role of neurotrophins and their receptors in human breast cancer. This family includes nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both synthetized as proneurotrophins (proNGF and proBDNF). (pro)NGF and (pro)BDNF initiate their biological effects by binding to both their specific receptors TrkA and TrkB, respectively, and the common receptor p75NTR. Currently, no data are available about their expression and potential role in canine mammary tumors. The aim of this study was to investigate expression of proNGF and BDNF as well as their receptors TrkA, TrkB, and p75NTR in canine mammary carcinomas, and to correlate them with clinicopathological parameters (grade, histological type, lymph node status, recurrence, and distant metastasis) and survival. Immunohistochemistry was performed on serial sections of 96 canine mammary carcinomas with antibodies against proNGF, BDNF, TrkA, TrkB, and p75NTR. Of the 96 carcinomas, proNGF expression was detected in 71 (74%), BDNF in 79 (82%), TrkA in 94 (98%), TrkB in 35 (37%), and p75NTR in 44 (46%). No association was observed between proNGF, BDNF, or TrkA expression and either clinicopathological parameters or survival. TrkB and p75NTR expression were associated with favorable clinicopathological parameters as well as better overall survival.

International audience; Purpose: F14512 is an epipodophyllotoxin derivative from etoposide, combined with a spermine moiety introduced as a cell delivery vector. The objective of this study was to compare the safety and antitumor activity of F14512 and etoposide phosphate in dogs with spontaneous non-Hodgkin lymphoma (NHL) and to investigate the potential benefit of F14512 in P-glycoprotein (Pgp) overexpressing lymphomas. Experimental Design: Forty-eight client-owned dogs with intermediate to high-grade NHL were enrolled into a randomized, double-blind trial of F14512 versus etoposide phosphate. Endpoints included safety and therapeutic efficacy. Results: Twenty-five dogs were randomized to receive F14512 and 23 dogs to receive etoposide phosphate. All adverse events (AEs) were reversible, and no treatment-related death was reported. Hematologic AEs were more severe with F14512 and gastrointestinal AEs were more frequent with etoposide phosphate. F14512 exhibited similar response rate and progression-free survival (PFS) as etoposide phosphate in the global treated population. Subgroup analysis of dogs with Pgp-overexpressing NHL showed a significant improvement in PFS in dogs treated with F14512 compared with etoposide phosphate. Conclusion: F14512 showed strong therapeutic efficacy against spontaneous NHL and exhibited a clinical benefice in Pgp-overexpressing lymphoma superior to etoposide phosphate. The results clearly justify the evaluation of F14512 in human clinical trials.

International audience; A 6-y-old neutered male ferret (Mustela putorius furo) was presented because of a 1-mo history of progressive weight loss, chronic cough, and hair loss. On clinical examination, the animal was coughing, slightly depressed, moderately hypothermic, and had bilateral epiphora. Thoracic radiography was suggestive of severe multinodular interstitial pneumonia. Abdominal ultrasound examination revealed hepatosplenomegaly and mesenteric and pancreaticoduodenal lymphadenopathy. Fine-needle aspiration of the pancreaticoduodenal lymph node, followed by routine Romanowsky and Ziehl-Neelsen stains, revealed numerous macrophages containing myriad acid-fast bacilli, leading to identification of mycobacteriosis. Autopsy and histologic examination confirmed the presence of disseminated, poorly defined, acid-fast, bacilli-rich granulomas in the pancreaticoduodenal and mesenteric lymph nodes, intestines, and lungs. Destaining of May-Grunwald/Giemsa-stained slides with alcohol, and then restaining with Ziehl-Neelsen, revealed acid-fast rods and avoided repeat tissue sampling without affecting the Ziehl-Neelsen stain quality and cytologic features. Tissue samples were submitted for a PCR assay targeting the heat shock protein gene (hsp65) and revealed 100% homology with Mycobacterium genavense. We emphasize the use of special stains and PCR for identification of this potential zoonotic agent.

Key Clinical Message An young alpaca was evaluated for bilateral progressive melting corneal ulcers and developped secondary bullous keratopathy during hospitalization. The tragic progression of melting ulcers in both eyes observed in our case leads us to recommend a rapid intensive medical therapy in young and debilitated alpacas presenting a corneal ulcer.

Pompe disease is a lysosomal storage disorder caused by acid-α-glucosidase (GAA) deficiency, leading to glycogen storage. The disease manifests as a fatal cardiomyopathy in infantile form. Enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history. The neurologic phenotype and the persistence of selective muscular weakness in some patients could be attributed to the central nervous system (CNS) storage uncorrected by ERT. GAA-KO 6neo/6neo mice were treated with a single intrathecal administration of adeno-associated recombinant vector (AAV) mediated gene transfer of human GAA at 1 month and their neurologic, neuromuscular, and cardiac function was assessed for 1 year. We demonstrate a significant functional neurologic correction in treated animals from 4 months onward, a neuromuscular improvement from 9 months onward, and a correction of the hypertrophic cardiomyopathy at 12 months. The regions most affected by the disease i.e. the brainstem, spinal cord, and the left cardiac ventricular wall all show enzymatic, biochemical and histological correction. Muscle glycogen storage is not affected by the treatment, thus suggesting that the restoration of muscle functionality is directly related to the CNS correction. This unprecedented global and long-term CNS and cardiac cure offer new perspectives for the management of patients. Electronic supplementary material The online version of this article (doi:10.1186/s40478-017-0464-2) contains supplementary material, which is available to authorized users.

A 15-year-old neutered female domestic shorthair cat was presented for weight loss, polydipsia/polyuria, and lethargy. A large fluctuant mass was palpated in the ventral right cervical region. Biochemistry results were consistent with primary hyperparathyroidism. Parathyroid hormone level in the fluid was higher to that observed in the plasma, consistent with a cystic parathyroid lesion. Right parathyroidectomy and thyroidectomy were performed without complications. Ionized calcium normalized within a few hours. Histopathology yielded a diagnosis of cystic parathyroid adenoma. Follow-up showed complete recovery of clinical signs and normalization of ionized calcium. This case shows an uncommon presentation of feline primary hyperparathyroidism secondary to a cystic parathyroid adenoma and is, to our knowledge, the first case presented with a large palpable mass in which parathyroid hormone concentration was measured. This report highlights the value of selective hormonal analyses of the cystic fluid to confirm the origin of the cystic lesion pre-operatively.Keywords: Adenoma, Calcium, Cyst, Hyperparathyroidism, Parathyroid hormone.

International audience; Rapid, sensitive, precise and accurate analysis of samples in their native in vivo environment is critical to better decipher physiological and physiopathological mechanisms. SpiderMass is an ambient mass spectrometry (MS) system designed for mobile in vivo and real-time surface analyses of biological tissues. The system uses a fibered laser, which is tuned to excite the most intense vibrational band of water, resulting in a process termed water-assisted laser desorption/ionization (WALDI). The water molecules act as an endogenous matrix in a matrix-assisted laser desorption ionization (MALDI)-like scenario, leading to the desorption/ionization of biomolecules (lipids, metabolites and proteins). The ejected material is transferred to the mass spectrometer through an atmospheric interface and a transfer line that is several meters long. Here, we formulate a three-stage procedure that includes (i) a laser system setup coupled to a Waters Q-TOF or Thermo Fisher Q Exactive mass analyzer, (ii) analysis of specimens and (iii) data processing. We also describe the optimal setup for the analysis of cell cultures, fresh-frozen tissue sections and in vivo experiments on skin. With proper optimization, the system can be used for a variety of different targets and applications. The entire procedure takes 1-2 d for complex samples.

CD44+/CD24–phenotype has been used to identify human and canine mammary cancer stem-like cells. In canine mammary tumors, CD44+/CD24–phenotype has been associated with high grade and lymph node infiltration. However, several studies have reported opposing results regarding the clinical significance of phenotypic groups formed by the combination of CD44 and CD24 in both human and canine mammary tumors. So far, no study has investigated the correlation between these phenotypes and survival in dogs. The aim of this study was to investigate the expression and distribution of CD44 and CD24 in canine mammary carcinomas and to correlate them with histological diagnosis and survival in a well-characterized cohort. Immunohistochemistry was performed in 96 mammary carcinomas with antibodies against CD44 and CD24. Expression of CD44+and CD44+/CD24–phenotype was detected in 75 of 96 (78%) and 63 of 96 (65.6%) carcinomas, respectively. Their expression was associated with tumor type, occurring more often in tubular complex carcinomas than in solid carcinomas. CD44+/CD24–phenotype was associated with a better overall survival ( P = .001). CD24+expression was detected in 52 of 96 tumors (54%) and CD44–/CD24+phenotype in 39 of 96 tumors (40.6%). Both were associated with poor clinicopathological parameters (high grade, and emboli). No correlation with overall survival was observed. CD44+/CD24–expression was associated with a better prognosis and occurred at high frequency and high level, indicating that this phenotype is not suitable to detect cancer stem cells in canine mammary carcinomas. Although further studies are needed, our results suggest that CD24 may constitute a valuable marker of poor prognosis for canine mammary carcinomas.

International audience; Histopathological diagnosis of biopsy samples and margin assessment of surgical specimens are challenging aspects in sarcoma. Using dog patient tissues, we assessed the performance of a recently developed technology for fast ex vivo molecular lipid-based diagnosis of sarcomas. The instrument is based on mass spectrometry (MS) molecular analysis through a laser microprobe operating under ambient conditions using excitation of endogenous water molecules. Classification models based on cancer/normal/necrotic, tumor grade, and subtypes showed a minimum of 97.63% correct classification. Specific markers of normal, cancer, and necrotic regions were identified by tandem MS and validated by MS imaging. Real-time detection capabilities were demonstrated by ex vivo analysis with direct interrogation of classification models.

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We assessed on dog sarcoma the performances of a newly developed technology, for fast ex-vivo diagnostic of tumors in the perspective to target in the future noninvasive in-vivo real-time diagnostic and excision margin definition intraoperatively, named Spidermass. The instrument is based on Mass Spectrometry (MS) molecular analysis through a laser microprobe operating under ambient condition using resonant excitation of endogenous water molecules Specificity of the molecular profiles generated with the instrument was evaluated using multivariate analysis for tumor typing or grading. Classification models based on cancer/normal, cancer/normal/necrotic, tumor grade, and cancer subtype showed at minimum of 97.63% correct classification. Specific markers of normal, cancer and necrotic regions were identified by tandem MS and validated by MS Imaging. Margins detection capabilities was studied, and compared to MS imaging and histological staining. Our results confirmed the high accuracy of the technology to differentiate between histological areas or sarcoma subtypes in real-time.

International audience; A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®.We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.

Respiratory pathogens represent a great burden for humanity and a potential source of new pandemics, as illustrated by the recent emergence of coronavirus disease 2019 (COVID-19). In recent decades, biotechnological advances have led to the development of numerous innovative therapeutic molecules and vaccine immunogens. However, we still lack effective treatments and vaccines against many respiratory pathogens. More than ever, there is a need for a fast, predictive, preclinical pipeline, to keep pace with emerging diseases. Animal models are key for the preclinical development of disease management strategies. The predictive value of these models depends on their ability to reproduce the features of the human disease, the mode of transmission of the infectious agent and the availability of technologies for monitoring infection. This review focuses on the use of non-human primates as relevant preclinical models for the development of prevention and treatment for human respiratory infections.

Erratum in : Publisher Correction: Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles. [Sci Rep. 2018]; International audience; Tuberculosis (TB) is a leading infectious cause of death worldwide. The use of ethionamide (ETH), a main second line anti-TB drug, is hampered by its severe side effects. Recently discovered "booster" molecules strongly increase the ETH efficacy, opening new perspectives to improve the current clinical outcome of drug-resistant TB. To investigate the simultaneous delivery of ETH and its booster BDM41906 in the lungs, we co-encapsulated these compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of this Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, "green" β-cyclodextrin (pCD) based NPs displayed the best physico-chemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mouse lungs using a Microsprayer®, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair in TB chemotherapy.