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1.
Open Biol ; 14(6): 230252, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38835241

RESUMEN

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the 'arms race' with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Humanos , Anticuerpos Antivirales/inmunología , Camélidos del Nuevo Mundo/inmunología , Epítopos/inmunología , Epítopos/química , Cricetinae , Unión Proteica , Modelos Moleculares
2.
bioRxiv ; 2024 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-38464327

RESUMEN

Objectives: Immunocompromised individuals are susceptible to severe COVID-19 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on SARS-CoV-2 infection in k18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection. Methods: Mice were immunosuppressed using cyclophosphamide and infected with a B lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered and viral load and viral sequence diversity was assessed. Results: Treatment of infected but immune compromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion (Ts/Tv) ratio, representative of A>G and C>U mutations and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir.Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristic of variants of concern (VOCs). Conclusions: Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimised public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model.

3.
iScience ; 27(1): 108763, 2024 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-38261926

RESUMEN

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for this respiratory pathogen.

4.
Viruses ; 15(8)2023 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-37631979

RESUMEN

The respiratory system is the main target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 19 (COVID-19) where acute respiratory distress syndrome is considered the leading cause of death. Changes in pulmonary blood vessels, among which an endothelialitis/endotheliitis has been particularly emphasized, have been suggested to play a central role in the development of acute lung injury. Similar vascular changes are also observed in animal models of COVID-19. The present study aimed to determine whether the latter are specific for SARS-CoV-2 infection, investigating the vascular response in the lungs of mice infected with SARS-CoV-2 and other respiratory viruses (influenza A and murine gammaherpesvirus) by in situ approaches (histology, immunohistology, morphometry) combined with RNA sequencing and bioinformatic analysis. Non-selective recruitment of monocytes and T and B cells from larger muscular veins and arteries was observed with all viruses, matched by a comparable transcriptional response. There was no evidence of endothelial cell infection in any of the models. Both the morphological investigation and the transcriptomics approach support the interpretation that the lung vasculature in mice mounts a stereotypic response to alveolar and respiratory epithelial damage. This may have implications for the treatment and management of respiratory disease in humans.


Asunto(s)
COVID-19 , Sistema Cardiovascular , Gammaherpesvirinae , Gripe Humana , Humanos , Animales , Ratones , SARS-CoV-2 , Modelos Animales de Enfermedad
5.
Virology ; 587: 109849, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37515945

RESUMEN

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 µg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811-1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.


Asunto(s)
Gammaherpesvirinae , Infecciones por Herpesviridae , Rhadinovirus , Animales , Ratones , Aminoácidos , Antivirales/metabolismo , Chlorocebus aethiops , Gammaherpesvirinae/genética , Ratones Endogámicos C57BL , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Rhadinovirus/genética , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo
6.
Nat Commun ; 14(1): 3583, 2023 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-37328472

RESUMEN

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.


Asunto(s)
COVID-19 , SARS-CoV-2 , Masculino , Animales , Cricetinae , Ratones , Antivirales/farmacología , Péptidos/farmacología , Anticuerpos , Mesocricetus , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/genética
7.
Emerg Microbes Infect ; 12(1): 2217940, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37219409

RESUMEN

Wild animals are naturally infected with a range of viruses, some of which may be zoonotic. During the human COVID pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this, we sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralization ability that are consistent with rats being exposed to SARS-CoV-2 and/or exposed to other viruses that result in cross-reactive antibodies.


Asunto(s)
COVID-19 , Virus , Humanos , Animales , Ratas , Ratones , SARS-CoV-2 , Roedores , Pandemias , Anticuerpos Antivirales
8.
Viruses ; 14(5)2022 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-35632761

RESUMEN

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, as well as after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally oriented regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and the possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates and broadly support the investigation of agents with adequate penetration into relevant regions of the CNS.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Sistema Nervioso Central , Tropismo Viral , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/complicaciones , Sistema Nervioso Central/fisiopatología , Sistema Nervioso Central/virología , Humanos , Ratones , Ratones Transgénicos , SARS-CoV-2/genética , Síndrome Post Agudo de COVID-19
9.
mSphere ; 6(4): e0021921, 2021 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-34287009

RESUMEN

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.


Asunto(s)
Infecciones por Coronavirus/virología , Microbiota/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Anciano , Animales , COVID-19/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2/genética
10.
Emerg Infect Dis ; 27(2): 517-528, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33496240

RESUMEN

The lack of population health surveillance for companion animal populations leaves them vulnerable to the effects of novel diseases without means of early detection. We present evidence on the effectiveness of a system that enabled early detection and rapid response a canine gastroenteritis outbreak in the United Kingdom. In January 2020, prolific vomiting among dogs was sporadically reported in the United Kingdom. Electronic health records from a nationwide sentinel network of veterinary practices confirmed a significant increase in dogs with signs of gastroenteric disease. Male dogs and dogs living with other vomiting dogs were more likely to be affected. Diet and vaccination status were not associated with the disease; however, a canine enteric coronavirus was significantly associated with illness. The system we describe potentially fills a gap in surveillance in neglected populations and could provide a blueprint for other countries.


Asunto(s)
Infecciones por Coronavirus/veterinaria , Coronavirus Canino , Brotes de Enfermedades , Enfermedades de los Perros/epidemiología , Vómitos/veterinaria , Animales , Enfermedades de los Perros/virología , Perros/virología , Reino Unido/epidemiología
11.
Viruses ; 12(10)2020 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-33066701

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Vacunas contra la COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , ADN Complementario/análisis , ADN Complementario/genética , ADN Viral/análisis , ADN Viral/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Epidemiología Molecular , Reacción en Cadena de la Polimerasa Multiplex , Pandemias , Neumonía Viral/diagnóstico , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Análisis de Secuencia
12.
J Extracell Vesicles ; 8(1): 1632100, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31275534

RESUMEN

Gram-negative bacteria naturally produce and secrete nanosized outer membrane vesicles (OMVs). In the human gastrointestinal tract, OMVs produced by commensal Gram-negative bacteria can mediate interactions amongst host cells (including between epithelial cells and immune cells) and maintain microbial homeostasis. This OMV-mediated pathway for host-microbe interactions could be exploited to deliver biologically active proteins to the body. To test this we engineered the Gram-negative bacterium Bacteroides thetaiotaomicron (Bt), a prominent member of the intestinal microbiota of all animals, to incorporate bacteria-, virus- and human-derived proteins into its OMVs. We then used the engineered Bt OMVs to deliver these proteins to the respiratory and gastrointestinal (GI)-tract to protect against infection, tissue inflammation and injury. Our findings demonstrate the ability to express and package both Salmonella enterica ser. Typhimurium-derived vaccine antigens and influenza A virus (IAV)-derived vaccine antigens within or on the outer membrane of Bt OMVs. These antigens were in a form capable of eliciting antigen-specific immune and antibody responses in both mucosal tissues and systemically. Furthermore, immunisation with OMVs containing the core stalk region of the IAV H5 hemagglutinin from an H5N1 strain induced heterotypic protection in mice to a 10-fold lethal dose of an unrelated subtype (H1N1) of IAV. We also showed that OMVs could express the human therapeutic protein, keratinocyte growth factor-2 (KGF-2), in a stable form that, when delivered orally, reduced disease severity and promoted intestinal epithelial repair and recovery in animals administered colitis-inducing dextran sodium sulfate. Collectively, our data demonstrates the utility and effectiveness of using Bt OMVs as a mucosal biologics and drug delivery platform technology.

13.
Dev Comp Immunol ; 100: 103411, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31202894

RESUMEN

Adipose tissue (AT) has wide functions as an active endocrine organ acting as a site of nutrient storage and thermogenesis. Recently it has been identified as having a key role in murine and human immunity and inflammation. Type 1 or type 2 immune responses and their respective cytokines have been linked to white or brown AT, respectively. Most dramatic is the involvement of type-2 innate lymphoid cells (ILC2s) in stimulating eosinophil recruitment via interleukin (IL)-13 which in turn stimulates alternative macrophage activation via IL-4/IL-13. Recruited leukocytes are capable of influencing the cellular composition and function of adipose tissue and present a route to combat human obesity, however these processes are poorly understood in ruminants. Here we have characterised the resident leukocytes populations within bovine mesenteric AT (MAT) and subcutaneous AT (SAT), compared with the corresponding mesenteric lymph node (MLN). Concurring with related studies, we find bovine AT has its own resident leukocyte populations where eosinophils and neutrophils dominate. Importantly the proportion of eosinophils or neutrophils corresponded to the adipocyte size found in both depots. Further exploration of this area may have important implications on the food production industry or could be applied to improve the course of pathogenesis during disease.


Asunto(s)
Bovinos/inmunología , Inmunidad Innata , Grasa Intraabdominal/citología , Leucocitos/inmunología , Grasa Subcutánea/citología , Adipocitos , Crianza de Animales Domésticos , Animales , Interleucina-13/inmunología , Interleucina-13/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Grasa Intraabdominal/inmunología , Recuento de Leucocitos , Leucocitos/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Macrófagos , Masculino , Mesenterio/citología , Mesenterio/inmunología , Grasa Subcutánea/inmunología
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