RESUMEN

In late 2019, the emergence of a novel coronavirus led to its identification as SARS-CoV-2, precipitating the onset of the COVID-19 pandemic. Many experimental and computational studies were performed on SARS-CoV-2 to understand its behavior and patterns. In this research, Molecular Dynamic (MD) simulation is utilized to compare the behaviors of SARS-CoV-2 and its Variants of Concern (VOC)-Alpha, Beta, Gamma, Delta, and Omicron-with the hACE2 protein. Protein structures from the Protein Data Bank (PDB) were aligned and trimmed for consistency using Chimera, focusing on the receptor-binding domain (RBD) responsible for ACE2 interaction. MD simulations were performed using Visual Molecular Dynamics (VMD) and Nanoscale Molecular Dynamics (NAMD2), and salt bridges and hydrogen bond data were extracted from the results of these simulations. The data extracted from the last 5 ns of the 10 ns simulations were visualized, providing insights into the comparative stability of each variant's interaction with ACE2. Moreover, electrostatics and hydrophobic protein surfaces were calculated, visualized, and analyzed. Our comprehensive computational results are helpful for drug discovery and future vaccine designs as they provide information regarding the vital amino acids in protein-protein interactions (PPIs). Our analysis reveals that the Original and Omicron variants are the two most structurally similar proteins. The Gamma variant forms the strongest interaction with hACE2 through hydrogen bonds, while Alpha and Delta form the most stable salt bridges; the Omicron is dominated by positive potential in the binding site, which makes it easy to attract the hACE2 receptor; meanwhile, the Original, Beta, Delta, and Omicron variants show varying levels of interaction stability through both hydrogen bonds and salt bridges, indicating that targeted therapeutic agents can disrupt these critical interactions to prevent SARS-CoV-2 infection.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , COVID-19 , Simulación de Dinámica Molecular , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Humanos , COVID-19/virología , COVID-19/metabolismo , Enlace de Hidrógeno , Sitios de Unión

RESUMEN

The involvement of neutrophils in the lungs during the recovery phase of coronavirus disease 2019 (COVID-19) is not well defined mainly due to the limited accessibility of lung tissues from COVID-19 survivors. The lack of an appropriate small animal model has affected the development of effective therapeutic strategies. We here developed a long COVID mouse model to study changes in neutrophil phenotype and association with lung injury. Our data shows persistent neutrophil recruitment and neutrophil extracellular trap formation in the lungs for up to 30 days post-infection which correlates with lung fibrosis and inflammation.

Asunto(s)

COVID-19 , Modelos Animales de Enfermedad , Trampas Extracelulares , Pulmón , Neutrófilos , SARS-CoV-2 , Animales , Trampas Extracelulares/inmunología , COVID-19/inmunología , COVID-19/complicaciones , Ratones , Neutrófilos/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , Pulmón/patología , Pulmón/inmunología , Pulmón/virología , Lesión Pulmonar/inmunología , Lesión Pulmonar/virología , Lesión Pulmonar/patología , Lesión Pulmonar/etiología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Infiltración Neutrófila/inmunología , Humanos , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/etiología

RESUMEN

Aging is identified as a significant risk factor for severe coronavirus disease-2019 (COVID-19), often resulting in profound lung damage and mortality. Yet, the biological relationship between aging, aging-related comorbidities, and COVID-19 remains incompletely understood. This study aimed to elucidate the age-related COVID19 pathogenesis using an Hutchinson-Gilford progeria syndrome (HGPS) mouse model, a premature aging disease model, with humanized ACE2 receptors. Pathological features were compared between young, aged, and HGPS hACE2 mice following SARS-CoV-2 challenge. We demonstrated that young mice display robust interferon response and antiviral activity, whereas this response is attenuated in aged mice. Viral infection in aged mice results in severe respiratory tract hemorrhage, likely contributing a higher mortality rate. In contrast, HGPS hACE2 mice exhibit milder disease manifestations characterized by minor immune cell infiltration and dysregulation of multiple metabolic processes. Comprehensive transcriptome analysis revealed both shared and unique gene expression dynamics among different mouse groups. Collectively, our studies evaluated the impact of SARS-CoV-2 infection on progeroid syndromes using a HGPS hACE2 mouse model, which holds promise as a useful tool for investigating COVID-19 pathogenesis in individuals with premature aging.

Asunto(s)

Envejecimiento Prematuro , Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , Progeria , SARS-CoV-2 , Animales , COVID-19/virología , COVID-19/patología , Envejecimiento Prematuro/virología , Envejecimiento Prematuro/genética , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , SARS-CoV-2/patogenicidad , Progeria/genética , Progeria/patología , Humanos , Pulmón/patología , Pulmón/virología , Femenino

RESUMEN

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , COVID-19 , Péptidos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Humanos , Animales , SARS-CoV-2/efectos de los fármacos , COVID-19/virología , Ratones , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Péptidos/farmacología , Péptidos/química , Péptidos/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Antivirales/farmacología , Antivirales/química , Línea Celular , Neumonía/tratamiento farmacológico , Neumonía/virología , Neumonía/prevención & control , Pulmón/virología , Pulmón/patología , Femenino

RESUMEN

The recently emerged BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 variants have a growth advantage. In this study, we explore the structural bases of receptor binding and immune evasion for the Omicron BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 sub-variants. Our findings reveal that BA.2.86 exhibits strong receptor binding, whereas its JN.1 sub-lineage displays a decreased binding affinity to human ACE2 (hACE2). Through complex structure analyses, we observed that the reversion of R493Q in BA.2.86 receptor binding domain (RBD) plays a facilitating role in receptor binding, while the L455S substitution in JN.1 RBD restores optimal affinity. Furthermore, the structure of monoclonal antibody (mAb) S309 complexed with BA.2.86 RBD highlights the importance of the K356T mutation, which brings a new N-glycosylation motif, altering the binding pattern of mAbs belonging to RBD-5 represented by S309. These findings emphasize the importance of closely monitoring BA.2.86 and its sub-lineages to prevent another wave of SARS-CoV-2 infections.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Anticuerpos Monoclonales , COVID-19 , Evasión Inmune , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , COVID-19/inmunología , COVID-19/virología , COVID-19/metabolismo , Sitios de Unión , Modelos Moleculares , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Mutación

RESUMEN

COVID-19 causes more severe and frequently fatal disease in patients with pre-existing comorbidities such as hypertension and heart disease. SARS-CoV-2 virus enters host cells through the angiotensin-converting enzyme 2 (ACE2), which is fundamental in maintaining arterial pressure through the renin-angiotensin system (RAS). Hypertensive patients commonly use medications such as angiotensin-converting enzyme inhibitors (ACEi), which can modulate the expression of ACE2 and, therefore, potentially impact the susceptibility and severity of SARS-CoV-2 infection. Here we assessed whether treatment of ACE2-humanized (K18-hACE2) mice with the ACEi Lisinopril affects lung ACE2 levels and the outcome of experimental COVID-19. K18-hACE2 mice were treated for 21 days with Lisinopril 10 mg/kg and were then infected with 105 PFU of SARS-CoV-2 (Wuhan strain). Body weight, clinical score, respiratory function, survival, lung ACE2 levels, viral load, lung histology, and cytokine (IL-6, IL-33, and TNF-α) levels were assessed. Mice treated with Lisinopril for 21 days showed increased levels of ACE2 in the lungs. Infection with SARS-CoV-2 led to massive decrease in lung ACE2 levels at 3 days post-infection (dpi) in treated and untreated animals, but Lisinopril-treated mice showed a fast recovery (5dpi) of ACE2 levels. Higher ACE2 levels in Lisinopril-treated mice led to remarkably higher lung viral loads at 3 and 6/7dpi. Lisinopril-treated mice showed decreased levels of the pro-inflammatory cytokines IL-6 and TNF-α in the serum and lungs at 6/7dpi. Marginal improvements in body weight, clinical score and survival were observed in Lisinopril-treated mice. No differences between treated and untreated infected mice were observed in respiratory function and lung histology. Lisinopril treatment showed both deleterious (higher viral loads) and beneficial (anti-inflammatory and probably anti-constrictory and anti-coagulant) effects in experimental COVID-19. These effects seem to compensate each other, resulting in marginal beneficial effects in terms of outcome for Lisinopril-treated animals.

RESUMEN

The SARS-CoV-2 pandemic and the emergence of novel virus variants have had a dramatic impact on public health and the world economy, underscoring the need for detailed studies that explore the high efficacy of additional vaccines in animal models. In this study, we confirm the pathogenicity of the SARS-CoV-2/Leiden_008 isolate (GenBank accession number MT705206.1) in K18-hACE2 transgenic mice. Using this isolate, we show that a vaccine consisting of capsid virus-like particles (cVLPs) displaying the receptor-binding domain (RBD) of SARS-CoV-2 (Wuhan strain) induces strong neutralizing antibody responses and sterilizing immunity in K18-hACE2 mice. Furthermore, we demonstrate that vaccination with the RBD-cVLP vaccine protects mice from both a lethal infection and symptomatic disease. Our data also indicate that immunization significantly reduces inflammation and lung pathology associated with severe disease in mice. Additionally, we show that the survival of naïve animals significantly increases when sera from animals vaccinated with RBD-cVLP are passively transferred, prior to a lethal virus dose. Finally, the RBD-cVLP vaccine has a similar antigen composition to the clinical ABNCOV2 vaccine, which has shown non-inferiority to the Comirnaty mRNA vaccine in phase I-III trials. Therefore, our study provides evidence that this vaccine design is highly immunogenic and confers full protection against severe disease in mice.

RESUMEN

Male cases diagnosed COVID-19 with more complications and higher mortality compared with females, and the overall consequences of male sex hormones and semen parameters deterioration were observed in COVID-19 patients, whereas the involvement and mechanism for spermatogenic cell remains unclear. The study was aimed to investigate the infection mode of S protein (D614G) pseudovirus (pseu-S-D614G) to spermatogenic cells, as well as the influence on cell growth. Both mouse spermatogonia (GC-1 cell, immortalized spermatogonia) and spermatocyte (GC-2 cell, immortalized spermatocytes) were used to detect the infection of pseu-S-D614G of SARS-CoV-2, and further explored the effect of SARS-CoV-2-spike protein (S-protein) and SARS-CoV-2-spike protein (omicron) (O-protein) on GC-1 cell apoptosis and proliferation. The data showed that the pseu-S-D614G invaded into GC-1 cells through either human ACE2 (hACE2) or human CD147 (hCD147), whereas GC-2 cells were insensitive to viral infection. In addition, the apoptosis and proliferation suppression inflicted by S-protein and O-protein on GC-1 cells was through Bax-Caspase3 signaling rather than arresting cell cycle progression. These findings suggest that CD147, apart from ACE2, may be a potential receptor for SARS-CoV-2 infection in testicular tissues, and that the apoptotic effect was induced in spermatogonia cells by S-protein or O-protein, eventually resulted in the damage to male fertility.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Apoptosis , Basigina , COVID-19 , SARS-CoV-2 , Espermatogonias , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Masculino , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Apoptosis/fisiología , Basigina/metabolismo , Línea Celular , Proliferación Celular , COVID-19/metabolismo , Espermatocitos/metabolismo , Espermatocitos/virología , Espermatogonias/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo

RESUMEN

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

Asunto(s)

Vacuna BCG , Encéfalo , COVID-19 , SARS-CoV-2 , Animales , Ratones , Vacuna BCG/administración & dosificación , COVID-19/inmunología , COVID-19/virología , SARS-CoV-2/fisiología , Encéfalo/patología , Encéfalo/virología , Carga Viral , Pulmón/patología , Pulmón/virología , Pulmón/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Ratones Transgénicos , Femenino

RESUMEN

Obesity is a risk factor for developing severe COVID-19. However, the mechanism underlying obesity-accelerated COVID-19 remains unclear. Here, we report results from a study in which 2-3-month-old K18-hACE2 (K18) mice were fed a western high-fat diet (WD) or normal chow (NC) over 3 months before intranasal infection with a sublethal dose of SARS-CoV2 WA1 (a strain ancestral to the Wuhan variant). After infection, the WD-fed K18 mice lost significantly more body weight and had more severe lung inflammation than normal chow (NC)-fed mice. Bulk RNA-seq analysis of lungs and adipose tissue revealed a diverse landscape of various immune cells, inflammatory markers, and pathways upregulated in the infected WD-fed K18 mice when compared with the infected NC-fed control mice. The transcript levels of IL-6, an important marker of COVID-19 disease severity, were upregulated in the lung at 6-9 days post-infection in the WD-fed mice when compared to NC-fed mice. Transcriptome analysis of the lung and adipose tissue obtained from deceased COVID-19 patients found that the obese patients had an increase in the expression of genes and the activation of pathways associated with inflammation as compared to normal-weight patients (n = 2). The K18 mouse model and human COVID-19 patient data support a link between inflammation and an obesity-accelerated COVID-19 disease phenotype. These results also indicate that obesity-accelerated severe COVID-19 caused by SARS-CoV-2 WA1 infection in the K18 mouse model would be a suitable model for dissecting the cellular and molecular mechanisms underlying pathogenesis.

Asunto(s)

COVID-19 , Pulmón , Obesidad , SARS-CoV-2 , Regulación hacia Arriba , COVID-19/genética , COVID-19/virología , COVID-19/metabolismo , COVID-19/patología , Animales , Obesidad/genética , Obesidad/metabolismo , Obesidad/complicaciones , Ratones , Humanos , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Dieta Alta en Grasa/efectos adversos , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , Modelos Animales de Enfermedad , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Índice de Severidad de la Enfermedad , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo

RESUMEN

Severe acute respiratory syndrome coronavirus 2 had devastating consequences for human health. Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID + EP), it induced high level of anti-S1 IgG and neutralizing antibodies (P < 0.0001), strong Th1-biased immune response as shown by IgG2a polarization (P < 0.01), increase in IFN-γ levels (P < 0.01), and increment in the ratio of IFN-γ secreting CD4+ (3.78-10.19%) and CD8+ (5.24-12.51%) T cells. Challenging K18-hACE2 transgenic mice showed that pcoSpikeD614G administered through IM and ID + EP routes conferred 90-100% protection and there was no sign of pneumonia. Subsequently, pcoSpikeD614G was evaluated as a promising DNA vaccine candidate and scale-up studies were performed. Accordingly, a large-scale production process was described, including a 36 h fermentation process of E. coli DH5α cells containing pcoSpikeD614G resulting in a wet cell weight of 242 g/L and a three-step chromatography for purification of the pcoSpikeD614G DNA vaccine.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Ratones Endogámicos BALB C , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunas de ADN , Vacunas de ADN/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Ratones , COVID-19/prevención & control , COVID-19/inmunología , Células HEK293 , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Femenino , Inmunogenicidad Vacunal , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología

RESUMEN

While an association between Parkinson's disease (PD) and viral infections has been recognized, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on PD progression remains unclear. Here, we demonstrate that SARS-CoV-2 infection heightens the risk of PD using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons and a human angiotensin-converting enzyme 2 (hACE2) transgenic (Tg) mouse model. Our findings reveal that SARS-CoV-2 infection exacerbates PD susceptibility and cellular toxicity in DA neurons pre-treated with human preformed fibrils (hPFFs). Additionally, nasally delivered SARS-CoV-2 infects DA neurons in hACE2 Tg mice, aggravating the damage initiated by hPFFs. Mice infected with SARS-CoV-2 display persisting neuroinflammation even after the virus is no longer detectable in the brain. A comprehensive analysis suggests that the inflammatory response mediated by astrocytes and microglia could contribute to increased PD susceptibility associated with SARS-CoV-2. These findings advance our understanding of the potential long-term effects of SARS-CoV-2 infection on the progression of PD.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas , Ratones Transgénicos , Enfermedad de Parkinson , SARS-CoV-2 , Animales , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/virología , Humanos , COVID-19/patología , COVID-19/virología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/virología , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Microglía/patología , Microglía/metabolismo , Microglía/virología , Células Madre Embrionarias Humanas/metabolismo , Astrocitos/patología , Astrocitos/virología , Astrocitos/metabolismo , Encéfalo/patología , Encéfalo/virología

RESUMEN

Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR - Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 × 105 PFU) or low (2.5 × 102 PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Antivirales , COVID-19 , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , SARS-CoV-2 , Animales , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Ratones , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Humanos , Pulmón/virología , Pulmón/patología , Tratamiento Farmacológico de COVID-19 , Queratina-18/genética , Carga Viral , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/farmacología , Técnicas de Sustitución del Gen , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Femenino

RESUMEN

Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019. In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virus-infected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105 PFU; however, 1×102 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.

RESUMEN

Objective: The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model. Methods: In this study, we established a mouse model to mimic the SARS-CoV-2 infection acute lung injury model via intratracheal injection of polyinosinic:polycytidylic acid (poly[I:C]) and SARS-CoV-2 recombinant spike protein (SP). After the different agents treatment, lung sections, bronchoalveolar lavage fluid (BALF) and fresh faeces were harvested. Then, H&E staining was used to examine symptoms of interstitial pneumonia. Flow cytometry was used to examine the change of immune cell populations. Multiplex cytokines assay was used to examine the inflammatory cytokines.16S rDNA high-throughput sequencing was used to examine the change of gut microbiome. Results: Our results showed that AGE and SFN significantly suppressed the symptoms of interstitial pneumonia, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated T cell populations in the mouse model. Furthermore, we also observed that the gut microbiome of genus Paramuribaculum were enriched in the AGE-treated group. Conclusion: Here, for the first time, we observed that these two novel, safe, and relatively inexpensive immunomodulatory agents exhibited the same effects on anti-inflammatory and immune responses as neutralizing monoclonal antibodies (mAbs) against interleukin 6 receptor (IL-6R), which have been suggested for treating COVID-19 patients. Our results revealed the therapeutic ability of these two immunomodulatory agents in a mouse model of SARS-CoV-2 acute lung injury by promoting anti-inflammatory and immune responses. These results suggest that AGE and SFN are promising candidates for the COVID-19 treatment.

Asunto(s)

Lesión Pulmonar Aguda , Enzima Convertidora de Angiotensina 2 , Antiinflamatorios , Tratamiento Farmacológico de COVID-19 , COVID-19 , Modelos Animales de Enfermedad , Agentes Inmunomoduladores , SARS-CoV-2 , Animales , Ratones , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/etiología , COVID-19/inmunología , SARS-CoV-2/inmunología , Agentes Inmunomoduladores/farmacología , Agentes Inmunomoduladores/uso terapéutico , Antiinflamatorios/uso terapéutico , Antiinflamatorios/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Isotiocianatos/farmacología , Isotiocianatos/uso terapéutico , Sulfóxidos , Humanos , Citocinas/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Pulmón/efectos de los fármacos , Masculino , Poli I-C , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico

RESUMEN

BACKGROUND: SARS-CoV-2 infection is considered as a relapsing inflammatory process with a dysregulation of IL-6 signalling. Classic IL-6 signalling is thought to represent a defence mechanism against pathogens. In contrast, IL-6 trans-signalling has pro-inflammatory effects. In severe COVID-19, therapeutic strategies have focused on global inhibition of IL-6, with controversial results. We hypothesized that specific blockade of IL-6 trans-signalling could inhibit inflammatory response preserving the host defence activity inherent to IL-6 classic signalling. METHODS: To test the role of the specific IL-6 trans-signalling inhibition by sgp130Fc in short- and long-term consequences of COVID-19, we used the established K18-hACE2 transgenic mouse model. Histological as well as immunohistochemical analysis, and pro-inflammatory marker profiling were performed. To investigate IL-6 trans-signalling in human cells we used primary lung microvascular endothelial cells and fibroblasts in the presence/absence of sgp130Fc. FINDINGS: We report that targeting IL-6 trans-signalling by sgp130Fc attenuated SARS-CoV-2-related clinical symptoms and mortality. In surviving mice, the treatment caused a significant decrease in lung damage. In vitro, IL-6 trans-signalling induced strong and persisting JAK1/STAT3 activation in endothelial cells and lung fibroblasts with proinflammatory effects, which were attenuated by sgp130Fc. Our data also suggest that in those cells with scant amounts of IL-6R, the induction of gp130 and IL-6 by IL-6:sIL-6R complex sustains IL-6 trans-signalling. INTERPRETATION: IL-6 trans-signalling fosters progression of COVID-19, and suggests that specific blockade of this signalling mode could offer a promising alternative to mitigate both short- and long-term consequences without affecting the beneficial effects of IL-6 classic signalling. These results have implications for the development of new therapies of lung injury and endotheliopathy in COVID-19. FUNDING: The project was supported by ISCIII, Spain (COV-20/00792 to MB, PI23/01351 to MARH) and the European Commission-Next generation EU (European Union) (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global, SGL2103029 to MB). PID2019-110587RB-I00 (MB) supported by MICIN/AEI/10.13039/501100011033/and PID2022-143034OB-I00 (MB) by MICIN/AEI/10.13039/501100011033/FEDER. MAR-H acknowledges support from ISCIII, Spain and the European Commission-Next generation EU (European Union), through CSIC's Global Health PTI.

Asunto(s)

COVID-19 , Receptor gp130 de Citocinas , Interleucina-6 , Ratones Transgénicos , SARS-CoV-2 , Transducción de Señal , Animales , Humanos , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Betacoronavirus , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/patología , COVID-19/metabolismo , Tratamiento Farmacológico de COVID-19 , Receptor gp130 de Citocinas/metabolismo , Receptor gp130 de Citocinas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Interleucina-6/metabolismo , Pulmón/patología , Pulmón/virología , Pulmón/metabolismo , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Neumonía Viral/patología , Neumonía Viral/metabolismo , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-6/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos

RESUMEN

BACKGROUND: Extracellular vesicles (EVs) are emerging as potential drug carriers in the fight against COVID-19. This study investigates the ability of EVs as drug carriers to target SARS-CoV-2-infected cells. METHODS: EVs were modified using Xstamp technology to carry the virus's RBD, enhancing targeting ability to hACE2+ cells and improving drug delivery efficiency. Characterization confirmed EVs' suitability as drug carriers. For in vitro tests, A549, Caco-2, and 4T1 cells were used to assess the targeting specificity of EVRs (EVs with membrane-surface enriched RBD). Moreover, we utilized an ex vivo lung tissue model overexpressing hACE2 as an ex vivo model to confirm the targeting capability of EVRs toward lung tissue. The study also evaluated drug loading efficiency and assessed the potential of the anti-inflammatory activity on A549 lung cancer cells exposed to lipopolysaccharide. Results demonstrate the successful construction of RBD-fused EVRs on the membrane-surface. In both in vitro and ex vivo models, EVRs significantly enhance their targeting ability towards hACE2+ cells, rendering them a safe and efficient drug carrier. Furthermore, ultrasound loading efficiently incorporates IL-10 into EVRs, establishing an effective drug delivery system that ameliorates the pro-inflammatory response induced by LPS-stimulated A549 cells. CONCLUSION: These findings indicate promising opportunities for engineered EVs as a novel nanomedicine carrier, offering valuable insights for therapeutic strategies against COVID-19 and other diseases.

RESUMEN

The SARS-CoV-2 betacoronavirus infects people through binding the human Angiotensin Receptor 2 (ACE2), followed by import into a cell utilizing the Transmembrane Protease, Serine 2 (TMPRSS2) and Furin cofactors. Analysis of the SARS-CoV-2 extracellular spike protein has suggested critical amino acids necessary for binding within a 197-residue portion, the receptor binding domain (RBD). A cell-based assay between a membrane tethered RBD-GFP fusion protein and the membrane bound ACE2-Cherry fusion protein allowed for mutational intersection of both RBD and ACE2 proteins. Data shows Omicron BA.1 and BA.2 variants have altered dependency on the amino terminus of ACE2 protein and suggests multiple epitopes on both proteins stabilize their interactions at the Nt and internal region of ACE2. In contrast, the H-CoV-NL63 RBD is only dependent on the ACE2 internal region for binding. A peptide inhibitor approach to this internal region thus far have failed to block binding of RBDs to ACE2, suggesting that several binding regions on ACE2 are sufficient to allow functional interactions. In sum, the RBD binding surface of ACE2 appears relatively fluid and amenable to bind a range of novel variants.

RESUMEN

It is widely accepted that the SARS-CoV-2 betacoronavirus infects humans through binding the human Angiotensin Receptor 2 (ACE2) that lines the nasal cavity and lungs, followed by import into a cell utilizing the Transmembrane Protease, Serine 2 (TMPRSS2) cofactor. ACE2 binding is mediated by an approximately 200-residue portion of the SARS-CoV-2 extracellular spike protein, the receptor binding domain (RBD). Robust interactions are shown using a novel cell-based assay between an RBD membrane tethered-GFP fusion protein and the membrane bound ACE2-Cherry fusion protein. Several observations were not predicted including, quick and sustained interactions leading to internalization of RBD fusion protein into the ACE2 cells and rapid downregulation of the ACE2-Cherry fluorescence. Targeted mutation in the RBD disulfide Loop 4 led to a loss of internalization for several variants tested. However, a secreted RBD did not cause ACE2 downregulation of ACE2-Cherry fluorescence. Thus, the membrane associated form of RBD found on the viral coat may have long-term system wide consequences on ACE2 expressing cells.

RESUMEN

COVID-19 is a global pandemic caused by the highly infectious SARS-CoV-2 virus. Efforts to combat SARS-CoV-2 infection include mass vaccination and development of monoclonal and convalescent plasma therapeutics that require precise measurements of correlative, functional neutralizing antibodies that prevent virus infection. Developing rapid, safe, easy-to-use, and high-quality neutralization assays are essential for the success of the massive effort. Here, we developed a vesicular stomatitis virus-based neutralization assay that was capable of quantifying varying degrees of neutralization in patient serum samples. This assay has two detection readouts, flow cytometry and live cell imaging. The two readout methods produced consistent values of all 50% neutralization titers, further enhancing measurement confidence on the assay. Moreover, the use of available reference standards such as the World Health Organization International Standard (NIBSC code 20/136) enables quantification and standardization of the pseudovirus neutralization assay with neutralizing antibody titers measured in International Units/mL. Quantitative and standardized neutralization assays are critical for reliable efficacy evaluation and comparison of numerous vaccines and therapeutics.

Asunto(s)

COVID-19 , Humanos , SARS-CoV-2 , Sueroterapia para COVID-19 , Pruebas Inmunológicas , Citometría de Flujo , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus