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The SARS-CoV-2 pandemic has caused unprecedented worldwide infections from persistent mutant variants with various degrees of infectivity and virulence. The elusiveness of a highly penetrant, worldwide vaccination strategy suggests that the complete eradication of SARS-CoV-2 is unlikely. Even with the advent of new antiviral agents, the disease burden worldwide continues to exceed current preventative and therapeutic strategies. Greater interest has been placed towards the development of affordable,broadly effective antiviral therapeutics. Here, we report that the small branched-chain fatty acid Valproic acid (VPA), approved for maintenance of seizure and bipolar disorder, has a novel anti- coronavirus activity that can be augmented with the addition of a long-chain, polyunsaturated omega-3 fatty acid, Docosahexaenoic acid (DHA). An EMR-based epidemiological study of patients tested for COVID-19 demonstrated a correlation exists between a reduced infection rate in patients treated withVPA of up to 25%, as well as a decreased risk of emergency room visits, hospitalization, ICU admission,and use of mechanical ventilation. In vitro studies have demonstrated that VPA modifies gene expression in MRC5 cells. Interestingly, VPA correlates with the inhibition of several SARS-CoV2 interacting genes and the greater inhibition of alpha-coronavirus HCoV-229E (a "common cold" virus) and SARS-CoV2. The VPA-DHA combination activates pre-existing intracellular antiviral mechanisms normally repressed by coronaviruses. Gene expression profiles demonstrate subtle differences in overall gene expression between VPA-treated and VPA-DHA-treated cells. HCoV-229E infection caused an intensely different response with a marked induction of multiple intracellular inflammatory genes. Changes in gene expression took at least 24 hours to manifest and most likely why prior drug screens failed to identify any antiviral VPA activity despite in silico predictions. This report demonstrates an interaction between HDAC inhibition and the potent activation of cellular antiviral responses. A foundation now exists for a low-cost, highly effective antiviral strategy when supplemented with DHA.
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Antivirales , COVID-19 , SARS-CoV-2 , Ácido Valproico , Ácido Valproico/farmacología , Ácido Valproico/uso terapéutico , Humanos , COVID-19/epidemiología , COVID-19/virología , SARS-CoV-2/efectos de los fármacos , Antivirales/uso terapéutico , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/uso terapéutico , Índice de Severidad de la Enfermedad , Masculino , Femenino , Persona de Mediana EdadRESUMEN
As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1-/- mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.
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Administración Intranasal , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Femenino , SARS-CoV-2/inmunología , SARS-CoV-2/genética , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ratones , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Cricetinae , Humanos , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Vacuna contra el Sarampión-Parotiditis-Rubéola/administración & dosificación , Virus del Sarampión/inmunología , Virus del Sarampión/genética , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Virus de la Parotiditis/inmunología , Virus de la Parotiditis/genética , Ratones Noqueados , Mesocricetus , Inmunoglobulina A/inmunología , Inmunoglobulina A/sangreRESUMEN
SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1Å resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.
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SARS-CoV-2 uses the double-membrane vesicles as replication organelles. However, how virion assembly occurs has not been fully understood. Here we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs have unusual electron-dense and dynamic inner structures, and their formation is driven by the accessory protein ORF3a via hijacking a specific subset of the trans-Golgi network (TGN) and early endosomal membranes. 3DB formation is conserved in related bat and pangolin coronaviruses yet lost during the evolution to SARS-CoV. 3DBs recruit the viral structural proteins spike (S) and membrane (M) and undergo dynamic fusion/fission to facilitate efficient virion assembly. A recombinant SARS-CoV-2 virus with an ORF3a mutant specifically defective in 3DB formation showed dramatically reduced infectivity for both extracellular and cell-associated virions. Our study uncovers the crucial role of 3DB in optimal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.
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The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
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Transporte Activo de Núcleo Celular , COVID-19 , Proteínas de Transporte Nucleocitoplasmático , ARN Mensajero , Proteínas de Unión al ARN , SARS-CoV-2 , Proteínas no Estructurales Virales , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Animales , COVID-19/virología , COVID-19/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Replicación Viral , Núcleo Celular/metabolismo , Células Vero , Virulencia , Chlorocebus aethiops , Células HEK293RESUMEN
Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.
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Lesión Renal Aguda , Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Muerte Celular Regulada , Humanos , Proteoma/metabolismo , Subtipo H3N2 del Virus de la Influenza A/fisiología , Replicación Viral/fisiología , Riñón/patología , Infecciones por Orthomyxoviridae/patologíaRESUMEN
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.
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COVID-19 , SARS-CoV-2 , Animales , Ratones , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Endorribonucleasas/metabolismo , Transducción de Señal , AntiviralesRESUMEN
A previously unknown coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was isolated in Wuhan, China in December 2019, from a patient with a respiratory disease linked to potential contact with wild animals [...].
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COVID-19 , Animales , Humanos , SARS-CoV-2 , Animales Salvajes , China/epidemiologíaRESUMEN
A previously unknown coronavirus, named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in the city of Wuhan, China, in December 2019 [...].
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Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still an ongoing global health crisis. Clinical data indicate that the case fatality rate (CFR) is age dependent, with a higher CFR percentage in the elderly population. We compared the pathogenesis of SARS-CoV-2 in young and aged K18-hACE2 transgenic mice. We evaluated morbidity, mortality, viral titers, immune responses, and histopathology in SARS-CoV-2-infected young and old K18-hACE2 transgenic mice. Within the limitation of having a low number of mice per group, our results indicate that SARS-CoV-2 infection resulted in slightly higher morbidity, mortality, and viral replication in the lungs of old mice, which was associated with an impaired IgM response and altered cytokine and chemokine profiles. Results of this study increase our understanding of SARS-CoV-2 infectivity and immuno-pathogenesis in the elderly population.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Anciano , Animales , Humanos , Ratones , COVID-19/inmunología , COVID-19/metabolismo , Citocinas , Modelos Animales de Enfermedad , Ratones Transgénicos , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/genética , Inmunoglobulina MRESUMEN
Following virus recognition of host cell receptors and viral particle/genome internalization, viruses replicate in the host via hijacking essential host cell machinery components to evade the provoked antiviral innate immunity against the invading pathogen. Respiratory viral infections are usually acute with the ability to activate pattern recognition receptors (PRRs) in/on host cells, resulting in the production and release of interferons (IFNs), proinflammatory cytokines, chemokines, and IFN-stimulated genes (ISGs) to reduce virus fitness and mitigate infection. Nevertheless, the game between viruses and the host is a complicated and dynamic process, in which they restrict each other via specific factors to maintain their own advantages and win this game. The primary role of the non-structural protein 1 (NS1 and Nsp1) of influenza A viruses (IAV) and the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively, is to control antiviral host-induced innate immune responses. This review provides a comprehensive overview of the genesis, spatial structure, viral and cellular interactors, and the mechanisms underlying the unique biological functions of IAV NS1 and SARS-CoV-2 Nsp1 in infected host cells. We also highlight the role of both non-structural proteins in modulating viral replication and pathogenicity. Eventually, and because of their important role during viral infection, we also describe their promising potential as targets for antiviral therapy and the development of live attenuated vaccines (LAV). Conclusively, both IAV NS1 and SARS-CoV-2 Nsp1 play an important role in virus-host interactions, viral replication, and pathogenesis, and pave the way to develop novel prophylactic and/or therapeutic interventions for the treatment of these important human respiratory viral pathogens.
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COVID-19 , Virus de la Influenza A , Humanos , Inmunidad Innata , Virus de la Influenza A/genética , Interferones/inmunología , SARS-CoV-2/metabolismo , Replicación ViralRESUMEN
The role of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 Spike (S) on disease pathogenesis was investigated. For this, we generated recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 S gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 strain genome. The recombinant viruses were characterized in vitro and in vivo. Viral entry, cell-cell fusion, plaque size, and the replication kinetics of the rWA1-Omi-S virus were markedly impaired when compared to the rWA1-D614G virus, demonstrating a lower fusogenicity and ability to spread cell-to-cell of rWA1-Omi-S. To assess the contribution of the Omicron BA.1 S protein to SARS-CoV-2 pathogenesis, the pathogenicity of rWA1-D614G and rWA1-Omi-S viruses was compared in a feline model. While the rWA1-D614G-inoculated cats were lethargic and showed increased body temperatures on days 2 and 3 post-infection (pi), rWA1-Omi-S-inoculated cats remained subclinical and gained weight throughout the 14-day experimental period. Animals inoculated with rWA1-D614G presented higher infectious virus shedding in nasal secretions, when compared to rWA1-Omi-S-inoculated animals. In addition, tissue replication of the rWA1-Omi-S was markedly reduced compared to the rWA1-D614G, as evidenced by lower viral load in tissues on days 3 and 5 pi. Histologic examination of the nasal turbinate and lungs revealed intense inflammatory infiltration in rWA1-D614G-inoculated animals, whereas rWA1-Omi-S-inoculated cats presented only mild to modest inflammation. Together, these results demonstrate that the S protein is a major virulence determinant for SARS-CoV-2 playing a major role for the attenuated phenotype of the Omicron virus. IMPORTANCE: We have demonstrated that the Omicron BA.1.1 variant presents lower pathogenicity when compared to D614G (B.1) lineage in a feline model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are over 50 mutations across the Omicron genome, of which more than two-thirds are present in the Spike (S) protein. To assess the role of the Omicron BA.1 S on virus pathogenesis, recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 Spike gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 were generated. While the Omicron BA.1 S promoted early entry into cells, it led to impaired fusogenic activity and cell-cell spread. Infection studies with the recombinant viruses in a relevant naturally susceptible feline model of SARS-CoV-2 infection here revealed an attenuated phenotype of rWA1-Omi-S, demonstrating that the Omi-S is a major determinant of the attenuated disease phenotype of Omicron strains.
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COVID-19 , Orthopoxvirus , SARS-CoV-2 , Animales , Gatos , COVID-19/virología , Fenotipo , SARS-CoV-2/clasificación , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virulencia , Factores de Virulencia/genéticaRESUMEN
Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.
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Arenaviridae , Coriomeningitis Linfocítica , Humanos , Arenaviridae/metabolismo , Línea Celular , Proteínas Quinasas/metabolismo , Interacciones Huésped-Patógeno , Virus de la Coriomeningitis Linfocítica/metabolismo , Proteínas Portadoras , Antivirales , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.
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Deltainfluenzavirus , Ácidos Neuramínicos , Receptores Virales , Animales , Bovinos , Membrana Celular/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/metabolismo , Orthomyxoviridae/metabolismo , Receptores Virales/metabolismo , Ácidos Siálicos/metabolismoRESUMEN
In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.
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Vacunas contra la COVID-19 , Vacunas contra la Influenza , Animales , Humanos , Ratones , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Formación de Anticuerpos , COVID-19/inmunología , COVID-19/prevención & control , Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/inmunología , Nucleoproteínas , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/química , Vacunas contra la COVID-19/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & controlRESUMEN
BACKGROUND: The continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza activity as novel broad-spectrum anti-influenza medications. METHODS: In this study, nitrogenous alkaloids were tested for their viral inhibitory activity against influenza A/H1N1 and A/H5N1 viruses. The cytotoxicity of tested alkaloids on MDCK showed a high safety range (CC50 > 200 µg/ml), permitting the screening for their anti-influenza potential. RESULTS: Herein, atropine sulphate, pilocarpine hydrochloride and colchicine displayed anti-H5N1 activities with IC50 values of 2.300, 0.210 and 0.111 µg/ml, respectively. Validation of the IC50 values was further depicted by testing the three highly effective alkaloids, based on their potent IC50 values against seasonal influenza A/H1N1 virus, showing comparable IC50 values of 0.204, 0.637 and 0.326 µg/ml, respectively. Further investigation suggests that colchicine could suppress viral infection by primarily interfering with IAV replication and inhibiting viral adsorption, while atropine sulphate and pilocarpine hydrochloride could directly affect the virus in a cell-free virucidal effect. Interestingly, the in silico molecular docking studies suggest the abilities of atropine, pilocarpine, and colchicine to bind correctly inside the active sites of the neuraminidases of both influenza A/H1N1 and A/H5N1 viruses. The three alkaloids exhibited good binding energies as well as excellent binding modes that were similar to the co-crystallized ligands. On the other hand, consistent with in vitro results, only colchicine could bind correctly against the M2-proton channel of influenza A viruses (IAVs). This might explicate the in vitro antiviral activity of colchicine at the replication stage of the virus replication cycle. CONCLUSION: This study highlighted the anti-influenza efficacy of biologically active alkaloids including colchicine. Therefore, these alkaloids should be further characterized in vivo (preclinical and clinical studies) to be developed as anti-IAV agents.
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Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Humanos , Colchicina/farmacología , Pilocarpina , Gripe Humana/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Estaciones del Año , Fitoquímicos/farmacología , Atropina , Antivirales/farmacologíaRESUMEN
The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten-like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile-filtered freeze-dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome-displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween-80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli-derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin-phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome-bound format prior to injection. Protein particles can readily be formed with sequent-divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of mice elicits antibodies that neutralize respective viral strains. When K18-hACE2 transgenic mice are immunized and challenged with ancestral SARS-CoV-2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates.
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Vacunas contra la COVID-19 , COVID-19 , Animales , Ratones , Nanovacunas , SARS-CoV-2 , Escherichia coli , Liposomas , COVID-19/prevención & control , LípidosRESUMEN
Mitochondrial dysfunction alters cellular metabolism, increases tissue oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, it is investigated whether the transfer of functional mitochondria into CD4+ T cells that are isolated from old mice (aged CD4+ T cells), can abrogate aging-associated mitochondrial dysfunction, and improve the aged CD4+ T cell functionality. The results show that the delivery of exogenous mitochondria to aged non-activated CD4+ T cells led to significant mitochondrial proteome alterations highlighted by improved aerobic metabolism and decreased cellular mitoROS. Additionally, mito-transferred aged CD4+ T cells showed improvements in activation-induced TCR-signaling kinetics displaying markers of activation (CD25), increased IL-2 production, enhanced proliferation ex vivo. Importantly, immune deficient mouse models (RAG-KO) showed that adoptive transfer of mito-transferred naive aged CD4+ T cells, protected recipient mice from influenza A and Mycobacterium tuberculosis infections. These findings support mitochondria as targets of therapeutic intervention in aging.