RESUMO
Respiratory syncytial virus (RSV) infections are a major cause of bronchiolitis and pneumonia in infants and older adults, for which there is no known correlate of protection. Increasing evidence suggests that Fc-mediated antibody effector functions have an important role, but little is known about the development, heterogeneity, and durability of these functional responses. In light of future vaccine strategies, a clear view of the immunological background and differences between various target populations is of crucial importance. In this study, we have assessed both quantitative and qualitative aspects of RSV-specific serum antibodies, including IgG/IgA levels, IgG subclasses, antibody-dependent complement deposition, cellular phagocytosis, and NK cell activation (ADNKA). Samples were collected cross-sectionally in different age groups (11-, 24-, and 46-month-old children, adults, and older adults; nâ =â 31-35 per group) and longitudinally following natural RSV infection in (older) adults (2-36 months post-infection; nâ =â 10). We found that serum of 24-month-old children induces significantly lower ADNKA than the serum of adults (Pâ <â 0.01), which is not explained by antibody levels. Furthermore, in (older) adults we observed boosting of antibody levels and functionality at 2-3 months after RSV infection, except for ADNKA. The strongest decrease was subsequently observed within the first 9 months, after which levels remained relatively stable up to three years post-infection. Together, these data provide a comprehensive overview of the functional landscape of RSV-specific serum antibodies in the human population, highlighting that while antibodies reach adult levels already at a young age, ADNKA requires more time to fully develop.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Lactente , Criança , Humanos , Idoso , Pré-Escolar , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Anticorpos Antivirais , Imunoglobulina G , Anticorpos NeutralizantesRESUMO
BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe acute lower respiratory tract infections in infants. Natural killer (NK) cells are important antiviral effector cells that likely encounter RSV in the presence of virus-specific (maternal) antibodies. As NK cells potentially contribute to immunopathology, we investigated whether RSV affects their antiviral effector functions. METHODS: We assessed the phenotype and functionality of primary neonatal and adult NK cells by flow cytometry after stimulation with RSV or RSV-antibody complexes. RESULTS: We demonstrate for the first time that RSV infects neonatal and adult NK cells in vitro. Preincubation of virus with subneutralizing concentrations of RSV-specific antibodies significantly increased the percentage of infected NK cells. Upon infection, NK cells were significantly more prone to produce interferon-γ, while secretion of the cytotoxicity molecule perforin was not enhanced. CONCLUSIONS: Our findings suggest that (antibody-enhanced) RSV infection of NK cells induces a proinflammatory rather than a cytotoxic response, which may contribute to immunopathology. Considering that most RSV vaccines currently being developed aim at inducing (maternal) antibodies, these results highlight the importance of understanding the interactions between innate effector cells and virus-specific antibodies.
Assuntos
Interações Hospedeiro-Patógeno , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Adulto , Anticorpos Bloqueadores/imunologia , Anticorpos Antivirais/imunologia , Células Cultivadas , Voluntários Saudáveis , Humanos , Recém-Nascido , Interferons/metabolismo , Células Matadoras Naturais/metabolismo , Perforina/metabolismo , Infecções por Vírus Respiratório SincicialRESUMO
The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.
Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/virologia , Inibidores Enzimáticos/farmacologia , Vírus da Febre Hemorrágica da Crimeia-Congo/efeitos dos fármacos , Febre Hemorrágica da Crimeia/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Ubiquitina/metabolismo , Proteínas Virais/antagonistas & inibidores , Antivirais/química , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Vírus da Febre Hemorrágica da Crimeia-Congo/enzimologia , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Ubiquitinação/efeitos dos fármacos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants. At this young age, infants typically depend on maternally transferred antibodies (matAbs) and their innate immune system for protection against infections. RSV-specific matAbs are thought to protect from severe illness, yet severe RSV disease occurs mainly below 6 months of age, when neutralizing matAb levels are present. To investigate this discrepancy, we asked if disease severity is related to antibody properties other than neutralization. Some antibody effector functions are mediated via their Fc binding region. However, it has been shown that this binding may lead to antibody-dependent enhancement (ADE) of infection or reduction of neutralization, both possibly leading to more disease. In this study, we first showed that high levels of ADE of RSV infection occur in monocytic THP-1 cells in the presence of RSV antibodies and that neutralization by these antibodies was reduced in Vero cells when they were transduced with Fc gamma receptors. We then demonstrated that antibodies from cotton rats with formalin-inactivated (FI)-RSV-induced pulmonary pathology were capable of causing ADE. Human matAbs also caused ADE and were less neutralizing in vitro in cells that carry Fc receptors. However, these effects were unrelated to disease severity because they were seen both in uninfected controls and in infants hospitalized with different levels of RSV disease severity. We conclude that ADE and reduction of neutralization are unlikely to be involved in RSV disease in infants with neutralizing matAbs.IMPORTANCE It is unclear why severity of RSV disease peaks at the age when infants have neutralizing levels of maternal antibodies. Additionally, the exact reason for FI-RSV-induced enhanced disease, as seen in the 1960s vaccine trials, is still unclear. We hypothesized that antibodies present under either of these conditions could contribute to disease severity. Antibodies can have effects that may lead to more disease instead of protection. We investigated two of those effects: antibody-dependent enhancement of infection (ADE) and neutralization reduction. We show that ADE occurs in vitro with antibodies from FI-RSV-immunized RSV-infected cotton rats. Moreover, passively acquired maternal antibodies from infants had the capacity to induce ADE and reduction of neutralization. However, no clear association with disease severity was seen, ruling out that these properties explain disease in the presence of maternal antibodies. Our data contribute to a better understanding of the impact of antibodies on RSV disease in infants.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Receptores de IgG/metabolismo , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vírus Sinciciais Respiratórios/imunologia , Índice de Gravidade de Doença , Animais , Anticorpos Antivirais/sangue , Anticorpos Facilitadores , Estudos de Casos e Controles , Chlorocebus aethiops , Feminino , Humanos , Lactente , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Monócitos/imunologia , Monócitos/patologia , Monócitos/virologia , Testes de Neutralização , Ratos , Receptores de IgG/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Sigmodontinae , Vacinação , Células Vero , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologiaRESUMO
Recent studies have revealed that proteases encoded by three very diverse RNA virus groups share structural similarity with enzymes of the Ovarian Tumor (OTU) superfamily of deubiquitinases (DUBs). The publication of the latest of these reports in quick succession prevented proper recognition and discussion of the shared features of these viral enzymes. Here we provide a brief structural and functional comparison of these virus-encoded OTU DUBs. Interestingly, although their shared structural features and substrate specificity tentatively place them within the same protease superfamily, they also show interesting differences that trigger speculation as to their origins.
Assuntos
Vírus de RNA/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Proteases Específicas de Ubiquitina/química , Proteínas Virais/químicaRESUMO
Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-ß mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.
Assuntos
Endopeptidases/metabolismo , Equartevirus/enzimologia , Fibroblastos/imunologia , Fibroblastos/virologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Papaína/metabolismo , Animais , Proteases Semelhantes à Papaína de Coronavírus , Endopeptidases/química , Endopeptidases/genética , Equartevirus/fisiologia , Células HEK293 , Vírus da Febre Hemorrágica da Crimeia-Congo/enzimologia , Cavalos , Humanos , Interferon beta/genética , Modelos Moleculares , Mutação/genética , Papaína/química , Papaína/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/imunologia , Especificidade por Substrato , Ubiquitina/química , Replicação Viral , Dedos de ZincoRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL(pro)) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL(pro) was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL(pro) domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL(pro), we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL(pro) to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL(pro) DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL(pro) domain was found to suppress IFN-ß promoter activation, PL(pro) variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL(pro), and not its proteolytic activity per se, in the inhibition of IFN-ß promoter activity. The ability to decouple the DUB activity of PL(pro) from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL(pro) as a viral DUB during MERS-CoV infection.
Assuntos
Tolerância Imunológica , Imunidade Inata , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Papaína/química , Papaína/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese , Mutação , Papaína/genética , Ubiquitina/químicaRESUMO
Respiratory pathogens can cause severe disease and even death, especially in the very young and very old. Studies investigating their prevalence often focus on individuals presenting to healthcare providers with symptoms. However, the design of prevention strategies, e.g. which target groups to vaccinate, will benefit from knowledge on the prevalence of, risk factors for and host response to these pathogens in the general population. In this study, upper respiratory samples (n = 1311) were collected cross-sectionally during winter from 11- and 24-month old children, their parents, and adults ≥60 years of age that were recruited irrespective of seeking medical care. Almost all children, approximately two-thirds of parents and a quarter of older adults tested positive for at least one pathogen, often in the absence of symptoms. Viral interference was evident for the combination of rhinovirus and respiratory syncytial virus. Attending childcare facilities and having siblings associated with increased pathogen counts in children. On average, children showed increased levels of mucosal cytokines compared to parents and especially proinflammatory molecules associated with the presence of symptoms. These findings may guide further research into transmission patterns of respiratory pathogens and assist in determining the most appropriate strategies for the prediction and prevention of disease.
Assuntos
Citocinas , Infecções Respiratórias , Estações do Ano , Humanos , Estudos Transversais , Países Baixos/epidemiologia , Lactente , Masculino , Feminino , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Infecções Respiratórias/imunologia , Prevalência , Pessoa de Meia-Idade , Adulto , Citocinas/metabolismo , Idoso , Pré-Escolar , Idoso de 80 Anos ou mais , Viroses/epidemiologia , Viroses/virologia , Viroses/imunologia , Vírus/isolamento & purificação , Vírus/classificação , Vírus/imunologiaRESUMO
Objectives: Increasing evidence suggests that Fc-mediated antibody effector functions have an important role in protection against respiratory viruses, including SARS-CoV-2. However, limited data are available on the potential differences in the development, heterogeneity and durability of these responses in children compared to adults. Methods: Here, we assessed the development of spike S1-specific serum antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD) and natural killer cell activation (ADNKA), alongside specific antibody binding concentrations (IgG, IgA and IgM) and IgG avidity in healthy adults (n = 38, 18-56 years) and children (n = 21, 5-16 years) following primary SARS-CoV-2 infection, with a 10-month longitudinal follow-up. Differences between groups were assessed using a nonparametric Kruskal-Wallis test with Dunn's multiple comparisons test. Results: We found similar (functional) antibody responses in children compared to adults, with a tendency for increased durability in children, which was statistically significant for ADCD (P < 0.05). While ADNKA was strongly reduced in both adults (P < 0.001) and children (P < 0.05) at the latest time point, ADCP remained relatively stable over time, possibly relating to an increase in avidity of the spike-specific antibodies (P < 0.001). Finally, the ADNKA capacity relative to antibody concentration appeared to decrease over time in both children and adults. Conclusion: In conclusion, our data provide novel insights into the development of SARS-CoV-2-specific antibody Fc-mediated effector functions in children and adults. An increased understanding of these characteristics in specific age populations is valuable for the future design of novel and improved vaccination strategies for respiratory viruses such as SARS-CoV-2.
RESUMO
A range of cell culture infection models have been used to study SARS-CoV-2 and perform antiviral drug research. Commonly used African green monkey Vero, human lung-derived Calu-3 and ACE2+TMPRSS2-expressing A549 cells, each have their limitations. Here, we describe human ACE2-expressing H1299 lung cells as a more efficient and robust model for SARS-CoV-2 research. These cells are as easy to handle as Vero cells, support SARS-CoV-2 replication to high titers, display a functional innate immune response and are suitable for plaque assays, microscopy, the production of (genetically stable) virus stocks and antiviral assays. H1299/ACE2-based (CPE reduction) assays can be performed without adding a P-gP drug efflux pump inhibitor, which is often required in Vero-based assays. Moreover, H1299/ACE2 cells allowed us to perform CPE reduction assays with omicron variants that did not work in Vero-based assays. In summary, H1299/ACE2 cells are a versatile infection model to study SARS-CoV-2 replication in the context of antiviral drug development and virus-host interaction studies.
Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , COVID-19 , SARS-CoV-2 , Replicação Viral , Humanos , SARS-CoV-2/fisiologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , COVID-19/virologia , Animais , Chlorocebus aethiops , Células Vero , Linhagem CelularRESUMO
The innate immune response constitutes the first line of defense against viral infection and is extensively regulated through ubiquitination. The removal of ubiquitin from innate immunity signaling factors by deubiquitinating enzymes (DUBs) therefore provides a potential opportunity for viruses to evade this host defense system. It was previously found that specific proteases encoded by the unrelated arteri- and nairoviruses resemble the ovarian tumor domain-containing (OTU) family of DUBs. In arteriviruses, this domain has been characterized before as a papain-like protease (PLP2) that is also involved in replicase polyprotein processing. In nairoviruses, the DUB resides in the polymerase protein but is not essential for RNA replication. Using both in vitro and cell-based assays, we now show that PLP2 DUB activity is conserved in all members of the arterivirus family and that both arteri- and nairovirus DUBs inhibit RIG-I-mediated innate immune signaling when overexpressed. The potential relevance of RIG-I-like receptor (RLR) signaling for the innate immune response against arterivirus infection is supported by our finding that in mouse embryonic fibroblasts, the production of beta interferon primarily depends on the recognition of arterivirus RNA by the pattern-recognition receptor MDA5. Interestingly, we also found that both arteri- and nairovirus DUBs inhibit RIG-I ubiquitination upon overexpression, suggesting that both MDA5 and RIG-I have a role in countering infection by arteriviruses. Taken together, our results support the hypothesis that arteri- and nairoviruses employ their deubiquitinating potential to inactivate cellular proteins involved in RLR-mediated innate immune signaling, as exemplified by the deubiquitination of RIG-I.
Assuntos
Infecções por Arterivirus/imunologia , Arterivirus/enzimologia , RNA Helicases DEAD-box/imunologia , Endopeptidases/imunologia , Febre Hemorrágica da Crimeia/imunologia , Imunidade Inata , Nairovirus/enzimologia , Proteínas Virais/imunologia , Animais , Arterivirus/química , Arterivirus/genética , Infecções por Arterivirus/enzimologia , Infecções por Arterivirus/virologia , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Febre Hemorrágica da Crimeia/enzimologia , Febre Hemorrágica da Crimeia/metabolismo , Febre Hemorrágica da Crimeia/virologia , Humanos , Camundongos , Camundongos Transgênicos , Nairovirus/química , Nairovirus/genética , Estrutura Terciária de Proteína , Transdução de Sinais , Ubiquitina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Respiratory syncytial virus (RSV) is a major cause of severe respiratory infection in infants and the elderly. The mechanisms behind severe RSV disease are incompletely understood, but a dysregulated immune response probably plays an important role. Platelets are increasingly being recognized as immune cells and are involved in the pathology of several viruses. The release of chemokines from platelets upon activation may attract, for example, neutrophils to the site of infection, which is a hallmark of RSV pathology. In addition, since RSV infections are sometimes associated with cardiovascular events and platelets express several known RSV receptors, we investigated the effect of RSV exposure on platelet degranulation. Washed human platelets were incubated with sucrose-purified RSV particles. P-selectin and CD63 surface expression and CCL5 secretion were measured to assess platelet degranulation. We found that platelets bind and internalize RSV particles, but this does not result in degranulation. Our results suggest that platelets do not play a direct role in RSV pathology by releasing chemokines to attract inflammatory cells.
RESUMO
Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Humanos , Países Baixos , Sistema Nervoso CentralRESUMO
Respiratory viruses including the respiratory syncytial virus (RSV) aggravate the global burden of virus-inflicted morbidity and mortality. Entry inhibitors are a promising class of antiviral drugs for combating these viruses, as they can prevent infection at the site of viral entry, i.e., the respiratory tract. Here we used a broad-spectrum entry inhibitor, composed of a ß-cyclodextrin backbone, functionalized with 11-mercapto-1-undecanesulfonate (CD-MUS) that is capable of neutralizing a variety of viruses that employ heparan sulfate proteoglycans (HSPG) to infect host cells. CD-MUS inactivates viral particles irreversibly by binding to viral attachment proteins through a multivalent binding mechanism. In the present study, we show that CD-MUS is well tolerated when administered to the respiratory tract of mice. Based on this, we developed an inhalable spray-dried powder formulation that fits the size requirements for lung deposition and disperses well upon use with the Cyclops dry powder inhaler (DPI). Using an in vitro dose-response assay, we show that the compound retained its activity against RSV after the spray drying process. Our study sets the stage for further in vivo studies, exploring the efficacy of pulmonary administered CD-MUS in animal models of RSV infection.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sinciciais Respiratórios , Animais , Vírus Sinciciais Respiratórios/metabolismo , Pós/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Administração por Inalação , Proteínas Virais/metabolismo , Inaladores de Pó SecoRESUMO
Nonpharmaceutical interventions (NPIs) to contain the SARS-CoV-2 pandemic drastically reduced human-to-human interactions, decreasing the circulation of other respiratory viruses, as well. Consequently, influenza virus circulation, which is normally responsible for 3 to 5 million hospitalizations per year globally, was significantly reduced. With the downscaling of the NPI countermeasures, there is a concern for increased influenza disease, particularly in individuals suffering from postacute effects of SARS-CoV-2 infection. To investigate this, we performed a sequential influenza H1N1 infection 4 weeks after an initial SARS-CoV-2 infection in ferrets. Upon H1N1 infection, ferrets that were previously infected with SARS-CoV-2 showed an increased tendency to develop clinical signs, compared to the control H1N1-infected animals. A histopathological analysis indicated only a slight increase for type II pneumocyte hyperplasia and bronchitis. Thus, the effects of the sequential infection appeared minor. However, ferrets were infected with B.1.351-SARS-CoV-2, the beta variant of concern, which replicated poorly in our model. The histopathology of the respiratory organs was mostly resolved 4 weeks after the SARS-CoV-2 infection, with only reminiscent histopathological features in the upper respiratory tract. Nevertheless, SARS-CoV-2 specific cellular and humoral responses were observed, confirming an established infection. On account of a modest trend toward the enhancement of the influenza disease, even upon a mild SARS-CoV-2 infection, our findings suggest that a stronger SARS-CoV-2 infection and its consequent, long-term effects could have a greater impact on the outcome of disease after a sequential influenza infection. Hence, the influenza vaccination of individuals suffering from postacute SARS-CoV-2 infection effects may be considered an avertible measure for such a scenario. IMPORTANCE During the COVID-19 pandemic, the use of face masks, social distancing, and isolation were effective not only in decreasing the circulation of SARS-CoV-2 but also in reducing other respiratory viruses, such as influenza. With fewer restrictions currently in place, influenza is slowly returning. In the meantime, people who are still suffering from long-COVID could be more vulnerable to an influenza virus infection and could develop a more severe influenza disease. This study provides directions to the effect of a previous SARS-CoV-2 exposure on influenza disease severity in a ferret model. This model is highly valuable to test sequential infections under controlled settings for translation to humans. We could not induce clear long-term COVID-19 effects, as the SARS-CoV-2 infections in the ferrets were mild. However, we still observed a slight increase in influenza disease severity compared to ferrets that had not encountered SARS-CoV-2 before. Therefore, it may be advisable to include long-COVID patients as a risk group for influenza vaccination.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Animais , Humanos , SARS-CoV-2 , Furões , Síndrome de COVID-19 Pós-Aguda , PandemiasRESUMO
Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1(pro)) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1(pro) domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2(pro), a downstream paralog that is conserved in the nsp3's of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1(pro) with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1(pro) was shown to cleave a peptide mimicking the cognate nsp2|nsp3 cleavage site. Like its PL2(pro) paralogs from several coronaviruses, TGEV PL1(pro) was also found to have deubiquitinating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2(pro) from other alphacoronaviruses, e.g., human coronaviruses 229E and NL63, our results unequivocally establish that these viruses employ two PL(pro)s with overlapping specificities toward both viral and cellular substrates.
Assuntos
Papaína/química , Papaína/metabolismo , Vírus da Gastroenterite Transmissível/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Coronavirus/enzimologia , Coronavirus/genética , Proteases Semelhantes à Papaína de Coronavírus , Cristalografia por Raios X , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Papaína/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Especificidade por Substrato , Vírus da Gastroenterite Transmissível/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais/genéticaRESUMO
Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nasal turbinates was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.
Assuntos
Brônquios/patologia , COVID-19/complicações , COVID-19/imunologia , Furões/imunologia , SARS-CoV-2/fisiologia , Administração Intranasal , Fatores Etários , Animais , Doenças Assintomáticas , Modelos Animais de Doenças , Furões/virologia , Humanos , Hiperplasia , Imunidade Celular , Imunidade Humoral , Injeção Intratimpânica , Masculino , Internalização do Vírus , Síndrome de COVID-19 Pós-AgudaRESUMO
The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95 % limit of detection (LOD95). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n = 13) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n = 6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was ≥96 % for all assays and the estimated LOD95 varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.
Assuntos
Infecções por Coronavirus , Coronavirus , Betacoronavirus , COVID-19 , Humanos , Pandemias , Pneumonia Viral , DNA Polimerase Dirigida por RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2RESUMO
Respiratory syncytial virus (RSV) is increasingly recognized for causing severe morbidity and mortality in older adults, but there are few studies on the RSV-induced immune response in this population. Information on the immunological processes at play during RSV infection in specific risk groups is essential for the rational and targeted design of novel vaccines and therapeutics. Here, we assessed the antibody and local cytokine response to RSV infection in community-dwelling older adults (≥60 years of age). During three winters, serum and nasopharyngeal swab samples were collected from study participants during acute respiratory infection and recovery. RSV IgG enzyme-linked immunosorbent assays (ELISA) and virus neutralization assays were performed on serum samples from RSV-infected individuals (n = 41) and controls (n = 563 and n = 197, respectively). Nasal RSV IgA and cytokine concentrations were determined using multiplex immunoassays in a subset of participants. An in vitro model of differentiated primary bronchial epithelial cells was used to assess RSV-induced cytokine responses over time. A statistically significant increase in serum neutralization titers and IgG concentrations was observed in RSV-infected participants compared to controls. During acute RSV infection, a statistically significant local upregulation of beta interferon (IFN-ß), IFN-λ1, IFN-γ, interleukin 1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), IL-6, IL-10, CXCL8, and CXCL10 was found. IFN-ß, IFN-λ1, CXCL8, and CXCL10 were also upregulated in the epithelial model upon RSV infection. In conclusion, this study provides novel insights into the basic immune response to RSV infection in an important and understudied risk population, providing leads for future studies that are essential for the prevention and treatment of severe RSV disease in older adults.IMPORTANCE Respiratory syncytial virus (RSV) can cause severe morbidity and mortality in certain risk groups, especially infants and older adults. Currently no (prophylactic) treatment is available, except for a partially effective yet highly expensive monoclonal antibody. RSV therefore remains a major public health concern. To allow targeted development of novel vaccines and therapeutics, it is of great importance to understand the immunological mechanisms that underlie (protection from) severe disease in specific risk populations. Since most RSV-related studies focus on infants, there are only very limited data available concerning the response to RSV in the elderly population. Therefore, in this study, RSV-induced antibody responses and local cytokine secretion were assessed in community-dwelling older adults. These data provide novel insights that will benefit ongoing efforts to design safe and effective prevention and treatment strategies for RSV in an understudied risk group.
Assuntos
Anticorpos Antivirais/sangue , Citocinas/análise , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Brônquios/citologia , Linhagem Celular , Chlorocebus aethiops , Citocinas/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células VeroRESUMO
OBJECTIVES: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody-mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection. Since antibodies also mediate natural killer (NK) cell activation, we investigated whether this functionality correlates with RSV disease. METHODS: We performed an observational case-control study including infants hospitalised for RSV infection, hernia surgery or RSV-negative respiratory viral infections. We determined RSV antigen-specific antibody levels in plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc-glycosylation of the RSV-specific antibodies by mass spectrometry. RESULTS: We found that RSV-specific maternal antibodies activate NK cells in vitro. While concentrations of RSV-specific antibodies did not differ between cases and controls, antibodies from infants hospitalised for severe respiratory infections (RSV and/or other) induced significantly less NK cell interferon-γ production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc-fucosylation of RSV-specific antibodies, but their glycosylation status did not significantly differ between cases and controls. CONCLUSION: Our results suggest that Fc-dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of using these properties to evaluate and improve the efficacy of novel vaccines.