Prevalence of human respiratory pathogens and associated mucosal cytokine levels in young children and adults: a cross-sectional observational study in the Netherlands during the winter of 2012/2013.

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.

Characterization of SARS-CoV-2 replication in human H1299/ACE2 cells: A versatile and practical infection model for antiviral research and beyond.

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.

Binding of respiratory syncytial virus particles to platelets does not result in their degranulation in vitro.

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.

Difference in respiratory syncytial virus-specific Fc-mediated antibody effector functions between children and adults.

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; n = 31-35 per group) and longitudinally following natural RSV infection in (older) adults (2-36 months post-infection; n = 10). We found that serum of 24-month-old children induces significantly lower ADNKA than the serum of adults (P < 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.

Development of an inhalable antiviral powder formulation against respiratory syncytial virus.

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.

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands.

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.

Influenza Infection in Ferrets with SARS-CoV-2 Infection History.

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.

Pathology and Immunity After SARS-CoV-2 Infection in Male Ferrets Is Affected by Age and Inoculation Route.

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.

Antibody and Local Cytokine Response to Respiratory Syncytial Virus Infection in Community-Dwelling Older Adults.

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.

Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.

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.

Natural killer cell activation by respiratory syncytial virus-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation.

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.

Pathogenesis of Respiratory Syncytial Virus Infection in BALB/c Mice Differs Between Intratracheal and Intranasal Inoculation.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease requiring hospitalization in infants. There are no market-approved vaccines or antiviral agents available, but a growing number of vaccines and therapeutics are in (pre)clinical stages of development. Reliable animal models are crucial to evaluate new vaccine concepts, but in vivo RSV research is hampered by the lack of well-characterized animal models that faithfully mimic the pathogenesis of RSV infection in humans. Mice are frequently used in RSV infection and vaccination studies. However, differences in the use of mouse strains, RSV subtypes, and methodology often lead to divergent study outcomes. To our knowledge, a comparison between different RSV inoculation methods in mice has not been described in the literature, even though multiple methods are being used across different studies. In this study, we evaluated various pathological and immunological parameters in BALB/c mice after intratracheal or intranasal inoculation with RSV-A2. Our study reveals that intranasal inoculation induces robust pathology and inflammation, whereas this is not the case for intratracheal inoculation. As immunopathology is an important characteristic of RSV disease in infants, these data suggest that in mice intranasal inoculation is a more appropriate method to study RSV infection than intratracheal inoculation. These findings will contribute to the rational experimental design of future in vivo RSV experiments.

Fc-Mediated Antibody Effector Functions During Respiratory Syncytial Virus Infection and Disease.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections and hospitalization in infants under 1 year of age and there is currently no market-approved vaccine available. For protection against infection, young children mainly depend on their innate immune system and maternal antibodies. Traditionally, antibody-mediated protection against viral infections is thought to be mediated by direct binding of antibodies to viral particles, resulting in virus neutralization. However, in the case of RSV, virus neutralization titers do not provide an adequate correlate of protection. The current lack of understanding of the mechanisms by which antibodies can protect against RSV infection and disease or, alternatively, contribute to disease severity, hampers the design of safe and effective vaccines against this virus. Importantly, neutralization is only one of many mechanisms by which antibodies can interfere with viral infection. Antibodies consist of two structural regions: a variable fragment (Fab) that mediates antigen binding and a constant fragment (Fc) that mediates downstream effector functions via its interaction with Fc-receptors on (innate) immune cells or with C1q, the recognition molecule of the complement system. The interaction with Fc-receptors can lead to killing of virus-infected cells through a variety of immune effector mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Antibody-mediated complement activation may lead to complement-dependent cytotoxicity (CDC). In addition, both Fc-receptor interactions and complement activation can exert a broad range of immunomodulatory functions. Recent studies have emphasized the importance of Fc-mediated antibody effector functions in both protection and pathogenesis for various infectious agents. In this review article, we aim to provide a comprehensive overview of the current knowledge on Fc-mediated antibody effector functions in the context of RSV infection, discuss their potential role in establishing the balance between protection and pathogenesis, and point out important gaps in our understanding of these processes. Furthermore, we elaborate on the regulation of these effector functions on both the cellular and humoral side. Finally, we discuss the implications of Fc-mediated antibody effector functions for the rational design of safe and effective vaccines and monoclonal antibody therapies against RSV.

Viral Infection of Human Natural Killer Cells.

Natural killer (NK) cells are essential in the early immune response against viral infections, in particular through clearance of virus-infected cells. In return, viruses have evolved multiple mechanisms to evade NK cell-mediated viral clearance. Several unrelated viruses, including influenza virus, respiratory syncytial virus, and human immunodeficiency virus, can directly interfere with NK cell functioning through infection of these cells. Viral infection can lead to immune suppression, either by downregulation of the cytotoxic function or by triggering apoptosis, leading to depletion of NK cells. In contrast, some viruses induce proliferation or changes in the morphology of NK cells. In this review article, we provide a comprehensive overview of the viruses that have been reported to infect NK cells, we discuss their mechanisms of entry, and describe the interference with NK cell effector function and phenotype. Finally, we discuss the contribution of virus-infected NK cells to viral load. The development of specific therapeutics, such as viral entry inhibitors, could benefit from an enhanced understanding of viral infection of NK cells, opening up possibilities for the prevention of NK cell infection.

Respiratory Syncytial Virus Infects Primary Neonatal and Adult Natural Killer Cells and Affects Their Antiviral Effector Function.

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.

In Vitro Enhancement of Respiratory Syncytial Virus Infection by Maternal Antibodies Does Not Explain Disease Severity in Infants.

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.

Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants.

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.

In vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease 2.

Arteriviruses are a family of positive-stranded RNA viruses that includes the prototypic equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV). Although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of PRRSV. The ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. We have recently shown that the deubiquitinase (DUB) activity of EAV papain-like protease 2 (PLP2) is important for the inhibition of innate immune activation during infection. A vaccine virus lacking PLP2 DUB activity may therefore be more immunogenic and provide improved protection against subsequent challenge than its DUB-competent counterpart. To test this hypothesis, twenty Shetland mares were randomly assigned to one of three groups. Two groups were vaccinated, either with DUB-positive (n=9) or DUB-negative (n=9) recombinant EAV. The third group (n=2) was not vaccinated. All horses were subsequently challenged with the virulent KY84 strain of EAV. Both vaccine viruses proved to be replication competent in vivo. In addition, the DUB-negative virus provided a similar degree of protection against clinical disease as its DUB-positive parental counterpart. Owing to the already high level of protection provided by the parental virus, a possible improvement due to inactivation of PLP2 DUB activity could not be detected under these experimental conditions. Taken together, the data obtained in this study warrant further in vivo investigations into the potential of using DUB-mutant viruses for the improvement of arterivirus vaccines.