Synergistic Detrimental Effects of Cigarette Smoke, Alcohol, and SARS-CoV-2 in COPD Bronchial Epithelial Cells.

Lung conditions such as COPD, as well as risk factors such as alcohol misuse and cigarette smoking, can exacerbate COVID-19 disease severity. Synergistically, these risk factors can have a significant impact on immunity against pathogens. Here, we studied the effect of a short exposure to alcohol and/or cigarette smoke extract (CSE) in vitro on acute SARS-CoV-2 infection of ciliated human bronchial epithelial cells (HBECs) collected from healthy and COPD donors. We observed an increase in viral titer in CSE- or alcohol-treated COPD HBECs compared to untreated COPD HBECs. Furthermore, we treated healthy HBECs accompanied by enhanced lactate dehydrogenase activity, indicating exacerbated injury. Finally, IL-8 secretion was elevated due to the synergistic damage mediated by alcohol, CSE, and SARS-CoV-2 in COPD HBECs. Together, our data suggest that, with pre-existing COPD, short exposure to alcohol or CSE is sufficient to exacerbate SARS-CoV-2 infection and associated injury, impairing lung defences.

Malondialdehyde acetaldehyde adduction of surfactant protein D attenuates SARS-CoV-2 spike protein binding and virus neutralization.

BACKGROUND: Over 43% of the world's population regularly consumes alcohol. Although not commonly known, alcohol can have a significant impact on the respiratory environment. Living in the time of the COVID-19 pandemic, alcohol misuse can have a particularly deleterious effect on SARS-CoV-2-infected individuals and, in turn, the overall healthcare system. Patients with alcohol use disorders have higher odds of COVID-19-associated hospitalization and mortality. Even though the detrimental role of alcohol on COVID-19 outcomes has been established, the underlying mechanisms are yet to be fully understood. Alcohol misuse has been shown to induce oxidative damage in the lungs through the production of reactive aldehydes such as malondialdehyde and acetaldehyde (MAA). MAA can then form adducts with proteins, altering their structure and function. One such protein is surfactant protein D (SPD), which plays an important role in innate immunity against pathogens. METHODS AND RESULTS: In this study, we examined whether MAA adduction of SPD (SPD-MAA) attenuates the ability of SPD to bind SARS-CoV-2 spike protein, reversing SPD-mediated virus neutralization. Using ELISA, we show that SPD-MAA is unable to competitively bind spike protein and prevent ACE2 receptor binding. Similarly, SPD-MAA fails to inhibit entry of wild-type SARS-CoV-2 virus into Calu-3 cells, a lung epithelial cell line, as well as ciliated primary human bronchial epithelial cells isolated from healthy individuals. CONCLUSIONS: Overall, MAA adduction of SPD, a consequence of alcohol overconsumption, represents one mechanism of compromised lung innate defense against SARS-CoV-2, highlighting a possible mechanism underlying COVID-19 severity and related mortality in patients who misuse alcohol.

Universal antibody targeting the highly conserved fusion peptide provides cross-protection in mice.

Influenza is a major public health concern causing millions of hospitalizations every year. The current vaccines need annual updating based on prediction of likely strains in the upcoming season. However, mismatches between vaccines and the actual circulating viruses can occur, reducing vaccine effectiveness significantly because of the remarkably high rate of mutation in the viral glycoprotein, hemagglutinin (HA). Clearly, it would be of great interest to determine the potential role of universally conserved epitopes in inducing protective immunity. Here, an antibody against the 14-aa fusion peptide sequence at the N-terminus of the HA2 subunit (Uni-1) was investigated for its ability to elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro and cross-protection against lethal infection in animals. Uni-1, known to neutralize influenza type A (IAV) in vitro, was found to induce strong ADCC against diverse influenza viruses, including human and avian IAVs and both lineages of type B (IBV). The ADCC effects against human IAVs by Uni-1 was comparable to ADCC induced by well-characterized antibodies, F10 and FI6V3. Importantly, mice treated with Uni-1 were protected against lethal challenge of IAV and IBV. These results revealed the versatile effector functions of this universal antibody against markedly diverse strains of both IAV and IBV.

The fusion peptide is the only universally conserved epitope in both IAV and IBVMono-specific universal antibody induces strong ADCC against human and avian IAVMono-specific universal antibody induces strong ADCC against IBV from both genetic lineages of IBVThe antibody has bi-functional effector functions against several influenza viruses.

SARS-CoV-2 Dysregulates Neutrophil Degranulation and Reduces Lymphocyte Counts.

SARS-CoV-2, the virus that causes COVID-19, has given rise to one of the largest pandemics, affecting millions worldwide. High neutrophil-to-lymphocyte ratios have been identified as an important correlate to poor recovery rates in severe COVID-19 patients. However, the mechanisms underlying this clinical outcome and the reasons for its correlation to poor prognosis are unclear. Furthermore, the mechanisms involved in healthy neutrophils acquiring a SARS-CoV-2-mediated detrimental role are yet to be fully understood. In this study, we isolated circulating neutrophils from healthy donors for treatment with supernates from infected epithelial cells and direct infection with SARS-CoV-2 in vitro. Infected epithelial cells induced a dysregulated degranulation of primary granules with a decrease in myeloperoxidase (MPO), but slight increase in neutrophil elastase release. Infection of neutrophils resulted in an impairment of both MPO and elastase release, even though CD16 receptor shedding was upregulated. Importantly, SARS-CoV-2-infected neutrophils had a direct effect on peripheral blood lymphocyte counts, with decreasing numbers of CD19+ B cells, CD8+ T cells, and CD4+ T cells. Together, this study highlights the independent role of neutrophils in contributing to the aberrant immune responses observed during SARS-CoV-2 infection that may be further dysregulated in the presence of other immune cells.

IFN-γ Attenuates Eosinophilic Inflammation but Is Not Essential for Protection against RSV-Enhanced Asthmatic Comorbidity in Adult Mice.

The susceptibility to respiratory syncytial virus (RSV) infection in early life has been associated with a deficient T-helper cell type 1 (Th1) response. Conversely, healthy adults generally do not exhibit severe illness from RSV infection. In the current study, we investigated whether Th1 cytokine IFN-γ is essential for protection against RSV and RSV-associated comorbidities in adult mice. We found that, distinct from influenza virus, prior RSV infection does not induce significant IFN-γ production and susceptibility to secondary Streptococcus pneumoniae infection in adult wild-type (WT) mice. In ovalbumin (OVA)-induced asthmatic mice, RSV super-infection increases airway neutrophil recruitment and inflammatory lung damage but has no significant effect on OVA-induced eosinophilia. Compared with WT controls, RSV infection of asthmatic Ifng-/- mice results in increased airway eosinophil accumulation. However, a comparable increase in eosinophilia was detected in house dust mite (HDM)-induced asthmatic Ifng-/- mice in the absence of RSV infection. Furthermore, neither WT nor Ifng-/- mice exhibit apparent eosinophil infiltration during RSV infection alone. Together, these findings indicate that, despite its critical role in limiting eosinophilic inflammation during asthma, IFN-γ is not essential for protection against RSV-induced exacerbation of asthmatic inflammation in adult mice.

Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages.

A quarter of all seasonal influenza cases are caused by type B influenza virus (IBV) that also dominates periodically. Here, we investigated a recombinant adenovirus vaccine carrying a synthetic HA2 representing the consensus sequence of all IBV hemagglutinins. The vaccine fully protected mice from lethal challenges by IBV of both genetic lineages, demonstrating its breadth of protection. The protection was not mediated by neutralizing antibodies but robust antibody-dependent cellular cytotoxicity and cell-mediated immune responses. Complete protection of the animals required the entire codon-optimized HA2 sequence that elicited a balanced immune response, whereas truncated vaccines without either the fusion peptide or the transmembrane domain reduced the efficacy of protection. Finally, the vaccines did not demonstrate any sign of disease exacerbation following lung pathology and morbidity monitoring. Collectively, these data suggest that it could be worth further exploring this prototype universal vaccine because of its considerable efficacy, safety, and breadth of protection.

Complex Roles of Neutrophils during Arboviral Infections.

Arboviruses are known to cause large-scale epidemics in many parts of the world. These arthropod-borne viruses are a large group consisting of viruses from a wide range of families. The ability of their vector to enhance viral pathogenesis and transmission makes the development of treatments against these viruses challenging. Neutrophils are generally the first leukocytes to be recruited to a site of infection, playing a major role in regulating inflammation and, as a result, viral replication and dissemination. However, the underlying mechanisms through which neutrophils control the progression of inflammation and disease remain to be fully understood. In this review, we highlight the major findings from recent years regarding the role of neutrophils during arboviral infections. We discuss the complex nature of neutrophils in mediating not only protection, but also augmenting disease pathology. Better understanding of neutrophil pathways involved in effective protection against arboviral infections can help identify potential targets for therapeutics.

Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-Cov-2 Infection In Vitro.

Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

Influenza Infection Induces Alveolar Macrophage Dysfunction and Thereby Enables Noninvasive Streptococcus pneumoniae to Cause Deadly Pneumonia.

Secondary Streptococcus pneumoniae infection is a significant cause of morbidity and mortality during influenza epidemics and pandemics. Multiple pathogenic mechanisms, such as lung epithelial damage and dysregulation of neutrophils and alveolar macrophages (AMs), have been suggested to contribute to the severity of disease. However, the fundamental reasons for influenza-induced susceptibility to secondary bacterial pneumonia remain unclear. In this study, we revisited these controversies over key pathogenic mechanisms in a lethal model of secondary bacterial pneumonia with an S. pneumoniae strain that is innocuous to mice in the absence of influenza infection. Using a series of in vivo models, we demonstrate that rather than a systemic suppression of immune responses or neutrophil function, influenza infection activates IFN-γR signaling and abrogates AM-dependent bacteria clearance and thereby causes extreme susceptibility to pneumococcal infection. Importantly, using mice carrying conditional knockout of Ifngr1 gene in different myeloid cell subsets, we demonstrate that influenza-induced IFN-γR signaling in AMs impairs their antibacterial function, thereby enabling otherwise noninvasive S. pneumoniae to cause deadly pneumonia.

PD-1 of Sigmodon hispidus: Gene identification, characterization and preliminary evaluation of expression in inactivated RSV vaccine-induced enhanced respiratory disease.

Sigmodon hispidus or cotton rat is an excellent animal model for studying human infections of respiratory viruses including respiratory syncytial virus (RSV), which is the leading cause of hospitalization in infants and causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research, no vaccine has been licensed whereas inactivated vaccines have been shown to induce severe adverse reaction in a clinical trial, with other forms of RSV vaccine also found to induce enhanced disease in preclinical animal studies. While arguably the cotton rat is the best small animal model for evaluation of RSV vaccines and antivirals, many important genes of the immune system remain to be isolated. Programmed cell death-1 (PD-1) plays an integral role in regulating many aspects of immunity by inducing suppressive signals. In this study, we report the isolation of mRNA encoding the cotton rat PD-1 (crPD-1) and characterization of the PD-1 protein. crPD-1 bound to its cognate ligand on dendritic cells and effectively suppressed cytokine secretion. Moreover, using the newly acquired gene sequence, we observed a decreased level of crPD-1 levels in cotton rats with enhanced respiratory disease induced by inactivated RSV vaccine, unraveling a new facet of vaccine-induced disease.

Chitosan alters inactivated respiratory syncytial virus vaccine elicited immune responses without affecting lung histopathology in mice.

Chitosan is a polysaccharide capable of augmenting immune responses with a proven safety record in animals and humans. These properties make it a potentially attractive agent for the prevention and treatment of infectious disease. Infection by respiratory syncytial virus (RSV) is the leading cause of serious lower respiratory disease in young children throughout the world. There is no licensed vaccine available against RSV whereas inactivated vaccine is known to cause enhanced respiratory disease instead of protection. Here, we investigated whether chitosan administered one or three days post-infection could protect animals against RSV infection and whether it could alter immune responses or immunopathology induced by inactivated RSV vaccine when administered twice before RSV infection. We found chitosan could modestly protect animals against RSV infection when given post-infection, while, in conjunction with inactivated RSV vaccine when given pre-infection, it could significantly reduce RSV infection in mice. Further mechanistic investigation revealed that chitosan enhanced antigen-specific immune responses through augmenting the induction of regulatory T cells, lung resident T cells and neutralizing antibodies while reversing Th2-skewed immune responses induced by inactivated RSV vaccine but, surprisingly, failing to reverse lung histopathology. Overall, this study sheds more light on the molecular mechanisms underlying inactivated RSV vaccine-induced disease.

Unveiling Integrated Functional Pathways Leading to Enhanced Respiratory Disease Associated With Inactivated Respiratory Syncytial Viral Vaccine.

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

Targeting CD40 enhances antibody- and CD8-mediated protection against respiratory syncytial virus infection.

Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. Here, the RSV fusion protein (F) was fused with CD40 ligand and delivered by an adenoviral vector into BALB/c mice where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule. In contrast to a formaldehyde-inactivated vaccine, the vectored vaccine effectively protected animals against RSV without inducing enhanced respiratory disease. This protection involved a robust induction of neutralizing antibodies and memory CD8 T cells, which were not observed in the inactivated vaccine group. Finally, the vectored vaccine was able to elicit long-lasting protection against RSV, one of the most challenging issues in RSV vaccine development. Further studies indicate that the long lasting protection elicited by the CD40 ligand targeted vaccine was mediated by increased levels of effector memory CD8 T cell 3 months post-vaccination.

Identification and characterisation of the CD40-ligand of Sigmodon hispidus.

Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses.

Identification of immunodominant CD8 epitope in the stalk domain of influenza B viral hemagglutinin.

Human infections by type B influenza virus constitute about 25% of all influenza cases. The viral hemagglutinin is comprised of two subunits, HA1 and HA2. While HA1 is constantly evolving in an unpredictable fashion, the HA2 subunit is highly conserved, making it a potential candidate for a universal vaccine. However, immunodominant epitopes in the HA2 subunit remain largely unknown. To delineate MHC Class I epitopes, we first identified 9-mer H-2Kd-restricted CD8 T cell epitopes in the HA2 domain by in silico analyses, followed by evaluating the immunodominance of these peptides in mice challenged with the virus. Of three peptides selected through in silico analysis, the universally conserved peptide, YYSTAASSL (B/HA2-190), possessed the highest predicted binding affinity to MHC Class I and was most effective in inducing IL-2 and TNF-α in mouse splenocytes. Importantly, the peptide demonstrated best capability of stimulating peptide-specific ex-vivo cytotoxicity against target cells. Taken together, this finding would be of value for assessment of cell-mediated immune responses elicited by vaccines based on the highly conserved HA2 stalk domain.

Immunopathogenesis associated with formaldehyde-inactivated RSV vaccine in preclinical and clinical studies.

INTRODUCTION: Respiratory syncytial virus (RSV) infection is responsible for one-third of deaths of acute lower respiratory infection in children less than one-year-old. The formaldehyde-inactivated RSV vaccine trial conducted in the 1960s predisposed the vaccinees to more serious RSV infection instead of protection. Better understanding of the underlying mechanism is of critical importance for better designing of safe and effective RSV vaccines. Areas covered: PubMed was searched to review immunopathology induced by RSV vaccines. We intend to dissect the differences in clinical and pathological manifestations of enhanced respiratory disease (ERD) in different animal models in comparison with humans. Formaldehyde-inactivated RSV vaccine causes ERD in both humans and animals, while RSV vaccine without formaldehyde treatment could also induce similar disease in animals, suggesting multiple pathways may be involved. Expert commentary: Identification of biomarkers pertinent to clinical evaluation should be further explored for safety assessment of RSV vaccines in human trials.

Development and applications of universal H7 subtype-specific antibodies for the analysis of influenza H7N9 vaccines.

H7N9 is a newly emerged avian influenza virus with a relatively high mortality rate in humans. At this time, there is no licensed vaccine for human protection. Development of analytical tools for H7N9 vaccine could facilitate vaccine development. Here, a universally conserved epitope in all H7 hemagglutinin (HA) sequences was identified through comprehensive bioinformatics analyses. The peptide epitope, RSGSSFYAEMK, (aa positions 149 to 159), is located on the head of the HA molecule. Antibodies generated against this universal H7 epitope were remarkably specific against H7 viral sequence with no detectable cross-reactivity to other HA subtypes. A new immunoblotting assay based on the universal H7 antibody was developed and compared with the traditional single radial immunodiffusion assay (SRID) for potency analyses of candidate H7N9 vaccines. This new assay was more sensitive and rapid compared to SRID. In addition to statistically acceptable precision and reproducibility, the new assay differs from many other alternative potency assays for influenza vaccine in that it is potentially stability-indicating, which is an important requirement for industry vaccine stability studies analyses. Furthermore, the robustness of this new assay was demonstrated by the quantitative determination of HA content in four H7N9 vaccines (split or inactivated) from different manufacturers.