Lung Virus Titers Research Articles

An Intramuscular DNA Vaccine for SARS-CoV-2 Decreases Viral Lung Load but Not Lung Pathology in Syrian Hamsters.

The 2019 novel coronavirus, SARS-CoV-2, first reported in December 2019, has infected over 102 million people around the world as of February 2021 and thus calls for rapid development of safe and effective interventions, namely vaccines. In our study, we evaluated a DNA vaccine against SARS-CoV-2 in the Syrian hamster model. Hamsters were vaccinated with a DNA-plasmid encoding the SARS-CoV-2 full length spike open reading frame (ORF) to induce host cells to produce spike protein and protective immune responses before exposure to infectious virus. We tested this vaccine candidate by both intranasal (IN) and intramuscular (IM) routes of administration and complexing with and without an in vivo delivery reagent. Hamsters receiving prime-boost-boost IM-only vaccinations recovered body weight quicker, had decreased lung viral loads, and increased SARS-CoV-2-specific antibody titers compared to control vaccinated animals but, surprisingly, lung pathology was as severe as sham vaccinated controls. The IM/IN combination group showed no efficacy in reducing lung virus titers or pathology. With increasing public health need for rapid and effective interventions, our data demonstrate that in some vaccine contexts, significant antibody responses and decreased viral loads may not be sufficient to prevent lung pathology.

Sorex araneus is a pathogenic microbial threat in commercial poultry farms

Rodents can be abundant around poultry houses, share their habitat with waterfowl and can readily enter poultry houses. Infective bacteria have been found in the feces, urine, and blood of Eurasian Shrew (Sorex Araneus) that live in poultry farms. 22 shrews were caught, and their guts were examined for harmful germs. In feces, Escherichia coli and Proteus species were found in 59.09% and 18.18% of cases, respectively. Salmonella spp. were found in 72.72% of feces and urine samples. Rodents can be abundant around poultry houses, share their habitat with waterfowl and can readily enter poultry houses. Different viruses and bacteria can replicate in rodents without adaptation, resulting in high viral titres in lungs and nasal turbinates, virus presence in nasal washes and saliva, and transmission to naive contact animals. Therefore, active viruses shedding by infected rodents may play a role in transmission to poultry. Making poultry houses rodent-proof and the immediate surroundings unattractive for rodents are recommended as preventive measures against possible any viruses and bacteria introduction. It is determined that common shrews serve as a reservoir for dangerous microorganisms, which can be avoided using effective shrew-eradication measures on poultry farms.

Roles of Fc receptor γ chain in inducing protective immune responses after F virus-like particles prime and FI-RSV boost against respiratory syncytial virus infection

Abstract The roles of Fc receptor (FcR) in protection or in inflammatory disease after RSV vaccination and infection remain unknown. Virus-like particles containing RSV fusion proteins (RSV F-VLPs) induce T-helper type 1 antibody responses and protection against RSV. Heterologous RSV F VLP prime and inactivated RSV (FI-RSV) boost vaccination was reported to be effective in providing protection without inflammatory disease. Here we investigated whether the FcRγ chain is important for immune protection by heterologous F VLP and FI-RSV vaccination using FcRγ chain knockout (−/−) mouse model. RSV F-VLP prime and FI-RSV boosted FcRγ−/− mice were not well protected, as shown by higher lung viral titers upon RSV virus challenge compared to RSV F-VLP prime-immunized wild-type mice. RSV F-VLP and FI-RSV immunization of FcRγ−/− mice induced significantly lower levels of neutralizing activity and interferon γ-producing CD8 T cells in bronchoalveolar lavage cells than those in in wild-type mice. In addition, FcRγ−/− mice displayed a trend of enhancing lung histopathology after RSV vaccination and infection. This study suggests that the FcR γ chain plays an important role in antiviral protection and CD8 T cell responses in RSV F-VLP and FI-RSV vaccination after RSV infections.

Rhinovirus C Infection Induces Type 2 Innate Lymphoid Cell Expansion and Eosinophilic Airway Inflammation.

Rhinovirus C (RV-C) infection is associated with severe asthma exacerbations. Since type 2 inflammation is an important disease mechanism in asthma, we hypothesized that RV-C infection, in contrast to RV-A, preferentially stimulates type 2 inflammation, leading to exacerbated eosinophilic inflammation. To test this, we developed a mouse model of RV-C15 airways disease. RV-C15 was generated from the full-length cDNA clone and grown in HeLa-E8 cells expressing human CDHR3. BALB/c mice were inoculated intranasally with 5 x 106 ePFU RV-C15, RV-A1B or sham. Mice inoculated with RV-C15 showed lung viral titers of 1 x 105 TCID50 units 24 h after infection, with levels declining thereafter. IFN-α, β, γ and λ2 mRNAs peaked 24-72 hrs post-infection. Immunofluorescence verified colocalization of RV-C15, CDHR3 and acetyl-α-tubulin in mouse ciliated airway epithelial cells. Compared to RV-A1B, mice infected with RV-C15 demonstrated higher bronchoalveolar eosinophils, mRNA expression of IL-5, IL-13, IL-25, Muc5ac and Gob5/Clca, protein production of IL-5, IL-13, IL-25, IL-33 and TSLP, and expansion of type 2 innate lymphoid cells. Analogous results were found in mice treated with house dust mite before infection, including increased airway responsiveness. In contrast to Rora fl/fl littermates, RV-C-infected Rora fl/fl Il7r cre mice deficient in ILC2s failed to show eosinophilic inflammation or mRNA expression of IL-13, Muc5ac and Muc5b. We conclude that, compared to RV-A1B, RV-C15 infection induces ILC2-dependent type 2 airway inflammation, providing insight into the mechanism of RV-C-induced asthma exacerbations.

Lianhuaqingwen capsule inhibits influenza-induced bacterial adhesion to respiratory epithelial cells through down-regulation of cell adhesion molecules

Ethnopharmacological relevanceInfluenza virus infection is widely believed to cause mild symptoms, but can lead to high mortality and severe disease complicated by secondary bacterial pneumonia. Traditional Chinese medicine (TCM) has been proposed as a promising agent to treat respiratory viral infections. A herbal formula Lianhuaqingwen capsule (LHQW) comprising two prescriptions: Maxing Shigan decoction and Yinqiao San, has been used clinically to treat respiratory infection with immune regulatory effects. However, little is known about the capacity of LHQW against influenza-induced secondary bacterial pneumonia. Aim of studyThis study aimed to evaluate the efficacy and underlying mechanism of LHQW on influenza A virus A/PR/8/34 (PR8) secondary methicillin-resistant Staphy-lococcus aureus (MRSA) infection. MethodsThe anti-adhesion activity of LHQW against PR8-induced MRSA infection was assessed in human lung epithelial (A549) cells and the effect of LHQW on the expression of intracellular adhesion molecule 1 (ICAM-1) was detected. Also, the mRNA expression levels of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation in PR8-infected A549 cells were determined. The body weight change, survivals, viral titers, colonies and the pathological parameters after LHQW treatment in severe pneumonia model have all been systematically determined. ResultsLHQW significantly reduced the adhesion of MRSA to PR8-infected A549 cells in a dose-dependent manner by suppressing the up-regulation of bacterial receptors. LHQW also markedly declined the overexpression of IL-6, IL-8, and TNF-α induced by LPS stimulated-A549 cells following influenza virus infection. Furthermore, the abnormal changes of lung index in dual-infection mice were relieved after administered with LHQW in preventive and therapeutic mode, but with no significantly difference (P > 0.05). LHQW could not effectively improve survival rate or prolong the survival time of mice (P > 0.05). LHQW (1000 mg/kg/d) administered prophylactically significantly decreased the lung viral titers (P < 0.05), slightly downregulated IL-6 but TNF-α, IL-1β levels and improved lung pathological inflammation including neutrophil infiltration, necrosis, which is consistent with the expression of inflammatory factors. ConclusionsLHQW inhibited influenza-induced bacterial adhesion by down-regulating the adhesion molecules with the improvement trend on severe pneumonia, indicating that it can be used as an adjuvant medication in severe viral-bacterial pneumonia therapy rather than as a single medication.

Roles of the Fc Receptor γ-Chain in Inducing Protective Immune Responses after Heterologous Vaccination against Respiratory Syncytial Virus Infection.

The roles of the Fc receptor (FcR) in protection or inflammatory disease after respiratory syncytial virus (RSV) vaccination and infection remain unknown. Virus-like particles containing RSV fusion proteins (RSV F-VLPs) induce T-helper type 1 antibody responses and protection against RSV. Heterologous RSV F-VLP prime and formalin-inactivated RSV (FI-RSV) boost vaccination has been reported to be effective in providing protection without inflammatory disease. Here, we investigated whether the FcRγ-chain is important for immune protection by the heterologous F-VLP and FI-RSV vaccination using FcRγ-chain knockout (−/−) mice. RSV F-VLP-primed and FI-RSV-boosted FcRγ −/− mice displayed less protective efficacy, as shown by higher lung viral titers upon RSV challenge, compared to RSV F-VLP-primed and FI-RSV-boosted immunized wild-type mice. RSV F-VLP and FI-RSV immunization induced lower levels of neutralizing activity and interferon-γ-producing CD8 T-cells in the bronchoalveolar lavage cells of FcRγ −/− mice than in those of wild-type mice. In addition, FcRγ −/− mice displayed a trend of enhancing lung histopathology after RSV vaccination and infection. This study suggests that the FcRγ-chain plays an important role in inducing antiviral protection and CD8 T-cell responses in RSV F-VLP prime and FI-RSV boost vaccination after RSV infections.

Epigraph hemagglutinin vaccine induces broad cross-reactive immunity against swine H3 influenza virus

Influenza A virus infection in swine impacts the agricultural industry in addition to its zoonotic potential. Here, we utilize epigraph, a computational algorithm, to design a universal swine H3 influenza vaccine. The epigraph hemagglutinin proteins are delivered using an Adenovirus type 5 vector and are compared to a wild type hemagglutinin and the commercial inactivated vaccine, FluSure. In mice, epigraph vaccination leads to significant cross-reactive antibody and T-cell responses against a diverse panel of swH3 isolates. Epigraph vaccination also reduces weight loss and lung viral titers in mice after challenge with three divergent swH3 viruses. Vaccination studies in swine, the target species for this vaccine, show stronger levels of cross-reactive antibodies and T-cell responses after immunization with the epigraph vaccine compared to the wild type and FluSure vaccines. In both murine and swine models, epigraph vaccination shows superior cross-reactive immunity that should be further investigated as a universal swH3 vaccine.

K18-hACE2 mice develop respiratory disease resembling severe COVID-19.

SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Additionally, we demonstrate that a mild disease course can be simulated by low dose infection with 102 TCID50 SARS-CoV-2, resulting in minimal clinical manifestation and near uniform survival. Taken together, these data support future application of this model to studies of pathogenesis and medical countermeasure development.

Protective cellular and mucosal immune responses following nasal administration of a whole gamma-irradiated influenza A (subtype H1N1) vaccine adjuvanted with interleukin-28B in a mouse model.

The use of gamma-irradiated influenza A virus (γ-Flu), retains most of the viral structural antigens, represent a promising option for vaccine development. However, despite the high effectiveness of γ-Flu vaccines, the need to incorporate an adjuvant to improve vaccine-mediated protection seems inevitable. Here, we examined the protective efficacy of an intranasal gamma-irradiated HIN1 vaccine co-administered with a plasmid encoding mouse interleukin-28B (mIL-28B) as a novel adjuvant in BALB/c mice. Animals were immunized intranasally three times at one-week intervals with γ-Flu, alone or in combination with the mIL-28B adjuvant, followed by viral challenge with a high lethal dose (10 LD50) of A/PR/8/34 (H1N1) influenza virus. Virus-specific antibody, cellular and mucosal responses, and the balance of cytokines in the spleen IFN-γ, IL-12, and IL-4) and in lung homogenates (IL-6 and IL-10) were measured by ELISA. The lymphoproliferative activity of restimulated spleen cells was also determined by MTT assay. Furthermore, virus production in the lungs of infected mice was estimated using the Madin-Darby canine kidney (MDCK)/hemagglutination assay (HA). Our data showed that intranasal immunization with adjuvanted γ-Flu vaccine efficiently promoted humoral, cellular, and mucosal immune responses and efficiently decreased lung virus titers, all of which are associated with protection against challenge. This combination also reduced IL-6 and IL-10 levels in lung homogenates. The results suggest that IL-28B can enhance the ability of the vaccine to elicit virus-specific immune responses and could potentially be used as an effective adjuvant.

Effects of Different Drug Combinations in Immunodeficient Mice Infected with an Influenza A/H3N2 Virus.

The prolonged treatment of immunosuppressed (IS) individuals with anti-influenza monotherapies may lead to the emergence of drug-resistant variants. Herein, we evaluated oseltamivir and polymerase inhibitors combinations against influenza A/H3N2 infections in an IS mouse model. Mice were IS with cyclophosphamide and infected with 3 × 103 PFU of a mouse-adapted A/Switzerland/9715293/2013 (H3N2) virus. Forty-eight hours post-infection, the animals started oseltamivir, favipiravir or baloxavir marboxil (BXM) as single or combined therapies for 10 days. Weight losses, survival rates and lung viral titers (LVTs) were determined. The neuraminidase (NA) and polymerase genes from lung viral samples were sequenced. All untreated animals died. Oseltamivir and favipiravir monotherapies only delayed mortality (the mean day to death (MDD) of 21.4 and 24 compared to 11.4 days for those untreated) while a synergistic improvement in survival (80%) and LVT reduction was observed in the oseltamivir/favipiravir group compared to the oseltamivir group. BXM alone or in double/triple combination provided a complete protection and significantly reduced LVTs. Oseltamivir and BXM monotherapies induced the E119V (NA) and I38T (PA) substitutions, respectively, while no resistance mutation was detected with combinations. We found that the multiple dose regimen of BXM alone provided superior benefits compared to oseltamivir and favipiravir monotherapies. Moreover, we suggest the potential for drug combinations to reduce the incidence of resistance.

Ganghuo Kanggan Decoction in Influenza: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation.

Background: Ganghuo Kanggan decoction (GHKGD) is a clinical experience prescription used for the treatment of viral pneumonia in the Lingnan area of China, and its clinical effect is remarkable. However, the mechanism of GHKGD in influenza is still unclear. Objective: To predict the active components and signaling pathway of GHKGD and to explore its therapeutic mechanism in influenza and to verified it in vivo using network pharmacology. Methods: The potential active components and therapeutic targets of GHKGD in the treatment of influenza were hypothesized through a series of network pharmacological strategies, including compound screening, target prediction and pathway enrichment analysis. Based on the target network and enrichment results, a mouse model of influenza A virus (IAV) infection was established to evaluate the therapeutic effect of GHKGD on influenza and to verify the possible molecular mechanism predicted by network pharmacology. Results: A total of 116 candidate active compounds and 17 potential targets were identified. The results of the potential target enrichment analysis suggested GHKGD may involve the RLR signaling pathway to reduce inflammation in the lungs. In vivo experiments showed that GHKGD had a protective effect on pneumonia caused by IAV-infected mice. Compared with the untreated group, the weight loss in the GHKGD group in the BALB/c mice decreased, and the inflammatory pathological changes in lung tissue were reduced (p < 0.05). The expression of NP protein and the virus titers in lung were significantly decreased (p < 0.05). The protein expression of RIG-I, NF-kB, and STAT1 and the level of MAVS and IRF3/7 mRNA were remarkably inhibited in GHKGD group (p < 0.05). After the treatment with GHKGD, the level of Th1 cytokines (IFN-γ, TNF-α, IL-2) was increased, while the expression of Th2 (IL-5, IL4) cytokines was reduced (p < 0.05). Conclusion: Through a network pharmacology strategy and in vivo experiments, the multi-target and multi-component pharmacological characteristics of GHKGD in the treatment of influenza were revealed, and regulation of the RLR signaling pathway during the anti-influenza process was confirmed. This study provides a theoretical basis for the research and development of new drugs from GHKGD.

In Vitro Profiling of Laninamivir-Resistant Substitutions in N3 to N9 Avian Influenza Virus Neuraminidase Subtypes and Their Association with In Vivo Susceptibility.

Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the in vitro susceptibility of multi-NAI-resistant AIVs is associated with in vivo susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3R292K) or Q136K (ma81K-N8Q136K) NA substitutions, which impart in vitro susceptibility only to LAN or OS, respectively. Both ma81K-N3R292K and ma81K-N8Q136K virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the in vitro susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of in vitro and in vivo NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs.IMPORTANCE The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that in vitro data predicted in vivo behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections.

The active GLP-1 analogue liraglutide alleviates H9N2 influenza virus-induced acute lung injury in mice

Influenza virus is responsible for significant morbidity and mortality worldwide. Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the major cause of death in influenza virus infected patients. Recent studies indicated that active glucagon like peptide-1 (GLP-1) encoded by glucagon (GCG) gene exerts anti-inflammatory functions. The aim of this study was to determine the potential role of active GLP-1 in H9N2 influenza virus-induced ALI/ARDS in mice. First, we uncovered that GCG mRNA expression levels and GCG precursor protein levels were significantly increased, but total GLP-1 and active GLP-1 levels were decreased in the lungs of H9N2-infected mice. Next, liraglutide, an active GLP-1 analogue, was used to treat infected mice and to observe its effects on H9N2 virus-induced ALI. Liraglutide treatment ameliorated the declined body weight, decreased food intake and mortality observed in infected mice. It also alleviated the severity of lung injury, including lowering lung index, decreasing inflammatory cell infiltration and lowing total protein levels in bronchoalveolar lavage fluid (BALF). In addition, liraglutide did not influence viral titers in infected lungs, but decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-α in BALF. These results indicated that liraglutide alleviated H9N2 virus-induced ALI in mice most likely due to lower levels of pro-inflammatory cytokines.

A Single Intranasal or Intramuscular Immunization with Chimpanzee Adenovirus Vectored SARS-CoV-2 Vaccine Protects Against Pneumonia in Hamsters

The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable or neutralizing the virus, antibody levels in serum were higher in hamsters immunized by an intranasal compared to intramuscular route. Accordingly, ChAd-SARS-CoV-2-S immunized hamsters were protected against a challenge with a high dose of SARS-CoV-2. After challenge, ChAd-SARS-CoV-2-S-immunized hamsters had less weight loss and showed reductions in viral RNA and infectious virus titer in both nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.Funding: This study was funded by NIH contracts and grants (R01 AI157155, 75N93019C00062, U01 AI151810, HHSN272201400018C, and HHSN272201700060C). J.B.C. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship.Conflict of Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, andCarnival Corporation and on the Scientific Advisory Board of Moderna and Immunome. TheDiamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, 548 GreenLight Biosciences Inc., AbbVie Inc., and Nano targeting & Therapy Biopharma Inc. M.S.D. and A.O.H.have filed a disclosure with Washington University for possible commercial development of ChAd SARS-CoV-2.

Examining effects of NS1 specific antibodies on sublethal influenza infection and secondary bacterial pneumonia in mice

Influenza is a highly contagious respiratory disease widespread throughout the world that causes disease in humans, birds and many mammalian species. Annually, around 20% of the global human gets sick with influenza so that more than 500,000 people die its various complications. Secondary bacterial pneumonia poses the peak threat during influenza infection, being most frequently caused by S. pneumoniae. Multiple studies in humans confirm the negative impact of influenza virus infection on subsequent outcome of bacterial pneumonia and provides insight into increased morbidity and mortality due to complicated influenza infection. In particular, the last 2009 influenza pandemic caused by H1N1 virus revealed that 25-56% cases of severe disease forms were associated with secondary pneumonia, among which 14-46% of them were fatal. Based on the aforementioned, it is of high priority to investigate a role of influenza virus proteins in developing of pathogen synergism in viral-bacterial pneumonia, particularly influenza virus non-structural protein NS1. The study objective was to examine effects of NS1-specific antibodies on course of influenza infection and secondary bacterial pneumonia in mice. For this, we used an experimental model of sublethal influenza infection followed by secondary Streptococcus pneumoniae bacterial pneumonia. Influenza A/Puerto Rico/8/34 (H1N1) virus and S. pneumoniae No. 3405 strain were used to simulate influenza infection. Rabbit serum containing antibodies against recombinant NS1 protein from A/Puerto Rico/8/34 virus and native rabbit serum (contain no specific antibodies) were used for vaccination. The study was carried out with female BALB/c mice, weighing 20-22 g. Protective activity of animal serum was assessed by using the three criteria: infection-related mortality, life expectancy and body weight change. The data obtained showed that passive transfer of antibodies specific to influenza virus NS1 protein did not lowered viral replication in sublethal murine model of influenza infection. Subsequent secondary bacterial pneumonia induced by S. pneumoniae revealed no protective effect of anti-NS1 protein antibodies assessed by measuring survival rate, lung viral and bacterial titers in treated vs. control mice.

Pharmacological Characterization of TP0591816, a Novel Macrocyclic Respiratory Syncytial Virus Fusion Inhibitor with Antiviral Activity against F Protein Mutants.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early childhood. However, no vaccines have yet been approved for prevention of RSV infection, and the treatment options are limited. Therefore, development of effective and safe anti-RSV drugs is needed. In this study, we evaluated the antiviral activity and mechanism of action of a novel macrocyclic anti-RSV compound, TP0591816. TP0591816 showed significant antiviral activities against both subgroup A and subgroup B RSV, while exerting no cytotoxicity. Notably, the antiviral activity of TP0591816 was maintained against a known fusion inhibitor-resistant RSV strain with a mutation in the cysteine-rich region or in heptad repeat B. Results of a time-of-addition assay and a temperature shift assay indicated that TP0591816 inhibited fusion of RSV with the cell membrane during viral entry. In addition, TP0591816 added after cell infection also inhibited cell-cell fusion. A TP0591816-resistant virus strain selected by serial passage had an L141F mutation, but no mutation in the cysteine-rich region or in heptad repeat B in the fusion (F) protein. Treatment with TP0591816 reduced lung virus titers in a dose-dependent manner in a mouse model of RSV infection. Furthermore, the estimated effective dose of TP0591816 for use against F protein mutants was thought to be clinically realistic and potentially tolerable. Taken together, these findings suggest that TP0591816 is a promising novel candidate for the treatment of resistant RSV infection.

Pharmacokinetic and pharmacodynamic analysis of baloxavir marboxil, a novel cap-dependent endonuclease inhibitor, in a murine model of influenza virus infection.

BackgroundBaloxavir acid, the active form of the orally available prodrug baloxavir marboxil, is a novel cap-dependent endonuclease inhibitor of influenza virus. Baloxavir marboxil has been shown to rapidly reduce virus titres compared with oseltamivir in clinical studies.ObjectivesWe investigated the relationship between pharmacokinetic (PK) parameters and antiviral activity of baloxavir acid based on virus titre reduction in lungs of infected mice.MethodsBALB/c mice infected with a sub-lethal dose of influenza A(H1N1), A(H1N1)pdm09, A(H3N2) or type B virus were treated on day 5 with oral baloxavir marboxil (0.5–50 mg/kg q12h), subcutaneous baloxavir acid (0.25–8 mg/kg/day), oseltamivir phosphate (5 or 50 eq mg/kg q12h) or other antivirals for 1 day. Lung virus titres were assessed 24 h after initial antiviral dosing. PK testing was performed at up to 24 h post-dosing of baloxavir marboxil or baloxavir acid in A/WSN/33-infected mice and the PK/pharmacodynamic (PD) relationship was evaluated for baloxavir acid.ResultsOral baloxavir marboxil administration showed dose-dependent virus titre reductions in lungs of mice infected with the different types/subtypes of influenza viruses 24 h post-dosing. Baloxavir marboxil at 15 mg/kg q12h resulted in ≥100-fold and ≥10-fold reductions in influenza A and B virus titres, respectively, compared with oseltamivir phosphate. PK/PD analysis showed that the plasma concentration at the end of the dosing interval (Cτ) or the plasma concentration at 24 h after initial dosing (C24) was the PK parameter predicting the virus titres at 24 h post-dosing of baloxavir acid.ConclusionsPK/PD analysis of baloxavir acid based on virus titre reduction in this mouse model could be helpful in predicting and maximizing virological outcomes in clinical settings.

Reduning plus ribavirin display synergistic activity against severe pneumonia induced by H1N1 influenza A virus in mice.

To investigate synergistic effect of Reduning (RDN) injection plus ribavirin against severe pneumonia induced by H1N1 influenza A virus in mice. We established a mouse model of severe pneumonia induced by influenza A virus by infecting Balb/c mice with CA07 virus. We randomly assigned the infected mice into four groups, and treated them with normal saline (NS group), RDN (injection, 86.6 mg/kg), ribavirin (injection, 66.6 mg/kg) or double Ribavirin plus RDN group, the same dosage as used in the single treatments) for 5 d. Lung index and lung pathology were recorded or calculated in terms of the curative effective. Cytokines, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome related protein including caspase-associated recruitment domain (CARD) domain Apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC), caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3), and reactive oxygen species were simultaneously investigated. RDN plus ribavirin treatment, not RDN or ribavirin alone, provided a significant survival benefit to the influenza A virus-infected mice. The combination treatment protected the mice against severe influenza infection by attenuating the severe lung injury. The combined treatment also reduced the viral titers in mouse lungs and lung index, downregulated their immunocytokine levels, including IL-1β and IL-18, and down regulated the NLRP3, especially the transcription and translation of caspase-1. Meanwhile NS group had significantly higher reactive oxygen species (ROS) expression which could was dramatically reduced by the treatment of RDN plus ribavirin. Our study showed that RDN combined with ribavirin could protect the mice, and reduce the lung immunopathologic damage caused by severe influenza pneumonia. The mechanism could be that it reduced ROS produce and inhibited NLRP3 inflammasome activation so that mainly lower the downstream inflammatory cytokines IL-1β and IL-18.

Short Communication: Oral Administration of Heat-killed Lactobacillus brevis KB290 in Combination with Retinoic Acid Provides Protection against Influenza Virus Infection in Mice.

Influenza virus type A (IAV) is a seasonal acute respiratory disease virus with severe symptoms, and an effective preventive measure is required. Despite many reports describing the potentially protective effects of lactic acid bacteria, few studies have investigated the effects of nutritional supplement combinations. This study reports the effect of the combined intake of heat-killed Lactobacillus brevis KB290 (KB290) and vitamin A (VA) on mice challenged with a sublethal dose of IAV. For 2 weeks, five groups of mice were fed either placebo, KB290, VA, or a combination of KB290 and VA (KB290+VA). After subsequent IAV challenge, bodyweight and general health were monitored for up to 2 weeks. Viral titres were determined in the lungs of animal subgroups euthanised at days 3, 7, and 14 after IAV challenge. A significant loss was observed in the bodyweights of IAV-infected animals from day 1 post-IAV challenge, whereas the mice fed KB290+VA did not lose any weight after IAV infection, indicating successful protection from the infection. Additionally, mice in the KB290+VA group showed the highest reduction in lung viral titres. In conclusion, the combination of KB290 and VA could be a useful food supplement relevant for protection against seasonal influenza virus infection in humans.

Human Metapneumovirus Induces Mucin 19 Which Contributes to Viral Pathogenesis.

Human Metapneumovirus (HMPV) remains one of the most common viral infections causing acute respiratory tract infections, especially in young children, elderly, and immunocompromised populations. Clinical symptoms can range from mild respiratory symptoms to severe bronchiolitis and pneumonia. The production of mucus is a common feature during HMPV infection, but its contribution to HMPV-induced pathogenesis and immune response is largely unknown. Mucins are a major component of mucus and they could have an impact on how the host responds to infections. Using an in vitro system and a mouse model of infection, we identified that Mucin 19 is predominantly expressed in the respiratory tract upon HMPV infection. Moreover, the lack of Muc19 led to an improved disease, lower lung viral titers and a decrease in the number of CD4+ T cells. These data indicate that mucin 19 contributes to the activation of the immune response to HMPV and to HMPV-induced pathogenesis.