Virus Neuraminidase Research Articles

Neuraminidase Inhibitor of Garcinia atroviridis L. Fruits and Leaves Using Partial Purification and Molecular Characterization.

Neuraminidase (NA) is an enzyme that prevents virions from aggregating within the host cell and promotes cell-to-cell spread by cleaving glycosidic linkages to sialic acid. The best-known neuraminidase is the viral neuraminidase, which present in the influenza virus. Thus, the development of anti-influenza drugs that inhibit NA has emerged as an important and intriguing approach in the treatment of influenza. Garcinia atroviridis L. (GA) dried fruits (GAF) are used commercially as seasoning and in beverages. The main objective of this study was to identify a new potential neuraminidase inhibitor from GA. A bioassay-guided fractionation method was applied to obtain the bioactive compounds leading to the identification of garcinia acid and naringenin. In an enzyme inhibition study, garcinia acid demonstrated the highest activity when compared to naringenin. Garcinia acid had the highest activity, with an IC50 of 17.34–17.53 µg/mL or 91.22–92.21 µM against Clostridium perfringens-NA, and 56.71–57.85 µg/mL or 298.32–304.31 µM against H1N1-NA. Based on molecular docking results, garcinia acid interacted with the triad arginine residues (Arg118, Arg292, and Arg371) of the viral neuraminidase, implying that this compound has the potential to act as a NA enzyme inhibitor.

Pre-existing antibodies directed against a tetramerizing domain enhance the immune response against artificially stabilized soluble tetrameric influenza neuraminidase

The neuraminidase (NA) is an abundant antigen at the surface of influenza virions. Recent studies have highlighted the immune-protective potential of NA against influenza and defined anti-NA antibodies as an independent correlate of protection. Even though NA head domain changes at a slightly slower pace than hemagglutinin (HA), NA is still subject to antigenic drift, and therefore an NA-based influenza vaccine antigen may have to be updated regularly and thus repeatedly administered. NA is a tetrameric type II membrane protein, which readily dissociates into dimers and monomers when expressed in a soluble form. By using a tetramerizing zipper, such as the tetrabrachion (TB) from Staphylothermus marinus, it is possible to stabilize soluble NA in its active tetrameric conformation, an imperative for the optimal induction of protective NA inhibitory antibodies. The impact of repetitive immunizations with TB-stabilized antigens on the immunogenicity of soluble TB-stabilized NA is unknown. We demonstrate that TB is immunogenic in mice. Interestingly, preexisting anti-TB antibodies enhance the anti-NA antibody response induced by immunization with TB-stabilized NA. This immune-enhancing effect was transferable by serum and operated independently of activating Fcγ receptors. We also demonstrate that priming with TB-stabilized NA antigens, enhances the NA inhibitory antibody responses against a heterosubtypic TB-stabilized NA. These findings have implications for the clinical development of oligomeric vaccine antigens that are stabilized by a heterologous oligomerizing domain.

Influenza virus entry and replication inhibited by 8-prenylnaringenin from Citrullus lanatus var. citroides (wild watermelon).

We previously demonstrated the anti‐influenza activity of Citrullus lanatus var. citroides (wild watermelon, WWM); however, the active ingredient was unknown. Here, we performed metabolomic analysis to evaluate the ingredients of WWM associated with antiviral activity. Many low‐molecular weight compounds were identified, with flavonoids accounting for 35% of all the compounds in WWM juice. Prenylated flavonoids accounted for 30% of the flavonoids. Among the measurable components of phytoestrogens in WWM juice, 8‐prenylnaringenin showed the highest antiviral activity. We synthesized 8‐prenylnaringenin and used liquid chromatography–mass spectrometry to quantitate the active ingredient in WWM. The antiviral activities of 8‐prenylnaringenin were observed against H1N1 and H3N2 influenza A subtypes and influenza B viruses. Moreover, 8‐prenylnaringenin was found to inhibit virus adsorption and late‐stage virus replication, suggesting that the mechanisms of action of 8‐prenylnaringenin may differ from those of amantadine and oseltamivir. We confirmed that 8‐prenylnaringenin strongly inhibited the viral entry of all the influenza virus strains that were examined, including those resistant to the anti‐influenza drugs oseltamivir and amantadine. This result indicates that 8‐prenylnaringenin may activate the host cell's defense mechanisms, rather than directly acting on the influenza virus. Since 8‐prenylnaringenin did not inhibit late‐stage virus replication of oseltamivir‐resistant strains, 8‐prenylnaringenin may interact directly with viral neuraminidase. These results are the first report on the anti‐influenza virus activity of 8‐prenylnaringenin. Our results highlight the potential of WWM and phytoestrogens to develop effective prophylactic and therapeutic approaches to the influenza virus.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

The effects of mutation on the drug binding affinity of Neuraminidase: case study of Capsaicin using steered molecular dynamics simulation.

The influenza virus is an important respiratory pathogen that causes many incidences of diseases and even death each year. One of the primary factors of this virus is the Neuraminidase surface protein, which causes the virus to leave the host cell and spread to new target cells. The main antiviral medication for influenza is designed as a protein inhibitor ligand that prevents further spread of the disease, and eventually relieves the emerged symptoms. The effectiveness of such inhibitory drugs is highly associated with their binding affinity. In this paper, the binding affinity of an herbal ligand of Capsaicin bound to Neuraminidase of the influenza virus is investigated using steered molecular dynamics (SMD) simulation. Since mutations of the virus directly impact the binding affinity of the inhibitory drugs, different mutations were generated by using Mutagenesis module. The rapid spread of infection during the avian influenza A/H5N1 epidemic has raised concerns about far more dangerous consequences if the virus becomes resistant to current drugs. Currently, oseltamivir (Tamiflu), zanamivir (Relenza), pramivir (Rapivab), and laninamivir (Inavir) are increasingly used to treat the flu. However, with the rapid evolution of the virus, some drug-resistant strains are emerging. Therefore, it is very important to seek alternative therapies and identify the roots of drug resistance. Obtained results demonstrated a reduced binding affinity for the applied mutations. This reduction in binding affinity will cause the virus mutation to become resistant to the drug, which will spread the disease and make it more difficult to treat. From a molecular prospect, this decrease in binding affinity is due to the loss of a number of effective bonds between the ligand and the receptor, which occurs with mutations of the wild-type (WT) species. The results of the present study can be used in the rational design of novel drugs that are compatible with specific mutations.

A rapid influenza diagnostic test based on detection of viral neuraminidase activity

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66–85.84%) and 95.39% (95% confidence interval: 94.32–96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.

A biochemiluminescent assay for rapid diagnosis of influenza.

A biochemiluminescent assay of influenza diagnosis is presented. The assay diagnoses influenza based on detection of the influenza viral neuraminidase activity. An instrument designed for the assay is also reported. This assay solves the problem that current influenza virus diagnosis assays are susceptible to virus mutation. A luciferase-based complex is synthesized as biochemiluminescent substrate. The substrate is cleaved to free luciferin with presence of influenza neuraminidase in specimen. Luciferase is oxidized to oxyluciferin with luciferin as catalyzer resulting in luminescence, which is proportional to the neuraminidase activity and measured by instrument. The instrument uses a photomultiplier tube as sensor, with 24 test channels. Fine optical arrangements enable the instrument with high sensitivity and accuracy. A total of 389 clinical specimens were collected to evaluate the performance of the assay in clinical settings. This assay had a sensitivity and specificity of 95.92% (95% confidence interval 91.38-98.12%) and 97.93% (95% confidence interval 95.26-99.11%), respectively, compared to the colloidal gold assay. As a biochemiluminscence assay, this assay is advantageous in sensitivity and specificity. It does not require any washing or separation steps, which makes the instrument simple in design and easy to operate or maintenance. The assay is suitable for the rapid diagnosis of influenza virus in point-of-care settings.

Induction of immunogenic response in Balb/c mice by virus-like particles designed using the influenza neuraminidase, hemagglutinin and matrix proteins

Induction of immunogenic response in Balb/c mice by virus-like particles designed using the influenza neuraminidase, hemagglutinin and matrix proteins

Phylogenetic and Mutational Analysis of Neuraminidase of Avian Influenza A (H9N2) Virus of B2- Sub Lineage, Isolated from Commercial Poultry Pakistan

Phylogenetic and Mutational Analysis of Neuraminidase of Avian Influenza A (H9N2) Virus of B2- Sub Lineage, Isolated from Commercial Poultry Pakistan

Synthesis and Neuraminidase Inhibitory Activity of Sialic Acid Analogues with Fluoro, Phosphono, and Sulfo Functionalities.

Methods to synthesize influenza virus inhibitors with fluoro, phosphono, and/or sulfo functional groups are described. The resulting sialic acid analogues are produced from the natural substrate N-acetylneuraminic acid as starting material. Fluorescent assay methods for inhibition of influenza neuraminidase and virus proliferation are also provided.

Computational modelling studies of some 1,3-thiazine derivatives as anti-influenza inhibitors targeting H1N1 neuraminidase via 2D-QSAR, 3D-QSAR, molecular docking, and ADMET predictions

BackgroundInfluenza virus disease remains one of the most contagious diseases that aided the deaths of many patients, especially in this COVID-19 pandemic era. Recent discoveries have shown that the high prevalence of influenza and SARS-CoV-2 coinfection can rapidly increase the death rate of patients. Hence, it became necessary to search for more potent inhibitors for influenza disease therapy. The present study utilized some computational modeling concepts such as 2D-QSAR, 3D-QSAR, molecular docking simulation, and ADMET predictions of some 1,3-thiazine derivatives as inhibitors of influenza neuraminidase (NA).ResultsThe 2D-QSAR modeling results showed GFA-MLR (R_{{text{train }}}^{2} = 0.9192, Q2 = 0.8767, R2adj = 0.8991, RMSE = 0.0959, R_{{{text{test}}}}^{2} = 0.8943, R_{{{text{pred}}}}^{2} = 0.7745) and GFA-ANN (R_{{text{train }}}^{2} = 0.9227, Q2 = 0.9212, RMSE = 0.0940, R_{{{text{test}}}}^{2} = 0.8831, R_{{{text{pred}}}}^{2} = 0.7763) models with the computed descriptors as ATS7s, SpMax5_Bhv, nHBint6, and TDB9m for predicting the NA inhibitory activities of compounds which have passed the global criteria of accepting QSAR model. The 3D-QSAR modeling was carried out based on the comparative molecular field analysis (CoMFA) and comparative similarity indices analysis (CoMSIA). The CoMFA_ES (R_{{text{train }}}^{2} = 0.9620, Q2 = 0.643) and CoMSIA_SED (R_{{text{train }}}^{2} = 0.8770, Q2 = 0.702) models were found to also have good and reliable predicting ability. The compounds were also virtually screened based on their binding scores via molecular docking simulations with the active site of the NA (H1N1) target receptor which also confirms their resilient potency. Four potential lead compounds (4, 7, 14, and 15) with the relatively high inhibitory rate (> 50%) and docking (> − 6.3 kcal/mol) scores were identified as the possible lead candidates for in silico exploration of improved anti-influenza agents.ConclusionThe drug-likeness and ADMET predictions of the lead compounds revealed non-violation of Lipinski’s rule and good pharmacokinetic profiles as important guidelines for rational drug design. Hence, the outcome of this research set a course for the in silico design and exploration of novel NA inhibitors with improved potency.

Innovated formulation of oseltamivir powder for suspension with stability study after reconstitution using a developed ion-pair reversed phase high-performance liquid chromatography method.

ABSTRACTOseltamivir is an antiviral neuraminidase inhibitor used for the treatment and prophylaxis of influenza infection with viruses A and B. The mechanism of oseltamivir antiviral activity is by inhibiting the activity of the viral neuraminidase enzyme present on the surface of the virus, which stops viral replication and infectivity. Oral suspensions of oseltamivir phosphate are dispensed orally capsules and suspension. However, the use of oral suspension for pediatric administration is preferable and is prepared as a powder for suspension. The reconstituted suspension degrades rapidly within a few days. The objective of this work is to establish a stable formulation of oseltamivir phosphate as a suspension and to assure the stability conditions for prolonged use after reconstitution in aqueous form. In addition, this required formulation should maintain a high rate of dissolution, which subsequently leads to higher bioavailability. In this study, oseltamivir forms an inclusion complex with the natural and safe polymer hydroxypropyl beta-cyclodextrin, which resembles a host because its structural cavity carries the oseltamivir molecule in the aqueous preparation and provides a protective property against environmental challengers. In addition, a high-performance liquid chromatography (HPLC) stability-indicating method of analysis has been developed using an ion-pair reversed-phase HPLC technique that is validated for precision, accuracy, reproducibility, and specificity for the determination of oseltamivir in suspension. The results of this work show the relatively long shelf life of the reconstituted oseltamivir oral powder for suspension in the new pediatric formulation, and the developed HPLC method was precisely suitable for stability study.

Antiviral activity of Epimedium koreanum Nakai water extract against influenza viruses

Epimedium koreanum Nakai (EKN) is a popular plant in Korean and Chinese medicine for treating a variety of ailments. The aqueous extract of EKN has a significant inhibitory impact on influenza A virus (IAV) infection by directly blocking viral attachment and having a virucidal effect, according to this study. Using fluorescent microscopy and fluorescence-activated cell sorting (FACS) with a green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus, we examined the effect of EKN on viral infection. By viral infection, EKN strongly suppresses GFP expression, and at a dosage of 100 µg/mL, EKN decreased GFP expression by up to 90% of the untreated infected control. Immunofluorescence and Western blot analyses against influenza viral proteins revealed that EKN decreased influenza viral protein expression in a dose-dependent manner. EKN inhibited the H1N1 influenza virus's hemagglutinin (HA) and neuraminidase (NA), preventing viral attachment to cells. Furthermore, EKN had a virucidal impact and inhibited the cytopathic effects of H1N1, H3N2 and influenza B virus infection. Finally, our findings show that EKN has the potential to be developed as a natural viral inhibitor against influenza virus infection.

Insights into the direct anti-influenza virus mode of action of Rhodiola rosea

Background: The anti-influenza A virus activities and contents of previously isolated most active flavonoids (rhodiosin and tricin) from a standardized hydro-ethanolic R. rosea root and rhizome extract (SHR-5®), did not fully explain the efficacy of SHR-5®. Moreover, the mode of antiviral action of SHR-5® is unknown.Purpose: To determine the anti-influenza viral principle of SHR-5® we evaluated i) the combined anti-influenza virus effect of rhodiosin and tricin, ii) the impact of its tannin-enriched fraction (TE), iii) its antiviral spectrum and mode of action, and iv) its propensity for resistance development in vitro.Methods: The combined anti-influenza virus effect of rhodiosin and tricin and the impact of TE were investigated with cytopathic effect (CPE)-inhibition assays in MDCK cells. A tannin-depleted fraction (TD) and TE were prepared by polyamide column chromatography and dereplicated by LC-MS. Plaque-reduction assays provided insights into the anti-influenza virus profile, the mode of action, and the propensity for resistance development of SHR-5®.Results: Our results i) did not reveal synergistic anti-influenza A virus effects of rhodiosin and tricin, but ii) proved a strong impact of TE mainly composed of prodelphinidin gallate oligomers. iii) TE inhibited the plaque-production of influenza virus A(H1N1)pdm09, A(H3N2), and B (Victoria and Yamagata) isolates (including isolates resistant to neuraminidase and/or M2 ion channel inhibitors) with 50% inhibitory concentration values between 0.12 - 0.53 µg/ml similar to SHR-5®. Mechanistic studies proved a virucidal activity, inhibition of viral adsorption, viral neuraminidase activity, and virus spread by SHR-5® and TE. iv) No resistance development was observed in vitro.Conclusion: For the first time a comprehensive analysis of the anti-influenza virus profile of a hydro-ethanolic R. rosea extract (SHR-5®) was assessed in vitro. The results demonstrating broad-spectrum multiple direct anti-influenza virus activities, and a lack of resistance development to SHR-5® together with its known augmentation of host defense, support its potential role as an adaptogen against influenza virus infection.

Investigation on the Binding Properties of N1 Neuraminidase of H5N1 Influenza Virus in Complex with Fluorinated Sialic Acid Analog Compounds—a Study by Molecular Docking and Molecular Dynamics Simulations

Worldwide, only two types of antiviral inhibitors (M2 ion channel protein inhibitor and Neuraminidase inhibitors) are approved to treat the influenza viral infection. But the mutation of amino acid sequence in the viral membrane proteins creates the viral resistance to existing antiviral drugs or inhibitors. So the corresponding antiviral drugs have to be reformulated to match these antigenic variations. Fluorination on the carbon–based molecule significantly enriches its biological properties. Hence this study is motivated to design the fluorinated sialic acid (SIA) analog inhibitors for the neuraminidase of H5N1 influenza A virus by substituting fluorine atom at different hydroxyls (O2, O4, O7, O8, and O9) of sialic acid. 100 ns molecular dynamics simulations are carried out for each protein–ligand complex system. NAMD pair interaction energy and MM–PBSA binding free energy calculations predict two possible binding modes for N1–SIA_F2, N1–SIA_F4, and N1–SIA_F7 complexes and single binding mode for N1–SIA_F8 and N1–SIA_F9 complexes. RMSD, RMSF, and hydrogen bonding analyses are used to understand the conformational flexibility and structural stability of each complex system. It has been concluded that the fluorinated sialic acid drug candidates SIA_F2 and SIA_F7 have better inhibiting potency against the N1 neuraminidase of H5N1 influenza virus.

Nanoscale Mapping of Recombinant Viral Proteins: From Cells to Virus-Like Particles.

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFlu-S). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

Prospects of using conservative linear B-cell epitopes of influenza virus A neuraminidase for induction of cross-protective immune response

BACKGROUND: Influenza is a dangerous, widespread infectious disease that takes thousands of lives during annual epidemics, and also causes significant damage to the countrys economy. The most effective means of fighting the influenza virus is vaccination of the population. Due to the variability of influenza viruses, the strain composition of influenza vaccines must be updated annually. In this regard, an urgent task is to improve the existing influenza vaccines in order to expand their spectrum of action. One of the promising approaches is the targeted induction of the humoral immune response to the conservative linear epitopes of influenza A virus neuraminidase.
 AIM: This project is aimed at assessing the immunogenicity and cross-protective activity of conserved neuraminidase epitopes in order to select promising targets for the targeted design of broad-spectrum influenza vaccines.
 MATERIALS AND METHODS: Peptides corresponding to linear B-cell epitopes of neuraminidase were chemically synthesized de novo. The peptides were conjugated with keyhole limpet hemocyanin. CBA mice were immunized and challenged with A/PR/8/34 (H1N1) and A/Philippines/2/1982 (H3N2) viruses at a dose of 3 LD50. The survival rate of the animals was assessed within 14 days after infection. The immunogenicity of the peptides was assessed in a standard enzyme-linked immunosorbent assay using the recombinant neuraminidase proteins of the viruses A/California/07/2009 (H1N1) and A/Hong Kong/4801/2014 (H3N2) as antigen.
 RESULTS: Immunization of neuraminidase with peptides MNPNQKIITIGS and ILRTQESEC, but not DNWKGSNRP, protected mice from lethality caused by the H1N1 and/or H3N2 virus. The protective potential of the peptides correlated with the levels of antineuraminidase antibodies after immunization.
 CONCLUSIONS: The presence of a cross-protective potential in two conserved linear B-cell epitopes of influenza A neuraminidase (MNPNQKIITIGS and ILRTQESEC) allows them to be recommended as a target for the development of a broad-spectrum influenza vaccine.

Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.

The antiviral effect of catechins on mumps virus infection

• The inhibitory effect and its mechanism of catechins on mumps virus infection. • Epigallocatechin gallate shows the highest antiviral potency. • A galloyl group in catechins is an important factor for the strong antiviral effect. • The antiviral effect can result from inhibition of the viral fusion activity. Catechins have been known to possess antiviral effects against influenza A and B viruses through the inhibition of both the viral receptor binding and viral sialidase activities. Here, we examined the antiviral effect of catechins on mumps virus infection. The addition of epigallocatechin gallate (EGCG), gallocatechin gallate, epicatechin gallate, or catechin gallate simultaneously with the mumps virus infection of Vero cells greatly inhibited the infection. EGCG showed the strongest antiviral effect among the catechins tested. These catechins did not exhibit an inhibitory effect against the receptor binding and sialidase activities in the hemagglutinin–neuraminidase of the mumps virus, which was different from a known mechanism for influenza A and B viruses. By contrast, these catechins inhibited the viral fusion activity. Our results indicate that catechins are a useful food-derived antiviral agent against the mumps virus.

Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase.

Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing foreign antigens. In this study, we generated and evaluated the live mucosal recombinant vaccine by integrating genes encoding influenza virus neuraminidase (NA) of the N2 subtype into the DNA of the probiotic strain Enterococcus faecium L3 (L3). We confirmed NA expression in the pili of L3 using immune electron microscopy. Mice were fed with a probiotic vaccine containing the NA gene (L3-NA) or pure L3. Oral administration of L3-NA caused detectable increase in virus-specific serum IgG and local IgA after the third feeding. Immunization with L3-NA increased the survival rate by 34% when the mice were infected using A(H1N1)pdm09 influenza virus after the third feeding. After S. pneumoniae post-influenza infection, the L3-NA-immunized mice were 50% more protected from lethality in comparison with L3-fed mice. Thus, a live probiotic vaccine candidate based on L3 induced the formation of systemic and local immunity and provide partial protection against complicated influenza.