Silver nanoparticle-modified melt-blown polypropylene: Antibacterial and antifungal properties and antiviral activity against SARS-CoV-2.

Melt-blown polymer fiber materials are frequently used in the face mask manufacturing. In the present work, a melt-blown polypropylene tape was modified by silver nanoparticles using chemical metallization. The silver coatings on the fiber surface consisted of crystallites 4-14 nm in size. For the first time, these materials were comprehensively tested for antibacterial, antifungal and antiviral activity. The silver-modified materials showed antibacterial and antifungal activities, especially at high concentrations of silver, and were found to be efficient against the SARS-CoV-2 virus. The silver-modified fiber tape can be used in the face mask manufacturing and as an antimicrobial and antiviral component in filters of liquid and gaseous media.

Glycyrrhizic acid derivatives as influenza A/H1N1 virus inhibitors.

This Letter describes the synthesis and antiviral activity study of some glycyrrhizic acid (GL) derivatives against influenza A/H1N1/pdm09 virus in MDCK cells. Conjugation of GL with l-amino acids or their methyl esters, and amino sugar (d-galactose amine) dramatically changed its activity. The most active compounds were GL conjugates with aromatic amino acids methyl esters (phenylalanine and tyrosine) (SI=61 and 38), and S-benzyl-cysteine (SI=71). Thus modification of GL is a perspective route in the search of new antivirals, and some of GL derivatives are potent as anti-influenza A/H1N1 agents.

A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice.

The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.

Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector.

The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

Camphor-based symmetric diimines as inhibitors of influenza virus reproduction.

Influenza is a continuing world-wide public health problem that causes significant morbidity and mortality during seasonal epidemics and sporadic pandemics. The purpose of the study was synthesis and investigation of antiviral activity of camphor-based symmetric diimines and diamines. A set of C2-symmetric nitrogen-containing camphor derivatives have been synthesized. The antiviral activity of these compounds was studied against rimantadine- and amantadine-resistant influenza virus A/California/7/09 (H1N1)pdm09 in MDCK cells. The highest efficacy in virus inhibiting was shown for compounds 2a-e with cage moieties bound by aliphatic linkers. The therapeutic index (selectivity index) for 2b exceeded that for reference compounds amantadine, deitiforin and rimantadine almost 10-fold. As shown by structure-activity analysis, the length of the linker has a dramatic effect on the toxicity of compounds. Compound 2e with -C12H24- linker exhibited the lowest toxicity (CTD50=2216µM). Derivatives of camphor, therefore, can be considered as prospective antiinfluenza compounds active against influenza viruses resistant to adamantane-based drugs.

Novel derivatives of usnic acid effectively inhibiting reproduction of influenza A virus.

Influenza virus is serious human pathogen leading to high morbidity and mortality all over the world. Due to high rate of mutation, it is able to fast development of drug resistance that makes necessary to search novel antivirals with broad range and alternative targets. In the present study we describe synthesis and anti-viral activity of novel derivatives of usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione). It is shown that anti-viral activity of usnic acid can be increased by side moieties introduction. The modification with chalcones appeared to be the most effective. Our study revealed that (-)-usnic acid exhibited higher antiviral activity than its (+)-enantiomer, but in the pairs of enantiomer derivatives such as enamines, pyrazoles and chalcones, the (+)-enantiomers were more potent inhibitors of the virus. For other groups of compounds the inhibiting activities of the enantiomers were comparable. Further optimization of the structure could therefore result in development of novel anti-influenza compound with alternative target and mechanism of virus-inhibiting action.

New quaternary ammonium camphor derivatives and their antiviral activity, genotoxic effects and cytotoxicity.

The synthesis and biological evaluation of a novel series of dimeric camphor derivatives are described. The resulting compounds were studied for their antiviral activity, cyto- and genotoxicity. Compounds 3a and 3d in which the quaternary nitrogen atoms are separated by the C5H10 and С9H18 aliphatic chain, exhibited the highest efficiency as an agent inhibiting the reproduction of the influenza virus A(H1N1)pdm09. The cytotoxicity data of compounds 3 and 4 revealed their moderate activity against malignant cell lines; compound 3f had the highest activity for the CEM-13 cells. These results show close agreement with the data of independent studies on toxicity of these compounds, in particular that the toxicity of compounds strongly depends on spacer length.

Antimycotic-antibiotic amphotericin B promotes influenza virus replication in cell culture.

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.

Anti-viral activity of (-)- and (+)-usnic acids and their derivatives against influenza virus A(H1N1)2009.

Influenza is a widespread respiratory infection. Every year it causes epidemics, quickly spreading from country to country, or even pandemics, involving a significant part of the human population of the earth. Being a highly variable infection, influenza easy accumulates the resistance mutations to many antivirals. Usnic acid, a dibenzofuran originally isolated from lichens belongs to the secondary metabolites and has a broad spectrum of biological activity. In humans, it can act as an anti-inflammatory, antimitotic, antineoplasic, antibacterial, and antimycotic agent. In this work we studied for the first time the antiviral activity of usnic acid and its derivatives against the pandemic influenza virus A(H1N1)pdm09. A total of 26 compounds representing (+) and (-) isomers of usnic acid and their derivates were tested for cytotoxicity and anti-viral activity in MDCK cells by microtetrazolium test and virus yield assay, respectively. Based on the results obtained, 50% cytotoxic dose (CTD(50)) and 50% effective dose (ED(50)) and selectivity index (SI) were calculated for each compound. Eleven of them were found to have SI higher than 10 (highest value 37.3). Absolute configuration was shown to have critical significance for the anti-viral activity. With minor exceptions, in the pair of enantiomers, (-)-usnic acid was more active comparing to (+)-isomer, but its biological activity was reversed after the usnic acid was chemically modified. Based on the obtained results, derivatives of usnic acid should be considered as prospective compounds for further optimization as anti-influenza substances.

Forced nonlinear resonance in a system of coupled oscillators.

We consider a resonantly perturbed system of coupled nonlinear oscillators with small dissipation and outer periodic perturbation. We show that for the large time t∼ɛ(-2) one component of the system is described for the most part by the inhomogeneous Mathieu equation while the other component represents pulsation of large amplitude. A Hamiltonian system is obtained which describes for the most part the behavior of the envelope in a special case. The analytic results agree with numerical simulations.

Antiviral properties, metabolism, and pharmacokinetics of a novel azolo-1,2,4-triazine-derived inhibitor of influenza A and B virus replication.

Influenza viruses of types A and B cause periodic pandemics in the human population. The antiviral drugs approved to combat influenza virus infections are currently limited. We have investigated an effective novel inhibitor of human influenza A and B viruses, triazavirine [2-methylthio-6-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazine-7(4I)-one] (TZV). TZV suppressed the replication of influenza virus in cell culture and in chicken chorioallantoic membranes, and it protected mice from death caused by type A and B influenza viruses. TZV was also effective against a rimantadine-resistant influenza virus strain and against avian influenza A virus H5N1 strains. The pharmacokinetic parameters and bioavailability of TZV were calculated after the administration of TZV to rabbits. The TZV metabolite AMTZV [2-methylthio-6-amino-1,2,4-triazolo[5,1-s]-1,2,4-triazin(e)-7(4I)-one] was discovered in IAK 293T and Huh7 cell cultures, a liver homogenate, and rabbit blood after intragastric administration of TZV. AMTZV was nontoxic and inactive as an inhibitor of influenza virus in cell culture. Most likely, this metabolite is a product of TZV elimination.

Synthesis and anti-viral activity of azolo-adamantanes against influenza A virus.

Chemotherapy and chemoprophylaxis of influenza is one of the most important directions of health protection activity. Due to the high rate of drug-resistant strains of influenza virus, there is a need for the search and further development of new potent antivirals against influenza with a broad spectrum of activity. In the present study, a set of di-, tri- and tetrazole derivatives of adamantane was efficiently prepared and their anti-influenza activities evaluated against rimantadine-resistant strain A/Puerto Rico/8/34. In general, derivatives of tetrazole possessed the highest virus-inhibiting activity. We demonstrated that several compounds of this set exhibited much higher activity than the currently used antiviral rimantadine, a compound of related structure. Moreover, we showed that these azolo-adamantanes were significantly less toxic. This study demonstrates that influenza viruses can be inhibited by adamantyl-azoles and thus have potential for developing antiviral agents with an alternate mechanism of action.

Safety and immunogenicity of a replication-deficient H5N1 influenza virus vaccine lacking NS1.

BACKGROUND: Traditional inactivated influenza vaccines are the type of vaccines that were most frequently developed for immunization against the highly pathogenic avian H5N1 influenza virus. However, clinical trials with inactivated influenza vaccines for H5N1 indicated that high doses and at least two immunizations are required for an effective immune response (Nicholson et al., 2001; Treanor, Campbell et al., 2006; Treanor, Schiff et al., 2006; Ehrlich et al., 2008). We investigated the safety and immunogenicity of a live attenuated H5N1 vaccine (delNS1-H5N1) lacking the interferon antagonist nonstructural protein 1 (NS1). METHODS: We conducted a double-blind, placebo-controlled, phase 1 study in healthy adult participants who were randomly assigned at a 2:1 ratio to receive two immunizations of delNS1-H5N1 vaccine at 6.8 log10 50% tissue culture infectious doses (TCID50)/subject or 7.5 log10 TCID50/subject, or placebo. RESULTS: Intranasal vaccination with the live attenuated delNS1-H5N1 vaccine was safe and well tolerated. The most common adverse events identified were symptoms associated with mild influenza infections, such as increased body temperature (>37.0 °C), pharyngeal erythema, rhinitis and throat irritation, and were reported within 7 days after the first immunization. delNS1-H5N1 was able to induce significant vaccine-specific serum antibody titers even at the lower dose level of 6.8 log10 TCID50/subject. Seroconversion occurred in 75% of study participants after only one immunization with 7.5 log10 TCID50/subject. Vaccine-specific local IgA responses were observed in 41.7% of individuals that showed serum antibody responses after 2nd immunization. CONCLUSIONS: We show that vaccination with a live attenuated H5N1 influenza vaccine lacking NS1 is safe and induces significant levels of vaccine-specific antibodies even after one immunization. The safety and immunogenicity data indicate that delNS1-H5N1 has the potential to fulfil the unmet need for an effective influenza vaccine in pandemic situations. (ClinicalTrials.gov identifier NCT03745274).

Photodynamic inactivation of influenza virus with fullerene C60 suspension in allantoic fluid.

BACKGROUND: Viruses are the most dangerous infectious contaminants of human donor blood and blood products. The purpose of the study was to investigate the virus-inactivating properties of fullerene suspension regarding influenza virus in allantoic fluid of chicken embryos. METHODS: Influenza virus was propagated in chicken embryos, water suspension of C60 fullerene was added to the allantoic fluid. The fluid was light-irradiated at constant oxygen flow through the specimen, and the dynamics of virus titer was studied by virus titration in MDCK cells. The morphology of virions was studied by electron microscopy (EM). RESULTS: Dramatic drop of infectious titer (8 to 1 log10 EID50) of the virus was observed within 2h after start of irradiation. No change of the titers was observed in control specimens without fullerene, or light, or oxygen. EM study revealed numerous defects of virions' morphology (destruction of outer membrane) leading to the loss of infectious properties of the virus. CONCLUSIONS: Water-insoluble fullerenes may be considered as a prospective way for inactivation of enveloped viruses in biological materials including blood products.

Safety and efficacy of p62 DNA vaccine ELENAGEN in a first-in-human trial in patients with advanced solid tumors.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy.

Interleukin-24 inhibits influenza A virus replication in vitro through induction of toll-like receptor 3 dependent apoptosis.

New anti-viral agents and strategies are urgently needed to fight rapidly mutating viruses, as vaccine programs cannot react fast enough to prevent pandemics. Recently, we have shown that interleukin-24 (IL-24) sensitizes tumor cells to toll-like receptor 3 (TLR3) mediated apoptosis. As influenza A virus stimulates the TLR3 receptor, we hypothesized that IL-24 might also exert an anti-viral effect. This study demonstrates that IL-24 reduces the titer of different influenza A virus subtypes independently of type I interferon in an apoptosis dependent manner. The anti-viral effect of IL-24 correlated with caspase-3 activation and could be blocked by a pan-caspase inhibitor and by small interfering RNA (siRNA) directed towards TLR3. Surprisingly, caspase-3 activation in influenza A virus/IL-24-stimulated cells correlated with the down-regulation of the B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia 1 (Mcl-1). Correspondingly, knockdown of Mcl-1 by siRNA enhanced caspase activation in influenza A virus infected cells and was furthermore linked to a reduction of viral titers. We conclude that IL-24 exerts an anti-viral role selectively purging virally infected cells by leading to a down-regulation of Mcl-1. Our findings might therefore represent the first step towards a new rational concept in the development of anti-viral strategies based on the induction of apoptosis.

Discovery of a new class of antiviral compounds: camphor imine derivatives.

A new class of compounds featuring a camphor moiety has been discovered that exhibits potent inhibitory activity against influenza A(H1N1)pdm09 and A(H5N1) viruses. The synthesized compounds were characterized by spectroscopic analysis; in addition the structures of compound 2 and 14 were elucidated by the X-ray diffraction technique. Structure-activity relationship studies have been conducted to identify the 1,7,7-trimethylbicyclo[2.2.1]heptanes2-ylidene group as the key functional group responsible for the observed antiviral activity. The most potent antiviral compound is imine 2 with therapeutic index more than 500.

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant region of hepatitis B core antigen: Broad protective efficacy of particles carrying four copies of M2e.

A long-term objective when designing influenza vaccines is to create one with broad cross-reactivity that will provide effective control over influenza, no matter which strain has caused the disease. Here we summarize the results from an investigation into the immunogenic and protective capacities inherent in variations of a recombinant protein, HBc/4M2e. This protein contains four copies of the ectodomain from the influenza virus protein M2 (M2e) fused within the immunodominant loop of the hepatitis B virus core antigen (HBc). Variations of this basic design include preparations containing M2e from the consensus human influenza virus; the M2e from the highly pathogenic avian A/H5N1 virus and a combination of two copies from human and two copies from avian influenza viruses. Intramuscular delivery in mice with preparations containing four identical copies of M2e induced high IgG titers in blood sera and bronchoalveolar lavages. It also provoked the formation of memory T-cells and antibodies were retained in the blood sera for a significant period of time post immunization. Furthermore, these preparations prevented the death of 75-100% of animals, which were challenged with lethal doses of virus. This resulted in a 1.2-3.5 log10 decrease in viral replication within the lungs. Moreover, HBc particles carrying only "human" or "avian" M2e displayed cross-reactivity in relation to human (A/H1N1, A/H2N2 and A/H3N2) or A/H5N1 and A(H1N1)pdm09 viruses, respectively; however, with the particles carrying both "human" and "avian" M2e this effect was much weaker, especially in relation to influenza virus A/H5N1. It is apparent from this work that to quickly produce vaccine for a pandemic it would be necessary to have several variations of a recombinant protein, containing four copies of M2e (each one against a group of likely influenza virus strains) with these relevant constructs housed within a comprehensive collection Escherichia coli-producers and maintained ready for use.

Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

Matrix 2 protein ectodomain (M2e) is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek). Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1) and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1) and A/Chicken/Kurgan/05/05 RG (H5N1) to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1). Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.

Live cold-adapted influenza A vaccine produced in Vero cell line.

The African green monkey kidney (Vero) cell line was used as a substrate for the development of a live cold-adapted (ca) reassortant influenza vaccine. For that purpose, a new master strain was generated by an adaptation of the wild type (wt) A/Singapore/1/57 virus to growth at 25 degrees C in a Vero cell line. The resulting cold-adapted (ca) muster strain A/Singapore/1/57ca showed temperature sensitive (ts) phenotype and was attenuated in animal models and protective in the challenge experiments in ferrets. Two vaccine candidates of influenza A(H1N1) and A(H3N2) subtypes (6/2 reassortants) inheriting six genes coding internal proteins from the new master strain and the surface antigens hemagglutinin (HA) and neuraminidase (NA) from the epidemic viruses were obtained by a standard method of genetic reassortment. All steps of the vaccine preparation were done exclusively in Vero cells, including the isolation of the epidemic viruses. Both vaccine strains were used for immunization of young adult volunteers in a limited clinical trial and appeared to be safe, well tolerated and immunogenic after intranasal administration.