Broad Spectrum Of Influenza Viruses Research Articles

Induction of Homosubtypic and Heterosubtypic Immunity to Influenza Viruses Using a Conserved Internal and External Proteins

The immunogenicity and protective properties of the designed recombinant fusion peptide of 3M2e and truncated nucleoprotein (trNP), originating from Influenza A virus, were investigated in the BALB/c mice model in comparison with the Mix protein (3M2e + trNP). The results were evaluated by antibody response, cytokine production, lymphocyte proliferation assay, and mortality rate after challenge with homologous (H1N1) and heterologous (H3N2) influenza viruses in BALB/c mice. The animals that received the chimer protein with or without adjuvant had more specific antibody responses and elicited memory CD4 T cells, and cytokines of Th1 and Th2 cells compared to the Mix protein. Moreover, the Mix protein, like the recombinant chimer protein, provided equal and effective protection against both homologous and heterologous challenges in mice. Nevertheless, the chimer protein demonstrated superior immune protection compared to the Mix protein. The percentage of survived animals in the adjuvanted protein group (78.4%) was less than the non-adjuvanted one (85.7%). However, the Mix protein plus Alum could induce protective immunity in only 57.1% and 42.8% of homologous and heterologous virus-challenged mice, respectively. Regarding the sufficient immunogenicity and protectivity of the chimer protein construct against influenza viruses, the findings of the study suggest that the chimer protein without a requirement of adjuvant can be used as an adequate vaccine formulation to protect against a broad spectrum of influenza viruses.

Binding inhibition of various influenza viruses by sialyllactose-modified trimer DNAs

Sialyllactose (SL)-modified trimer DNAs with a similar SL presentation as their binding sites on influenza virus hemagglutinin (HA) trimer were designed and synthesized. These trimer DNAs showed high affinity for various influenza viruses, including A/Puerto Rico/08/34 (H1N1), A/Beijing/262/95 (H1N1), A/Yokohama/77/2008 (H1N1), and A/Panama/2007/99 (H3N2). Thus, presentation of SL residues on three vertexes of the scaffold as well as sialic acid binding sites on the HA trimer regardless of a tri-branched or triangular scaffold are important for high affinity for influenza viruses. These compounds have the potential for use in detection and as inhibitors of a broad spectrum of influenza viruses.

P-266 - Molecular determinants of the influenza fusion peptide activity

The emergence of an influenza pandemic is one of the biggest health threats of our time and there is an urgent need to develop vaccines and drugs against a broad spectrum of influenza viruses (IV). A promising strategy to combat IV is to inactivate the fusion process between the viral and host membranes, which is mediated by the surface protein hemagglutinin (HA). During this process, the N-terminal region of HA, known as fusion peptide (FP), inserts into the host membrane. Although it has been shown that the FP plays a crucial role in the fusion process, the molecular effect of the peptide remains unclear. To analyse the molecular determinants underlying the IV FP, we used state of the art simulation techniques, including metadynamics and constant pH molecular dynamics. The simulation results were combined spectroscopic methods to analyse the peptide's affinity for lipid membranes and its ability to promote lipid-mixing. This allowed us to obtain a detailed molecular characterization of the peptide's conformational properties and its effect on the host membrane. Our work also sheds light into the effect of mutations and external conditions, such as pH, on the FP activity. These results can be useful for the design of novel therapies against this devastating pathogen.

Evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (FLU-v) developed by SEEK: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase IIb trial

BackgroundCurrent influenza vaccines, based on antibodies against surface antigens, are unable to provide protection against newly emerging virus strains which differ from the vaccine strains. Therefore the population has to be re-vaccinated annually. It is thus important to develop vaccines which induce protective immunity to a broad spectrum of influenza viruses. This trial is designed to evaluate the immunogenicity and safety of FLU-v, a vaccine composed of four synthetic peptides with conserved epitopes from influenza A and B strains expected to elicit both cell mediated immunity (CMI) and humoral immunity providing protection against a broad spectrum of influenza viruses.MethodsIn a single-center, randomized, double-blind and placebo-controlled phase IIb trial, 222 healthy volunteers aged 18–60 years will be randomized (2:2:1:1) to receive two injections of a suspension of 500 μg FLU-v in saline (arm 1), one dose of emulsified 500 μg FLU-v in Montanide ISA-51 and water for injection (WFI) followed by one saline dose (arm 2), two saline doses (arm 3), or one dose of Montanide ISA-51 and WFI emulsion followed by one saline dose (arm 4). All injections will be given subcutaneously. Primary endpoints are safety and FLU-v induced CMI, evaluated by cytokine production by antigen specific T cell populations (flow-cytometry and ELISA). Secondary outcomes are measurements of antibody responses (ELISA and multiplex), whereas exploratory outcomes include clinical efficacy and additional CMI assays (ELISpot) to show cross-reactivity.DiscussionBroadly protective influenza vaccines able to provide protection against multiple strains of influenza are urgently needed. FLU-v is a promising vaccine which has shown to trigger the cell-mediated immune response. The dosages and formulations tested in this current trial are also estimated to induce antibody response. Therefore, both cellular and humoral immune responses will be evaluated.Trial registrationEudraCT number 2015–001932-38; retrospectively registered clinicaltrials.gov NCT02962908 (November 7th 2016).

Rupestonic acid derivative YZH-106 suppresses influenza virus replication by activation of heme oxygenase-1-mediated interferon response

Given the limitation of available antiviral drugs and vaccines, there remains to be a pressing need for novel anti-influenza drugs. Rupestonic acid derivatives were reported to have an anti-influenza virus activity, but their mechanism remains to be elucidated. Herein, we aim to evaluate the antiviral activity of YZH-106, a rupestonic acid derivative, against a broad-spectrum of influenza viruses and to dissect its antiviral mechanisms. Our results demonstrated that YZH-106 exhibited a broad-spectrum antiviral activity against influenza viruses, including drug-resistant strains in vitro. Furthermore, YZH-106 provided partial protection of the mice to Influenza A virus (IAV) infection, as judged by decreased viral load in lungs, improved lung pathology, reduced body weight loss and partial survival benefits. Mechanistically, YZH-106 induced p38 MAPK and ERK1/2 phosphorylation, which led to the activation of erythroid 2-related factor 2 (Nrf2) that up-regulated heme oxygenase-1 (HO-1) expression in addition to other genes. HO-1 inhibited IAV replication by activation of type I IFN expression and subsequent induction of IFN-stimulated genes (ISGs), possibly in a HO-1 enzymatic activity-independent manner. These results suggest that YZH-106 inhibits IAV by up-regulating HO-1-mediated IFN response. HO-1 is thus a promising host target for antiviral therapeutics against influenza and other viral infectious diseases.

From neuraminidase inhibitors to conjugates: a step towards better anti-influenza drugs?

For the treatment of seasonal flu and possible pandemic infections the development of new anti-influenza drugs that have good bioavailability against a broad spectrum of influenza viruses including the resistant strains is needed. In this review, we summarize previous methods for the structural modification of zanamivir, a potent neuraminidase inhibitor that has rare drug resistance, in order to develop effective anti-influenza drugs. We also report recent research into the design of multivalent zanamivir drugs and bifunctional zanamivir conjugates, some of which have shown better efficacy in animal experiments. As a step towards developing improved antivirals, conjugating anti-influenza drugs with anti-inflammatory agents can improve oral bioavailability and also exert synergistic effect in influenza therapy.

A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses

Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

New class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets.

BackgroundThe urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype H1N1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian H5N1 viruses, and indeed the evolving swine-origin H1N1 influenza pandemic. A recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including H1, H2, H5, H6 and H9 subtypes) has the potential to prevent and treat influenza in humans. Here we report the latest efficacy data for a representative antibody of this novel class.Methodology/Principal FindingsWe evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody CR6261 against lethal challenge with the highly pathogenic avian H5N1 virus in ferrets, the optimal model of human influenza infection. Survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macro- and microscopic pathology were investigated. Prophylactic administration of 30 and 10 mg/kg CR6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented H5N1-induced lung pathology. When administered therapeutically 1 day after challenge, 30 mg/kg CR6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage.Conclusions/SignificanceThese data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza.

Broad and potent anti-influenza virus spectrum of epigallocatechin-3-O-gallate-monopalmitate

Numerous influenza pandemics in the last century resulted in the death of millions of people worldwide. With the current and future threats of influenza pandemics development of new antiviral compounds remains a great demand. However, currently only a limited number of drugs are available for the treatment and prophylaxis of influenza. A neuraminidase (NA) inhibitor, oseltamivir phosphate, is the most commonly used antiviral drug. However, a number of reports suggest the emergence of oseltamivir phosphate resistance in new seasonal influenza viruses and highly pathogenic avian influenza (H5N1) (for example, de Jong et al, 2005). (–)-Epigallocatechin-3-O-gallate (EGCG: 1), a major component of green tea plant (Camellia sinensis), has been recognized to possess antiviral properties. Reports on the anti-influenza activity of 1 found that it inhibited virus adsorption (Nakayama et al, 1993), as well as acidification of endosomes and lysosomes (Imanishi et al, 2002). Such virus inhibition activity is different from other current NA or proton pump inhibitors, suggesting that 1 can be developed into a new class of antiviral compounds that are effective against current drug resistant influenza strains. However, 1 has not been used as an antiviral compound because of its poor lipid membrane permeability and low chemical stability. It was previously reported that the introduction of long alkyl chain groups to 1 improved its lipid membrane permeability (Tanaka et al, 1998), while protection of its hydroxyl groups with acyl groups increased its chemical stability under physiological conditions (Lam et al, 2004). Recently, we reported a method to synthesize EGCG-fatty acid monoesters using lipase-catalyzed transesterification and demonstrated that EGCG-fatty acids monoesters possessed improved influenza virus inhibitory effect against influenza A/PR8/34/(H1N1) in an alkyl length dependent manner (Mori et al, 2008). Here, we investigated the spectrum of influenza virus inhibition activity of EGCG (1) and EGCG-C-16 (2). As shown in Figures 1a, 2 is a mixture of four regio-isomers and the ratio of each regio-isomer 2a:2b:2c:2d is 38:35:7:20, respectively. Because the B-ring-modified esters (2a and 2b) and D-ring-modified esters (2c and 2d) showed exactly the same antiviral activities (data not shown), we used the mixture of four regio-isomers (2a-d) in the following assays. Figure 1. A. Chemical structure of EGCG (1) and EGCG-C16 (2). 2 is a mixture of four regio-isomers (2a-d). 2a: R2=R3= R4=H, R1= CO(CH)14CH3, 2b: R1=R3=R4=H, R2= CO(CH)14CH3, 2c: R1=R2=R4=H, R3= CO(CH)14CH3, 2d: R1=R2=R3=H, R4= CO(CH)14CH3. B. Avian influenza virus ... A series of human influenza viruses, an experimental strain (A/Puerto Rico/8/34/(H1N1)), vaccine strains (A/Beijing/262/95/(H1N1), A/Panama/2007/99/(H3N2), and B/Yamanashi/166/98/), drug-resistant strains (Yokohama/77/2008/(H1N1) OPR: oseltamivir phosphate-resistant (OPR), Yokohama/63/2007/(H1N1) AR: amantadine-resistant (AR), A/Yokohama/91/2008/(H1N1) OPR/AR: (OPR/AR) and avian pathogenic influenza (A/Duck/Hong Kong/342/78/(H5N2)), were directly incubated with 1 or 2 for 30 min prior to inoculation into MDCK cells. The cells were inoculated with virus, with or without the compounds, for 1hr, and the direct virus inhibitory effects were assessed by a plaque formation assay at 54hr post-incubation. The plaque inhibition activity was calculated relative to no compound. The cytotoxicity of compounds on MDCK cells were evaluated by the MTT cell proliferation assay. Briefly, the cells were incubated with 1 or 2 for 24hr for the MTT assay. The results of the plaque inhibition assay and MTT assay are expressed as mean ± standard error of three independent experiments. We also investigated the virus inhibition activity of 1 and 2 on avian influenza A/Duck/Hong Kong/342/78 (H5N2) virus in ovo using 11-day-old chicken embryonated eggs (n=12) inoculated with compound-treated or untreated viruses. As shown in Table 1, both 1 and 2 showed broad virus inhibitory effects on MDCK cells. The EC50 values of 2 on these viruses were between 10 to 61nM, which were approximately 7.1 to 44-fold lower than those of 1. The CC50 value of 2 was 82μM, which was only 3.1-fold lower than that of 1 (255 μM). Thus, the SI value of 2 was improved 2.2 to 14-fold compared to 1. Table 1. Direct virus inhibitory effect of 1 and 2 based on the plaque formation assay With respect to the avian influenza virus inhibition assay in ovo, virus treated with 1, zanamivir, or oseltamivir phosphate showed a moderate viral inhibitory effect (Figures 1b). However, 2 almost completely inhibited the infection (Figures. 1b), and the efficacy was retained even at 0.1μM (data not shown). In summary, 2 inhibited human and avian influenza A and B viruses, including drug-resistant viruses. 2 was found to be more effective than neuraminidase inhibitors, and strongly inhibited the infection of avian influenza (H5N2) virus in chicken embryonated eggs. This unique viral inhibitory action has the potential to be utilized to effectively control a broad spectrum of influenza viruses.

Robust Sequence Selection Method Used To Develop the FluChip Diagnostic Microarray for Influenza Virus

DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to identify regions of conservation in the influenza virus genome. From these regions of conservation, capture and label sequences capable of discriminating between different viral types and subtypes were selected. The salient features of the method were the use of phylogenetic trees for data reduction and the selection of a relatively small number of capture and label sequences capable of identifying a broad spectrum of influenza viruses. A detailed experimental evaluation of the selected sequences is described in a companion paper. The software is freely available under the General Public License at http://www.colorado.edu/chemistry/RGHP/software/.