Influenza Virus Hemagglutination Research Articles

Production of a promising modular proteinaceous self-assembled delivery system for vaccination.

Recently, there have been enormous advances in nano-delivery materials, especially safer and more biocompatible protein-based nanoparticles. Generally, proteinaceous nanoparticles (such as ferritin and virus-like particles) are self-assembled from some natural protein monomers. However, to ensure their capability of assembly, it is difficult to upgrade the protein structure through major modifications. Here, we have developed an efficient orthogonal modular proteinaceous self-assembly delivery system that could load antigens with an attractive coupling strategy. In brief, we constructed a nanocarrier by fusing two orthogonal domains-a pentameric cholera toxin B subunit and a trimer forming peptide-and an engineered streptavidin monomer for binding biotinylated antigens. After successfully preparing the nanoparticles, the receptor-binding domain of SARS-CoV-2 spike protein and influenza virus haemagglutination antigen are used as model antigens for further evaluation. We found that the biotinylated antigen is able to bind to the nanoparticles with high affinity and achieve efficient lymph node drainage when loaded on the nanoparticles. Then, T cells are greatly activated and the formation of germinal centers is observed. Experiments of two mouse models demonstrate the strong antibody responses and prophylactic effects of these nanovaccines. Thus, we establish a proof-of-concept for the delivery system with the potential to load diverse antigen cargos to generate high-performance nanovaccines, thereby offering an attractive platform technology for nanovaccine preparation.

Non-epidermidis coagulase-negative Staphylococcus isolated from farm animals can inhibit the hemagglutinating activity of Newcastle disease virus and bovine parainfluenza virus type 3

The Embp protein of Staphylococcus epidermidis inhibits the hemagglutination of the H1N1 influenza virus and protects birds from a viral respiratory infection. Several species of Coagulase-negative Staphylococcus (CoNS) are present in the respiratory cavity, particularly in nostrils. We hypothesize that non-epidermidis CoNS found in animals can have the same function as observed in S. epidermidis. Thirty Non-epidermidis CoNS isolates were obtained from poultry, sheep, goat, pig, and dairy cow nostrils. Haemagglutination inhibition (HI) activity was assayed in bacteria-free supernatants from non-epidermidis CoNS against Newcastle disease virus (NDV) and bovine parainfluenza virus type 3 (BPIV). In 13 of the 30 strains (43.3 %), bacteria-free supernatants showed HI activity for NDV and BPIV-3. Staphylococcus xylosus supernatants from poultry (one isolate), sheep (two isolates), goat (one isolate), and dairy cow (three isolates) had the highest frequency of HI activity on NDV and BPIV-3, followed by Staphylococcus sp. supernatants from goat (one isolate), dairy cow (two isolates), and finally Staphylococcus equorum, Staphylococcus chromogens and Staphylococcus gallinarum supernatants with single isolation from poultry, pig and poultry, respectively. Nine isolates had the homologous gene to the embp gene of S. epidermidis, and it was associated with HI activity in the studied viruses. By Pulsed‐field gel electrophoresis, S. xylosus isolates showed to be different clones and related to the origin of isolation and HI activity. These results demonstrate that non-epidermidis CoNS supernatants from different animals and origins have the ability of HI on NDV and BPIV-3, indicating that not only S. epidermidis has the same function.

Investigation on side-product formation during the synthesis of a lactoferrin-derived lactam-bridged cyclic peptide.

Bovine lactoferrin C-lobe is able to prevent both influenza virus hemagglutination and cell infection. In particular, it was demonstrated that the fragment 418SKHSSLDCVLRP429 is a potent antiviral peptide. Therefore, we tried to increase the stability of this fragment through side-chain lactam cyclization of the peptide, S[KHSSLD]CVLRP (1). However, classic strategy involving solid-supported cyclization of the linear precursor, containing orthogonal allyl/alloc-based protection for the key amino and carboxyl residues, did not provide the desired cyclic peptide. Here, we report the identification of problematic stretches during the sequence assembly process and the optimization of the different parameters involved in the construction of 1. Results indicated a significant influence of β-protecting group of both aspartic acid and adjacent cysteine residues on the formation of side products. Therefore, the identification of suitable β-protecting groups of these residues allowed us to optimize the synthesis of designed lactam-bridged cyclic peptide.

Lactoferrin-derived Peptides Active towards Influenza: Identification of Three Potent Tetrapeptide Inhibitors

Bovine lactoferrin is a biglobular multifunctional iron binding glycoprotein that plays an important role in innate immunity against infections. We have previously demonstrated that selected peptides from bovine lactoferrin C-lobe are able to prevent both Influenza virus hemagglutination and cell infection. To deeper investigate the ability of lactoferrin derived peptides to inhibit Influenza virus infection, in this study we identified new bovine lactoferrin C-lobe derived sequences and corresponding synthetic peptides were synthesized and assayed to check their ability to prevent viral hemagglutination and infection. We identified three tetrapeptides endowed with broad anti-Influenza activity and able to inhibit viral infection in a concentration range femto- to picomolar. Our data indicate that these peptides may constitute a non-toxic tool for potential applications as anti-Influenza therapeutics.

Chemoenzymatic synthesis and characterization of N-glycolylneuraminic acid-carrying sialoglycopolypeptides as effective inhibitors against equine influenza virus hemagglutination.

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2→3)- and α(2→6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a-c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.

Biological and protective properties of immune sera directed to the influenza virus neuraminidase.

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. The neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.

Bovine lactoferrin-derived peptides as novel broad-spectrum inhibitors of influenza virus

Bovine lactoferrin (bLf) is a multifunctional glycoprotein that plays an important role in innate immunity against infections, including influenza. Here we have dissected bLf into its C- and N-lobes and show that inhibition of influenza virus hemagglutination and cell infection is entirely attributable to the C-lobe and that all major virus subtypes, including H1N1 and H3N2, are inhibited. By far-western blotting and sequencing studies, we demonstrate that bLf C-lobe strongly binds to the HA2 region of viral hemagglutinin, precisely the highly conserved region containing the fusion peptide. By molecular docking studies, three C-lobe fragments were identified which inhibited virus hemagglutination and infection at fentomolar concentration range. Besides contributing to explain the broad anti-influenza activity of bLf, our findings lay the foundations for exploiting bLf fragments as source of potential anti-influenza therapeutics.

In vitro inhibition of influenza virus infection by a crude extract from Isatis indigotica root resulting in the prevention of viral attachment

Isatis indigotica root (IIR) has been widely used as a Chinese medicinal herb to treat regular seasonal influenza over the long history of traditional Chinese medicinal practice. However, its inhibitory activities against influenza virus infections along with the associated mechanisms have not been investigated comprehensively. In this study, the chemical nature, mode of action and in vitro anti-influenza activities of a crude extract (G2) of IIR were characterized. The extract was found to inhibit different subtypes of human or avian influenza viruses at various magnitudes of activity (IC50 0.39‑4.3 mg/ml) in vitro, including A/PR/8/34 (H1N1), A/FM/1/47 (H1N1), A/Aichi/2/68 (H3N2), seasonal influenza (A/Guangzhou/GIRD/02/09 H1N1, B/Guangzhou/GIRD/08/09), novel swine-originating influenza (A/Guangzhou/GIRD/07/09, H1N1), A/Duck/Guangdong/09 (H6N2), A/Duck/Guangdong/94 (H7N3) and A/Chicken/Guangdong/96 (H9N2), while G2 was inactive against respiratory syncytial virus (RSV), adenovirus 3 (ADV3), parainfluenza virus 3 (PIV3) and enterovirus 71 (EV71). An apparent virus titer reduction was detected when the influenza viruses were pretreated with G2, and it was also shown that G2 exhibited inhibitory effects on influenza virus hemagglutination. In addition, G2 played a role in the early stages of infection, which did not easily result in the emergence of virus drug resistance. Thus, G2 may affect the attachment of influenza virus by interfering with the viral particles, thereby preventing the binding of influenza virus to the host cell surface.

In Vitro inhibitory activity of Alpinia katsumadai extracts against influenza virus infection and hemagglutination

BackgroundAlpinia katsumadai (AK) extracts and fractions were tested for in vitro antiviral activities against influenza virus type A, specially human A/PR/8/34 (H1N1) and avian A/Chicken/Korea/MS96/96 (H9N2), by means of time-of-addition experiments; pre-treatment, simultaneous treatment, and post treatment.ResultsIn pre-treatment assay, the AK extracts and AK fractions did not show significant antiviral activity. During the simultaneous treatment assay, one AK extract and five AK fractions designated as AK-1 to AK-3, AK-5, AK-10, and AK-11 showed complete inhibition of virus infectivity against A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). The 50% effective inhibitory concentrations (EC50) of these one AK extracts and five AK fractions with exception of the AK-9 were from 0.8 ± 1.4 to 16.4 ± 4.5 μg/mL against A/PR/8/34 (H1N1). The two AK extracts and three AK fractions had EC50 values ranging from <0.39 ± 0.4 to 2.3 ± 3.6 μg/mL against A/Chicken/Korea/MS96/96 (H9N2). By the hemagglutination inhibition (HI) assay, the two AK extracts and five AK fractions completely inhibited viral adsorption onto chicken RBCs at less than 100 μg/mL against both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). Interestingly, only AK-3 was found with inhibition for both viral attachment and viral replication after showing extended antiviral activity during the post treatment assay and quantitative real-time PCR.ConclusionsThese results suggest that AK extracts and fractions had strong anti-influenza virus activity that can inhibit viral attachment and/or viral replication, and may be used as viral prophylaxis.

Curcumin inhibits influenza virus infection and haemagglutination activity

Curcumin (diferuloylmethane) is a widely used spice and colouring agent in food. Accumulated evidence indicates that curcumin is associated with a great variety of pharmacological activities, including an anti-microbial effect. In this study, the anti-influenza activity of curcumin was evaluated. Our results demonstrated that treatment with 30 μM curcumin reduced the yield of virus by over 90% in cell culture. The EC 50 determined using plaque reduction assays was approximately 0.47 μM (with a selective index of 92.5). Time of drug addition experiments demonstrated curcumin had a direct effect on viral particle infectivity that was reflected by the inhibition of haemagglutination; this effect was observed in H1N1 as well as in H6N1 subtype. In contrast to amantadine, viruses did not develop resistance to curcumin. Furthermore, by comparison of the antiviral activity of structural analogues, the methoxyl groups of curcumin do not play a significant role in the haemagglutinin interaction.

Clarithromycin Inhibits Progeny Virus Production from Human Influenza Virus-Infected Host Cells

In vitro effects of macrolide clarithromycin (CAM) on influenza A virus-infected cells were examined using plaque reduction assay by treating cells either before or after viral adsorption. The significant inhibitory effect on influenza virus infection was detected only when the cells were treated with CAM after viral adsorption. The predominant inhibitory effect was observed during 4-7th hour after viral adsorption using viral production assay. CAM did not exhibit inhibitory effects on influenza virus hemagglutination, membrane fusion and viral sialidase activities. These findings indicate that CAM acts on a middle to late stage of the viral replication cycle resulting in inhibition of progeny virus production from the infected cells.

Chemoenzymatic synthesis and application of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination

Sialoglycopeptide (SGP) is referred as the glycopeptide in hen’s egg yolk, which has an N-linked, complex-type, disialyl biantennary oligosaccharide with an α-(2→6)-sialyl N-acetyllactosamine residue. The residue is known as a binding ligand of type-A human influenza virus hemagglutinin. We describe herein a simple synthesis of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination that makes use of the natural source ingredient, SGP, and the transglycosylation activity of endo-β- N-acetylglucosaminidase from Mucor hiemalis (Endo-M). Its inhibitiory activity for influenza virus hemagglutination is 40 times higher than that of SGP, and its competitive inhibition is determined to be over 300 times higher than that of fetuin. These results indicate that a sialoglycopolymer having a multivalent sialo-oligosaccharide could potentially be used for the prevention of influenza virus infection.

Influence of glycosylation on micelle-stabilizing ability and biological properties of C-terminal fragments of cow's κ-casein

Abstract C-terminal fragment of bovine κ-casein contains glycosidic residues. There are several glycoforms of κ-casein containing different kinds and numbers of glycosidic residues. Such heterogeneity affects properties of this protein and its fragments. The C-terminal fragment of cow's κ-casein (residues: 106–169) is the main factor stabilizing casein micelles. Glycosidic moieties connected to this fragment enhance the ability of κ-casein to stabilize micelles and also the resistance of this protein to the action of proteolytic enzymes and high temperature in simple model systems. κ-Casein, its C-terminal fragment (macropeptide or glycomacro-peptide) or products of its proteolysis can inhibit proliferation of lymphocytes B, binding Cholera toxin to its receptor, hemagglutination of influenza virus, adhesion of bacteria to cell surface, acid secretion in the stomach, as well as stimulate the release of cholecystokinin in the intestinal cells and the growth of Lactococcus lactis bacteria. Glycosidic moieties may act as an information carrier enabling recognition of compounds (e.g. components of cells) interacting with κ-casein, glycomacropeptide or its fragments.

The immunotargeting approach to adjuvant-independent immunization with influenza haemagglutinin

By conjugating the bromelain fragment of the influenza virus haemagglutinin (BHA) to a mouse monoclonal antibody specific for a species-crossreactive, framework determinant on class II major histocompatibility complex molecules, the adjuvant-independent immunotargeting approach could be compared with more conventional immunizations involving Freund's complete adjuvant (FCA) and alum, in different species. Both rabbits and ferrets made significant virus-blocking IgG responses to the immunotargeted BHA in the absence of adjuvant, and no adverse reactions were observed with the conjugate immunizations. As measured by enzyme-linked immunosorbent assay, these responses were equal to those observed with alum, and were approximately one-quarter of the level seen with FCA. In terms of influenza virus haemagglutination inhibition, immunotargeting appeared to be more effective than alum, but less effective than FCA. Collectively, the results indicate that immunotargeting may provide a safe and effective alternative to the use of adjuvants in the development of defined subunit vaccines.

4-O-acetyl-N-acetylneuraminic acid in the N-linked carbohydrate structures of equine and guinea pig α2-macroglobulins, potent inhibitors of influenza virus infection

To investigate the molecular basis of the differential ability of human, equine, and guinea pig α2-macroglobulins to inhibit hemagglutination and infectivity of a human influenza virus, A/Memphis/102/72 (H3N2), the structures of oligosaccharides released from the three glycoproteins by hydrazinolysis were analyzed comparatively. Approximately seven to eight sugar chains were released from each subunit of two potent inhibitors (equine and guinea pig α2-macroglobulins) and a weak inhibitor (human α2-macroglobulin). More than 70% of the oligosaccharides contained sialic acids in all three cases. Structural analysis of these sialo-oligosaccharides revealed that all of the three glycoproteins contain biantennary oligosaccharides with one and two sialic acids as major sugar chains (70 ∼ 80% of total sugar chains). Four percent of the biantennary oligosaccharides from equine sample, 10% of those from guinea pig, and 24% of those from human contain a fucosylated trimannosyl core. No triantennary oligosaccharide was detected in equine α2-macroglobulin. However, human and guinea pig α2-macroglobulins contain both fucosylated and nonfucosylated triantennary oligosaccharides. All sialic acid residues occur as the Sia α2→6Gal group. The one unique feature of the carbohydrate groups of equine and guinea pig α2-macroglobulins was the presence of 4-O-Ac-Neu5Ac as 30–50% of the total sialic acids, while human α2-macroglobulin contained only Neu 5Ac. However, 4-O-Ac-Neu5Ac is not responsible for the potent inhibition of influenza virus infection and hemagglutination as will be described in the accompanying paper.

Synthesis of C-terminal peptide fragments of the heavy chain of hemagglutinin of influenza virus of subtypes H1 and H3

It is proposed to use the chromogenic 2-[4-phenylazo)benzylsulfonyl]ethyl group, which can be eliminated by organic bases in aprotic solvents, for the protection of a C-terminal carboxy group in the synthesis of peptides. The synthesis of a number of 10–16-membered peptides corresponding to C-terminal fragments of the heavy chain of the hemagglutination of influenza virus of subtypes H1 and H3 has been performed with the use of this group.

Inhibition of influenza virus hemagglutination by animal sera.

Inhibition of influenza virus hemagglutination by animal sera.

Characterization of visna virus envelope neuraminic acid

Visna virus particles inhibit influenza virus hemagglutination in an assay for neuraminic acid-containing viruses. Pretreatment of visna virus with neuraminidase abolished hemagglutination inhibition activity but did not significantly affect attachment, infectivity, or virus-induced cell fusion in sheep choroid plexus cell monolayers.

Human respiratory tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial secretions, and sputum of patients with bronchitis and cystic fibrosis

Mucous glycoproteins were isolated by agarose gel filtration from nonpurulent tracheobronchial secretions and purulent sputum which had been reduced, carboxymethylated and, in the case of purulent secretions, treated with deoxyribonuclease. The solubilized and purified glycoproteins were fractionated on diethylaminoethyl cellulose into two major (I, II) and two minor (Ia, III) blood group active components. Components I and II had similar carbohydrate and amino acid compositions which were typical for human blood group substances. These two components did differ in several respects. Component I contained 1.4–2.6% sulfate and did not inhibit influenza virus hemagglutination while component II contained 7.1–7.8% sulfate and was a potent inhibitor of virus hemagglutination. Component II also migrated more rapidly on sodium dodecyl sulfate-3.3% acrylamide gel electrophoresis. Components I and II in purulent secretions displayed only minor compositional differences from their counterparts in nonpurulent secretions. Component II was more abundant in two sputum samples from subjects with cystic fibrosis than in purulent bronchitic secretions or in nonpurulent secretions.

Sialylatin of glycoproteins of murine mammary tumor virus, murine leukemia virus, and Mason-Pfizer monkey virus

Neuraminidase treatment of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus resulted in loss of their capacity to inhibit hemagglutination of influenza virus. Hemagglutination-inhibition activity of these RNA tumor viruses could be restored by in vitro resialylation catalyzed by sialyl transferase. The major glycoprotein in the intact envelope of desialylated and, to some extent, native virions could be specificallly labeled in vitro with CMP-(14C) sialic acid. These studies further characterize the individual glycoproteins of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus.