Hemagglutinin Peptide Research Articles

Plant-derived virus-like particle vaccines drive cross-presentation of influenza A hemagglutinin peptides by human monocyte-derived macrophages

A growing body of evidence supports the importance of T cell responses to protect against severe influenza, promote viral clearance, and ensure long-term immunity. Plant-derived virus-like particle (VLP) vaccines bearing influenza hemagglutinin (HA) have been shown to elicit strong humoral and CD4+ T cell responses in both pre-clinical and clinical studies. To better understand the immunogenicity of these vaccines, we tracked the intracellular fate of a model HA (A/California/07/2009 H1N1) in human monocyte-derived macrophages (MDMs) following delivery either as VLPs (H1-VLP) or in soluble form. Compared to exposure to soluble HA, pulsing with VLPs resulted in ~3-fold greater intracellular accumulation of HA at 15 min that was driven by clathrin-mediated and clathrin-independent endocytosis as well as macropinocytosis/phagocytosis. At 45 min, soluble HA had largely disappeared suggesting its handling primarily by high-degradative endosomal pathways. Although the overall fluorescence intensity/cell had declined 25% at 45 min after H1-VLP exposure, the endosomal distribution pattern and degree of aggregation suggested that HA delivered by VLP had entered both high-degradative late and low-degradative static early and/or recycling endosomal pathways. At 45 min in the cells pulsed with VLPs, HA was strongly co-localized with Rab5, Rab7, Rab11, MHC II, and MHC I. High-resolution tandem mass spectrometry identified 115 HA-derived peptides associated with MHC I in the H1-VLP-treated MDMs. These data suggest that HA delivery to antigen-presenting cells on plant-derived VLPs facilitates antigen uptake, endosomal processing, and cross-presentation. These observations may help to explain the broad and cross-reactive immune responses generated by these vaccines.

H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology between an H1N1 hemagglutinin and two hypocretin sequences has been reported.T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched healthy controls.HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T cell cross-reactivity was not detected.These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin peptides presented by HLA-DQ6 is involved in the development of narcolepsy.

Conserved HA-peptide NG34 formulated in pCMV-CTLA4-Ig reduces viral shedding in pigs after a heterosubtypic influenza virus SwH3N2 challenge.

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.

Highly conserved hemagglutinin peptides of H1N1 influenza virus elicit immune response.

In the current study, two highly conserved (> 90%) H1N1 hemagglutinin peptides STDTVDTVLEKNVTVTHSVNL (H1) and KVNSVIEKMNTQFTAVGKEF (H2) containing multiple T-cell epitopes have been assessed for their immunogenic potential in vitro, subjecting peripheral blood mononuclear cells from healthy volunteers to repetitive stimulation of chemically synthesised H1 and H2 peptides, and measuring their interferon (IFN)-γ level (ELISA) and proliferation (MTT assay). Further, these peptides were analysed for their binding affinity with 18 different human leukocyte antigen (HLA) class I and II by means of molecular docking. All seven samples tested for H1- and H2-induced IFN-γ secretion were found to have enhanced IFN-γ production. Six (H1) and five (H2) samples have shown proliferative response compared to unstimulated cells. Peptide-induced IFN-γ secretion and proliferation in healthy samples represent the immunogenic potential of these peptides. Further, molecular docking results reveal that the peptides have comparable binding energy to that of native bound peptide for both HLA classes which indicates that these peptides have the capability to be presented by different HLA molecules required for T-cell response. Hence, these conserved immunogenic hemagglutinin peptides are potential candidates for influenza vaccine development.

Interaction of Nevirapine with the Peptide Binding Groove of HLA-DRB1*01:01 and Its Effect on the Conformation of HLA-Peptide Complex.

Human leukocyte antigen (HLA)-DRB1*01:01 has been shown to be involved in nevirapine-induced hepatic hypersensitivity reactions. In the present study, in silico docking simulations and molecular dynamics simulations were performed to predict the interaction mode of nevirapine with the peptide binding groove of HLA-DRB1*01:01 and its possible effect on the position and orientation of the ligand peptide derived from hemagglutinin (HA). In silico analyses suggested that nevirapine interacts with HLA-DRB1*01:01 around the P4 pocket within the peptide binding groove and the HA peptide stably binds on top of nevirapine at the groove. The analyses also showed that binding of nevirapine at the groove will significantly change the inter-helical distances of the groove. An in vitro competitive assay showed that nevirapine (1000 μM) increases the binding of the HA peptide to HLA-DRB1*01:01 in an allele-specific manner. These results indicate that nevirapine might interact directly with the P4 pocket and modifies its structure, which could change the orientation of loaded peptides and the conformation of HLA-DRB1*01:01; these changes could be distinctively recognized by T-cell receptors. Through this molecular mechanism, nevirapine might stimulate the immune system, resulting in hepatic hypersensitivity reactions.

Exploring the nature of the H-bonds between the human class II MHC protein, HLA-DR1 (DRB*0101) and the influenza virus hemagglutinin peptide, HA306-318, using the quantum theory of atoms in molecules

The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HCaliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) >> P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.

Probing the metastable state of influenza hemagglutinin

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.

A lipidated peptide of Mycobacterium tuberculosis resuscitates the protective efficacy of BCG vaccine by evoking memory T cell immunity

BackgroundThe current BCG vaccine induces only short-term protection against Mycobacterium tuberculosis (Mtb), suggesting its failure to generate long-lasting memory T cells. Previously, we have demonstrated that a self-adjuvanting peptide of Mtb (L91), successfully generated enduring memory Th1 cells. Consequently, we investigated if L91 was able to recuperate BCG potency in perpetuating the generation of memory T cells and protection against Mtb infected mice.MethodsIn the present study, we evaluated the potency of a self adjuvanting Mtb peptide vaccine L91 in invigorating BCG immune response against Mtb in mice. Female BALB/c mice were immunized with BCG. Later, they were boosted twice with L91 or an antigenically irrelevant lipidated influenza virus hemagglutinin peptide (LH). Further, PBMCs obtained from BCG vaccinated healthy subjects were cultured in vitro with L91. T cell responses were determined by surface markers and intracellular cytokine staining. Secretion of cytokines was estimated in the culture supernatants (SNs) by ELISA.ResultsCompared to the BCG-vaccinated controls, L91 booster significantly enhanced the percentage of memory Th1 cells and Th17 cells and reduced the mycobacterial burden in BCG primed and L91-boosted (BCG-L91) group, even after 229 days of BCG vaccination. Further, substantial augmentation in the central (CD44hiCD62LhiCD127hi) and effector memory (CD44hiCD62LloCD127lo) CD4 T cells was detected. Furthermore, greater frequency of polyfunctional Th1 cells (IFN-γ+TNF-α+) and Th17 cells (IFN-γ+IL-17A+) was observed. Importantly, BCG-L91 successfully prevented CD4 T cells from exhaustion by decreasing the expression of PD-1 and Tim-3. Additionally, augmentation in the frequency of Th1 cells, Th17 cells and memory CD4 T cells was observed in the PBMCs of the BCG-vaccinated healthy individuals following in vitro stimulation with L91.ConclusionsOur study demonstrated that L91 robustly reinvigorate BCG potency to invoke enduring protection against Mtb. This novel vaccination stratagem involving BCG-priming followed by L91-boosting can be a future prophylactic measure to control TB.

Evaluation of T and B memory cell responses elicited by the pandemic H1N1 vaccine in HIV-infected and HIV-uninfected individuals

This study was to compare B and T memory cells elicited by a single dose monovalent 2009 influenza A (H1N1) vaccine (strain A/California/7/2009 H1N1) in HIV+ and HIV- groups, and to analyze the impact of the prior seasonal vaccines to the immunogenicity of this vaccine. Blood samples were collected before vaccination (day 0) and at days 28 and 180. Participants were categorized into HIV-/LAIV, HIV-/TIV and HIV+/TIV subgroups according to the trivalent live-attenuated or inactivated (LAIV or TIV) seasonal influenza vaccines they received previously. The IgG+ memory B cells (BMem) and IFNγ+ T cells were measured against antigens including the H1N1 vaccine, the hemagglutinin (HA) and neuraminidase (NA) proteins or peptide pools of the pandemic and the seasonal H1N1 strains, respectively. Overall BMem responses increased significantly at day 28 but returned to baseline by day 180 in all three subgroups. The average frequency of the H1N1-specific BMem at day 28 for the HIV-/LAIV, HIV-/TIV and HIV+/TIV groups was 2.14%, 1.26% and 1.67%, respectively, and the average fold change was 14.39, 3.81 and 3.93, respectively. The differences of BMem between HIV-/LAIV and the two TIV subgroups were significant. For the IFNγ response, the overall spot counts ranged widely between 0 and 958/106 PBMCs. The group average spot counts to H1N1 vaccine was 89, 102, and 30 at day 28 for HIV-/LAIV, HIV-/TIV and HIV+/TIV subgroups, respectively. The average increase of IFNγ response at day 28 vs day 0 in all three subgroups did not reach 2-fold. Participants with a prior LAIV seasonal vaccine, as compared to a TIV seasonal vaccine, responded significantly better to the monovalent H1N1 vaccine. Excluding LAIV participants, no difference was seen between the HIV+ and HIV- subject groups in terms of BMem. The BMem response declined at 6months.

1H NMR Study of “Host–Guest” Interactions of Micellar Assemblies from Amphiphilic Invertible Polymers and Peptides

Abstract“Host–guest” interactions between self‐assembled micellar nanostructures from amphiphilic invertible polymers (AIPs) and two peptides, swine‐origin Influenza A surface protein Hemagglutinin and research antibody peptide V5, are investigated by changing the polymer concentration and polymer/peptide ratio in the aqueous solution. Formation of mixed micellar assemblies from AIP and each peptide is revealed using detailed 1H NMR spectroscopic study. Peptide molecule localization in the micellar assemblies depends on the specific interactions between amino acid functional groups of the peptide and polymer fragments. The resulting mixed micellar structures can be considered as a promising material toward delivery and stimuli‐responsive sustained release of peptides.

Organization and dynamics of Trp14 of hemagglutinin fusion peptide in membrane mimetic environment

The fusion peptide of influenza hemagglutinin (HA) is important for cellular entry of influenza virus. Previous studies showed that HA fusion peptide assumes a bent structure in membrane environment, which is extremely important for its fusion-promoting activity. In this work, we have measured the organization and dynamics of the tryptophan (Trp14) residue in presence of sodium dodecyl sulfate (SDS) to investigate the conformational flexibility of tryptophan in membrane-mimetic environment. The steady state and time-resolved fluorescence measurements were employed to investigate the location and rotational flexibility of the tryptophan residue in the SDS micelles. We have calculated the apparent rotational correlation time of Trp14 from the fluorescence anisotropy and fluorescence lifetime data, and apparent rotational correlation time shows a substantial increase in presence of SDS. We have further measured the red edge excitation shift (REES) of Trp14 fluorescence in absence and in presence of two different concentrations of SDS. A large magnitude of REES at post-micellar concentration of SDS indicates the polar confined environment of Trp14 residue in the micellar milieu. Taken together, our results confirm that the tryptophan is located in a motionally restricted interfacial region of micelles. The interfacial location and slow dynamics of Trp14 might be important to adopt the appropriate structure of HA fusion peptide in membrane environment.

Encoded Silicon-Chip-Based Platform for Combinatorial Synthesis and Screening.

Solid-supported chemical libraries have proven useful for the rapid and cost-effective discovery of bioactive compounds. However, traditional on-bead screening involves time-intensive chemical characterization of hit compounds and high false positive rates. Herein, we report a new platform for encoded chemical synthesis and solid-supported screening using p-Chips, microsized silicon microtransponders capable of storing and emitting unique numerical identifiers (IDs). By encoding the structures of library members using p-Chip IDs, we can track compound identities throughout both split-and-pool synthesis and protein binding assays without destructive cleavage. Thanks to the numerical IDs, our p-Chip platform can provide binding constants for library members simply by stripping and reprobing with different protein concentrations, unlike traditional on-bead assays. To showcase these features, we synthesized a library of 108 hemagglutinin (HA) peptide variants using split-and-pool approach, and measured EC50s for each variant directly on p-Chips. On-chip EC50s obtained from these studies showed excellent correlation (80%) with those obtained using traditional ELISA methods. Our screen also yielded a false positive rate of 14%, markedly superior to that reported for conventional bead-based binding studies (66-96%).1-9 On the basis of these results, we believe the p-Chip platform has the potential to improve the effectiveness of solid-supported high-throughput screening by a significant margin.

Mucosal vaccination of conserved sM2, HA2 and cholera toxin subunit A1 (CTA1) fusion protein with poly gamma-glutamate/chitosan nanoparticles (PC NPs) induces protection against divergent influenza subtypes

To develop a safe and effective mucosal vaccine that broad cross protection against seasonal or emerging influenza A viruses, we generated a mucosal influenza vaccine system combining the highly conserved matrix protein-2 (sM2), fusion peptide of hemagglutinin (HA2), the well-known mucosal adjuvant cholera toxin subunit A1 (CTA1) and poly-γ-glutamic acid (γ-PGA)-chitosan nanoparticles (PC NPs), which are safe, natural materials that are able to target the mucosal membrane as a mucosal adjuvant. The mucosal administration of sM2HA2CTA1/PC NPs could induce a high degree of systemic immunity (IgG and IgA) at the site of inoculation as well as at remote locations and also significantly increase the levels of sM2- or HA2-specific cell-mediated immune response. In challenge tests in BALB/c mice with 10 MLD50 of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird/Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005 (H7N3) or A/Chicken/Korea/116/2004(H9N2) viruses, the recombinant sM2HA2CTA1/PC NPs provided cross protection against divergent lethal influenza subtypes and also the protection was maintained up to six months after vaccination. Thus, sM2HA2CTA1/PC NPs could be a promising strategy for a universal influenza vaccine.

Bivalent kinetic binding model to surface plasmon resonance studies of antigen-antibody displacement reactions

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This is beneficial because displacement of high molecular weight receptors is detected instead of low molecular weight ligand as in classical binding analysis. Thus, detection limit is potentially lowered.Using the influenza haemagglutinin (HA) peptide binding to mono or bivalent anti-haemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and anti-Haemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy.Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibodies. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding. It became obvious that rebinding is in any case not necessary to understand the binding kinetics of our model system HA/anti-HA. Maximum decline of SPR response could be used to determine ligand concentrations in analyte.

The length of the N-terminal region of a photocleavable peptide bound to DR1 determines the kinetics of photocleavable peptide fragment release

Abstract MHC class II molecules (MHCII) bind peptides and present them to CD4+ T cells. As prepared as a recombinant protein, DR1 (a MHCII) is in equilibrium between peptide-receptive and peptide-averse conformations. In order to dissect which step of the peptide binding reaction is being affected in DR1 mutants with altered peptide affinity, we wanted to generate DR1 fully in the peptide-receptive conformation. To generate this form, we used a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII proteins. We used variants of a well-characterized peptide derived from influenza hemagglutinin (HA), with different truncations at the N-terminal end, expecting to generate peptides with different affinity for DR1. This was confirmed by higher IC50 values obtained for the shorter peptides compared to the longer ones. All of the studied peptides were cleaved as expected after UV treatment as shown by mass spectrometry. However, when DR1-photocleavable peptide complexes were exposed to UV light, they generated different amounts of peptide-receptive DR1 measured by their ability to bind fluorescently-labeled HA peptide. The shorter the peptide, the more peptide-receptive DR1 was generated. This suggests that minimizing the interaction between photocleavable peptide and DR1 allows faster dissociation of the photo-generated fragments, and thus faster association of an incoming peptide. These results showed that the kinetics of photocleavable peptide fragment release can interfere with the kinetics of subsequent reactions.

Artificial antigen-presenting cells expressing HLA class II molecules as an effective tool for amplifying human specific memory CD4(+) T cells.

Owing to their multiple immune functions, CD4(+) T cells are of major interest for immunotherapy in chronic viral infections and cancer, as well as for severe autoimmune diseases and transplantation. Therefore, standardized methods allowing rapid generation of a large number of CD4(+) T cells for adoptive immunotherapy are still awaited. We constructed stable artificial antigen-presenting cells (AAPCs) derived from mouse fibroblasts. They were genetically modified to express human leukocyte antigen (HLA)-DR molecules and the human accessory molecules B7.1, Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3). AAPCs expressing HLA-DR1, HLA-DR15 or HLA-DR51 molecules and loaded with peptides derived from influenza hemagglutinin (HA), myelin basic protein (MBP) or factor VIII, respectively, activated specific CD4(+) T-cell clones more effectively than Epstein-Barr virus (EBV)-transformed B cells. We also showed that AAPCs were able to take up and process whole Ag proteins, and present epitopes to specific T cells. In primary cultures, AAPCs loaded with HA peptide allowed generation of specific Th1 lymphocytes from healthy donors as demonstrated by tetramer and intracellular cytokine staining. Although AAPCs were less effective than autologous peripheral blood mononuclear cells (PBMCs) to stimulate CD4(+) T cells in primary culture, AAPCs were more potent to reactivate and expand memory Th1 cells in a strictly Ag-dependent manner. As the availability of autologous APCs is limited, the AAPC system represents a stable and reliable tool to achieve clinically relevant numbers of CD4(+) T cells for adoptive immunotherapy. For fundamental research in immunology, AAPCs are also useful to decipher mechanisms involved in the development of human CD4 T-cell responses.

Immune suppressive activity of the influenza fusion peptide

Immune suppressive domains have been identified in retro and filoviral fusion proteins. Such domains constitute small peptide motifs that are evolutionarily very well preserved within each group. We here test the hypothesis that such preservation reflects a dual selection pressure for both immune suppression and membrane fusion activity in influenza viruses for which no immune suppressive peptide motifs have been identified. We identified a conserved motif in the fusion peptide of influenza hemagglutinin as a candidate for an immune suppressive domain using comparative and phylogenetic analysis. This peptide was indeed found to exhibit immune suppressive activity in several in vitro assays. Similar to the previously reported peptides from retro and filoviruses the influenza peptide had immune suppressive activity when presented as a dimer but not as a monomer.

Kallistatin ameliorates influenza virus pathogenesis by inhibition of kallikrein-related peptidase 1-mediated cleavage of viral hemagglutinin.

Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses.

Highly Pathogenic Avian Influenza A(H5N8) Virus from Waterfowl, South Korea, 2014

To the Editor: To date, 18 hemagglutinin (HA) subtypes and 11 neuraminidase (NA) subtypes have been identified in influenza A viruses (1–4). Influenza A viruses containing HA subtypes 1–16 circulate in aquatic birds (1,2), whereas those harboring HA subtypes 17 and 18 are found in bats (3,4). On January 18, 2014, the government of South Korea reported an outbreak of highly pathogenic avian influenza A(H5N8) virus in breeding ducks in the southern part of Jeollabuk-Do Province (5). More than 12 million poultry have since been culled, but the spread of the virus continues in duck and chicken farms. We report the genetic characterization of this virus. On February 15, 2014, a total of 200 fecal samples were collected from waterfowl in the Pungse River in Chungnam Province, which is geographically close to Jeollabuk-Do Province. All samples were inoculated into hens’ eggs, and influenza A viruses were confirmed by PCR by using influenza A–specific nucleoprotein (NP) primers. We obtained 1 isolate, A/waterfowl/Korea/S005/2014 (H5N8), and sequenced the full regions of all 8 genes as described (6). These sequences were deposited into GenBank under accession nos. {type:entrez-nucleotide-range,attrs:{text:KJ511809-KJ511816,start_term:KJ511809,end_term:KJ511816,start_term_id:629688568,end_term_id:629688575}}KJ511809-KJ511816. We conducted a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi, http://platform.gisaid.org/epi3/frontend#4ead5c) to identify the closest gene sequences to those of A/waterfowl/Korea/S005/2014 (H5N8) (Table). Sequences for polymerase basic (PB) 2 (99% homology), HA (97% homology), and NP (99% homology) genes were closely related to those of A/wild duck/Shandong/628/2011 (H5N1). Sequences for PB1 (99% homology), polymerase acidic subunit (PA) (98% homology), matrix (M) (99% homology), and nonstructural (NS) (99% homology) genes were closely related to those of A/duck/Jiangsu/1-15/2011 (H4N2). Sequences for the NA (98% homology) gene were closely related to that of A/duck/Jiangsu/k1203/2010 (H5N8). Phylogenic analysis showed that all 8 genes of A/waterfowl/Korea/S005/2014 (H5N8) belonged to the Eurasian lineage, and that the HA gene clustered with clade 2.3.4 (Technical Appendix Figure 1). Table Nucleotide homology of genes of influenza virus strain A/waterfowl/Korea/S005/2014 (H5N8) to the closest related influenza virus strains* We further analyzed the amino acid sequences of the virus isolate (online Technical Appendix Table 1). Positions 138 and 160 of the HA protein (H3 numbering) contained an alanine (A) residue, which was previously found to be related to enhanced binding to the human influenza receptor (7). The connecting peptide of HA contained an insertion of 4 basic amino acids (arginine-arginine-arginine-lysine), which is the same as in the HA of A/duck/Korea/Buan2/2014 (H5N8), an isolate from a duck farm in South Korea (GenBank accession no. {type:entrez-nucleotide-range,attrs:{text:KJ413839.1-KJ413846.1,start_term:KJ413839.1,end_term:KJ413846.1,start_term_id:589826076,end_term_id:589826093}}KJ413839.1-KJ413846.1). Aspartic acid was found in M1 at position 30 and alanine at position 215; this combination has been connected with increased virulence in mice (8). The NS1 sequence contained serine at position 42, which is related to the enhanced pathogenicity in mice, but a truncation of the amino acids at positions 218–230 that has been linked with reduced pathogenicity in mice (9) was not identified. Asparagine was identified at position 31 of M2, which is the same in M2 of A/duck/Korea/Buan2/2014 (H5N8) and confers resistance to amantadine and rimantadine (10). Because all 8 genes of A/waterfowl/Korea/S005/2014 (H5N8) are closely related to those of the A/duck/Korea/Buan2/2014 (H5N8) isolate that was obtained from a duck farm, it is likely that A/waterfowl/Korea/S005/2014 (H5N8) originated from infected waterfowl that had visited poultry on an infected farm (Technical Appendix Figure 1). Our laboratory has studied the feces of wild birds in Chungnam Province since 2009, surveying >20,000 fecal samples from wild birds in this area each year, but we had not previously isolated avian influenza A(H5N8) virus from any samples. The genetic analysis of the A/waterfowl/Korea/S005/2014 (H5N8) isolate indicates that this novel strain may have been created by the reassortment of PB2, HA, and NP segments from H5N1-like avian influenza virus; PB1, PA, M, and NS segments from H4N2-like avian influenza virus; and NA segments from H5N8-like avian influenza virus (Technical Appendix Figure 2). Most genes of the virus we isolated are related to those of avian influenza viruses isolated in China, but the HA gene of A/waterfowl/Korea/S005/2014 (H5N8) showed only 97% homology to the closest HA gene in GenBank, which indicates that this gene may have been created in poultry in South Korea. To our knowledge, no outbreak of this virus in poultry farms in China has been reported, and we found no previous reports in the literature that migratory birds could carry the virus. Taken together, our data suggest that A/waterfowl/Korea/S005/2014 (H5N8) may have been reassorted in a duck farm in South Korea. Technical Appendix: Phylogenetic analysis, schematic diagram, and identification of amino acids of influenza virus strain A/waterfowl/Korea/S005/2014 (H5N8). Click here to view.(1.2M, pdf)

Conserved peptides containing overlapping CD4+ and CD8+ T-cell epitopes in the H1N1 influenza virus: an immunoinformatics approach.

Pandemic threats of the H1N1 influenza virus have drawn attention to developing a universal vaccine against circulating and future strains of this virus. An immunoinformatics study was conducted to identify conserved peptides containing CD4+ and CD8+ T-cell epitopes from all the hemagglutinin (HA) and neuraminidase (NA) protein sequences available until February 2013 to cover the seasonal as well as the pandemic strains of the H1N1 virus. In the present study, six different immunoinformatics prediction programs were used in order to define the epitopes. Five conserved peptides of HA and six of NA protein were obtained that contained overlapping CD4+ and CD8+ T-cell epitopes. These identified peptides have a binding affinity for a large number of major histocompatibility complex (MHC) alleles. WHGSNRPWVSF of NA protein is a new peptide whose T-cell response has not been previously reported. Population coverage studies have shown that these peptide fragments have the capacity to induce a potent immune response among individuals from different populations around the world. Hence, these HA and NA peptides may be considered as interesting candidates for vaccine design.