Influenza Hemagglutinin Research Articles

High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory™, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression.

Baculovirus-based expression of proteins in insect cell cultures has emerged as a powerful technology to produce complex protein biologics for many applications ranging from multiprotein complex structural biology to manufacturing of therapeutic proteins including virus-like particles (VLPs). VLPs are protein assemblies that mimic live viruses but typically do not contain any genetic material, and therefore are safe and attractive alternatives to life attenuated or inactivated viruses for vaccination purposes. MultiBac is an advanced baculovirus expression vector system (BEVS) which consists of an engineered viral genome that can be customized for tailored applications. Here we describe the creation of a MultiBac-based VLP-factory™, based on the M1 capsid protein from influenza, and its application to produce in a parallelized fashion an array of influenza-derived VLPs containing functional mutations in influenza hemagglutinin (HA) thought to modulate the immune response elicited by the VLP.

Neuraminidase activity and specificity of influenza A virus are influenced by haemagglutinin-receptor binding

ABSTRACT Influenza virus haemagglutinin (HA) and neuraminidase (NA) are involved in the recognition and modulation of sialic acids on the cell surface as the virus receptor. Although the balance between two proteins functions has been found to be crucial for viral fitness, the interplay between the proteins has not been well established. Herein we present evidence for interplay between influenza HA and NA, which may affect the balance between two glycoprotein functions. NA enzymatic activities against sialoglycans were promoted by the presence of HA, which is in accordance with the level of co-existing HA. Such activity enhancement was lost when the HA-receptor binding properties were abolished by low-pH treatment or by mutations at the HA receptor binding domain. Sialidase activities of NA-containing virus-like particles and native influenza viruses were detected using different NA-assays and sialic acid substrates. Most pronounced HA-mediated NA enhancement was found when intact virions were confronted with multivalent surface-anchored substrates, which mimics the physiological conditions on cell membranes. Using recombinant viruses with altered HA bindings preference between α2,3- and α2,6-linked sialic acids, we also found that NA function against different substrates is correlated with the HA-receptor specificity. The effect of HA-receptor specificities on NA functions, together with the HA-mediated NA enhancement, may play a role in virus evasion of the mucus barrier, as well as in cross-species adaptation. Our data also indicate the importance of using multivalent substrates in future studies of NA functions.

An integrated device for the rapid and sensitive detection of the influenza hemagglutinin.

Influenza is a viral respiratory tract infection responsible for up to 5 million cases of severe infection and nearly 600 000 deaths worldwide each year. While treatments for influenza exist, diagnostics for the virus at the point of care are limited in their sensitivity and ability to differentiate between subtypes. We have developed an integrated two-dimensional paper network (2DPN) for the detection of the influenza virus by the surface glycoprotein, hemagglutinin. The hemagglutinin assay was developed using proteins computationally designed to bind with high affinity to the highly-conserved sialic acid binding site. The integrated 2DPN uses a novel geometry that allows automated introduction of an enzymatic amplification reagent directly to the detection zone. This assay was integrated into a prototype device and demonstrated successful detection of clinically relevant virus concentrations spiked into 70 μL of virus-free pediatric nasal swab samples. Using this novel geometry, we found improved assay performance on the device (compared to a manually-operated dipstick method), with a sensitivity of 4.45 × 102 TCID50 per mL on device.

Occurrence of Influenza Hemagglutinin Antibodies in the Polish Population during the Epidemic Season 2017/18.

This study seeks to define the level of antihemagglutinin antibodies, using the hemagglutination inhibition assay (HAI), in the serum of patients, stratified into seven age groups, in Poland during the influenza epidemic season of 2017/18. A quadrivalent influenza vaccine has been introduced in Poland as of this epidemic season, making it possible for the first time to conduct the analysis for four antigens: A/Michigan/45/2015 (H1N1) pdm09, A/Hong Kong/4801/2014 (H3N2), B/Brisbane/60/2008 - Victoria lineage, and B/Phuket/3073/2013 - Yamagata lineage. We found that the level of individual antihemagglutinin antibodies was different among the seven age groups studied; with the highest in patients of 5-9years and 10-14years of age. Interestingly, the protection factor, defined as the percentage of people with the level of antihemagglutinin antibodies of at least 1:40 after vaccination or due to a previous infection, was the highest for the antigen A/Hong Kong/4801/2014 (H3N2) in the same age groups (74% and 75%, respectively). Taking into account the dismal 3.6% of the vaccinated population in Poland, these findings point toward the sustained presence of an immune system response in patients after a prior influenza virus infection.

Effects of environmental conditions before gene transfer on the amount of influenza hemagglutinin transiently expressed in <I>Nicotiana benthamiana</I> leaves

Effects of environmental conditions before gene transfer on the amount of influenza hemagglutinin transiently expressed in <I>Nicotiana benthamiana</I> leaves

Monoclonal antibodies to hemagglutinin of influenza b viruses victoria evolutionary lineage.

Co-circulation of two evolutionary distinct lineages of influenza virus in one epidemic season has led to development specifc reagents for rapid identifcation and typing of new isolates. Panel of MAbs to hemagglutinin of influenza virus B/Brisbane/46/15 belonging to Victoria evolutionary lineage was developed. All MAbs reacted in ELISA with B/Victoria-like strains only. There were no interactions with heterologous influenza viruses of B/Yamagata lineage, seasonal and potentially pandemic influenza A viruses. All MAbs reacted in hemagglutination inhibition and virus neutralization. MAbs interacted in hemagglutination inhibition only with B/Victoria-like viruses, but did not interacted B/Yamagata-like strains. Neutralization and hemagglutination inhibition studies of viruses isolated before 1983 with MAbs revealed that MAbs 6E11, 9G5, 9B5 and 6A4 had the ability to interact with the virus B/ Russia/69 which may evidence that B strains of early isolation period (before lineage separation) have common epitope with recent Victoria lineage viruses. MAbs 7C8, 7G9, 7H8 and 8D11 were directed to a conserved epitope (or epitopes) specifc for influenza hemagglutinin viruses of B/Victoria group. The presence of differences in the effectiveness of the interaction of MAbs 6A9, 7G9 and 8A8 in hemagglutination inhibition test allows the identifcation and differentiation of strains isolated in chicken embryos and MDCK cell culture. Thus, the developed MAbs can be successfully used for identifcation and antigenic analysis of B/Victoria-like strains.

Oral vaccination with influenza hemagglutinin combined with human pulmonary surfactant-mimicking synthetic adjuvant SF-10 induces efficient local and systemic immunity compared with nasal and subcutaneous vaccination and provides protective immunity in mice

We reported previously that a synthetic mucosal adjuvant SF-10, which mimics human pulmonary surfactant, delivers antigen to mucosal dendritic cells in the nasal cavity and promotes induction of humoral and cellular immunity. The aim of the present study was to determine the effects of oral administration of antigen combined with SF-10 (antigen-SF-10) on systemic and local immunity. Oral administration of ovalbumin, a model antigen, combined with SF-10 enhanced ovalbumin uptake into intestinal antigen presenting MHC II+CD11c+ cells and their CD11b+CD103+ and CD11b+CD103- subtype dendritic cells, which are the major antigen presenting subsets of the intestinal tract, more efficiently compared to without SF-10. Oral vaccination with influenza hemagglutinin vaccine (HAv)-SF-10 induced HAv-specific IgA and IgG in the serum, and HAv-specific secretory IgA and IgG in bronchoalveolar lavage fluid, nasal washes, gastric extracts and fecal material; their levels were significantly higher than those induced by subcutaneous HAv or intranasal HAv and HAv-SF-10 vaccinations. Enzyme-linked immunospot assay showed high numbers of HAv-specific IgA and IgG antibody secreting cells in the gastrointestinal and respiratory mucosal lymphoid tissues after oral vaccination with HAv-SF-10, but no or very low induction following oral vaccination with HAv alone. Oral vaccination with HAv-SF-10 provided protective immunity against severe influenza A virus infection, which was significantly higher than that induced by HAv combined with cholera toxin. Oral vaccination with HAv-SF-10 was associated with unique cytokine production patterns in the spleen after HAv stimulation; including marked induction of HAv-responsive Th17 cytokines (e.g., IL-17A and IL-22), high induction of Th1 cytokines (e.g., IL-2 and IFN-γ) and moderate induction of Th2 cytokines (e.g., IL-4 and IL-5). These results indicate that oral vaccination with HAv-SF-10 induces more efficient systemic and local immunity than nasal or subcutaneous vaccination with characteristically high levels of secretory HAv-specific IgA in various mucosal organs and protective immunity.

A unique nanoparticulate TLR9 agonist enables a HA split vaccine to confer FcγR-mediated protection against heterologous lethal influenza virus infection.

The development of a universal influenza vaccine that can provide a robust and long-lasting protection against a broader range of influenza virus strains is a global public health priority. One approach to improve vaccine efficacy is to use an adjuvant to boost immune responses to the target antigens; nevertheless, the role of adjuvants in the context of influenza vaccines is not fully understood. We have previously developed the K3-schizophyllan (SPG) adjuvant, which is composed of nanoparticulated oligodeoxynucleotides K3, a TLR9 agonist, with SPG, a non-agonistic β-glucan ligand of Dectin-1. In this study, K3-SPG given with conventional influenza hemagglutinin (HA) split vaccine (K3-SPG HA) conferred protection against antigenically mismatched heterologous virus challenge. While K3-SPG HA elicited robust cross-reactive HA-specific IgG2c and CD8 T-cell responses, CD8 T-cell depletion had no impact on this cross-protection. In contrast, K3-SPG HA was not able to confer protection against heterologous virus challenge in FcRγ-deficient mice. Our results indicated that FcγR-mediated antibody responses induced by the HA antigen and K3-SPG adjuvant were important for potent protection against antigenically mismatched influenza virus infection. Thus, we demonstrated that the K3-SPG-adjuvanted vaccine strategy broadens protective immunity against influenza and provides a basis for the development of next-generation influenza vaccines.

A particle-filter framework for robust cryo-EM 3D reconstruction.

Single-particle electron cryomicroscopy (cryo-EM) involves estimating a set of parameters for each particle image and reconstructing a 3D density map; robust algorithms with accurate parameter estimation are essential for high resolution and automation. We introduce a particle-filter algorithm for cryo-EM, which provides high-dimensional parameter estimation through a posterior probability density function (PDF) of the parameters given in the model and the experimental image. The framework uses a set of random support points to represent such a PDF and assigns weighting coefficients not only among the parameters of each particle but also among different particles. We implemented the algorithm in a new program named THUNDER, which features self-adaptive parameter adjustment, tolerance to bad particles, and per-particle defocus refinement. We tested the algorithm by using cryo-EM datasets for the cyclic-nucleotide-gated (CNG) channel, the proteasome, β-galactosidase, and an influenza hemagglutinin (HA) trimer, and observed substantial improvement in resolution.

Lectin microarray analyses reveal host cell-specific glycan profiles of the hemagglutinins of influenza A viruses

Glycan structures on hemagglutinin (HA) of influenza A viruses have been analyzed previously to understand their significance. However, the formerly established methods using mass spectrometry present disadvantages such as procedure complexity, sensitivity, and throughput. Our study has established a novel method for analyzing glycan profiles of HA using lectin microarray techniques. We successfully obtained glycan profiles of HA starting from 1 ml of the 106 TCID50 samples through simple antigen enrichment using optimized immunoprecipitation. The profiles were reasonably consistent with known glycan structures of HA. Next, we compared glycan profiles of the HAs prepared from chicken embryos, MDCK, Vero, and A549 cells, and demonstrated the host cell-specific HA glycan profiles. Notably, the HA from MDCK cells was α1–3 galactosylated. Our method provides a highly sensitive and simple procedure for glycan profiling of the viral glycoproteins, thereby paving way for direct glycan analyses of human- and animal-derived virions.

GVHD Is Sustained By T Cell Maintenance within Target Tissues: Insights from Clonal Tracking and Parabiosis

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloSCT). In GVHD, donor T cells recognize recipient tissues as non-self and mount a broad attack that results in multi-organ damage. While there has been some success in diminishing the incidence of GVHD, less progress has been made in treating established or steroid-refractory disease without severe global immunosuppression. This is in part due to a lack of understanding of the underlining mechanisms that sustain GVHD despite chronic T cell antigen exposure.To address this, we developed a mouse GVHD model that allows us to track the progeny of single alloreactive T cell clones. We used this model to test hypotheses on GVHD maintenance: 1) GVHD is driven by the continuous output and trafficking of alloreactive T cells from secondary lymphoid tissues (SLT) into GVHD target organs; and 2) once tissues are seeded with alloreactive T cells from SLT, GVHD is maintained locally within affected tissues. We reasoned that if GVHD is maintained by the continuous SLT output of T cells, the progeny of single alloreactive clones in a target tissue would come into equilibrium with those in SLT and other target tissues. Alternatively, if there is a degree of local tissue GVHD maintenance, our model predicts that clonal progeny should be unequally distributed and not in equilibrium with SLT.We first tested these possibilities using a GVHD model wherein BALB/c RAG2-/- TCR transgenic (Tg) CD4 T cells (TS1) that recognize the S1 peptide derived from influenza hemagglutinin (HA) induce GVHD in BALB/c RAG2-/- mice that ubiquitously express HA (HA104 mice). We generated TS1 TCR Tg mice on 9 congenic backgrounds based on the expression of CD45.1/2, Thy1.1/2 and GFP. We transferred 500 naïve TS1 cells of 1 clonotype (to induce GVHD) along with single naïve TS1 cells from the remaining 8 clonotypes and BALB/c RAG2-/- bone marrow (BM) into lethally irradiated HA104 mice. We recovered a total of 432 single-cell derived TS1 clones (72% of input clones) from tissues of 79 mice, analyzed 7-35 days after transfer. We enumerated the TS1 clonal composition of each tissue (expressed as the % of all TS1 of that tissue) and found disparate clonal distribution across tissues within individual mice. For example, in a representative mouse analyzed at day 33 (Figure 1), the fractions of a single TS1 clone were relatively high in the colon (1.4%) and small intestine intraepithelial lymphocyte (IEL) (4.6%) when compared to the spleen (0.07%), BM (0.02%) and liver (0.03%). These data support that TS1 clones are not equally distributed among tissues and are not in equilibrium with SLT, suggesting that GVHD is at least in part maintained locally. We also analyzed TS1 clonal frequency distribution in a second model. BALB/c RAG2-/- BM and polyclonal T cells were transplanted into F1 (BALB/c HA104xB10.D2) recipients along with 8 single distinct TS1 cells. In this system, GVHD is induced by polyclonal BALB/c cells and TS1 cells are trackers of reactivity to HA. Preliminary experiments also indicate unequal distributions of TS1 clones across tissues in individual mice.In a second approach to test whether GVHD is maintained locally, irradiated HA104 mice were reconstituted with either 500 Thy1.1 or 500 Thy1.2 TS1 cells and CD45.1 or CD45.2 BALB/c RAG2-/- BM. One partner also received congenic TS1 single cells. We performed parabiosis of mice from one group to the other 21-28 days later. We analyzed 4 pairs 4 weeks post-joining, looking first at the blood to establish a baseline for TS1 crossover from the parabiotic partner. In blood, 18.9% ±1.9 of all TS1 were derived from the partner. Importantly, relative to equilibration in blood, there were far fewer partner-derived TS1 cells in all other tissues (Figure 2). Only a few single cell-derived TS1 clones were detected in the partner mouse at very low frequencies, even when they were dominant in tissues of the corresponding partner. Together, these data indicate that once GVHD is established, local maintenance dominates over new TS1 entry. Consistent with this, TS1 cells incorporate BrdU in vivo even at late time points. We are combining proliferation and clonality data at multiple timepoints to develop a mathematical model of GVHD establishment and maintenance. We are also extending our observations in a polyclonal GVHD model wherein the progeny from single alloreactive CD8 cells can be enumerated. DisclosuresShlomchik:NapaJen: Consultancy.

Diminished B-Cell Response After Repeat Influenza Vaccination.

Annual vaccination with influenza vaccines is recommended for protection against influenza in the United States. Past clinical studies and meta-analysis, however, have reported conflicting results on the benefits of annual vaccination. B-cell responses elicited following repeat influenza vaccinations over multiple seasons have not been examined in detail. We analyzed the B-cell and antibody (Ab) responses in volunteers vaccinated yearly, from 2010 or 2011 through 2014, with seasonal trivalent inactivated influenza vaccines. Statistical analyses were designed to help correct for possible bias due to reduced sample size in the later years of the study. We show that, after the second annual vaccination, the frequency of vaccine-specific plasmablasts and the binding reactivity of plasmablast-derived polyclonal Abs are reduced and do not increase in subsequent years. Similar trends are observed with the serum hemagglutination inhibition Ab response after each annual vaccination, as well as the binding reactivity of plasmablast-derived polyclonal Abs to the hemagglutinin of influenza A virus vaccine components, even with changes in the seasonal vaccine components during the study. Our findings indicate a diminished B-cell response to annual vaccination with seasonal trivalent influenza vaccine. These results emphasize the need for developing improved strategies to enhance the immunogenicity and efficacy of annual influenza vaccination.

GoCTF: Geometrically optimized CTF determination for single-particle cryo-EM

Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1 Å to 3.7 Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use.

2554. Safety and Immunogenicity of NasoVAX, a Novel Intranasal Influenza Vaccine

BackgroundNasoVAX is a replication-deficient adenovirus-based vaccine designed to express influenza hemagglutinin in nasal epithelial cells when given as a nasal spray. In preclinical studies, NasoVAX was associated with divergent strain protection. Prior preclinical and clinical studies with the vector demonstrated lack of impact from baseline adenovirus immunity.MethodsSixty healthy adults were randomized to an A/California 2009-based monovalent NasoVAX formulation at doses of 109, 1010, or 1011 viral particles or saline placebo, all given as a 0.25 mL nasal spray in each nostril. Subjects were followed for safety, including solicited local and systemic side effects. Immune measures included hemagglutination inhibition (HAI) and neutralizing antibody (MN) at days 1, 15, 29, 90, and 180, and γ-interferon ELISpot at day 1 and 8. A parallel cohort of 20 similar subjects were dosed with Fluzone® injectable influenza vaccine containing an A/California 2009 component and had assessments at the same timepoints. The laboratory was blind to treatment assignment for these comparator samples.ResultsNasoVAX was well tolerated with no serious adverse events and no fever. Solicited symptoms such as nasal congestion, sore throat, and headache did not increase with dose and were not statistically different than placebo. Available immune response data are shown below.GroupNasoVAX (109 vp)NasoVAX (1010 vp)NasoVAX (1011 vp)Fluzone®PlaceboSeroprotection Rate at Day 29 (≥1:40 HAI)80%100%100%95%53%(95% CIs)(51.9%, 95.7%)(78.2%, 100.0%)(78.2%, 100.0%)(75.1%, 99.9%)(26.6%, 78.7%)MN Responder Rate at Day 29 (2-fold rise)40%47%73%70%0%(95% CIs)(16.3%, 67.7%)(21.3%, 73.4%)(44.9%, 92.2%)(45.7%, 88.1%)(0.0%, 21.8%)Median ELISpot Day 8 SFC/106 PBMC58.012.0307.555.50.0(95% CIs)(5.31, 110.69)(0.0, 60.36)(2.15, 612.78)(4.12, 106.87)(0.0, 38.49)ConclusionNasoVAX intranasal influenza vaccine was well tolerated and elicited comparable antibody responses and nearly 6-fold higher cellular immune responses than a licensed injectable vaccine.Disclosures S. Tasker, Altimmune, Inc: Employee and Shareholder, Salary. V. Krishnan, Altimmune, Inc.: Employee and Shareholder, Salary. S. Bart, Altimmune, Inc.: Research Contractor, fee for research services. A. Suyundikov, Altimmune, Inc.: Employee, Salary. P. G. Booth, Altimmune, Inc.: Research Contractor, fee for research services. A. Wight O’Rourke, Altimmune, Inc.: Employee and Shareholder, Salary. J. Zhang, Altimmune, Inc.: Employee and Shareholder, Salary. B. Georges, Altimmune, Inc.: Employee and Shareholder, Salary. S. Roberts, Altimmune, Inc.: Employee and Shareholder, Salary.

Immune Correlates of Protection Induced by Virus-Like Particles Containing 2009 H1N1 Pandemic Influenza HA, NA or M1 Proteins

ABSTRACTInfluenza virus-like particle (VLPs) vaccines are a promising alternative to conventional egg-based vaccines. Evaluation of vaccine efficacy induced by HA-M1 VLPs, NA-M1 VLPs or M1 VLPs against virus challenge infection would provide important insight into vaccine design strategy. In this study, we generated VLPs containing hemagglutinin (HA), neuraminidase (NA) or M1 proteins derived from the A/California/04/09. Mice were immunized intramuscularly with HA-M1, NA-M1 or M1 VLPs and protective immunity was evaluated by assessing lung virus loads against low (5LD50) or high (100LD50) lethal dose of homologous virus challenges. High levels of virus-specific serum IgG antibody responses were induced in mice after HA-M1 VLPs immunization, whereas low or no IgG antibody responses were detected from immunization with NA-M1 VLPs or M1 VLPs, independently. Mice that were immunized with HA-M1 VLPs showed below the limit of detection on lung virus loads against low dose (5LD50) of challenge and significant reduction against high dose (100LD50) of challenge infection. Mice that were immunized with NA-M1 or M1 VLPs also displayed reduced lung viral loads compared to naïve control. In vitro cultures of cells from mouse spleen and bone marrow revealed that HA-M1 VLPs and NA-M1 VLPs induced higher levels of antibody-secreting cell (ASC) responses compared to naïve control, whereas M1 VLPs showed no ASC responses. HA-M1, NA-M1 or M1 VLPs immunization demonstrated varying degree of protection with respect to body weight changes and survival rates, which are consistent with the levels of antibody responses in sera and ASC responses from spleen and bone marrow.

Characterization of the innate stimulatory capacity of plant-derived virus-like particles bearing influenza hemagglutinin

Cell-mediated immunity is an important component of immediate and long-term anti-viral protection. Dendritic cells (DCs) are essential for the induction of cell-mediated immunity by instructing the activation and differentiation of antigen-specific T cell responses. Activated DCs that express co-stimulatory molecules and pro-inflammatory cytokines are necessary to promote the development of type 1 immune responses required for viral control. Here we report that plant-derived virus-like particles (VLPs) bearing influenza hemagglutinins (HA) directly stimulate mouse and human DCs. DCs exposed to H1- and, to a lesser extent, H5-VLPs in vitro rapidly express co-stimulatory molecules and produce pro-inflammatory cytokines including IL-12, IL-6 and TNFα. Furthermore, these VLPs support the activation and differentiation of antigen-specific T cell responses. Mechanistically, H1-VLPs stimulate the activation of kinases typically activated downstream of pattern recognition receptors including AKT, p38, and p42/44 ERK. In vivo, immunization with plant-derived VLPs induce the accumulation of both cDC1s and cDC2 in the draining lymph node and a corresponding increase in T and B cells. VLPs devoid of HA protein activate DCs, suggesting they are intrinsically immunostimulatory. Together, the results demonstrate that these candidate plant-derived VLP vaccines have an inherent and direct stimulatory effect on DCs and can enhance the ability of DCs to promote Type 1 immune responses.

Genetic characteristics of hemagglutinin and neuraminidase of avian influenza A (H7N9) virus in Guizhou province, 2014-2017

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.

Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

Avenues to Characterize the Interactions of Extended N-Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case.

Long‐chain multiantenna N‐glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X‐ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N‐glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N‐glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N‐glycan in the presence of HK/68 hemagglutinin is described.

Avenues to Characterize the Interactions of Extended N‐Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case

AbstractLong‐chain multiantenna N‐glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X‐ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N‐glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N‐glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N‐glycan in the presence of HK/68 hemagglutinin is described.