Influenza Hemagglutinin Protein Research Articles

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Prevention and treatment of HPV-related cancer through a mRNA vaccine expressing APC-targeting antigen.

Persistent human papillomavirus (HPV) infection is associated with multiple malignancies. Developing therapeutic vaccines to eliminate HPV-infected and malignant cells holds significant value. In this study, we introduced a lipid nanoparticle encapsulated mRNA vaccine expressing tHA-mE7-mE6. Mutations were introduced into E6 and E7 of HPV to eliminate their tumourigenicity. A truncated influenza haemagglutinin protein (tHA), which binds to the CD209 receptor on the surface of dendritic cells (DCs), was fused with mE7-mE6 in order to allow efficient uptake of antigen by antigen presenting cells. The tHA-mE7-mE6 (mRNA) showed higher therapeutic efficacy than mE7-mE6 (mRNA) in an E6 and E7+ tumour model. The treatment resulted in complete tumour regression and prevented tumour formation. Strong CD8+ T-cell immune response was induced, contributing to preventing and curing of E6 and E7+ tumour. Antigen-specific CD8+ T were found in spleens, peripheral blood and in tumours. In addition, the tumour infiltration of DC and NK cells were increased post therapy. In conclusion, this study described a therapeutic mRNA vaccine inducing strong anti-tumour immunity in peripheral and in tumour microenvironment, holding promising potential to treat HPV-induced cancer and to prevent cancer recurrence.

Targeting host-virus interactions: in silico analysis of the binding of human milk oligosaccharides to viral proteins involved in respiratory infections

Respiratory viral infections, a major public health concern, necessitate continuous development of novel antiviral strategies, particularly in the face of emerging and re-emerging pathogens. In this study, we explored the potential of human milk oligosaccharides (HMOs) as broad-spectrum antiviral agents against key respiratory viruses. By examining the structural mimicry of host cell receptors and their known biological functions, including antiviral activities, we assessed the ability of HMOs to bind and potentially inhibit viral proteins crucial for host cell entry. Our in silico analysis focused on viral proteins integral to host-virus interactions, namely the hemagglutinin protein of influenza, fusion proteins of respiratory syncytial and human metapneumovirus, and the spike protein of SARS-CoV-2. Using molecular docking and simulation studies, we demonstrated that HMOs exhibit varying binding affinities to these viral proteins, suggesting their potential as viral entry inhibitors. This study identified several HMOs with promising binding profiles, highlighting their potential in antiviral drug development. This research provides a foundation for utilizing HMOs as a natural source for designing new therapeutics, offering a novel approach in the fight against respiratory viral infections.

Evolution and Phylodynamics of the Hemagglutinin Protein of Influenza A/(H1N1)pdm09 Virus Isolates from India from 2009 to 2020.

Influenza A/(H1N1)pdm09 virus evolves through continuous antigenic variation in both surface antigens, such as hemagglutinin (HA) and neuraminidase (NA) proteins, which affect its pathogenicity, the effectiveness of the host immune response, and drug resistance. This study reports the evolution and dynamics of 527 HA protein sequences of influenza A/(H1N1)pdm09 Indian isolates submitted from 2009 to 2020. These isolates were aligned with a reference sequence and 22 sequences representing different clades using MEGA X, and subjected to phylogenetic analysis. The strains were predominantly grouped in clades 6B.1 and 6B.2. Prediction of glycosylation sites using the BioEdit and NetNglyc servers showed 12 glycosylation sites distributed in both the stem and globular head regions of HA. Functional evaluation showed that there were 22 deleterious mutations that could affect the function of HA. In addition, 403 unique mutations were distributed across various isolates, indicating the dynamics of antigenic variation in Indian isolates. These results provide an understanding of the frequency, phylodynamics, and impact of mutations in Indian isolates of influenza A/(H1N1)pdm09 relative to global isolates. Monitoring the genomic evolution of the virus will support studies on strain selection for vaccine development and devising control and prevention measures to manage this respiratory infection.

Safety and immunogenicity of orally administered poxvirus vectored constructs in the white-footed mouse (Peromyscus leucopus).

Globally, zoonotic spillover is becoming more frequent and represents a growing public health concern. Reservoir-targeted vaccination offers an intriguing alternative to traditional vaccine practices by establishing protection in wild populations that maintain the natural pathogen cycle. As an important pathogen reservoir, Peromyscus leucopus Rafinesque or the white-footed mouse has been the target of several experimental vaccines. However, strategies are limited by the method of administration, need for repeated dosing, or safety of constructs in the field. To address these concerns, we evaluated two highly attenuated poxviruses, raccoonpox virus (RCN) and modified vaccinia Ankara (MVA) virus as potential oral vaccine vectors in white-footed mice. Following oral administration, P. leucopus showed no adverse signs. A single oral dose elicited robust immune responses in mice to the foreign influenza hemagglutinin protein expressed by poxvirus vaccine vectors. Serum hemagglutinin inhibition antibody titers were detected by day 7 post immunization and persisted until study termination (77days post immunization). This study establishes the safety and immunogenicity of recombinant MVA and RCN poxviruses in P. leucopus and demonstrates the suitability of these vectors as part of a reservoir-targeted vaccine strategy for white-footed mice.

Influenza virus reduces ubiquitin E3 ligase MARCH10 expression to decrease ciliary beat frequency.

Respiratory viruses, such as influenza, decrease airway cilia function and expression, which leads to reduced mucociliary clearance and inhibited overall immune defense. Ubiquitination is a posttranslational modification using E3 ligases, which plays a role in the assembly and disassembly of cilia. We examined the role of membrane-associated RING-CH (MARCH) family of E3 ligases during influenza infection and determined that MARCH10, specifically expressed in ciliated epithelial cells, is significantly decreased during influenza infection in mice, human lung epithelial cells, and human lung tissue. Cellular depletion of MARCH10 in differentiated human bronchial epithelial cells (HBECs) using CRISPR/Cas9 showed a decrease in ciliary beat frequency. Furthermore, MARCH10 cellular knockdown in combination with influenza infection selectively decreased immunoreactive levels of the ciliary component, dynein axonemal intermediate chain 1. Cellular overexpression of MARCH10 significantly decreased influenza hemagglutinin protein levels in the differentiated HBECs and knockdown of MARCH10 increased IL-1β cytokine expression, whereas overexpression had the reciprocal effect. These findings suggest that MARCH10 may have a protective role in airway pulmonary host defense and innate immunity during influenza infection.

2142. Safety and immunogenicity of COVID Influenza Combination Vaccine

Abstract Background We developed a COVID-Influenza Combination (CIC) vaccine, comprising recombinant SARS-CoV-2 Spike (rS) and quadrivalent influenza hemagglutinin (HA) protein nanoparticles (qNIV), and Matrix-MTM adjuvant. rS/Matrix-M previously demonstrated efficacy against COVID-19 in Phase 3 trials, while qNIV/Matrix-M previously demonstrated induction of broadly cross-reactive antibodies. Here we report preliminary safety and immunogenicity results of a first-ever Phase 1/2 CIC dose-finding trial. Methods Seropositive (COVID-19 vaccinated ≥ 8 weeks prior) participants (N=642) aged 50-70 years were randomized equally, to receive two intramuscular doses, 56 days apart, to 1 of 14 different dose/formulations of CIC using a design of experiments approach (dose range: rS 2.5-22.5ug, HA 5-60ug; and 50ug Matrix-M), or to 1 of 2 reference formulations of either standalone rS with Matrix-M [2 doses] or qNIV with Matrix-M [1 dose only]. Pre- and post-vaccination (Days 0, 28, 56, 70, 84, 182) immunogenicity assessments including SARS-CoV-2 anti-S IgG and influenza HAI antibodies to vaccine-homologous strains. Reactogenicity was assessed 7 days following each dose, and safety outcomes assessed through Day 70. Multiple regression was used to create predictive models to assess antibody response surfaces and for dose optimization. Results All CIC formulations were well tolerated, with a reactogenicity and safety profile generally comparable to standalone rS or qNIV. Regression modelling of post-first dose responses revealed that both rS and HA antigens in a CIC formulation modestly interfered with each other, however, interference was overcome with dose adjustment across a range of rS/HA doses. Specifically, higher rS dose ( >20ug), in a dose dependent fashion, overcame HA interference, closely matching standalone rS IgG reference responses (GMEU 16,818), whereas lower, intermediate HA dose overcame rS interference, closely matching standalone HA reference HAI responses for H3N2 (GMT 145), H1N1 (GMT 134), and B-Victoria (GMT 66); while modestly (at least 34%) lower than the reference B-Yamagata response (GMT 101). Conclusion CIC formulations were well tolerated and immunogenic, with various dose combinations achieving response comparable to standalone vaccines. Disclosures Vivek Shinde, MD, MPH, Novavax Inc.: Stocks/Bonds Wayne Woo, MS, Novavax Inc.: Stocks/Bonds Sharon Liu, n/a, Novavax Inc.: Stocks/Bonds Sara Cook, n/a, Novavax Inc.: Stocks/Bonds Zenaida Santiago, n/a, Novavax Inc.: Stocks/Bonds Susan Neal, n/a, Novavax Inc.: Stocks/Bonds Joyce S. Plested, n/a, Novavax Inc.: Stocks/Bonds Tim Vincent, n/a, Novavax Inc.: Stocks/Bonds Mingzhu Zhu, n/a, Novavax Inc.: Stocks/Bonds Shane Cloney-Clark, n/a, Novavax Inc.: Stocks/Bonds Iksung Cho, MS, Novavax Inc.: Stocks/Bonds Lou Fries, n/a, Novavax Inc.: Stocks/Bonds Greg Glenn, n/a, Novavax Inc.: Stocks/Bonds.

The potential of a universal influenza virus-like particle vaccine expressing a chimeric cytokine.

The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.

Transmembrane domain of IFITM3 is responsible for its interaction with influenza virus HA2 subunit.

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits influenza virus infection by blocking viral membrane fusion, but the exact mechanism remains elusive. Here, we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin (HA). The restriction of IFITM3 on HA-mediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses. Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA2 subunit but not HA1 subunit of H5 and H7 virus. Truncated analyses showed that the transmembrane domain of the IFITM3 and HA2 subunit might play an important role in their interaction. Finally, this interaction of IFITM3 was also verified with HA2 subunits from other subtypes of influenza A virus and influenza B virus. Overall, our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain, providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.

Lotus (Nelumbo nucifera Gaertn.) leaf water extracts suppress influenza a viral infection via inhibition of neuraminidase and hemagglutinin

• Lotus ( Nelumbo nucifera Gaertn.) leaf inhibits influenza virus infection. • Lotus leaf directly blocks IAV binding onto the cells. • Lotus leaf suppresses neuraminidase and hemagglutinin of IAV. • Lotus leaf prevents cytopathic effect by H1N1 and H3N2 IAV. • Isoquercitrin in the lotus leaf is the main active component for an anti-IAV efficacy. Lotus ( Nelumbo nucifera Gaertn.) is a perennial aquatic plant, that has been used in folk to treat various diseases. Here, we first demonstrate the water extract of lotu s leaf (WLL) has a strong inhibitory effect on influenza A virus (IAV) infection by inhibiting neuraminidase (NA) and hemagglutinin (HA). To investigate the effect of WLL on IAV infection, we used green fluorescent protein (GFP)-tagged Influenza A/PR/8/34, fluorescence-activated cell sorting (FACS) analysis, and plaque reduction assay. WLL significantly repressed viral infection, dose-dependently. Immunofluorescence (IF) analysis confirmed that WLL reduces influenza HA, NA, M2, and NP viral proteins expression. WLL strongly inhibited HA and reduced NA activity of IAV. Among six components in WLL, isoquercitrin exerted potent antiviral efficacy. Additionally, WLL potently inhibited the cytopathic effect caused by H1N1 or H3N2 IAV. In conclusion, our results suggest that WLL could be used as a natural viral inhibitor for H1N1 and H3N2 influenza viral infection.

A large-scale systematic survey reveals recurring molecular features of public antibody responses to SARS-CoV-2

SummaryGlobal research to combat the COVID-19 pandemic has led to the isolation and characterization of thousands of human antibodies to the SARS-CoV-2 spike protein, providing an unprecedented opportunity to study the antibody response to a single antigen. Using the information derived from 88 research publications and 13 patents, we assembled a dataset of ∼8,000 human antibodies to the SARS-CoV-2 spike protein from >200 donors. By analyzing immunoglobulin V and D gene usages, complementarity-determining region H3 sequences, and somatic hypermutations, we demonstrated that the common (public) responses to different domains of the spike protein were quite different. We further used these sequences to train a deep-learning model to accurately distinguish between the human antibodies to SARS-CoV-2 spike protein and those to influenza hemagglutinin protein. Overall, this study provides an informative resource for antibody research and enhances our molecular understanding of public antibody responses.

Induction of immunogenic response in Balb/c mice by virus-like particles designed using the influenza neuraminidase, hemagglutinin and matrix proteins

Induction of immunogenic response in Balb/c mice by virus-like particles designed using the influenza neuraminidase, hemagglutinin and matrix proteins

Combined delivery of TLR2 and TLR7 agonists by Nanostructured lipid carriers induces potent vaccine adjuvant activity in mice

Toll-like receptor (TLR) agonists are promising adjuvants and the combination of TLR agonists enhance immune responses by providing synergistic immune activity via triggering different signalling pathways. However, systematic cytotoxicity due to the immediate release of such immune potentiators from the site of injection hampers its clinical performance. Nanostructured lipid carriers (NLCs) offer a possibility to incorporate multiple TLR agonists with high encapsulation efficiency and slow drug release. Herein, we synthesized NLCs from didodecyldimethylammonium bromide (D12DAB) and oleic acid and used these to co-encapsulate a Pam2CS derivative (T-2, TLR2 agonist) with an imidazoquinoline derivative (T-7, TLR7 agonist) as a combination vaccine adjuvant. Hydrodynamic diameter and zeta potential of the prepared NLCs were found to be in the range of 200–500 nm and 23–27 mV, respectively. Spherical shape and size of prepared NLCs were also assessed through Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) analysis. In-vitro release studies of T-7 demonstrated sustained release and the addition of lipopeptide T-2 augmented encapsulation efficiency (from 84 to 92.9%) with a slight trigger in the release percentage. All NLC formulations were screened in TLR2/1, TLR2/6, TLR7 and TLR8 reporter cell lines and loaded NLC formulation showed high TLR2 and TLR7 agonistic activity. Adjuvant potency was evaluated through intramuscular immunization of female C57BL/6 mice with recombinant hepatitis B surface antigen and influenza hemagglutinin protein. T-2 and T-7 loaded NLCs induced good protective efficacy in mice challenged with a lethal dose of influenza virus.

96. Relative Vaccine Effectiveness Against Influenza-Related and Any Respiratory-Related Hospital Encounter During the 2019/20 High Influenza Activity Period: A Comprehensive Real-World Analysis to Compare Quadrivalent Cell-based and Egg-based Influenza Vaccines

Abstract Background Changes in the influenza hemagglutinin protein during replication of influenza in eggs during vaccine production may contribute to low vaccine effectiveness (VE). This phenomenon, egg adaptation, can explain VE differences between egg-based (QIVe-SD) and cell-based (QIVc) quadrivalent influenza vaccines. This research evaluated the relative vaccine effectiveness (rVE) of QIVc versus QIVe-SD in the reduction of influenza-related and any respiratory-related hospitalizations/emergency room (ER) visits among subjects 4-64 years old during the 2019/20 influenza season. Methods A retrospective cohort analysis was conducted among subjects 4-64 years old vaccinated with QIVc or QIVe-SD using administrative claims data in the U.S. (IQVIA PharMetrics® Plus). The adjusted number of events and rates of influenza-related hospitalizations/ER visits and respiratory-related hospitalizations/ER visits were assessed using inverse probability of treatment weighting (IPTW). Poisson regression was used to estimate relative vaccine effectiveness (rVE). In the main analysis, the study period was from Aug 4, 2019 to Mar 7, 2020 (ending early to avoid any influenza outcome misclassification with COVD-19 infection). In the assessment of the high influenza activity period (HIAP), the analysis period was restricted to Dec 8, 2019 to Mar 7, 2020. Results During the 2019/20 influenza season, 1,150,134 recipients of QIVc and 3,924,819, of QIVe-SD were identified following IPTW. In the main analysis, adjusted results show that QIVc was associated with a significantly higher rVE compared to QIVe-SD against influenza-related hospitalizations/ER visits (5.3% [95%CI: 0.5%-9.9%]) and respiratory-related hospitalizations/ER visits (8.2% [95%CI: 6.5%-9.8%]). Similarly, in the HIAP analysis, QIVc was associated with a significantly higher rVE compared to QIVe-SD for influenza-related hospitalizations/ER visits (5.7% [95%CI: 0.8%-10.4%]) and respiratory-related hospitalizations/ER visits (7.3% [95%CI: 5.4%-9.2%]). Conclusion QIVc was more effective in preventing influenza-related and respiratory-related hospitalizations/ER visits compared to QIVe-SD, using either a broad influenza season definition or restricting to the HIAP. Disclosures Victoria Divino, PhD, Seqirus (Consultant) Maarten Postma, Dr., Seqirus (Consultant) Stephen I. Pelton, MD, Seqirus (Consultant) Joaquin F. Mould-Quevedo, PhD, Seqirus (Employee) Ruthwik Anupindi, PhD, Seqirus (Consultant) Mitchell DeKoven, PhD, Seqirus (Consultant) myron J. levin, MD, GSK group of companies (Employee, Research Grant or Support)

Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9

A new strain of Influenza A Virus (IAV), so-called "H7N9 Avian Influenza", is the first strain of this virus in which a human is infected by transmitting the N9 of influenza virus. Although continuous human-to-human transmission has not been reported, the occurrence of various H7N9-associated epidemics and the lack of production of strong antibodies against H7N9 in humans warn of the potential for H7N9 to become a new pandemic. Therefore, the need for effective vaccination against H7N9 as a life-threatening viral pathogen has become a major concern. The current study reports the design of a multi-epitope vaccine against Hemagglutinin (HA) and Neuraminidase (NA) proteins of H7N9 Influenza A virus by prediction of Cytotoxic T lymphocyte (CTL), Helper T lymphocyte (HTL), IFN-γ and B-cell epitopes. Human β-defensin-3 (HβD-3) and pan HLA DR-binding epitope (PADRE) sequence were considered as adjuvant. EAAAK, AAY, GPGPG, HEYGAEALERAG, KK and RVRR linkers were used as a connector for epitopes. The final construct contained 777 amino acids that are expected to be a recombinant protein of about ~ 86.38 kDa with antigenic and non-allergenic properties after expression. Modeled protein analysis based on the tertiary structure validation, docking studies, and molecular dynamics simulations results like Root-mean-square deviation (RMSD), Gyration, Root-mean-square fluctuation (RMSF) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) showed that this protein has a stable construct and capable of being in interaction with Toll-like receptor 7 (TLR7), TLR8 and m826 antibody. Analysis of the obtained data the demonstrates that suggested vaccine has the potential to induce the immune response by stimulating T and Bcells, and may be utilizable for prevention purposes against Avian Influenza A (H7N9).

Classification of Influenza Hemagglutinin Protein Sequences using Convolutional Neural Networks.

The Influenza virus can be considered as one of the most severe viruses that can infect multiple species with often fatal consequences to the hosts. The Hemagglutinin (HA) gene of the virus can be a target for antiviral drug development realised through accurate identification of its sub-types and possible the targeted hosts. This paper focuses on accurately predicting if an Influenza type A virus can infect specific hosts, and more specifically, Human, Avian and Swine hosts, using only the protein sequence of the HA gene. In more detail, we propose encoding the protein sequences into numerical signals using the Hydrophobicity Index and subsequently utilising a Convolutional Neural Network-based predictive model. The Influenza HA protein sequences used in the proposed work are obtained from the Influenza Research Database (IRD). Specifically, complete and unique HA protein sequences were used for avian, human and swine hosts. The data obtained for this work was 17999 human-host proteins, 17667 avian-host proteins and 9278 swine-host proteins. Given this set of collected proteins, the proposed method yields as much as 10% higher accuracy for an individual class (namely, Avian) and 5% higher overall accuracy than in an earlier study. It is also observed that the accuracy for each class in this work is more balanced than what was presented in this earlier study. As the results show, the proposed model can distinguish HA protein sequences with high accuracy whenever the virus under investigation can infect Human, Avian or Swine hosts.

Mosaic Hemagglutinin-Based Whole Inactivated Virus Vaccines Induce Broad Protection Against Influenza B Virus Challenge in Mice.

Influenza viruses undergo antigenic changes in the immuno-dominant hemagglutinin (HA) head domain, necessitating annual re-formulation of and re-vaccination with seasonal influenza virus vaccines for continuing protection. We previously synthesized mosaic HA (mHA) proteins of influenza B viruses which redirect the immune response towards the immuno-subdominant conserved epitopes of the HA via sequential immunization. As ~90% of current influenza virus vaccines are manufactured using the inactivated virus platform, we generated and sequentially vaccinated mice with inactivated influenza B viruses displaying either the homologous (same B HA backbones) or the heterologous (different B HA backbones) mosaic HAs. Both approaches induced long-lasting and cross-protective antibody responses showing strong antibody-dependent cellular cytotoxicity (ADCC) activity. We believe the B virus mHA vaccine candidates represent a major step towards a universal influenza B virus vaccine.

Decreased vaccine protection of egg-based influenza vaccine in the elderly and nonhemagglutinin-focused immunity.

Severe influenza illness or death is a serious concern among the elderly population despite vaccination. To investigate how the adaptive immune response after vaccination varies with the patient's age, Jung et al., in a recent issue of the JCI, extensively analyzed the serum antibody response in different age groups after immunization with the egg-based influenza vaccine Fluzone. As expected, the immune response in young adults was dominated by antibodies targeting the influenza hemagglutinin (HA) protein. On the contrary, the serological repertoire of elderly donors was characterized by cross-reactive (CR) antibodies recognizing non-HA antigens. Surprisingly, a substantial fraction of these CR antibodies targeted sulfated glycans typical of egg-produced proteins, which does not provide protection against human influenza viruses. Overall, these findings are of great value in understanding suboptimal immunity after influenza vaccination and shaping future vaccine efforts that will achieve increased protection in the elderly.

Natural influenza infection produces a greater diversity of humoral responses than vaccination in immunosuppressed transplant recipients

The humoral immune response to influenza virus infection is complex and may be different compared to the antibody response elicited by vaccination. We analyzed the breadth of IgG and IgA responses in solid organ transplant (SOT) recipients to a diverse collection of 86 influenza antigens elicited by natural influenza A virus (IAV) infection or by vaccination. Antibody levels were quantified using a custom antigen microarray. A total of 120 patients were included: 80 IAV infected (40 A/H1N1 and 40 A/H3N2) and 40 vaccinated. Based on hierarchical clustering analysis, infection with either H1N1 or H3N2 virus showed a more diverse antibody response compared to vaccination. Similarly, H1N1-infected individuals showed a significant IgG response to 27.9% of array antigens and H3N2-infected patients to 43.0% of antigens, whereas vaccination elicited a less broad immune response (7.0% of antigens). Immune responses were not exclusively targeting influenza hemagglutinin (HA) proteins but were also directed against conserved influenza antigens. Serum IgA responses followed a similar profile. This study provides novel data on the breadth of antibody responses to influenza. We also found that the diversity of response is greater in influenza-infected rather than vaccinated patients, providing a potential mechanistic rationale for suboptimal vaccine efficacy in this population.

Hemagglutination Inhibition (HAI) antibody landscapes after vaccination with H7Nx virus like particles.

A systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-binding antibodies against co-circulating H7 influenza strains. A panel of eight H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013. VLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain. This study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.