The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain.

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

Structure of an H3N2 influenza virus nucleoprotein.

Influenza A viruses of the H1N1 and H3N2 subtypes are responsible for seasonal epidemic events. The influenza nucleoprotein (NP) binds to the viral genomic RNA and is essential for its replication. Efforts are under way to produce therapeutics and vaccines targeting the NP. Despite this, no structure of an NP from an H3N2 virus has previously been determined. Here, the structure of the A/Northern Territory/60/1968 (H3N2) influenza virus NP is presented at 2.2 Šresolution. The structure is highly similar to those of the A/WSN/1933 (H1N1) and A/Hong Kong/483/97 (H5N1) NPs. Nonconserved amino acids are widely dispersed both at the sequence and structural levels. A movement of the 73-90 RNA-binding loop is observed to be the key difference between the structure determined here and previous structures. The data presented here increase the understanding of structural conservation amongst influenza NPs and may aid in the design of universal interventions against influenza.

The cleavage of spike protein НА0→НА1/HA2 by trypsin permits activation of the M2 channel without its proteolytic cleavage in the influenza A virus.

M2 plays numerous regulatory roles in influenza A virus infection confirming the old adage: "a little body often harbors a great sense". The comment here demonstrates that a small viral protein M2, having 14 kD m.w. and situating in the virion at a minor amount of only about 40 molecules per virus particle is resistant to trypsin at concentrations initiating the HA0 cleavage and virus infectivity activation. A mechanism involving a programmed disassembly by cascade-type transmembrane signaling of the HA-M2-M1-RNP cooperation during virus entry into the infected cell is proposed.

The native structure of the assembled matrix protein 1 of influenza A virus.

Influenza A virus causes millions of severe cases of disease during annual epidemics. The most abundant protein in influenza virions is matrix protein 1 (M1), which mediates virus assembly by forming an endoskeleton beneath the virus membrane1. The structure of full-length M1, and how it oligomerizes to mediate the assembly of virions, is unknown. Here we determine the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We find that the C-terminal domain of M1 is disordered in solution but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerizing M1 into linear strands that coat the interior surface of the membrane of the assembling virion. In the M1 polymer, five histidine residues-contributed by three different monomers of M1-form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore reveal mechanisms of influenza virus assembly and disassembly.

Quantification of Multivalent Interactions between Sialic Acid and Influenza A Virus Spike Proteins by Single-Molecule Force Spectroscopy.

Multivalency is a key principle in reinforcing reversible molecular interactions through the formation of multiple bonds. The influenza A virus deploys this strategy to bind strongly to cell surface receptors. We performed single-molecule force spectroscopy (SMFS) to investigate the rupture force required to break individual and multiple bonds formed between synthetic sialic acid (SA) receptors and the two principal spike proteins of the influenza A virus (H3N2): hemagglutinin (H3) and neuraminidase (N2). Kinetic parameters such as the rupture length (χß) and dissociation rate (koff) are extracted using the model by Friddle, De Yoreo, and Noy. We found that a monovalent SA receptor binds to N2 with a significantly higher bond lifetime (270 ms) compared to that for H3 (36 ms). By extending the single-bond rupture analysis to a multibond system of n protein-receptor pairs, we provide an unprecedented quantification of the mechanistic features of multivalency between H3 and N2 with SA receptors and show that the stability of the multivalent connection increases with the number of bonds from tens to hundreds of milliseconds. Association rates (kon) are also provided, and an estimation of the dissociation constants (KD) between the SA receptors to both proteins indicate a 17-fold higher binding affinity for the SA-N2 bond with respect to that of SA-H3. An optimal designed multivalent SA receptor showed a higher binding stability to the H3 protein of the influenza A virus than to the monovalent SA receptor. Our study emphasizes the influence of the scaffold on the presentation of receptors during multivalent binding.

Structural transitions in influenza haemagglutinin at membrane fusion pH.

Infection by enveloped viruses involves fusion of their lipid envelopes with cellular membranes to release the viral genome into cells. For HIV, Ebola, influenza and numerous other viruses, envelope glycoproteins bind the infecting virion to cell-surface receptors and mediate membrane fusion. In the case of influenza, the receptor-binding glycoprotein is the haemagglutinin (HA), and following receptor-mediated uptake of the bound virus by endocytosis1, it is the HA that mediates fusion of the virus envelope with the membrane of the endosome2. Each subunit of the trimeric HA consists of two disulfide-linked polypeptides, HA1 and HA2. The larger, virus-membrane-distal, HA1 mediates receptor binding; the smaller, membrane-proximal, HA2 anchors HA in the envelope and contains the fusion peptide, a region that is directly involved in membrane interaction3. The low pH of endosomes activates fusion by facilitating irreversible conformational changes in the glycoprotein. The structures of the initial HA at neutral pH and the final HA at fusion pH have been investigated by electron microscopy4,5 and X-ray crystallography6-8. Here, to further study the process of fusion, we incubate HA for different times at pH 5.0 and directly image structural changes using single-particle cryo-electron microscopy. We describe three distinct, previously undescribed forms of HA, most notably a 150 Å-long triple-helical coil of HA2, which may bridge between the viral and endosomal membranes. Comparison of these structures reveals concerted conformational rearrangements through which the HA mediates membrane fusion.

Early prediction of antigenic transitions for influenza A/H3N2.

Influenza A/H3N2 is a rapidly evolving virus which experiences major antigenic transitions every two to eight years. Anticipating the timing and outcome of transitions is critical to developing effective seasonal influenza vaccines. Using a published phylodynamic model of influenza transmission, we identified indicators of future evolutionary success for an emerging antigenic cluster and quantified fundamental trade-offs in our ability to make such predictions. The eventual fate of a new cluster depends on its initial epidemiological growth rate--which is a function of mutational load and population susceptibility to the cluster--along with the variance in growth rate across co-circulating viruses. Logistic regression can predict whether a cluster at 5% relative frequency will eventually succeed with ~80% sensitivity, providing up to eight months advance warning. As a cluster expands, the predictions improve while the lead-time for vaccine development and other interventions decreases. However, attempts to make comparable predictions from 12 years of empirical influenza surveillance data, which are far sparser and more coarse-grained, achieve only 56% sensitivity. By expanding influenza surveillance to obtain more granular estimates of the frequencies of and population-wide susceptibility to emerging viruses, we can better anticipate major antigenic transitions. This provides added incentives for accelerating the vaccine production cycle to reduce the lead time required for strain selection.

Changes in the Receptor-Binding Properties of H3N2 Viruses during Long-Term Circulation in Humans.

It was previously shown that hemagglutinin residues Thr155, Glu158, and Ser228 are crucial for the recognition of Neu5Gc. In this study, we demonstrated that the ability to bind the Neu5Gc-terminated receptor is related to the amino acid 145: viruses of years 1972-1999 with Lys145 bind to the receptor, whereas viruses with Asn145 do not. Sporadic appearance and disappearance of the ability to bind Neu5Gc oligosaccharides and the absence of Neu5Gc in the composition of human glycoconjugates indicate the non-adaptive nature of this ability. It was previously shown that unlike H1N1 viruses, H3N2 viruses of years 1968-1989 did not distinguish between Neu5Acα2-6Galß1-4Glc (6'SL) and Neu5Acα2-6Galß1-4GlcNAc (6'SLN). H3N2 viruses isolated after 1993 have acquired the ability to distinguish between 6'SL and 6'SLN, similarly to H1N1 viruses. We found that the affinity for 6'SLN has gradually increased from 1992 to 2003. After 2003, the viruses lost the ability to bind a number of sialosides, including 6'SL, that were good receptors for earlier H3N2 viruses, and retained high affinity for 6'SLN only, which correlated with the acquisition of new glycosylation sites at positions 122, 133, and 144, as well as Glu190Asp and Gly225Asp substitutions, in hemagglutinin. These substitutions are also responsible for the receptor-binding phenotype of human H1N1 viruses. We conclude that the convergent evolution of the receptor specificity of the H1N1 and H3N2 viruses indicates that 6'SLN is the optimal natural human receptor for influenza viruses.

Generation of Stable Influenza Virus Hemagglutinin through Structure-Guided Recombination.

Hemagglutinin (HA) is the major surface antigen of influenza virus and the most promising influenza vaccine immunogen. In 2013, the devastating H7N9 influenza virus was identified in China, which induced high mortality. The HA of this virus (H7) is relatively unstable, making it challenging to produce an effective vaccine. To improve the stability of HA protein from H7N9 influenza virus for better vaccine antigens without impairing immunogenicity, we recombined the HA from H7N9 (H7) with a more stable HA from H3N2 (H3) by structure-guided recombination, resulting in six chimeric HAs, FrA-FrF. Two of these chimeric HAs, FrB and FrC, exhibited proper hemagglutination activity and presented improved thermal stability compared to the original H7. Mice immunized with FrB and FrC elicited H7-specific antibodies comparable to those induced by parental H7, and the antisera collected from these immunized mice successfully inhibited H7N9 infection in a microneutralization assay. These results suggest that our structural-recombination approach can create stabilizing chimeric antigens while maintaining proper immunogenicity, which may not only benefit the construction of more stable HA vaccines to fight against H7N9 infection, but also facilitate effective vaccine improvements for other influenza viruses or infectious pathogens. In addition, this study also demonstrates the potential for better engineering of multimeric protein complexes like HA to achieve improved function, which are often immunologically or pharmaceutically important but difficult to modify.

The 2nd sialic acid-binding site of influenza A virus neuraminidase is an important determinant of the hemagglutinin-neuraminidase-receptor balance.

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.

Application of an immunoglobulin Y-alkaline phosphatase bioconjugate as a diagnostic tool for influenza A virus.

The diagnosis of influenza A virus is essential since it can be confused with influenza A like illness and lead to inaccurate drug prescription. In this study, the M2e peptide, a strategic antigen that is conserved in all virus subtypes, was used as a diagnostic marker of influenza A. For the first time, M2e-specific IgY antibody was covalently conjugated to alkaline phosphatase (ALP) enzyme in the presence of glutaraldehyde. The antibody-enzyme bioconjugate was characterized by fluorescence and Fourier-transform infrared spectroscopy. Subsequently, the diagnostic value of this bioconjugate was evaluated by direct sandwich ELISA using nasopharyngeal swab samples positive/negative for H1N1 and H3N2, which were previously analyzed by rRT-PCR for influenza. In conclusion, the M2e-specific IgY-ALP bioconjugate demonstrated positive results for Influenza A in samples that were diagnosed as Influenza A via the RT-PCR method.

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.

Inactivation of human and avian influenza viruses by potassium oleate of natural soap component through exothermic interaction.

An influenza epidemic is still a problem despite the development of vaccines and anti-influenza drugs. Preventive measures such as handwashing are fundamental and important for counteracting influenza virus infection. In this study, we clarified the anti-influenza virus effects of surfactants, which are the main components of hand soaps for hand washing: potassium oleate (C18:1), sodium laureth sulfate (LES) and sodium lauryl sulfate (SDS). For a human influenza virus strain (H3N2), C18:1 reduced the infectivity by 4 logs or more, whereas LES and SDS reduced the infectivity by 1 log or less. Similar results were obtained when an avian influenza virus strain (H5N3) was used. The interaction between the surfactant and virus was then investigated by isothermal titration calorimetry. The LES-virus system showed a positive value of enthalpy changes (ΔH), meaning an exothermic interaction that indicated a hydrophobic interaction. In contrast, both the C18:1-virus system and the SDS-virus system showed negative values of ΔH, meaning an endothermic interaction that indicated an electrical interaction. The ΔH value of the C18:1-virus system was much higher than that of the SDS-virus system. A mixture of C18:1 and HA proteins similarly showed negative values of ΔH. These results indicate that influenza virus inactivation by a hydrophobic interaction of a surfactant with the viral envelope is insufficient to prevent infection, whereas inactivation by an electrical interaction of a surfactant with HA proteins is sufficient to prevent influenza virus infection.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

A Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants.

Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HA's functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature-but only when the measurements are made on a viral strain similar to the ones being studied in nature.

Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin.

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.

Isolation of an Egg-Adapted Influenza A(H3N2) Virus without Amino Acid Substitutions at the Antigenic Sites of Its Hemagglutinin.

Antigenic changes in the hemagglutinin protein of recent A(H3N2) viruses often arise when these viruses adapt to their egg host. By serial egg passages of a cell-propagated virus, we successfully isolated an egg-adapted influenza A(H3N2) virus, A/Saitama/103/2014, without amino acid substitutions at the antigenic sites of its hemagglutinin protein but with multiple substitutions in its neuraminidase protein. Antigenic analysis of this egg-adapted A/Saitama/103/2014 virus indicated that its antigenicity did not differ from that of the World Health Organization prototype cell-propagated vaccine virus: A/Hong Kong/4801/2014. Our results suggest that this strategy may facilitate egg-based vaccine production without antigenic alterations in hemagglutinin by egg adaptation.

Three mutations switch H7N9 influenza to human-type receptor specificity.

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.

Residues F103 and M106 within the influenza A virus NS1 CPSF4-binding region regulate interferon-stimulated gene translation initiation.

Influenza A virus (IAV) non-structural protein 1 (NS1) suppresses host innate immune responses by inhibiting type I interferon (IFN) production. We provide evidence that residues F103 and M106 in the CPSF4-binding domain of A/HK/1/68 [H3N2] NS1 contribute to post-transcriptional inhibition of antiviral IFN-stimulated genes (ISGs), thereby suppressing an antiviral type I IFN response. Recombinant (r) IAVs encoding F103L and M106I mutations in NS1 replicate to significantly lower viral titers in human A549 lung epithelial cells and primary type II alveolar cells. In A549 cells, rIAVs encoding these mutant NS1s induce higher levels of IFN-ß production and are more sensitive to the antiviral effects of IFN-ß treatment. qPCR characterization of polysomal mRNA, in the presence or absence of IFN-ß treatment, identified a greater proportion of heavy polysome-associated ISGs including EIF2AK2, OAS1, and MxA in A549 cells infected with rIAVs encoding these CPSF4-binding mutant NS1s, in contrast to rIAV encoding wildtype NS1.