The 2009 pandemic H1N1 hemagglutinin stalk remained antigenically stable after circulating in humans for a decade

An H1N1 influenza virus caused a pandemic in 2009 and descendants of this virus continue to circulate seasonally in humans. Upon infection with the 2009 H1N1 pandemic strain (pH1N1), many humans produced antibodies against epitopes in the hemagglutinin (HA) stalk. HA stalk-focused antibody responses were common among pH1N1-infected individuals because HA stalk epitopes were conserved between the pH1N1 strain and previously circulating H1N1 strains. Here, we completed a series of experiments to determine if the pH1N1 HA stalk has acquired substitutions since 2009 that prevent the binding of human antibodies. We identified several amino acid substitutions that have accrued in the pH1N1 HA stalk from 2009-2019. We completed enzyme-linked immunosorbent assays, absorption-based binding assays, and surface plasmon resonance experiments to determine if these substitutions affect antibody binding. Using sera collected from 230 humans (aged 21-80 years), we found that pH1N1 HA stalk substitutions that have emerged since 2009 do not affect antibody binding. Our data suggest that the HA stalk domain of pH1N1 viruses remained antigenically stable after circulating in humans for a decade.

Broadly-neutralizing antibodies that bind to the influenza hemagglutinin stalk domain enhance the effectiveness of neuraminidase inhibitors via Fc-mediated effector functions

The conserved hemagglutinin stalk domain is an attractive target for broadly effective antibody-based therapeutics and next generation universal influenza vaccines. Protection provided by hemagglutinin stalk binding antibodies is principally mediated through activation of immune effector cells. Titers of stalk-binding antibodies are highly variable on an individual level, and tend to increase with age as a result of increasing exposures to influenza virus. In our study, we show that stalk-binding antibodies cooperate with neuraminidase inhibitors to protect against influenza virus infection in an Fc-dependent manner. These data suggest that the effectiveness of neuraminidase inhibitors is likely influenced by an individual's titers of stalk-binding antibodies, and that neuraminidase inhibitors may enhance the effectiveness of future stalk-binding monoclonal antibody-based treatments.

Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

Structure-Guided Creation of an Anti-HA Stalk Antibody F11 Derivative That Neutralizes Both F11-Sensitive and -Resistant Influenza A(H1N1)pdm09 Viruses

The stalk domain of influenza virus envelope glycoprotein hemagglutinin (HA) constitutes the axis connecting the head and transmembrane domains, and plays pivotal roles in conformational rearrangements of HA for virus infection. Here we characterized molecular interactions between the anti-HA stalk neutralization antibody F11 and influenza A(H1N1)pdm09 HA to understand the structural basis of the actions and modifications of this antibody. In silico structural analyses using a model of the trimeric HA ectodomain indicated that the F11 Fab fragment has physicochemical properties, allowing it to crosslink two HA monomers by binding to a region near the proteolytic cleavage site of the stalk domain. Interestingly, the F11 binding allosterically caused a marked suppression of the structural dynamics of the HA cleavage loop and flanking regions. Structure-guided mutagenesis of the F11 antibody revealed a critical residue in the F11 light chain for the F11-mediated neutralization. Finally, the mutagenesis led to identification of a unique F11 derivative that can neutralize both F11-sensitive and F11-resistant A(H1N1)pdm09 viruses. These results raise the possibility that F11 sterically and physically disturbs proteolytic cleavage of HA for the ordered conformational rearrangements and suggest that in silico guiding experiments can be useful to create anti-HA stalk antibodies with new phenotypes.

Identification of Quinolinones as Antivirals Against Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a re-emerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time of addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that endowed VEEV resistance to this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of CHIKV to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses, and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

Sequences in the stalk domain regulate autoinhibition and ciliary tip localization of the immotile kinesin-4 KIF7

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by autoinhibition as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for autoinhibition of KIF7 whereas the adjacent regulatory coiled coil (rCC) that contributes to autoinhibition of the motile kinesin-4's KIF21A and KIF21B, is not sufficient for KIF7 autoinhibition. Disease-associated mutations in the inhCC relieve autoinhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC and could be increased by disease-associated mutations regardless of the protein's autoinhibition state. These findings suggest that loss of KIF7 autoinhibition and/or altered cilium tip localization can contribute to pathogenesis of human disease.

Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies

ABSTRACT Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA-Gaussia princeps luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models. IMPORTANCE Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.

Introduction of Cysteines in the Stalk Domain of Recombinant Influenza Virus N1 Neuraminidase Enhances Protein Stability and Immunogenicity in Mice

Influenza virus surface glycoproteins represent the main targets of the immune system during infection and vaccination. Current influenza virus vaccines rely mostly on the hemagglutinin, requiring a close match between the vaccine and circulating strains. Recently, the neuraminidase (NA) has become an attractive target; however low immunogenicity and stability in vaccine preparations remain an obstacles. Here, we took advantage of the hypervariable stalk domain of the NA to introduce cysteines at different positions and to produce more stable multimeric forms of the protein. We generated 11 N1 constructs and characterized the proteins by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and by testing their enzymatic activity and representation of antigenic epitopes. Moreover, we evaluated their potential to induce a protective immune response in vivo and characterized the polyclonal antibody responses of immunized mice. We observed that the introduction of cysteines at certain positions led to the formation of stable N1 dimers, which are capable of inducing a strong antibody response characterized by neuraminidase inhibiting activity and protection of mice from high dose viral challenge. Overall, our results provide evidence for the feasibility of introducing stalk modifications to enhance the stability and immunogenicity of NA-based recombinant antigens.

Mutations in the Hemagglutinin Stalk Domain Do Not Permit Escape from a Protective, Stalk-Based Vaccine-Induced Immune Response in the Mouse Model

ABSTRACT Current seasonal influenza virus vaccines target regions of the hemagglutinin (HA) head domain that undergo constant antigenic change, forcing the painstaking annual reformulation of vaccines. The development of broadly protective or universal influenza virus vaccines that induce cross-reactive, protective immune responses could circumvent the need to reformulate current seasonal vaccines. Many of these vaccine candidates target the HA stalk domain, which displays epitopes conserved within and across influenza virus subtypes, including those with pandemic potential. While HA head-mediated antigenic drift is well understood, the potential for antigenic drift in the stalk domain is understudied. Using a panel of HA stalk-specific monoclonal antibodies (MAbs), we applied selection pressure to the stalk domain of A/Netherlands/602/2009 (pdmH1N1) to determine fitness and phenotypes of escape mutant viruses (EMVs). We found that HA stalk MAbs with lower cross-reactivity caused single HA stalk escape mutations, whereas MAbs with broader cross-reactivity forced multiple mutations in the HA. Each escape mutant virus greatly decreased mAb neutralizing activity, but escape mutations did not always ablate MAb binding or Fc-Fc receptor-based effector functions. Escape mutant viruses were not attenuated in vitro but showed attenuation in an in vivo mouse model. Importantly, mice vaccinated with a chimeric HA universal vaccine candidate were protected from lethal challenge with EMVs despite these challenge viruses containing escape mutations in the stalk domain. Our study indicates that while the HA stalk domain can mutate under strong MAb selection pressure, mutant viruses may have attenuated phenotypes and do not evade a polyclonal, stalk-based vaccine-induced response. IMPORTANCE Broadly protective or universal influenza virus vaccines target viral epitopes that appear to be conserved. However, it is unclear whether the virus will be able to escape once immunological pressure is applied to these epitopes through vaccination of large proportions of the population. Studies that investigate the fitness and antigenic characteristics of viruses that escape immunological pressure on these conserved epitopes are therefore urgently needed.