Exploiting the Affimer platform against influenza A virus

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are short-lived due to the high mutation rates of IAV. Currently, monoclonal antibodies (mAbs) have emerged as a promising approach to tackle future IAV pandemics. Additionally, several antibody-like alternatives exist that aim to improve upon mAbs. Affimers, one such alternative, benefit from a short development time, high expression levels in E. coli, and complete animal-free production. Here we exploited the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Starting from a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV H3 subtype infection in vitro. Two of these Affimers were characterised in detail: they exhibited binding affinities to the target H3 HA protein in the nM range and bound specifically to the HA1 head domain. Cryo-EM employing a novel spray approach to prepare cryo-grids allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that the mode of inhibition of these Affimers is based on blocking the interaction of HA to the host-cell receptor - sialic acid. Additionally, these Affimers inhibited IAV strains closely related to the one employed for Affimer isolation. Overall, these results support the use of Affimers as an alternative to existing targeted therapies for IAV and pave the way for their use as diagnostic reagents. Importance Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections, but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like, bacterially made proteins. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterised the mechanism of inhibition of the Affimers by challenging with related influenza virus strains. We additionally characterised an HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

[1]  D. Burton,et al.  Fully synthetic platform to rapidly generate tetravalent bispecific nanobody–based immunoglobulins , 2023, Proceedings of the National Academy of Sciences of the United States of America.

[2]  A. Gruber,et al.  The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants , 2022, Nature biotechnology.

[3]  J. Lai,et al.  Abstract 1815: AVA6000, a novel Precision medicine, targeted to the tumor microenvironment via Fibroblast Activation Protein (FAP) mediated cleavage , 2022, Cancer Research.

[4]  O. Watson,et al.  Global impact of the first year of COVID-19 vaccination: a mathematical modelling study , 2022, The Lancet Infectious Diseases.

[5]  Meiwan Chen,et al.  A roadmap to pulmonary delivery strategies for the treatment of infectious lung diseases , 2022, Journal of Nanobiotechnology.

[6]  A. Usher.  The global COVID-19 treatment divide , 2022, The Lancet.

[7]  D. Sheward,et al.  A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo , 2022, Nature Communications.

[8]  T. Harder,et al.  Influenza A Viruses and Zoonotic Events—Are We Creating Our Own Reservoirs? , 2021, Viruses.

[9]  F. Sobott,et al.  On-grid and in-flow mixing for time-resolved cryo-EM , 2021, Acta crystallographica. Section D, Structural biology.

[10]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[11]  D. Sheward,et al.  A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo , 2021, bioRxiv.

[12]  Lijun Rong,et al.  Small Molecule Inhibitors of Influenza Virus Entry , 2021, Pharmaceuticals.

[13]  A. Caflisch,et al.  Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs , 2021, bioRxiv.

[14]  D. Bernstein,et al.  A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial , 2020, Nature Medicine.

[15]  J. Ravetch,et al.  The role of IgG Fc receptors in antibody-dependent enhancement , 2020, Nature Reviews Immunology.

[16]  J Gomez-Blanco,et al.  DeepEMhancer: a deep learning solution for cryo-EM volume post-processing , 2020, Communications Biology.

[17]  P. Millner,et al.  Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests , 2020, PLoS neglected tropical diseases.

[18]  B. Kelley.  Developing therapeutic monoclonal antibodies at pandemic pace , 2020, Nature Biotechnology.

[19]  Diana C. F. Monteiro,et al.  Sample deposition onto cryo-EM grids: from sprays to jets and back , 2020, Acta crystallographica. Section D, Structural biology.

[20]  I. Wilson,et al.  Broadly protective human antibodies that target the active site of influenza virus neuraminidase , 2019, Science.

[21]  Jasenko Zivanov,et al.  Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1 , 2019, bioRxiv.

[22]  Michael J. McPherson,et al.  Affimer-enzyme-inhibitor switch sensor for rapid wash-free assays of multimeric proteins. , 2019, ACS sensors.

[23]  M. Byrne,et al.  Affimer reagents as tools in diagnosing plant virus diseases , 2019, Scientific Reports.

[24]  Yun Zhang,et al.  Targeting Hemagglutinin: Approaches for Broad Protection against the Influenza A Virus , 2019, Viruses.

[25]  Diana C. F. Monteiro,et al.  A cryo-EM grid preparation device for time-resolved structural studies , 2019, bioRxiv.

[26]  N. Bragazzi,et al.  The Spanish Influenza Pandemic: a lesson from history 100 years after 1918 , 2019, Journal of preventive medicine and hygiene.

[27]  Poul Nissen,et al.  Namdinator – automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps , 2018, bioRxiv.

[28]  I. Wilson,et al.  A multifunctional human monoclonal neutralizing antibody that targets a unique conserved epitope on influenza HA , 2018, Nature Communications.

[29]  R. Lerner,et al.  A complex epistatic network limits the mutational reversibility in the influenza hemagglutinin receptor-binding site , 2018, Nature Communications.

[30]  Conrad C. Huang,et al.  UCSF ChimeraX: Meeting modern challenges in visualization and analysis , 2018, Protein science : a publication of the Protein Society.

[31]  Caitlin E. Mullarkey,et al.  Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice , 2017, Nature Communications.

[32]  Ji-hye Lee,et al.  Conformational modulation of influenza virus hemagglutinin: characterization and in vivo efficacy of monomeric form , 2017, Scientific Reports.

[33]  Song-Liang Wang,et al.  Evolution of Influenza A Virus by Mutation and Re-Assortment , 2017, International journal of molecular sciences.

[34]  Mona Wong-Barnum,et al.  COSMIC2: A Science Gateway for Cryo-Electron Microscopy Structure Determination , 2017, PEARC.

[35]  Joseph H. Davis,et al.  Addressing preferred specimen orientation in single-particle cryo-EM through tilting , 2017, Nature Methods.

[36]  Filomena Esteves,et al.  Affimer proteins are versatile and renewable affinity reagents , 2017, eLife.

[37]  M. Hussain,et al.  Drug resistance in influenza A virus: the epidemiology and management , 2017, Infection and drug resistance.

[38]  F. Krammer,et al.  Universal influenza virus vaccines and therapeutic antibodies. , 2017, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[39]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[40]  P. Collins,et al.  Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes , 2016, Cell.

[41]  Cinque S. Soto,et al.  Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses , 2016, Cell.

[42]  Andrew Rambaut,et al.  Origins of the 2009 H1N1 influenza pandemic in swine in Mexico , 2016, eLife.

[43]  Tri Giang Phan,et al.  Challenges and opportunities for non-antibody scaffold drugs. , 2015, Drug discovery today.

[44]  Kai Zhang,et al.  Gctf: Real-time CTF determination and correction , 2015, bioRxiv.

[45]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[46]  Caitlin E. Mullarkey,et al.  Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones , 2015, Journal of Virology.

[47]  Vidya Subramanian,et al.  Broadly neutralizing influenza hemagglutinin stem-specific antibody CR8020 targets residues that are prone to escape due to host selection pressure. , 2014, Cell host & microbe.

[48]  Y. Iba,et al.  Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus , 2014, Nature Communications.

[49]  Y. Iba,et al.  Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses , 2014, Journal of Virology.

[50]  Qing Zhu,et al.  A Broadly Neutralizing Human Monoclonal Antibody Directed against a Novel Conserved Epitope on the Influenza Virus H3 Hemagglutinin Globular Head , 2014, Journal of Virology.

[51]  J. Nettleship,et al.  Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications , 2014, Protein engineering, design & selection : PEDS.

[52]  Antoine M. van Oijen,et al.  Mechanisms of Hemagglutinin Targeted Influenza Virus Neutralization , 2013, PloS one.

[53]  P. Palese,et al.  Influenza virus hemagglutinin stalk-based antibodies and vaccines. , 2013, Current opinion in virology.

[54]  N. Cox,et al.  Pandemic Influenza Planning, United States, 1978–2008 , 2013, Emerging infectious diseases.

[55]  John Steel,et al.  Cross-neutralization of influenza A viruses mediated by a single antibody loop , 2012, Nature.

[56]  N. S. Laursen,et al.  Highly Conserved Protective Epitopes on Influenza B Viruses , 2012, Science.

[57]  Yoshiyuki Suzuki,et al.  Evidence for N-Glycan Shielding of Antigenic Sites during Evolution of Human Influenza A Virus Hemagglutinin , 2012, Journal of Virology.

[58]  Anil Wipat,et al.  Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant. , 2011, Protein engineering, design & selection : PEDS.

[59]  Martin H. Koldijk,et al.  A Highly Conserved Neutralizing Epitope on Group 2 Influenza A Viruses , 2011, Science.

[60]  Surender Khurana,et al.  Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin , 2011, Proceedings of the National Academy of Sciences.

[61]  J. Taubenberger,et al.  Influenza: The Once and Future Pandemic , 2010, Public health reports.

[62]  Michael Busby,et al.  Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant , 2010, Protein engineering, design & selection : PEDS.

[63]  R. Hai,et al.  Broadly Protective Monoclonal Antibodies against H3 Influenza Viruses following Sequential Immunization with Different Hemagglutinins , 2010, PLoS pathogens.

[64]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[65]  G. Morris.  Pandemic Influenza Planning , 2009 .

[66]  P. Chames,et al.  THEMED SECTION: VECTOR DESIGN AND DRUG DELIVERY REVIEW Therapeutic antibodies: successes, limitations and hopes for the future , 2022 .