key: cord-0327782-43h0dlf2 authors: Antanasijevic, Aleksandar; Schulze, Autumn J.; Reddy, Vijay S.; Ward, Andrew B. title: High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions date: 2022-01-31 journal: bioRxiv DOI: 10.1101/2022.01.31.478566 sha: f6dd4717c85dcbdd5c658bee49518c3ddbd14a99 doc_id: 327782 cord_uid: 43h0dlf2 Non-polio enteroviruses (NPEVs) cause serious illnesses in young children and neonates including aseptic meningitis, encephalitis, neonatal sepsis and inflammatory muscle disease, among others. Over 100 serotypes have been described to date but except for the EV-A71, there are no available vaccines or therapeutics against NPEVs. Efforts towards rationally designed pan-NPEV vaccines would greatly benefit from structural biology methods for rapid and comprehensive evaluation of vaccine candidates and elicited antibody responses. Towards this goal, we tested if electron-microscopy-based polyclonal epitope mapping (EMPEM), a method where structural analysis is performed using serum-derived polyclonal antibodies (pAbs), can be applied to an NPEV. EMPEM was performed on immune complexes featuring CV-A21 viral particles and CV-A21-specific pAbs isolated from mice. Notably, this is the first example of structural analysis of polyclonal immune complexes comprising whole virions. We introduce a data processing workflow that allows reconstruction of different pAbs at near-atomic resolution. The analysis resulted in identification of several antibodies targeting two immunodominant epitopes, near the 3-fold and 5-fold axis of symmetry; the latter overlapping with the receptor binding site. These results demonstrate that EMPEM can be applied to map broad-spectrum polyclonal immune responses against intact virions and define potentially cross-reactive epitopes. Herein, we introduce an EMPEM pipeline for rapid structural analysis of pAb responses elicited 78 by enteroviruses using whole viral particles. Our data processing workflow enabled reconstruction of 79 immune complexes featuring pAbs at near atomic resolution. In a case study using the Coxsackievirus A21 80 (CV-A21), we discovered two immunodominant sites on the surface of CV-A21 readily targeted by 81 antibodies; one in immediate proximity to the receptor binding site. Overall, we demonstrate the feasibility 82 of this approach and provide valuable insights for future vaccine design efforts. For this proof-of-concept study we selected CV-A21 viral strain responsible for a substantial 87 proportion of enterovirus-associated acute respiratory tract infections in humans (Couch, Douglas, Assembled immune complexes (or free virions) were first characterized using negative stain EM 117 (nsEMPEM; Figure 1C ). pAbs isolated from the control group of mice did not bind to CV-A21, based on We next relaxed atomic models into the pAbC-1, pAbC-3 and pAbC-5 cryoEMPEM maps. Given to the symmetry-related neighboring binding site. In theory, all 3 epitopes surrounding the 3-fold symmetry 187 axis and all 5 epitopes at the 5-fold symmetry axis could be simultaneously occupied with antibodies. We speculate that the relatively high immunogenicity of Site-1 ad Site-2 epitope clusters is due to multivalent 189 binding to IgG and IgM forms of antibodies; as previously described for human papillomavirus (Schiller & 190 Lowy, 2018). Site-2 is particularly interesting because it is in immediate proximity to the receptor binding site 192 (RBS) for CV-A21 ( Figure 3C ). While antibodies targeting Site-1 are distal from the RBS and it is unlikely 193 that they would interfere with receptor binding as Fabs ( Figure 3D ), antibodies against Site-2 sterically 194 block ICAM-1 from accessing the RBS ( Figure 3E ) and may be able to suppress viral entry. We then looked Using EMPEM we can study polyclonal antibody responses in a longitudinal manner enabling us to 240 understand the hierarchy of immunodominant epitopes in different CV strains and to identify optimal 241 vaccine candidates for eliciting the most desirable antibody responses. Antibodies targeting epitopes near the 5-fold axis can sterically block access to the receptor binding 243 site and thereby prevent the virus from infecting cells. This feature is highly desirable, but the relatively 244 low level of sequence conservation in this area is problematic as elicited antibodies are unlikely to cross-245 react with other coxsackievirus strains and may sterically compete with more desirable antibodies. Coxsackievirus A21 particles were propagated in H1-HeLa cells. The virions were harvested from lysed 256 cells 2 days after infection, clarified by centrifugation, 0.2 µm filtered, and inactivated by incubation with 257 100 µg/mL formaldehyde at 37 ºC for 3 days, followed by low-speed centrifugation to remove cell debris. The supernatant was pelleted through a 30% sucrose cushion via ultracentrifugation at 175,000 g for 14 259 hours at 4 ºC. The resuspended pellet was sedimented through a 15-45% (w/v) sucrose density gradient and For cryoEM the immune complexes were assembled by combining 300 µg of the purified, inactivated CV-300 A21 viral particles with 8 mg of Fab/Fc from the mice that received CV-A21 virus as immunogen. Further 301 processing was done as described in the previous paragraph. Samples featuring CV-A21 particles (free or as part of an immune complex) were diluted in TBS to 50 305 μg/ml and loaded onto in-house made carbon-coated 400-mesh copper grids (glow-discharged at 15 mA 306 for 25 s). The sample was blotted off after 10 s and the grids were stained with 2% (w/v) uranyl-formate 307 for 60 s. Grids were imaged on a Tecnai F20 electron microscope (FEI) equipped with a TemCam F416 308 CMOS camera (TVIPS). The microscope operates at 200 kV. The imaging defocus was set to 1.5 µm and 309 the total electron dose adjusted to 25 e -/Å 2 . The magnification was set to 62,000 X, with the resulting pixel 2018). CTF estimation was performed using GCTF (Zhang, 2016) . Particles were picked manually and 335 subjected to 1 round of 2D classification to remove bad picks. Selected particles were subjected to a round 336 of 3D refinement with icosahedral symmetry imposed. Low resolution map of the CV capsid was used as 337 an initial model for the initial 3D refinement/classification steps. A tight solvent mask around the CV capsid 338 was applied to remove the signal contributions from pAbs and internal viral components during refinement. Pre-refined particles were symmetry-expanded (icosahedral symmetry). 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Turner, Charles A. Bowman and Jean-Christophe 386 Ducom (The Scripps Research Institute) for their help with different aspects of EM experiments. The Competing Interests The authors have no special interests to declare.