High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions

Abstract

Non-polio enteroviruses (NPEVs) cause serious illnesses in young children and neonates including aseptic meningitis, encephalitis, and inflammatory muscle disease, among others. While over 100 serotypes have been described to date, vaccine only exists for EV-A71. 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 introduced a cryo-electron-microscopy-based approach for structural analysis of virus- or vaccine-elicited polyclonal antibodies (pAbs) in complex with whole NPEV virions. We demonstrated the feasibility using coxsackievirus A21 and reconstructed five structurally distinct pAbs bound to the virus. The pAbs targeted two immunodominant epitopes, one overlapping with the receptor binding site. These results demonstrate that our method can be applied to map broad-spectrum polyclonal immune responses against intact virions and define potentially cross-reactive epitopes.

Influence of Amino Acid Substitutions in Capsid Proteins of Coxsackievirus B5 on Free Chlorine and Thermal Inactivation

“…This step was necessary to ensure that the virus sample can be handled and imaged under biosafety level 1 conditions. The samples were washed with PBS using an Amicon Ultra centrifugal unit (MWCO: 100 kDa, Merck Millipore) and subjected to size-exclusion chromatography to further purify the viral fraction as described elsewhere . Size-exclusion chromatography was performed using a HiPrep 16/60 Sephacryl S-500 HR column (Cytiva) running in tris-based buffer (25 mM tris-HCl, 150 mM NaCl, pH 7.5).…”

“…Grids were prepared as described before . Briefly, 3 μL of the purified CVB5F.cas.genogroupB at 4.5 mg mL –1 concentration was loaded onto Quantifoil R 1.2/1.3 grids (EMS), which were previously glow-discharged for 30 s in a GloCube Plus device (Quorum Technologies).…”

Influence of amino acid substitutions in capsid proteins of coxsackievirus B5 on free chlorine and thermal inactivation

“…The supernatant was collected and amended with paraformaldehyde at 100 μg mL -1 and incubated at 4°C for five days to fix the samples. The fixed samples were washed with PBS using an Amicon Ultra centrifugal unit (MWCO: 100 kDa, Merck Millipore) and subjected to size exclusion chromatography to further purify the viral fraction as described elsewhere 59 . Size-exclusion chromatography was performed using a HiPrep 16/60 Sephacryl S-500 HR column (Cytiva) running in tris-based buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5).…”

“…Grids were prepared as described before 59 . Briefly, 3 µL of the purified CVB5 mutant virus sample at 4.5 mg mL -1 concentration was loaded onto Quantifoil R 1.2/1.3 grids (EMS), that were previously glow-discharged for 30 s in a GloCube Plus device (Quorum Technologies).…”

“…Grids were prepared as described before 59 . Briefly, 3 µL of the purified CVB5 mutant virus sample at 4.5 mg mL -1 concentration was loaded onto Quantifoil R 1.…”