Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell–cell fusion

Figures

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  Fig. 1.. The micro-fusion inhibition test (mFIT). Samples containing antibodies are incubated with effector cells (HEK293T Lenti rLuc-GFP 1–7) expressing the viral glycoprotein (vGP) of interest. The antibody–effector cell mix is then co-cultured with target cells (HEK293T Lenti rLuc-GFP 8–11) expressing the corresponding vGP’s cellular receptor and incubated for 18–24 h. In (a) the presence of fusion-inhibitory neutralizing antibodies (nAbs) prevents the reconstitution of the rLuc-GFP reporter in fused cells, while in (b) the absence of specific neutralizing antibodies (non-nAbs), allows vGP-mediated cell–cell fusion to occur. Subsequent mixing of the target and effector cell cytoplasm leads to reconstitution of the split reporter and increased GFP and luciferase signals. This figure was generated using modified images from SMART Servier Medical Art By Servier, used under CC BY 3.0, https://smart.servier.com/, accessed June 2020.

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  Fig. 2.. Examining the neutralization of cell–cell fusion by monoclonal antibodies in human RSV mFITs. (a) Molecular surface representation of RSV F trimer in the pre-fusion (left; PDB 4MMV) and post-fusion (right; PDB 3RRR) forms with antigenic sites coloured as follows: site ø, pink; site I, orange; site II, purple; site III, turquoise; site IV, red; site V, blue. RSV F-specific mAbs are annotated and coloured according to the corresponding antigenic binding site. Ψ represents human mAbs, while γ represents murine mAbs. Molecular graphics and analyses were performed in UCSF’s ChimeraX program. (b) Murine mAbs (1 : 160 working dilution) and (c) human mAbs (5, 2.5 and 1.25 µg ml−1) were tested in hRSV-F mFITs. mAb 19, positive control; mAb16, specific negative control; MeVH, non-specific negative control (Table S1). Data are expressed as a percentage of the average luciferase readings seen in no-sera/negative controls with 50 or 90 % inhibition (IC50 and IC90) lines indicated. Error bars represent mean±sd.

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  Fig. 3.. Using NiV mFITs to characterize neutralization of cell–cell fusion by monoclonal antibodies and sera from NiV vaccinated pigs. (a) Molecular surface representation of NiV-F pre-fusion trimer (left, PDB 5EVM) and NiV G dimer (right, PDB 2VWD). The stalk domain of G (residues 62–117) is modelled after parainfluenza virus 5 stalk (PDB 4JF7). The 5B3 epitope on Nipah F is coloured red, and the m102.4 epitope on Nipah G is coloured blue. Molecular graphics and analyses performed with UCSF ChimeraX. Antibodies against NiV-F- and NiV-G-specific mAbs were tested in a NiV-FG (b) mFIT (20, 2 and 0.2 µg ml−1) and (c) mVNT using NiV viral pseudotypes (10, 1 and 0.1 µg ml−1). A negative control, RSV F mAb (101F), was also included. (d) Sera from individual representative NiV mcsF- or NiV sG-vaccinated pigs were tested longitudinally in a NiV-FG mFIT (1 : 5 working dilution) and by (e) mVNT using NiV viral pseudotypes. (f) An xy scatter plot illustrating the correlation between mFIT results (% reduction) and mVNTs (IC90) from the immunogenicity study performed in pigs (Table S2). A linear line of regression is shown together with the calculated R 2 value. Data are expressed as a percentage of…

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  Fig. 4.. Examining neutralization of fusion by monoclonal antibodies and convalescent patient plasma in SARS-CoV-2 mFITs. (a) Molecular surface representation of SARS-CoV-2 and SARS-CoV spike trimers, with the S230 binding epitope highlighted in orange, and an RBD alignment between the two spikes shown. Molecular graphics and analyses performed with UCSF ChimeraX. A soluble ACE2-Fc and a mAb targeting SARS-CoV, S230, were tested in (b) SARS-CoV-2 and SARS-CoV mFITs (100, 50, 25 and 12.5 µg ml−1) and (c) mVNT using SARS-CoV-2 and SARS-CoV viral pseudotypes (50, 25, 12.5 and 6.25 µg ml−1). Convalescent human plasma from COVID-19 recovered patients and a negative plasma pool from healthy donors were tested in a (d) SARS-CoV-2 mFIT (1 : 20 final dilution) and by (e) mVNT using SARS-CoV-2 viral pseudotypes. (f) An xy scatter plot illustrating the correlation between IC50 results from (d) and (e). A linear line of regression is shown together with the calculated R 2 value and the Pearson’s correlation factor, R, all calculated from the tabulated data (under). Data are expressed as a percentage of the average luciferase readings seen in no-sera/negative controls with 50 or 90 % inhibition (IC50…