Fig. 3: A complement-mediated mechanism of enhancement for SARS-CoV-2 neutralisation titres.

Use of transmission electron microscopy (TEM), compstatin, and ACE2 inhibition assays to determine the mechanism of complement-enhanced neutralisation. a TEM was used to identify possible viral aggregation and/or lysis following incubation with immune sera (using representative data from OPTIC sample 8) and pooled human plasma (PHP) or heat-inactivated (HI)-PHP. Each biological sample was tested in duplicate with a total of 136 images captured across three magnifications. No clear difference was observed between the conditions tested. The black arrows indicate examples of the SARS-CoV-2 (VIC01) particles. b Microneutralisation assays with compstatin or a control peptide showed the effects of C3 inhibition. Each spot represents the mean value of 6 replicates across duplicate assays and error bars show the standard error. The addition of PHP with either compstatin or the control peptide significantly increased SARS-CoV-2 neutralisation. A further increase in neutralisation was observed with the use of the control peptide, which was significant in 2/3 samples using a two-way ANOVA with Tukey’s multiple comparisons test. Exact p values for samples 2 (media vs. control, p = <0.0001; media vs. compstatin, p = 0.0015; compstatin vs. control, p = 0.0032), 8 (media vs. control, p = <0.0001; media vs. compstatin, p = <0.0001; compstatin vs. control, p = 0.2562), and 10 (media vs. control, p = <0.0001; media vs. compstatin, p = <0.0001; compstatin vs. control, p = 0.0012) c Human ACE2 competition assays were supplemented with either PHP or HI-PHP to measure the effect of complement on ACE2 binding to various SARS-CoV-2 spike proteins. The presence of complement significantly enhanced ACE2 inhibition for all antigens tested. Each spot represents duplicate values of 4 OPTIC serum samples and the error bars show the standard error. Sample dilutions of either 1:10 or 1:100 are shown dependent on whether the observations were within the limits of detection. Significance was determined using paired, two-sided T-tests for each antigen. Exact p values are 0.0004 (BA.2.12.1), <0.0001 (BA.2.75), 0.0001 (BA.2-1…), 0.0041 (B.1.1.529), 0.0256 (Wuhan), 0.0302 (B.1.617.2;AY.4), 0.0035 (B.1.1.7), 0.0085 (B.1.351), 0.0334 (BA.5). The results were analysed and presented using GraphPad Prism (Version 10). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Antigen “BA.2-1…” includes: BA.2; BA.2.1; BA.2.2; BA.2.3; BA.2.5; BA.2.6; BA.2.7; BA.2.8; BA.2.10; BA.2.12. Antigen “B.1.1.529” includes: B.1.1.529; BA.1; BA.1.15. Source data are provided as a Source Data file.