IgV somatic mutation of human anti-SARS-CoV-2 monoclonal antibodies governs neutralization and breadth of reactivity

Abstract

Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we report a possibly novel approach to rapid generation of potent broadly neutralizing human anti-SARS-CoV-2 Abs. We isolated SARS-CoV-2 spike protein-specific memory B cells by panning from the blood of convalescent subjects after infection with SARS-CoV-2 and sequenced and expressed Ig genes from individual B cells as human mAbs. All of 43 human mAbs generated in this way neutralized SARS-CoV-2. Eighteen of the forty-three human mAbs exhibited half-maximal inhibitory concentrations (IC50) of 6.7 × 10-12 M to 6.7 × 10-15 M for spike-pseudotyped virus. Seven of the human mAbs also neutralized (with IC50 < 6.7 × 10-12 M) viruses pseudotyped with mutant spike proteins (including receptor-binding domain mutants and the S1 C-terminal D614G mutant). Neutralization of the Wuhan Hu-1 founder strain and of some variants decreased when coding sequences were reverted to germline, suggesting that potency of neutralization was acquired by somatic hypermutation and selection of B cells. These results indicate that infection with SARS-CoV-2 evokes high-affinity B cell responses, some products of which are broadly neutralizing and others highly strain specific. We also identify variants that would potentially resist immunity evoked by infection with the Wuhan Hu-1 founder strain or by vaccines developed with products of that strain, suggesting evolutionary courses that SARS-CoV-2 could take.

Keywords: COVID-19; Immunoglobulins; Immunology; Molecular biology.

Figure 1. Properties of spike-specific Ig sequences.

Immune repertoire profiling data were generated by single-cell sequencing using the 10× platform on B cells that were panned for binding to the SARS-CoV-2 spike protein (as detailed in Methods). Additional data on Ab heavy chain gene rearrangements were generated on bulk PBMC gDNA (see Methods). (A) Frequency of spike-specific single-cell sequenced clones separated by Ig isotype. Each color indicates a subject, and the height of each bar represents the frequency of spike-specific clones expressing the given isotype in that subject. (B) String plot of spike-binding clones that were also found in the bulk blood libraries. Each row represents a clone, each column represents a subject, and the intensity of each cell shows the copy number fraction of the associated spike-specific clone in the blood.

Figure 2. Comparison of the spike-specific VH repertoire and the VH repertoire in the blood.

IgH sequences were obtained from nonpanned blood B cells by NGS and from spike-panned B cells, as explained in the legend of Figure 1. (A) Number of clones using the top 50 most frequent VH genes in spike-specific clones and in all clones found in the blood. Each column indicates a VH gene, blue bars indicate the frequency of the given VH (by clone) in the blood, and the oranges bar indicate the frequency among spike-specific clones. (B) Fold change of VH gene frequencies in spike-enriched Ig clones as compared with VH gene frequencies in the blood. The height of each bar indicates the ratio of VH gene usage of spike-specific clones to that of other clones in the blood with the same VH. Red indicates a higher frequency in spike-binding clones and blue indicates lower frequency in spike-binding blood clones

Figure 3. Somatic mutation of spike-specific VH genes according to the subjects’ level of virus neutralizing Abs in plasma.

The frequency of mutated IgG (A) or IgA (B) VH sequences in relation to germline in subjects grouped by the level of virus neutralizing Abs. ID₅₀ ≤1:50, low neutralizing; 1:50 <ID₅₀ < 1:200, mid neutralizing; ID₅₀ >1:200, high neutralizing. Analysis was by 2-tailed Mann Whitney test function of Prism 9. Data represent mutations in the variable exons of all single-cell IgG or IgA spike-panned B cell clones. Mean ± SEM.

Figure 4. Neutralization of virus pseudotyped with SARS-CoV-2 Wuhan Hu-1 spikes by human mAbs.

Highly mutated mAbs were cloned and expressed on 293T/17 cells. The supernatants were collected, and the amount of IgG was measured by ELISA. Serially diluted Abs were incubated with viruses pseudotyped with the reference (Wuhan Hu-1) or mutant spikes, and infection of 293T-ACE2 cells was assessed by luminescence. The Ab concentration that inhibits 50% of infection (IC₅₀) was calculated for each sample. (A) The neutralization curves are depicted for 40 of 43 mAbs isolated to illustrate the breath of neutralization potency. Curves were obtained from 10 serial dilutions measured in duplicate. (B) The IC₅₀ for each of the isolated mAbs. Error bars represent mean ± SEM. (C) The neutralization curve of mAb 19 with SARS-CoV-2 live virus, Washington USA-WA1/2020 clinical isolate, and SARS-CoV-2 Wuhan Hu-1 spike-pseudotyped virus. Results were obtained from 8 serial dilutions. (D) The linear correlation of IC₅₀ and number of mutations in the VH and VL genes for the 11 most potent mAbs. Analysis was by the simple linear regression followed by Spearman’s r test functions of Prism 9. (E) Neutralization curves of viruses pseudotyped with SARS-CoV-2 Wuhan Hu-1 spike of mAbs 5, 13, 15, and 20 mutated and in germline configuration. Neutralization data curves were analyzed by the nonlinear fit function of Prism 9 and the absolute IC₅₀ was calculated when possible. Data reflect typical plots from at least 2 independent experiments.

Figure 5. Neutralization of virus pseudotyped with SARS-CoV-2 mutant spikes by spike-specific highly mutated human mAbs.

(A) Neutralization curves of viruses pseudotyped with SARS-CoV-2 variant spikes for mAbs 1, 2, 5, 12, 13, 15, and 20. (B) mAb 50% inhibitory concentration (IC₅₀) for SARS-CoV-2 variant spikes. Mean ± SEM. (C) The monoclonal IgG1 Ab IC₅₀ for SARS-CoV-2 spikes encoding the following mutations: H49Y, S247R, V367F, R408I, H519Q, A520S, and D614G. Data were analyzed by the nonlinear fit function of Prism 9, and the absolute IC₅₀ was calculated when possible. Data reflect a typical plot from 3 independent experiments. Colors reflect IC₅₀: lowest values are shown in red in a gradient to the highest values, shown in green.

Figure 6. Neutralization of virus pseudotyped with SARS-CoV-2 mutant spikes by mature mAb and in germline configuration.

The neutralization curves of viruses pseudotyped with SARS-CoV-2 H49Y, A520S and D614G spikes of mAbs 5, 13, 15, and 20 mutated and in germline configuration are shown. Data were analyzed by the nonlinear fit function of Prism 9, and the absolute IC₅₀s calculated when possible. Data reflect a typical plot from 3 independent experiments.