Tocilizumab-treated convalescent COVID-19 patients retain the cross-neutralization potential against SARS-CoV-2 variants

Abstract

Although tocilizumab treatment in severe and critical coronavirus disease 2019 (COVID-19) patients has proven its efficacy at the clinical level, there is little evidence supporting the effect of short-term use of interleukin-6 receptor blocking therapy on the B cell sub-populations and the cross-neutralization of SARS-CoV-2 variants in convalescent COVID-19 patients. We performed immunological profiling of 69 tocilizumab-treated and non-treated convalescent COVID-19 patients in total. We observed that SARS-CoV-2-specific IgG1 titers depended on disease severity but not on tocilizumab treatment. The plasma of both treated and non-treated patients infected with the ancestral variant exhibit strong neutralizing activity against the ancestral virus and the Alpha, Beta, and Delta variants of SARS-CoV-2, whereas the Gamma and Omicron viruses were less sensitive to seroneutralization. Overall, we observed that, despite the clinical benefits of short-term tocilizumab therapy in modifying the cytokine storm associated with COVID-19 infections, there were no modifications in the robustness of B cell and IgG responses to Spike antigens.

Keywords: Health sciences; Immunology; Virology; treatment.

Graphical abstract

Figure 1

Impact of tocilizumab treatment on anti-S total IgG and IgG1 titers (A–D) (A) Anti-S total IgG and IgG1 were measured by ELISA in the plasma of non-treated and tocilizumab-treated COVID-19 patients recruited until October 2020. The titers of mild/moderate (n = 16), severe (n = 12), and critical (n = 13) untreated patients, and of severe (n = 8) and critical (n = 9) tocilizumab-treated patients sampled 3 and 6 months after symptoms, were analyzed. The area under the curve (AUC) is represented according to the day post-symptoms onset. See also Figure S1. (B) IgG1 titers were measured as previously. (C) Anti-S total IgG was analyzed 1M–3M after symptoms. The AUC is represented according to disease severity. (D) IgG1 titers were measured as previously. Data are represented as mean ± SEM. The data were analyzed by an unpaired nonparametric Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01.

Figure 2

Sensitivity of SARS-CoV-2 variants to neutralization by plasma from convalescent patients with different disease severity and treatment status The neutralizing activities of the plasma from COVIMUNE cohort of convalescent individuals from the first wave of COVID-19 (infected with the wild-type strain) against the indicated viral isolates are expressed as PRNT50 titers. (A–C) (A) Neutralizing activity of plasma from non-treated individuals with mild/moderate (n = 16), severe (n = 12), or critical (n = 13) COVID-19 disease. (B) Neutralizing activity of plasma from tocilizumab-treated individuals with severe (left panel, n = 8) and critical (right panel, n = 9) disease 3 months after symptoms. (C) Neutralizing activity of plasma from tocilizumab-treated individuals with severe (left panel, n = 8) and critical (right panel, n = 9) disease 6 months after symptoms. Data are representative of two independent experiments. Two-sided Friedman test with Dunn’s multiple comparison was performed between each viral strain. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. LOD, limit of detection. The threshold of detection was at a value of 40. The sera that did not neutralize at the dilution of 1/40 were set under the limit of detection.

Figure 3

Tocilizumab does not affect the B cell phenotype of COVID-19 patients B cell characterization of peripheral blood mononuclear cell of healthy donors (HD) (n = 4) and mild/moderate (n = 9), non-treated severe/critical (n = 9), and tocilizumab-treated severe/critical patients (n = 6) was analyzed by flow cytometry. Patients recruited from May 2020 to April 2021 were included. Representative patient samples of the different groups are displayed. (A) Gating strategy of the B cell characterization of the convalescent patients. (B) Unswitched memory IgD⁺ (USM; CD27⁺IgD⁺), switched memory IgD⁻(SWM; CD27⁺IgD⁻), naive (CD27⁻IgD⁺), and double-negative (DN) (CD27⁻IgD^-) B cells. (C) The ASCs/plasmablasts were identified as CD27⁺CD38^hi cells. (D–H) The following subset frequencies were analyzed: (D) ASCs, (E) naive B cells, (F) double-negative B cells (DN), (G) memory B cells (USM, unswitched memory), and (H) memory B cells (SWM, switched memory). The data were analyzed by a multi-comparison Kruskal-Wallis test followed by a Dunn’s post-hoc test. ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SEM.