SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition

Background Following SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic. Methods The study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA. Results The frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups. Conclusions The antibody responses to the spike protein, but not the anti-RBD B cell responses diverge between convalescent males and females who develop PCC. Our findings also suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens.

Supplementary Figure S2.Gating strategy to quantify RBD-specific B cells.

Supplementary Figure S3: D614G-RBD specific B cell frequencies in convalescent PCC and No-PCC groups at 3 months post-infection.
Data presented in Fig. 2 were segregated by sex and compared.No significant differences were observed between females and males in the frequencies of D614G RBD reactive B cell subsets as evaluated by Mann Whitney's test.Anti-omicron IgG responses in uninfected (U), No-PCC and PCC groups were determined by ELISA.
The groups were compared by Mann Whitney's test.(https://rdocumentation.org/packages/ggbiplot/versions/0.55).Immune parameters for which more than 50% of the samples had values at both 1-and 3-month timepoints were utilized.These parameters included sex, number of symptoms at the acute infection timepoint, BMI, anti-D614G RBD specific B cell subsets (CD19 + , CD19 + CD20 + , IgD + , IgD -, naïve, DN, SM and USM), anti-D614G RBD IgG, antispike IgG, anti-spike IgG3, anti-spike IgA, anti-nucleocapsid IgG and anti-nucleocapsid IgA.Excluded from the data were data points with no values as well as those obtained after vaccination.PC1 and PC2 were plotted and a normal data ellipse for each group was generated.

Supplementary
. Anti-omicron RBD specific IgG responses in uninfected and convalescent individuals with or without PCC.Plasma samples were collected during routine clinical visit at 3 months post PCR-positive diagnosis.
responses in PCC and No-PCC groups among males and females.Correlation matrices were generated for the indicated parameters for the convalescent groups with or without PCC at 3 months post-infection.Nonparametric Spearman's correlation coefficient values (numbers) and their significance (asterisks) are indicated.* p<0.05, ** p<0.01, *** p<0.001.Asterisks are color coded to as visual aids to denote specific comparisons described in the text.The white and black numbers and asterisks are only used to contrast with the background color.The number of samples and exact p values are shown in the Supplementary Table S5.Supplementary Figure S6.PCA analyses of clinical parameters, B cell reactivities and antibody responses to SARS-CoV-2 antigens in PCC and No-PCC groups at 1 and 3 months post-infection.23.5% explained var .)standardized PC2(14.7%explained var.) Figure S7.Correlation matrix for anti-RBD, spike and nucleocapsid antibody responses in PCC and No-PCC groups at 1-month post-infection.Correlation matrix for antibody responses at 1-month post infection in (a) females No-PCC, (b) males No-PCC, (c) females PCC and (d) males PCC groups.RBD refers to D614G-RBD.The numbers in the squares indicate the Spearman coefficient value.The p values are indicated in the figure as asterisks (* , ** p<0.01, *** p<0.001) and are given in Supplementary Table S6.Due to the limited quantity of samples in which B cell reactivities were measured, these parameters were not included in the correlation matrix and are given in Supplementary Fig. S8.Supplementary Figure S8.Correlation matrix for B cell responses at 1-month post infection in females and males with and without PCC.The numbers in the squares indicate the Spearman coefficient value.The p values are indicated in the figure as asterisks ( * p<0.05, ** p<0.01, *** p<0.001) and are given in Supplementary Table S6.For simplicity they are indicated in the figure with asterisks.Supplementary Fig. 10.Correlation matrix for antibody responses between 1-and 3-months and 3-and 6-months post infection in females and males with and without PCC.RBD refers to RBD-numbers in the squares indicate the Spearman coefficient value.The p values are indicated in the figure as asterisks ( * p<0.05, ** p<0.01, *** p<0.001) and are given in Supplementary