Deficient neutralizing antibody response and specific lack of RBD-responsive B cells in elderly long-term convalescent patients from severe COVID-19

Abstract

[Background] Severe COVID-19 is defined by admission to intensive care units with respiratory support. This condition increases with age. To investigate in the possible mechanisms by which severe COVID-19 occurs, we compared the response of specific antibodies against the viral antigens RBD, Spike (S), nuclear (N), and membrane (Mpro) and the neutralizing titers in plasma of elderly patients convalescent from severe COVID-19 with convalescent patients from mild disease.

[Methods] Plasma was collected from cohort 1: 20 healthy donors (vaccinated and unvaccinated), cohort 2: 40 elderly long-term convalescent patients from severe COVID-19 (mean: 73 years old, 60/40 ratio men/women, and 10 months after infection), and cohort 3: 60 convalescent from mild COVID-19 patients, who were vaccinated (mean: 42 years old, 30/70 ration men/women, 8 months after mild infection, and 5.7 months after last vaccination shoot). To compare reactivity against native S protein, we used a sensitive method to measure specific IgG1 and IgA in plasma by flow cytometry. We assessed the neutralization titers in plasma by infection assays in HEK-293T cells or Vero cells expression ACE-2, using S- and GFP-expressing pseudotyped viruses, and quantified IgG, IgA, and IgM antibodies against RBD, S, N, and mPro antigens using the Multiplex Serological SARS-CoV-2 assay (Immunostep). To evaluate the presence of specific T and B cells specific for S and RBD antigens, we used kits from Miltenyi Biotech and analyzed the expression of activation antigens after cell stimulation with these antigens by flow cytometry.

[Results] Significant levels of IgG and IgA against S protein were found in the plasma of convalescent patients from cohorts 2 and 3, with no differences in total anti-S antibody response between the two cohorts. Interestingly, anti-RBD IgG levels were found to be extremely low in plasma samples from cohort 2, but not in plasma samples from cohort 3. In accordance with these results, the neutralizing titers (IC50) found were very low in the plasma samples from cohort 2, compared to those from cohort 3. Analysis of the presence of RBD- and S-specific B cells present in peripheral blood mononuclear cells (PBMCs) of these cohorts revealed significantly lower levels of RBD-specific B cells, but of not S-specific B lymphocytes, in the PBMCs from cohort 2 cells but not in those from cohort 3. Furthermore, lower B cell activation, as demonstrated by CD25 expression, was observed in PBMCs from cohort 2 compared to those from cohort 3 after their stimulation with RBD, but not with S, N or Mpro proteins. These results together indicate the specific lack of RBD-specific B cells in cohort 2 patients.

[Conclusions] The low neutralizing capacity observed in the plasma of elderly long-term convalescent patients, who recovered from severe COVID-19 (cohort 2) correlates with low specific levels of anti-RBD antibodies and reduced levels of RBD-responsive B cells. These results could help explain the severity of COVID-19 in patients from cohort 2 compared to those from cohort 3, who had a mild disease. Future experiments will evaluate the presence of neutralizing antibodies in cohort 2 patients after vaccination.