Streptococcus pneumoniae colonization associates with impaired adaptive immune responses against SARS-CoV-2

Background Although recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, cases of coinfection with Streptococcus pneumoniae in patients with coronavirus disease 2019 (COVID-19) during hospitalization have been reported infrequently. This apparent contradiction may be explained by interactions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pneumococci in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses. Methods Here, we investigated the relationship of these 2 respiratory pathogens in 2 distinct cohorts of health care workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and patients with moderate to severe disease who presented to the hospital. We assessed the effect of coinfection on host antibody, cellular, and inflammatory responses to the virus. Results In both cohorts, pneumococcal colonization was associated with diminished antiviral immune responses, which primarily affected mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients. Conclusion Our findings suggest that S. pneumoniae impair host immunity to SARS-CoV-2 and raise the question of whether pneumococcal carriage also enables immune escape of other respiratory viruses and facilitates reinfection. Trial registration ISRCTN89159899 (FASTER study) and ClinicalTrials.gov NCT03502291 (LAIV study).

Supplemental figure 4. Gating strategy for B cell subset analysis using a representative PBMC sample. Identification of the B cells based on the following steps from left to right: 1) Time dot-plot to determine the quality of stability of the acquisition 2) Adequate adjustment of the gate of lymphocytes 3) Exclusion of doublets with the identification of singlets improving the accuracy of the analysis 4) Selection of the viable lymphocytes 5) Identification of the B cells based on CD19+ expression. Analysis within B cells (CD19+) subset. Subsequently, we identified plasma blasts (CD27+CD38++) and non-plasma cells (CD38-) based on the expression of CD27 (memory cell marker) vs. CD38 (plasma cell marker). From non-plasma cells we identified the naïve, memory B cells (mBC) and the double negative population based on the expression of IgD vs. CD27. Finally, to delineate the SARS-CoV-2 specific B cells against S1 and S2, we analysed within the B cells (CD19+) and memory B cells (mBC) the percentage of expression S1 and S2 proteins conjugated with biotin and labelled with Streptavidin (BV785 and PE, respectively). Abbreviations: FMO (Fluorescence Minus One).

Supplemental figure 5. Gating strategy for T cell subset analysis (CD4+ and CD8+) by flow cytometry using a representative PBMC sample. A)
Identification of the T cellular subsets CD4 and CD8 based on the following steps: 1) Time dot -plot to determine the quality of stability of the acquisition 2) Adequate adjustment of the gate of lymphocytes 3) Exclusion of doublets with the identification of singlets improving the accuracy of the analysis 4) Selection of the viable lymphocytes 5) Identification of the T cells based on CD3+ expression 6) Identification of the TCD4+ and TCD8+ within CD3+ cells. B) Analysis within TCD4+ cells subset. Representative dot-pots of cytokine production (IFN-, TNF- and IL2) following SARS-CoV-2 Spike (S) protein stimulation (2µg/mL) and SEB (Staphylococcus Enterotoxin B) as positive control compared to mock (unstimulated) within CD4+ cells. TCD4+ cells were also stimulated SARS-CoV-2 with S1 subunit (S1) and Nucleocapsid (N) (not shown) (C) Analysis within TCD8+ cells subset. Representative dot-pots of cytokine production (IFN-, TNF- and IL2) following SARS-CoV-2 Spike protein stimulation (2µg/mL) and SEB (Staphylococcus Enterotoxin B) as positive control compared to mock (unstimulated) within CD8+ cells. CD8+ cells were also stimulated SARS-CoV-2 with S1 subunit (S1) and Nucleocapsid (N) (not shown).
Supplemental Table 1

Supplemental table 4. Summary and specifications of the multiparametric flow cytometry panel used for T cell analysis by flow cytometry.
We developed a multiparametric flow cytometry panel composed with extracellular (CD3, CD4 and CD8) and intracellular (cytokines) monoclonal antibodies (from Biolegend and BD Biosciences) including a viability dye (Thermofisher) to assess SARS-CoV-2 specific T cells in HCW and patients with SARS-CoV-2 infection by flow cytometry. Electronic compensation was set using CompBeads (BD Biosciences), Arc beads (ThermoFisher) and using the Spectral Flow automated unmixing software (Aurora, Cytek Biosciences) according to manufacturer's instructions. Abbreviations (NA, Not applicable).

Upper respiratory tract and blood sample collection
HCWs were asked to provide a self-collected flocked combined nasal and throat (NT) swab (Amies, MWE, UK) twice per week. The NT sample were collected by swabbing their throat (tonsil area) first and then a separate swab was used for the nose. Both swabs were combined in the same tube.In

Serum isolation and handling
Whole blood was collected in serum separator tubes (SST BD Vacutainer tubes, USA) and centrifuged for 10min at 1200g for serum separation in a biosafety level 3 facility. The serum was then aliquoted and stored at -80 o C until further use. Prior to use in assays, serum samples were treated with 1% Triton-X-100 for 1h in room temperature(1).
Respiratory samples handling NP swabs collected from patients were transferred to LSTM diagnostic laboratory and processed immediately. Self-collected NP swabs from HCWs were stored at -80 o C till further use. Throat swabs in STGG, saliva raw and saliva in STGG, SAM filters were stored at -80 o C till further use. Where appropriate, respiratory secretion samples were treated with 1% Triton-X-100 for 1h at room temperature prior to use in SARS-CoV-2 ELISAs or cytokine beads assay.