LIMITED INDUCTION OF LUNG-RESIDENT MEMORY T CELLS BY SARS-CoV-2 mRNA VACCINATION

Background: Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients after mild and severe infection, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Method(s): We obtained cross-sectional paired blood and lung biopsy samples from patients (n=30) undergoing lung resection for various reasons and assigned them to one of four groups: I.) uninfected unvaccinated individuals (n=5), II.) unvaccinated long-term SARS-CoV-2 convalescent individuals (between 6.0-10.5 months post-infection;n=9), III.) uninfected and long-term vaccinated individuals (between 6.0-7.7 months after the second or third dose;n=10), and IV.) uninfected and short-term vaccinated individuals (between 1.3-1.8 months after the third or fourth dose;n=6). We determined the presence of SARS-CoV-2-specific CD4+ and CD8+ T cells in blood and lung samples after exposure of cells to M, N, and S peptide pools, followed by flow cytometry to detect TRM cells expressing interferon (IFN)gamma and/or CD107a, as a degranulation marker. Result(s): We found that the frequency of CD4+ T cells secreting IFNgamma in response to S-peptides was variable but detectable in blood and lung up to 8 months after mRNA vaccination. Moreover, the IFNgamma response of CD4+ T cells in the lung of mRNA-vaccinated patients was similar to the response found in convalescent patients. However, in mRNA-vaccinated patients, lung responses presented less frequently with a TRM phenotype compared to convalescent infected individuals and, strikingly, polyfunctional CD107a+ IFNgamma+ TRM were virtually absent in vaccinated patients. Conclusion(s): mRNA vaccines might induce memory responses within the lung parenchyma in some patients, potentially contributing to the overall disease control. However, the robust and broad TRM response established in convalescent-infected individuals may offer advantages at limiting disease if the virus is not blocked by initial mechanisms of protection, such as neutralization. Our results warrant investigation of mucosal vaccine-induced resident T cell responses in establishing superior site-specific protective immunity.

Function, homing, and residency of T-cell immune responses against SARS-CoV-2

Background: In order to inform vaccine development on the correlates of protection against SARS-CoV-2, we performed detailed phenotypic and functional analyses in clinically-defined groups of patients recruited during the first wave of SARS-CoV-2 infection, including the assessment of Resident Memory T cells (TRM) in lung of convalescent patients. Methods: Blood samples from 46 participants diagnosed with acute COVID-19 (14 symptomatic non-hospitalized;20 mild-hospitalized and 12 severehospitalized) were obtained 7-16 days after symptoms onset. Lung biopsies were obtained from three convalescent patients 1 to 7.5 months after initial infection. The phenotype and functional capabilities of SARS-CoV-2-specific CD4+ and CD8+T cells were measured by FACS after stimulation with a pool of overlapping SARS-CoV-2 viral peptides (M, N and S). Results: Pattern variations associated with viral-specific T cell responses where based on two factors, the targeted viral protein and the cohort of patients assessed. Overall, stimulation with M and N viral peptides induced a Th1 profile exemplified by IFNg production in CD4+T cells and degranulation in CD8+T cells respectively, whereas S peptides induced a Th2 profile exemplified by IL-4. Hospitalized patients showed increased IFNg secretion in CD4+T cells in response to any viral protein compared to non-hospitalized patients (p=0.020 for M and S peptides in the mild group;p=0.004 for M, p=0.011 for N and p=0.007 for S peptides in the severe group;Figure 1) and IL-4 secretion in CD8+T cells in response to S peptides (p=0.004 and p=0.003 for mild and severe patients, respectively). In contrast, the expression of IL-10, which was mostly expressed in CCR7+ cells, was significantly increased in CD4+T cells from non-hospitalized patients after stimulation with M peptides when compared to the mild COVID-19 group (p=0.035). Importantly, SARS-CoV-2 specific T cell responses with a biased TRM profile were detected up to 7.5 months after infection in the lung of convalescent patients. However, tissue responses strongly differed from blood. Conclusion: Our results suggest that a balanced anti-inflammatory antiviral response promoted by non-spike proteins may be key to favor infection resolution without major complications. Further, while immune responses migrate and establish in the lung as resident memory T cells, the magnitude and profile of the lung SARS-Cov-2 specific T cells strongly differ from the response detected in blood.

Identification of repurposing drugs against SARS-CoV-2 using human lung tissues

Background: No effective drugs against SARS-CoV-2 infection are available. Screening of therapeutic candidates is primarily performed using immortalized cell lines. However, primary cell targets might show intrinsic differences in the expression profile of relevant host proteins, required for viral replication that could significantly affect the activity and potency of antivirals. Thus, the development of more physiological models for antiviral drug screening are urgently needed. Methods: Lung tissue was obtained from routinely thoracic surgical resections and was immediately digested before experiment set up. Cell populations and expression of ACE2 were characterized by FACS, and cell targets for SARS-CoV-2 were identified using a VSV∗ΔG(GFP)-S pseudotyped virus. 39 repurposing drugs previously identified by in silico models as potential viral entry inhibitors were tested using a VSV∗ΔG(Luc)-S virus. Cytotoxic concentration (CC50) and inhibitory concentration (IC5O) values were calculated using a non-linear regression dose-response curve and were compared to drug activity in VeroE6 cells. Results: Alveolar type II (AT-II) cells, the main cell target for SARS-CoV-2 infection in lungs, were identified within a fraction of cells characterized by CD45-, CD31-, EpCAM+ and HLA-DR+, (∼0.01-0.5% of viable cells). Using a VSV∗ΔG(GFP)-S virus we showed that viral entry was occurring in cells compatible with an AT-II phenotype, and infection was efficiently blocked with an anti-ACE2 antibody (Figure 1). Despite low and variable numbers of AT-II targets, antiviral assays using VSV∗ΔG(Luc)-S were highly sensitive and reproducible (CV of 17%). Compared with VeroE6 cells, IC50 values trended to be higher in tissues. Moreover, we found that 12.8% of the tested compounds had discordant results, where 10.25% of the drugs showed some antiviral effect in lung cell suspensions but no activity in VeroE6 and 3.9% showed only antiviral effect in VeroE6. Modulation of ACE2 expression by some of these compounds was also highly discordant between the cell line and lung tissue. Cepharantine (IC50=6μM, CC50=14μM) and Ergoloid (IC50=4.3μM, CC50=24μM) were identified as the most active entry inhibitors in lung cell suspension. Conclusion: The use of lung tissue for the screening of antiviral compounds represents a valid physiological and relevant model, which evidences intrinsic discrepancies with cell lines. Importantly, we identified repurposing drugs against SARS-CoV-2 with potential for clinical testing.