IMPAIRED CYTOTOXIC IMMUNE RESPONSE and EBV REACTIVATION in PATIENTS with LONG COVID19

Background: About 10% of individuals with mild infection with SARS-CoV-2 suffer from Long COVID-19, defined as signs and symptoms developed during or following COVID-19 that continue for more than twelve weeks and cannot be explained by an alternative diagnosis. In this study, we analyzed the ADCC response and the reactivation of CMV and EBV in Long COVID-19 syndrome, in comparison with patients who completely recovered from mild COVID-19 Methods: 30 patients with Long COVID-19 (Long COVID-19) and 20 individuals who suffered mild COVID-19 and were completely recovered (Recovered) were recruited for this study. Specific anti-SARS-CoV-2 IgG titers were analyzed by direct ELISA and their neutralizing capability was measured by using pseudovirus neutralization assay. Phenotype of CD4+ and CD8+ T cells, NK, NKT, and B cells in peripheral blood was analyzed by flow cytometry. ADCC activity was analyzed using rituximab-coated Raji cells as target. EBV and CMV reactivation in plasma was analyzed by qPCR. Results: 1) 86.6% and 55.50% participants were female in Long COVID-19 and Recovered cohorts, respectively. Median age at COVID-19 diagnosis was 42y(IQR 37-46) and 45y (IQR 28-57), respectively. 2) Similar levels of CD4+ T cells were observed in both groups. However, Tregs were increased 2.8-fold in Long COVID-19 participants (p=0.0007). 3) CD8+ T cells, CD8+TCRγδ and CD8+TCRγδ were increased 1.3-(p=0.0005), 2.0-(p=0.049), and 2.5-fold (p=0.005) in Long COVID-19 individuals. 4) Expression of CD56 in NK cells and CD3-CD56+CD16+ cells were increased 1.7-(p=0.0005) and 1.7-fold (p=0.032) in Long COVID-19, respectively. 5) Specific anti-SARS-CoV-2 IgG titers were increased 2.3-fold in Long COVID-19 individuals (p=0.02) and their neutralizing capacity was increased 4.2-fold (p=0.034) in this cohort. However, ADCC activity was decreased 1.4-fold (p=0.0044). 6) Resting memory B cells were increased 2.3-fold during Long COVID-19, whereas plasmablasts were reduced 3.1-fold. 7) EBV was reactivated in 33.3% of Long COVID-19 individuals (p<0.0001), whereas CMV was not reactivated in any individual. Conclusion: Despite high levels of neutralizing antibodies and cytotoxic immune populations, an impaired antibody-dependent cytotoxic activity was observed in PBMCs from individuals with Long COVID-19. This defective cytotoxic immune response may impede viral clearance, which may also contribute to EBV reactivation observed in these individuals, thereby influencing on the persistent COVID-19 symptoms.

Experience of a Private Hospital in the Treatment of COVID-19 Pneumonia in Veracruz, Mexico

Background. Large mortality rates have been reported in the Mexican public health system, however in the experiences of private hospitals that have resources and infrastructure this is lower compared to the national average. Methods. Descriptive and retrospective study. Adult patients treated for pneumonia due COVID-19 from April to December 2020 are entered into the study. Its general characteristics such as gender and age, comorbidities, influenza vaccination history, clinical characterization, laboratory and tomographic diagnosis of sars cov2 pneumonia are studied, as well as the drug and oxygen therapy treatments received and finally, its evolution and clinical outcome. Results. 132 patients were studied, of which 51% were female. The main age groups affected were 65 and over (43.9%), 50-59 years (20.4%) and 25-44 years (16.6%). The main comorbidities found were: arterial hypertension (43.9%), Diabetes mellitus 2 (33.3%), heart disease (11.3%) and obesity (10.6%). 95.4% of the patients were not vaccinated against influenza. The main symptoms reported were: fever (92%), cough (87%), dyspnea (76%) and headache (52%). The diagnosis was confirmed with RT-PCR in 63%, reporting negative RT-PCR in 36%;the antigen test was positive in 1%. Regarding the findings of the chest computed tomography, CORADS 5 was reported in 30%, CORADS 6 in 3% and CORADS 4 in 20%. The main treatments used in patients with severe inflammatory pneumonia were: steroids (98%), enoxaparin (100%), tocilizumab (20%), baricitinib (60%), direct oral anticoagulants (10%), fibroquel (5%). 60% were treated with a combination of two or more drugs. The main oxygenation contributions were: 20% nasal tips - mask/reservoir, 60% high flow nasal cannula, 20% mechanical ventilation. In 95% the prone position was indicated. Regarding the clinical evolution, 65.1% were towards improvement, 17.4% died, 12.1% requested transfer to another unit and 5.3% requested voluntary discharge. Overall mortality was 17%. Conclusion. A hospital strategy that has the necessary resources and infrastructure as well as openness to the use of medication with emergency approvals for its use or off-label indications, can help limit morbidity and mortality in vulnerable populations and manifest risk factors such as Mexican population.

Characterization of the Humoral and Cellular Immune Response Against the Natural Infection By Sars-Cov-2 in Oncohematological Patients with Post-COVID19 Autologous Transplantation

Background: Oncohematological patients may have a lower immune response against SARS-CoV-2, both to natural infection and to vaccines. Most studies have focused on the analysis of the humoral response, which means that the information available on the cellular response against SARS-CoV-2 in these patients is limited. Current recommendations include vaccination against SARS-CoV-2 in patients undergoing autologous hematopoietic stem cell transplantation (AHSTC), regardless of whether they have been previously exposed to the virus. These recommendations are based on previous studies with other vaccines. Therefore, it is necessary to analyze the immune response that is developed in these patients in order to make specific recommendations for COVID-19 vaccination. Objective: To study the humoral and cellular immune response before and after AHSTC in patients with oncohematological neoplasms who were exposed to SARS-CoV-2 before the transplantation. Materials & methods: Nine patients with previous exposure to SARS-CoV-2 who underwent AHSTC (Table 1) and 8 healthy donors who recovered from mild COVID-19 were recruited from Hospital Ramón y Cajal and Primary Healthcare Center Pedro Laín Entralgo (Madrid, Spain), respectively. Specific direct cellular cytotoxicity (DCC) of PBMCs from these patients against Vero E6 cells infected with pseudotyped SARS-CoV-2 was determined. The activation of caspase-3 in Vero cells was measured after 1 hour of co-culture with PBMCs, in which cytotoxic cell populations were analyzed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was analyzed by quantifying the binding of Annexin V to rituximab-coated Raji cells as targets of PBMCs. Results: 1) 66% of AHSTC patients did not develop detectable levels of IgGs against SARS-CoV-2 (Fig. 1). In 33% of these patients with detectable IgG, the titers decreased after AHSTC, as well as their neutralizing capacity (Fig. 1B and C). 2) AHSTC patients showed increased levels of immature B cells (9.5-fold;p=0.0586) and plasmablasts (28.8-fold), in comparison with healthy donors who had mild COVID-19, while naive and resting memory B cells decreased 1.7- and 6.9-fold, respectively. 3) Specific DCC against SARS-CoV-2-infected cells increased 1.5-fold in comparison with healthy donors (Fig. 2A). Cytotoxic populations with NK phenotypes (CD3-CD56+CD16+), NKT (CD3+CD56+CD16+), and CD8+ T cells (CD3+CD8+TCRγδ+) increased 1.9- (p=0.0311), 1.9- (p=0.0592), and 1.6-fold, respectively (Fig. 2B). ADCC increased 2.1-fold in PBMCs from AHSTC patients in comparison with healthy donors (p = 0.0592). Conclusions: Our data show for the first time how the humoral and cellular immune response against the natural infection by SARS-Cov-2 may be modified in patients who were subsequently subjected to AHSTC. Although the humoral response may be reduced after AHSTC, the specific cellular response showed an increased cytotoxic activity. These results could be extrapolated to patients who were vaccinated against COVID-19 prior to AHSTC. Therefore, this information could be useful to define the recommendations for COVID-19 vaccination after AHSTC. [Formula presented] Disclosures: Garcia-Gutiérrez: Pfizer: Research Funding;Incyte: Consultancy;Novartis: Consultancy;Bristol-Myers Squibb: Consultancy.

Characterization of the Early Humoral and Cellular Response Developed in Oncohematological Patients Post-Vaccination with One Dose Against COVID-19

[Formula presented] Background: Oncohematological patients present a variable immune response against many vaccines, due to the immunodeficiency caused by the disease and its treatment. The experience of vaccination against COVID-19 in oncohematological patients is low and mostly limited to studies of humoral immunity. However, the humoral and cellular immune responses between different oncohematological diseases (OHD) have not been compared. Objective: To compare the humoral and cellular immune responses in four groups of patients with OHD after receiving the first dose of one COVID-19 vaccine. Materials & methods: We recruited 53 patients in four groups according to diagnosis: Chronic Lymphatic Leukemia (CLL) (n=14), Chronic Myeloid Leukemia (CML) (n=11), Multiple Myeloma (MM) (n=15), and Allogeneic Hematopoietic Stem Cell Transplantation (ASCT) (n=13) (Table 1). Samples were collected prior to vaccination and 3 weeks after receiving one dose of COMIRNATY (BioNTech-Pzifer), mRNA-1273 (Moderna), or AZD1222 (AstraZeneca). Twenty-six healthy donors with similar vaccination pattern were recruited. IgG titers against SARS-CoV-2 were quantified by Euroimmun-Anti-SARS-CoV-2 ELISA. Direct cellular cytotoxicity (DCC) was determined against Vero E6 cells infected with pseudotyped SARS-CoV-2, measuring caspase-3 activation after co-culture with PBMCs, in which cytotoxic populations were phenotyped by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) analyses were performed using Annexin V on Raji cells as a target. Results: 1) Early humoral response against COVID-19 vaccination in patients with CML was 5.1- (p<0.0001), 2.8- (p=0.0027), and 3.2-fold (p<0.0001) higher than in patients with CLL, MM and HSCT, respectively, and 3.5-fold higher than in healthy donors (p=0.0460) (Fig. 1). 84% of CLL patients did not develop detectable IgG titers. Individuals with OHD developed lower titres of neutralizing antibodies than healthy donors. 2) Unspecific ADCC was overall reduced in patients with OHD, mostly in individuals with ASCT (3.2-fold lower (p<0,0001)), whereas ADCC was reduced 2.2- (p<0.0001), 1.8- (p=0.0040), and 2.2-fold (p<0.0001) in individuals with CLL, CML and MM, respectively (Fig. 2A). However, specific DCC was increased 4.7-, 8.1- (p=0.0189), and 2.1-fold, respectively, in PBMCs from patients with CLL, MM, or ASCT, in comparison with healthy donors, whereas patients with CML showed a very similar response than healthy donors (Fig. 2B). 3) Levels of CD3+CD8+TCRγδ+ T cells were increased 2.2-, 2.1-, 2.7-, and 4.3-fold (p=0.0394) in patients with CLL, CML, MM, and ASCT, respectively, in comparison with healthy donors. CD3+CD8-TCRγδ+ T cells were also increased in patients with OHD, expressing high levels of the degranulation marker CD107a. However, the levels of CD3-CD56+CD107a+ NK cells were reduced 4.2- (p=0.0003) and 3.6-fold (p=0.0010) in PBMCs from patients with MM and ASCT, respectively, in comparison with healthy donors. Conclusions: We found significant differences in the early humoral immune response after one single dose of COVID-19 vaccine depending on the OHD analyzed. It was observed for the first time that the early cytotoxic immune response is efficient in all groups of patients, although superior in those who were not exposed to ASCT. Most cytotoxic activity relied on CD8+ T cells. These data can be useful to determine the efficacy of COVID-19 vaccines in patients with OHD. [Formula presented] Disclosures: Garcia Gutierrez: BMS: Consultancy, Honoraria, Research Funding;Novartis: Consultancy, Honoraria, Research Funding;Incyte: Consultancy, Honoraria, Research Funding;Pfizer: Consultancy, Honoraria, Research Funding.

Temporary layoffs, short-time work and COVID-19: the case of a dual labour market

Purpose: This paper aims to study the type of short-time work (STW) schemes implemented in Spain to preserve jobs and workers’ incomes during the COVID-19 crisis and the corresponding labour market outcomes. Design/methodology/approach: A dynamic macroeconomic model of job creation and destruction of the search and matching type in a dual labour market. Findings: The model shows that the availability of STW schemes does not necessarily prevent a large increase in unemployment and job destruction. The quantitative effects depend on the degree of subsidization of payroll taxes and on the design of the policy. A scenario with a moderate degree of subsidization and where the subsidy is independent of the reduction in hours worked is the least harmful for both welfare and fiscal deficit. The cost of such a strategy is a higher unemployment rate. Concerning heterogeneous effects, the unemployed are the ones who experience the strongest distributional changes. Originality/value: The effectiveness of STW schemes in dual labour markets using a search and matching model in the context of the COVID-19 crisis has not been analysed elsewhere. The literature has emphasized the importance of dynamics, labour market institutions and workers’ heterogeneity to understand workforce adjustment decisions in the face of temporary shocks to de- mand especially when firms’ human capital is relevant. These elements are present in the model. In addition, this paper computes welfare and distributional effects and the cost of these policies. © 2021, Victoria Osuna and José Ignacio García Pérez.

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.