ABSTRACT
Background. Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16 - NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset. Methods. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n =15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n =7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis. Results. The CD56bright CD16 - NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n =8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p< 0.05, ns: not significant Figure 2. NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. * *: p< 0.01, * * *: p< 0.001, ns: not significant. Conclusion. We demonstrate and characterize a nonclassical population of CD56dim CD16 - NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19.
ABSTRACT
Purpose: Solid organ transplant recipients (SOTR) develop weak antibody responses after SARS-CoV-2 vaccination. Published data on neutralizing activity of plasma, a better measure of protection, in SOTR following an additional dose of SARSCoV- 2 vaccine is limited. Method(s): Plasma was longitudinally collected from SOTR following initial COVID- 19 vaccination. Neutralizing activity against SARS-CoV-2 was assessed using the cPass Neutralization Antibody Detection Kit (GenScript, Biotech). ELISAs were performed against SARS-CoV-2 proteins (S1, N, RBD), CMV (glycoprotein B), Influenza A H1N1 (nucleoprotein), HSV-1, EBV glycoprotein (gp350), and tetanus toxoid for comparison. Result(s): Demographic and clinical characteristics are summarized in table 1. No participants had evidence of COVID-19 infection as IgG titers to SARS-CoV-2 N protein were low. Neutralizing activity against SARS-CoV-2 RBD was observed in 39.6% of individuals (N=21/53) ~93 days after initial vaccination. Participants with neutralizing activity were more likely to have received a liver transplant (47.6% vs 6.25%, p=0.001), and less likely to be on an anti-metabolite (52.4% vs. 87.5%, p=0.009) or triple immunosuppression (14.3% vs. 53.1%, p=0.008). After an additional vaccine dose, 78.1% (N=25/32) of participants developed neutralizing activity with significant increases in viral neutralization (figure 1, median 36.8% [95%CI 18.9-64.6] to 97.2% [95%CI 74.0-98.9], p<0.0001). Participants with low neutralizing activity demonstrated adequate antibody titers to other microbial antigens (figure 2). Conclusion(s): An additional dose of SARS-CoV-2 vaccine increased the number of SOTR with neutralizing activity and the magnitude of the seroresponse. SOTR with low neutralizing activity maintain humoral responses to other microbial antigens suggesting the diminished seroresponse might be related to inhibition of new B cell responses.
ABSTRACT
Purpose : To systematically investigate ocular changes in autopsied eyes from fatal cases of Coronavirus disease 2019 (COVID-19) and to investigate the localization of severe acute respiratory syndrome coronavirus (SARS-CoV-2) within ocular structures. Methods : Macroscopic and microscopic histopathological evaluation was performed and the localization of SARS-CoV-2 RNA within ocular tissues investigated using an in situ hybridization (ISH) technique in 13 eyes. Contralateral eyes were freshly dissected, and droplet digital polymerase chain reaction (ddPCR) assay was performed on ocular fluids and tissues to quantify SARS-CoV-2 RNA. Results : A total of 21 fatal COVID-19 cases were included (mean age, 60.2 years [range, 27- 91 years];23.8% female). Histopathological abnormalities include vascular changes (61.9%), cytoid bodies (52.4%), and retinal edema (23.8%) with minimal inflammation (0.09%) were observed. Non-CMV viral inclusions were identified in one eye. No CMV positivity was detected. Of the 21 contralateral eyes tested by ddPCR, 14 tested positive for SARS-CoV-2. Using ddPCR and ISH, SARS-CoV-2 localization was observed in the following ocular tissues and fluid: cornea (27.3%), aqueous (26.3%), lens (54.5%), vitreous (15.0%), retina (22.2%), choroid/sclera (47.4%), and optic nerve (50.0%). The choroid/sclera, optic nerve and lens were the most frequent ocular structures found to be ddPCR positive. Evidence of replication was detected in four cases. Conclusions : Our results suggest that SARS-CoV-2 localizes to intraocular tissues. However, histological changes observed are likely a secondary hemodynamic change rather than primary effect of the virus.