Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Pathogenic neutrophilia drives acute respiratory distress syndrome in severe COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing COVID-19 pandemic have caused [~]40 million cases and over 648,000 deaths in the United States alone. Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African-American (AA) patients in some areas, yet targeted studies within this demographic are scant. Multi-omics single-cell analyses of immune profiles from airways and matching blood samples of Black/AA patients revealed low viral load, yet pronounced and persistent pulmonary neutrophilia with advanced features of cytokine release syndrome and acute respiratory distress syndrome (ARDS), including exacerbated production of IL-8, IL-1{beta}, IL-6, and CCL3/4 along with elevated levels of neutrophil elastase and myeloperoxidase. Circulating S100A12+/IFITM2+ mature neutrophils are recruited via the IL-8/CXCR2 axis, which emerges as a potential therapeutic target to reduce pathogenic neutrophilia and constrain ARDS in severe COVID-19. Graphical AbstractThe lung pathology due to severe COVID-19 is marked by a perpetual pathogenic neutrophilia, leading to acute respiratory distress syndrome (ARDS) even in the absence of viral burden. Circulating mature neutrophils are recruited to the airways via IL-8 (CXCL8)/CXCR2 chemotaxis. Recently migrated neutrophils further differentiate into a transcriptionally active and hyperinflammatory state, with an exacerbated expression of IL-8 (CXCL8), IL-1{beta} (IL1B), CCL3, CCL4, neutrophil elastase (NE), and myeloperoxidase (MPO) activity. Airway neutrophils and recruited inflammatory monocytes further increase their production of IL-8 (CXCL8), perpetuating lung neutrophilia in a feedforward loop. MdCs and T cells produce IL-1{beta} and TNF, driving neutrophils reprogramming and survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/446468v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@81fd3aorg.highwire.dtl.DTLVardef@181e63org.highwire.dtl.DTLVardef@172fedcorg.highwire.dtl.DTLVardef@ba55a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Protracted yet coordinated differentiation of long-lived SARS-CoV-2-specific CD8+ T cells during COVID-19 convalescence

CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc322-331, a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38- parameter CyTOF phenotyping on tetramer-identified Nuc322-331-specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS- CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322-331-specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a [~]6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.

Distinctive features of SARS-CoV-2-specific T cells predict recovery from severe COVID-19

Although T cells are likely players in SARS-CoV-2 immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe COVID-19. We analyzed T cells from longitudinal specimens of 34 COVID-19 patients with severities ranging from mild (outpatient) to critical culminating in death. Relative to patients that succumbed, individuals that recovered from severe COVID-19 harbored elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 displayed elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of severe COVID-19 patients, these results support a model whereby lung-homing T cells activated through bystander effects contribute to immunopathology, while a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/21250054v2_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@c82ec8org.highwire.dtl.DTLVardef@778d7forg.highwire.dtl.DTLVardef@ea9130org.highwire.dtl.DTLVardef@1e21805_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIDysfunctional spike-specific T cells are characteristic of severe COVID-19 C_LIO_LISpike-specific CD127+ Th1 cells are increased in survivors of severe COVID-19 C_LIO_LISpike-specific Tregs and IL6+ CD8+ T cells are increased in fatal COVID-19 C_LIO_LIEscalation of activated lung-homing CXCR4+ T cells associates with fatal COVID-19 C_LI BRIEF SUMMARYBy conducting CyTOF on total and SARS-CoV-2-specific T cells from longitudinal specimens spanning the entire spectrum of COVID-19 diseases, Neidleman et al. demonstrate that spike-specific Th1 cells capable of IL7-dependent homeostatic proliferation predict survival from severe COVID-19, while Tregs and IL6+ CD8+ T cells recognizing spike predict fatal outcome. Fatal COVID-19 is characterized by escalating activation of bystander CXCR4+ T cells in the lungs. Boosting SARS-CoV-2-specific CD4+ T effector responses while diminishing CXCR4-mediated homing may help recovery from severe disease.

SARS-CoV-2-specific T cells exhibit unique features characterized by robust helper function, lack of terminal differentiation, and high proliferative potential

Convalescing COVID-19 patients mount robust T cell responses against SARS-CoV-2, suggesting an important role for T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells, and predominantly Tcm with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can homeostatically proliferate, and can persist for over two months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection, and support an important role for SARS-CoV-2-specific T cells in host control of COVID-19.