Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing (preprint)

Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

Prime hAd5 Spike + Nucleocapsid Vaccination Induces Ten-Fold Increases in Mean T-Cell Responses in Phase 1 Subjects that are Sustained Against Spike Variants (preprint)

ABSTRACT In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 ‘A’ strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine ( NCT04591717 ; https://clinicaltrials.gov/ct2/show/NCT04591717 ), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

Th1 Dominant Nucleocapsid and Spike Antigen-Specific CD4+ and CD8+ Memory T Cell Recall Induced by hAd5 S-Fusion + N-ETSD Infection of Autologous Dendritic Cells from Patients Previously Infected with SARS-CoV-2 (preprint)

To address the need for a safe, efficacious vaccine against SARS-CoV-2 infection with the critical properties of enabling both blocking viral entry into cells and clearing virus from cells already infected, we have developed a bivalent, human adenovirus serotype 5 (hAd5) SARS-CoV-2 S-Fusion + N-ETSD vaccine that is currently in clinical testing. This vaccine uses the next-generation hAd5 [E1-, E2b-, E3-] platform previously used successfully in cancer patients with pre-existing adenovirus immunity, engineered to express both SARS-CoV-2 spike (S) protein modified to improve the generation of neutralizing antibodies to block entry of the virus, and nucleocapsid (N) protein with an Enhanced T cell Stimulation Domain (ETSD) to activate CD4+ and CD8+ T cells to clear the virus and block replication by killing infected cells. The targeting of N to endosomes and lysosomes to enhance CD4+ and CD8+ T-cell responses distinguishes our vaccine. In our previously reported pre-clinical studies we showed that in mice, the hAd5 S-Fusion + N-ETSD vaccine elicits both humoral and T-cell responses that are robust and T helper cell 1 (Th1) dominant. Here we report that the hAd5 S-Fusion + N-ETSD vaccine is recognized by anti-sera and T cells from previously SARS-CoV-2 infected patients, and that the presence of N is vital for T-cell recall. The findings presented herein: i. demonstrate specific recognition of hAd5 S-Fusion + N-ETSD infected cells by plasma antibodies from previously SARS-CoV-2 infected patients, but not antibodies from virus-naive subjects; ii. show enhanced binding of plasma SARS-CoV-2 antibodies from previously infected patients to monocyte-derived dendritic cells (MoDCs) expressing the hAd5 S-Fusion + N-ETSD vaccine as compared to hAd5 S-Fusion alone; iii. reveal N-ETSD localizes to vesicles associated with MHC class II antigen presentation, including endosomes, lysosomes, and autophagosomes in MoDCs; iv. demonstrate endosome/lysosome-targeted N-ETSD elicits higher interferon-gamma T-cell responses than cytoplasm-localized N; and v. N-ETSD alone or in the hAd5 S-Fusion + N-ETSD construct induces both CD4+ and CD8+ T cell memory recall. This recognition of hAd5 S-Fusion + N-ETSD vaccine antigens by T cells from previously SARS-CoV-2 infected patients, together with the ability of this vaccine candidate to elicit de novo immune responses in naive mice suggests that it re-capitulates the natural immune response to SARS-CoV-2 to activate both B and T cells towards viral neutralization and recognition of infected cells, critical for prevention of COVID-19 disease. Intriguingly, our hAd5 S-Fusion + N-ETSD T-cell biased vaccine has the potential to not only provide protection for uninfected individuals, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.

Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C) (preprint)

Initially, the global outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spared children from severe disease. However, after the initial wave of infections, clusters of a novel hyperinflammatory disease have been reported in regions with ongoing SARS-CoV-2 epidemics. While the characteristic clinical features are becoming clear, the pathophysiology remains unknown. Herein, we report on the immune profiles of eight Multisystem Inflammatory Syndrome in Children (MIS-C) cases. We document that all MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with normal isotype-switching and neutralization capability. We further profiled the secreted immune response by high-dimensional cytokine assays, which identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1) and mucosal immune dysregulation (IL-17A, CCL20, CCL28). Mass cytometry immunophenotyping of peripheral blood revealed reductions of mDC1 and non-classical monocytes, as well as both NK- and T-lymphocytes, suggesting extravasation to affected tissues. Markers of activated myeloid function were also evident, including upregulation of ICAM1 and Fc{gamma}R1 in neutrophil and non-classical monocytes, well-documented markers in autoinflammation and autoimmunity that indicate enhanced antigen presentation and Fc-mediated responses. Finally, to assess the role for autoimmunity secondary to infection, we profiled the auto-antigen reactivity of MIS-C plasma, which revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal and immune-cell antigens. All patients were treated with anti-IL6R antibody or IVIG, which led to rapid disease resolution tracking with normalization of inflammatory markers. One Sentence SummaryThis study maps the cellular and serological immune dysfunction underlying a novel pediatric inflammatory syndrome associated with SARS-CoV-2.