Antiviral innate immunity is diminished in the upper respiratory tract of severe COVID-19 patients (preprint)

Understanding early innate immune responses to coronavirus disease 2019 (COVID-19) is crucial to developing targeted therapies to mitigate disease severity. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection elicits interferon expression leading to transcription of IFN-stimulated genes (ISGs) to control viral replication and spread. SARS-CoV-2 infection also elicits NF-{kappa}B signaling which regulates inflammatory cytokine expression contributing to viral control and likely disease severity. Few studies have simultaneously characterized these two components of innate immunity to COVID-19. We designed a study to characterize the expression of interferon alpha-2 (IFNA2) and interferon beta-1 (IFNB1), both type-1 interferons (IFN-1), interferon-gamma (IFNG), a type-2 interferon (IFN-2), ISGs, and NF-{kappa}B response genes in the upper respiratory tract (URT) of patients with mild (outpatient) versus severe (hospitalized) COVID-19. Further, we characterized the weekly dynamics of these responses in the upper and lower respiratory tracts (LRTs) and blood of severe patients to evaluate for compartmental differences. We observed significantly increased ISG and NF-{kappa}B responses in the URT of mild compared with severe patients early during illness. This pattern was associated with increased IFNA2 and IFNG expression in the URT of mild patients, a trend toward increased IFNB1-expression and significantly increased STING/IRF3/cGAS expression in the URT of severe patients. Our by-week across-compartment analysis in severe patients revealed significantly higher ISG responses in the blood compared with the URT and LRT of these patients during the first week of illness, despite significantly lower expression of IFNA2, IFNB1, and IFNG in blood. NF-{kappa}B responses, however, were significantly elevated in the LRT compared with the URT and blood of severe patients during peak illness (week 2). Our data support that severe COVID-19 is associated with impaired interferon signaling in the URT during early illness and robust pro-inflammatory responses in the LRT during peak illness.

Influenza vaccination reveals and partly reverses sex dimorphic immune imprints associated with prior mild COVID-19 (preprint)

Viral infections can have profound and durable functional impacts on the immune system. There is an urgent need to characterize the long-term immune effects of SARS-CoV-2 infection given the persistence of symptoms in some individuals and the continued threat of novel variants including the recent rapid acceleration in infections. As the majority of COVID-19 patients experienced mild disease, here we use systems immunology approaches to comparatively assess the post-infection immune status (mean: 151 [5th - 95th percentile: 58 - 235] days after diagnosis) and subsequent innate and adaptive responses to seasonal influenza vaccination (as an "immune challenge") in 33 previously healthy individuals after recovery from mild, non-hospitalized COVID-19, as compared to 40 age- and sex-matched healthy controls with no history of COVID-19. Sex-specific, temporally stable shifts in signatures of metabolism, T-cell activation, and innate immune/inflammatory processes suggest that mild COVID-19 can establish new post-infection immunological set-points. COVID-19-recovered males had an increase in CD71hi B-cells (including influenza-specific subsets) before vaccination and more robust innate, influenza-specific plasmablast, and antibody responses after vaccination compared to healthy males. Intriguingly, by day 1 post-vaccination in COVID-19-recovered subjects, the expression of numerous innate defense/immune receptor genes (e.g., Toll-like receptors) in monocytes increased and moved away from their post-COVID-19 repressed state toward the pre-vaccination baseline of healthy controls, and these changes tended to persist to day 28 in females, hinting that the acute inflammatory responses induced by vaccination could partly reset the immune states established by prior mild COVID-19. Our study reveals sex-dimorphic immune imprints and in vivo functional impacts of mild COVID-19 in humans, suggesting that prior COVID-19 could change future responses to vaccination and in turn, vaccines could help reset the immune system after COVID-19, both in an antigen-agnostic manner.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children (preprint)

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

Epitope Diversity and SARS-CoV-2 Variants Neutralizing Capacity of SARS-CoV-2 Hyperimmune Intravenous Immunoglobulins for Treatment of COVID-19 (preprint)

Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike-based epitope-mapping demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to a different extent by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 ( < 2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher doses achieved by larger volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.Funding: The research work described in this manuscript was supported by FDA Medical Countermeasures Initiative (MCMi) grant # OCET 2021-1565 and NIH-NIAID IAA #AAI20040. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Declaration of Interest: The authors declare no competing interests.Ethical Approval: This study was approved by Food and Drug Administration’s Research Involving Human Subjects Committee (RIHSC #2020-04-02). This study complied with all relevant ethical regulations for work with human participants, and informed consent was obtained. Samples were collected from patients who provided informed consent to participate in the study.

Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19 (preprint)

Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 (<2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.

Antibody repertoire induced by SARS-CoV-2 spike protein immunogens (preprint)

Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody response generated following vaccination by these vaccine modalities. To better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various SARS-CoV-2 spike protein antigens: S-ectodomain (S1+S2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (aa 16-685), the receptor-binding domain (RBD) (aa 319-541), and the S2 domain (aa 686-1213 as control). Antibody response was analyzed by ELISA, Surface Plasmon Resonance (SPR) against different Spike proteins in native conformation, and a pseudovirion neutralization assay to measure the quality and function of the antibodies elicited by the different Spike antigens. All three antigens (S1+S2 ectodomain, S1 domain, and RBD) generated strong neutralizing antibodies against SARS-CoV-2. Vaccination induced antibody repertoire was analyzed by SARS-CoV-2 spike Genome Fragment Phage Display Libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD and S2 domains. Furthermore, these analyses demonstrated that surprisingly the RBD immunogen elicited a higher antibody titer with 5-fold higher affinity antibodies to native spike antigens compared with other spike antigens. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease. One Sentence SummarySARS-CoV-2 Spike induced immune response