Evidence for Aerosol Transfer of SARS-CoV-2-Specific Humoral Immunity.

Infectious particles can be shared through aerosols and droplets formed as the result of normal respiration. Whether Abs within the nasal/oral fluids can similarly be shared between hosts has not been investigated. The circumstances of the SARS-CoV-2 pandemic facilitated a unique opportunity to fully examine this provocative idea. The data we show from human nasal swabs provides evidence for the aerosol transfer of Abs between immune and nonimmune hosts.

B cells promote CD8 T cell primary and memory responses to subunit vaccines.

The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called "helper" cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Magnetic Enrichment of SARS-CoV-2 Antigen-Binding B Cells for Analysis of Transcriptome and Antibody Repertoire

The ongoing COVID-19 pandemic has had devastating health impacts across the globe. The development of effective diagnostics and therapeutics will depend on the understanding of immune responses to natural infection and vaccination to the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While both B-cell immunity and T-cell immunity are generated in SARS-CoV-2-infected and vaccinated individuals, B-cell-secreted antibodies are known to neutralize SARS-CoV-2 virus and protect from the disease. Although interest in characterizing SARS-CoV-2-reactive B cells is great, the low frequency of antigen-binding B cells in human blood limits in-depth cellular profiling. To overcome this obstacle, we developed a magnetic bead-based approach to enrich SARS-CoV-2-reactive B cells prior to transcriptional and antibody repertoire analysis by single-cell RNA sequencing (scRNA-seq). Here, we describe isolation of SARS-CoV-2 antigen-binding B cells from two seropositive donors and comparison to nonspecific B cells from a seronegative donor. We demonstrate that SARS-CoV-2 antigen-binding B cells can be distinguished on the basis of transcriptional profile and antibody repertoire. Furthermore, SARS-CoV-2 antigen-binding B cells exhibit a gene expression pattern indicative of antigen experience and memory status. Combining scRNA-seq methods with magnetic enrichment enables the rapid characterization of SARS-CoV-2 antigen-binding B cells.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus.

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T cell-based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). C-C chemokine receptor type 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendritic cells (DCs), and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127hi, KLRG-1lo, OX40+ve CD62L+ve, and mucosally imprinted CD69+ve CD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs induced by CCR2 deficiency was linked to dampened expression of T-bet but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses, and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens, including IAV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IMPORTANCE While antibody-based immunity to influenza A virus (IAV) is type and subtype specific, lung- and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of antiviral lung-resident memory T cells following intranasal vaccination. These findings suggest that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.

Risk Factors of SARS-CoV-2 Antibodies in Arapahoe County First Responders-The COVID-19 Arapahoe SErosurveillance Study (CASES) Project.

OBJECTIVES: Define the seroprevalence and risk factors for SARS-CoV-2 antibodies in Arapahoe County, Colorado first responders (eg, law enforcement, human services, fire departments). METHODS: Two hundred sixty four first responders were enrolled June to July 2020. SARS-CoV-2 seropositivity was defined as detection of immunoglobulin G (IgG) antibodies to both spike receptor binding domain and nucleocapsid in venous blood by validated enzyme-linked immunosorbent assay. We compared risk factors for being seropositive versus seronegative. RESULTS: 4% (11/264) were SARS-CoV-2 seropositive. Seropositive participants were significantly more likely to have lung disease (% seropositive, % seronegative; P-value) (36%, 8%; P = 0.01), prior SARS-CoV-2/COVID-19 testing (36%, 8%; P ≤ 0.01), a prior positive result (18%, less than 1%), and to believe they previously had COVID-19 (64%, 15%; P < 0.01). Only 15% of those believing they had COVID-19 had anti-SARS-CoV-2 antibodies. CONCLUSIONS: Human services employees and individuals with lung disease are at SARS-CoV-2 exposure risk. Few individuals believed they had COVID-19 had prior exposure.

Development and Validation of a Multiplex Microsphere Immunoassay Using Dried Blood Spots for SARS-CoV-2 Seroprevalence: Application in First Responders in Colorado, USA.

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R = 0.83, P < 0.0001 and R = 0.87, P < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R = 0.76, P = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (P < 0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.

Conserved and Novel Mouse CD8 T Cell Epitopes within SARS-CoV-2 Spike Receptor Binding Domain Protein Identified following Subunit Vaccination.

The prior existence of human ACE2 protein-expressing mice used to study SARS-CoV and the rapid development of mouse-adapted virus strains have allowed the study of SARS-CoV-2 in mice, even as we are still learning about its natural pathology in humans. With myriad genetically altered strains on the C57BL/6 background and the abundance of immunological reagents available to interrogate its immune responses, the C57BL/6 mice may provide useful insight into the immunology of SARS-CoV-2 infection and vaccination. To conduct more detailed studies on their T cell responses to vaccines and infection, the epitopes eliciting those responses must be characterized in further detail. In this study, we mapped CD8 T cell epitopes within the receptor binding domain of the SARS-CoV-2 spike protein in C57BL/6 mice. Our study identified five major CD8 T cell epitopes in immunized C57BL/6 mice, including one, VVLSFELL, presented by H-2Kb and common between SARS-CoV and SARS-CoV-2.

Down but far from out: The durability of SARS-CoV-2 immunity after asymptomatic infection.

The dynamics of immune responses in asymptomatic SARS-CoV-2-infected subjects remain to be fully characterized. The work presented in this issue of JEM by Le Bert et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202617) sheds some light on these issues and ultimately provides some degree of confidence in the magnitude and persistence of immunity over time after asymptomatic infection with SARS-CoV-2.

Programming Multifaceted Pulmonary T Cell Immunity by Combination Adjuvants.

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 TRM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 TRM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2.