CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment (preprint)

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization (preprint)

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and to develop, validate, improve, and implement serological testing and associated technologies. SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Function is more reliable than quantity to follow up the humoral response to the Receptor Binding Domain of SARS- CoV-2 Spike Protein after Natural Infection or COVID-19 vaccination (preprint)

On this work we report that despite of a decline in the total anti-Spike antibodies the neutralizing antibodies remains at a similar level for an average of 98 days in a longitudinal cohort of 59 Hispanic/Latino exposed to SARS-CoV-2. We are also reporting that the percentage of neutralization correlates with the IgG titers and that in the first collected samples, IgG1 was the predominant isotype (62.71%), followed by IgG4 (15.25%), IgG3 (13.56%), and IgG2 (8.47%) during the tested period. The IgA was detectable in 28.81% of subjects. Only 62.71% of all subjects have detectable IgM in the first sample despite of confirmed infection by a molecular method. Our data suggests that 100% that seroconvert make detectable neutralizing antibody responses measured by a surrogate viral neutralization test. We also found that the IgG titers and neutralizing activity were higher after the first dose in 10 vaccinated subjects out of the 59 with prior infection compare to a subgroup of 21 subjects naive to SARS-CoV-2. One dose was enough but two were necessary to reach the maximum percentage of neutralization in subjects with previous natural infection or naive to SARS-CoV-2 respectively. Like the pattern seen after the natural infection, after the second vaccine dose, the total anti-S antibodies and titers declined but not the neutralizing activity which remains at same levels for more than 80 days after the first vaccine dose. That decline, however, was significantly lower in pre-exposed individuals which denotes the contribution of the natural infection priming a more robust immune response to the vaccine. Also, our data indicates that the natural infection induces a more robust humoral immune response than the first vaccine dose in unexposed subjects. However, the difference was significant only when the neutralization was measured but not by assessing the total anti-S antibodies or the IgG titers. This work is an important contribution to understand the natural immune response to the novel coronavirus in a population severely hit by the virus. Also provide an invaluable data by comparing the dynamic of the immune response after the natural infection vs. the vaccination and suggesting that a functional test is a better marker than the presence or not of antibodies. On this context our results are also highly relevant to consider standardizing methods that in addition to serve as a tool to follow up the immune response to the vaccines may also provide a correlate of protection.

The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera. (preprint)

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.

Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay (preprint)

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2 (preprint)

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.