Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection.

Although SARS-CoV-2 infects the upper respiratory tract, we know little about the amount, type, and kinetics of antibodies (Ab) generated in the oral cavity in response to COVID-19 vaccination. We collected serum and saliva samples from participants receiving two doses of mRNA COVID-19 vaccines and measured the level of anti-SARS-CoV-2 Ab. We detected anti-Spike and anti-Receptor Binding Domain (RBD) IgG and IgA, as well as anti-Spike/RBD associated secretory component in the saliva of most participants after dose 1. Administration of a second dose of mRNA boosted the IgG but not the IgA response, with only 30% of participants remaining positive for IgA at this timepoint. At 6 months post-dose 2, these participants exhibited diminished anti-Spike/RBD IgG levels, although secretory component-associated anti-Spike Ab were more stable. Examining two prospective cohorts we found that participants who experienced breakthrough infections with SARS-CoV-2 variants had lower levels of vaccine-induced serum anti-Spike/RBD IgA at 2-4 weeks post-dose 2 compared to participants who did not experience an infection, whereas IgG levels were comparable between groups. These data suggest that COVID-19 vaccines that elicit a durable IgA response may have utility in preventing infection. Our study finds that a local secretory component-associated IgA response is induced by COVID-19 mRNA vaccination that persists in some, but not all participants. The serum and saliva IgA response modestly correlate at 2-4 weeks post-dose 2. Of note, levels of anti-Spike serum IgA (but not IgG) at this timepoint are lower in participants who subsequently become infected with SARS-CoV-2. As new surges of SARS-CoV-2 variants arise, developing COVID-19 booster shots that provoke high levels of IgA has the potential to reduce person-to-person transmission.

Convergent CDR3 homology amongst Spike-specific antibody responses in convalescent COVID-19 subjects receiving the BNT162b2 vaccine.

Convalescent coronavirus disease 2019 (COVID-19) subjects who receive BNT162b2 develop robust antibody responses against SARS-CoV-2. However, our understanding of the clonal B cell response pre- and post-vaccination in such individuals is limited. Here we characterized B cell phenotypes and the BCR repertoire after BNT162b2 immunization in two convalescent COVID-19 subjects. BNT162b2 stimulated many B cell clones that were under-represented during SARS-CoV-2 infection. In addition, the vaccine generated B cell clusters with >65% similarity in CDR3 VH and VL region consensus sequences both within and between subjects. This result suggests that the CDR3 region plays a dominant role adjacent to heavy and light chain V/J pairing in the recognition of the SARS-CoV-2 spike protein. Antigen-specific B cell populations with homology to published SARS-CoV-2 antibody sequences from the CoV-AbDab database were observed in both subjects. These results point towards the development of convergent antibody responses against the virus in different individuals.

Persistence of T Cell and Antibody Responses to SARS-CoV-2 Up to 9 Months after Symptom Onset.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces T cell, B cell, and Ab responses that are detected for several months in recovered individuals. Whether this response resembles a typical respiratory viral infection is a matter of debate. In this study, we followed T cell and Ab responses in 24 mainly nonhospitalized human subjects who had recovered from PCR-confirmed SARS-CoV-2 infection at two time points (median of 45 and 145 d after symptom onset). Ab responses were detected in 95% of subjects, with a strong correlation between plasma and salivary anti-spike (anti-S) and anti-receptor binding domain IgG, as well as a correlation between circulating T follicular helper cells and the SARS-CoV-2-specific IgG response. T cell responses to SARS-CoV-2 peptides were determined using intracellular cytokine staining, activation markers, proliferation, and cytokine secretion. All study subjects had a T cell response to at least one SARS-CoV-2 Ag based on at least one T cell assay. CD4+ responses were largely of the Th1 phenotype, but with a lower ratio of IFN-γ- to IL-2-producing cells and a lower frequency of CD8+:CD4+ T cells than in influenza A virus (IAV)-specific memory responses within the same subjects. Analysis of secreted molecules also revealed a lower ratio of IFN-γ to IL-2 and an altered cytotoxic profile for SARS-CoV-2 S- and nucleocapsid-specific responses compared with IAV-specific responses. These data suggest that the memory T cell phenotype after a single infection with SARS-CoV-2 persists over time, with an altered cytokine and cytotoxicity profile compared with long-term memory to whole IAV within the same subjects.

Intranasal HD-Ad vaccine protects the upper and lower respiratory tracts of hACE2 mice against SARS-CoV-2.

BACKGROUND: The ongoing COVID-19 pandemic has resulted in 185 million recorded cases and over 4 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. RESULTS: Here, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) produces a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. CONCLUSION: Our approaches provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

SPEEDS: A portable serological testing platform for rapid electrochemical detection of SARS-CoV-2 antibodies.

The COVID-19 pandemic has resulted in a worldwide health crisis. Rapid diagnosis, new therapeutics and effective vaccines will all be required to stop the spread of COVID-19. Quantitative evaluation of serum antibody levels against the SARS-CoV-2 virus provides a means of monitoring a patient's immune response to a natural viral infection or vaccination, as well as evidence of a prior infection. In this paper, a portable and low-cost electrochemical immunosensor is developed for the rapid and accurate quantification of SARS-CoV-2 serum antibodies. The immunosensor is capable of quantifying the concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against the SARS-CoV-2 spike protein in human serum. For IgG and IgM, it provides measurements in the range of 10.1 ng/mL - 60 µg/mL and 1.64 ng/mL - 50 µg/mL, respectively, both with an assay time of 13 min. We also developed device stabilization and storage strategies to achieve stable performance of the immunosensor over 24-week storage at room temperature. We evaluated the performance of the immunosensor using COVID-19 patient serum samples collected at different time points after symptom onset. The rapid and sensitive detection of IgG and IgM provided by our immunosensor fulfills the need of rapid COVID-19 serological testing for both point-of-care diagnosis and population immunity screening.

SARS-CoV-2-Reactive Mucosal B Cells in the Upper Respiratory Tract of Uninfected Individuals.

SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease.

Detection and Neutralization of SARS-CoV-2 Using Non-conventional Variable Lymphocyte Receptor Antibodies of the Evolutionarily Distant Sea Lamprey.

SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2.

A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies.

Better diagnostic tools are needed to combat the ongoing COVID-19 pandemic. Here, to meet this urgent demand, we report a homogeneous immunoassay to detect IgG antibodies against SARS-CoV-2. This serological assay, called SATiN, is based on a tri-part Nanoluciferase (tNLuc) approach, in which the spike protein of SARS-CoV-2 and protein G, fused respectively to two different tNLuc tags, are used as antibody probes. Target engagement of the probes allows reconstitution of a functional luciferase in the presence of the third tNLuc component. The assay is performed directly in the liquid phase of patient sera and enables rapid, quantitative and low-cost detection. We show that SATiN has a similar sensitivity to ELISA, and its readouts are consistent with various neutralizing antibody assays. This proof-of-principle study suggests potential applications in diagnostics, as well as disease and vaccination management.

Detection of SARS-CoV-2 Viral Particles Using Direct, Reagent-Free Electrochemical Sensing.

The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.

Systematic Examination of Antigen-Specific Recall T Cell Responses to SARS-CoV-2 versus Influenza Virus Reveals a Distinct Inflammatory Profile.

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. In this study, we investigated human T cell recall responses to fully glycosylated spike trimer, recombinant N protein, as well as to S, N, M, and E peptide pools in the early convalescent phase and compared them with influenza-specific memory responses from the same donors. All subjects showed SARS-CoV-2-specific T cell responses to at least one Ag. Both SARS-CoV-2-specific and influenza-specific CD4+ T cell responses were predominantly of the central memory phenotype; however SARS-CoV-2-specific CD4+ T cells exhibited a lower IFN-γ to TNF ratio compared with influenza-specific memory responses from the same donors, independent of disease severity. SARS-CoV-2-specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. Most SARS-CoV-2-convalescent subjects also produced IFN-γ in response to seasonal OC43 S protein. We observed granzyme B+/IFN-γ+, CD4+, and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ T cell responses predominating over CD8+ T cell responses. Peripheral T follicular helper (pTfh) responses to S or N strongly correlated with serum neutralization assays as well as receptor binding domain-specific IgA; however, the frequency of pTfh responses to SARS-CoV-2 was lower than the frequency of pTfh responses to influenza virus. Overall, T cell responses to SARS-CoV-2 are robust; however, CD4+ Th1 responses predominate over CD8+ T cell responses, have a more inflammatory profile, and have a weaker pTfh response than the response to influenza virus within the same donors, potentially contributing to COVID-19 disease.

Enhancing the performance of paper-based electrochemical impedance spectroscopy nanobiosensors: An experimental approach.

Accurate, rapid, and low-cost molecular diagnostics is essential in managing outbreaks of infectious diseases, such as the pandemic of coronavirus disease 2019 (COVID-19). Accordingly, microfluidic paper-based analytical devices (µPADs) have emerged as promising diagnostic tools. Among the extensive efforts to improve the performance and usability of diagnostic tools, biosensing mechanisms based on electrochemical impedance spectroscopy (EIS) have shown great promise because of their label-free operation and high sensitivity. However, the method to improve EIS biosensing on µPADs is less explored. Here, we present an experimental approach to enhancing the performance of paper-based EIS biosensors featuring zinc oxide nanowires (ZnO NWs) directly grown on working electrodes (WEs). Through a comparison of different EIS settings and an examination of ZnO-NW effects on EIS measurements, we show that ZnO-NW-enhanced WEs function reliably with Faradaic processes utilizing iron-based electron mediators. We calibrate paper-based EIS biosensors with different morphologies of ZnO NWs and achieve a low limit of detection (0.4 pg ml-1) in detecting p24 antigen as a marker for human immunodeficiency virus (HIV). Through microscopic imaging and electrochemical characterization, we reveal that the morphological and the electrochemical surface areas of ZnO-NW-enhanced WEs indicate the sensitivities and sensing ranges of the EIS nanobiosensors. Finally, we report that the EIS nanobiosensors are capable of differentiating the concentrations (blank, 10 ng ml-1, 100 ng ml-1, and 1 µg ml-1) of IgG antibody (CR3022) to SARS-CoV-2 in human serum samples, demonstrating the efficacy of these devices for COVID-19 diagnosis. This work provides a methodology for the rational design of high-performance EIS µPADs and has the potential to facilitate diagnosis in pandemics.

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients.

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.

A simple protein-based surrogate neutralization assay for SARS-CoV-2.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.