The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Distinct features of SARS-CoV-2 humoral immunity against Omicron breakthrough infection.

BACKGROUND: SARS-CoV-2 Omicron breakthrough infection (Omicron-BTI) after vaccination has been frequently observed. A more detailed understanding of the humoral immunity against Omicron-BTI is required. METHODS: We measured strain-specific live-virus based neutralizing activity, anti-spike IgG, and anti-receptor-binding domain (RBD) IgG titers in individuals with Omicron/BA.1-BTI and directly compared them with controls with diverse combinations of wild-type (WT) mRNA vaccination and infection history. RESULTS: Omicron-BTI individuals showed markedly higher neutralizing titers against all the WT, Delta, and Omicron strains in convalescent sera, compared with unvaccinated Omicron-infection individuals with only Omicron neutralizing activity. Similar tendencies were found in strain-specific anti-spike and anti-RBD IgG titers. The Omicron-specificity (BA.1/WT neutralizing ratio), Omicron-neutralizing efficiency per antibody unit, and anti-Omicron RBD-directivity of anti-spike antibodies in Omicron-BTI individuals were all significantly lower than those in unvaccinated Omicron-infection individuals, but they were equivalent to or higher than those in uninfected vaccinees. The induction of Omicron-specific neutralizing activity after Omicron-BTI was not weakened for eight months from the last vaccination. CONCLUSIONS: These findings suggest that cross-reactive vaccine-induced immunity was intensively stimulated following Omicron breakthrough infection, which contributed to Omicron neutralization. Measuring SARS-CoV-2 variant-specific antibody levels as well as neutralizing activity is useful for evaluating humoral immunity after breakthrough infection in the current situation of antigenic gaps between vaccinated and epidemic (Omicron sub-lineages) strains.

Humoral and cellular responses to repeated COVID-19 exposure in multiple sclerosis patients receiving B-cell depleting therapies: a single-center, one-year, prospective study.

Multiple sclerosis patients treated with anti-CD20 therapy (aCD20-MS) are considered especially vulnerable to complications from SARS-CoV-2 infection due to severe B-cell depletion with limited viral antigen-specific immunoglobulin production. Therefore, multiple vaccine doses as part of the primary vaccination series and booster updates have been recommended for this group of immunocompromised individuals. Even though much less studied than antibody-mediated humoral responses, T-cell responses play an important role against CoV-2 infection and are induced efficiently in vaccinated aCD20-MS patients. For individuals with such decoupled adaptive immunity, an understanding of the contribution of T-cell mediated immunity is essential to better assess protection against CoV-2 infection. Here, we present results from a prospective, single-center study for the assessment of humoral and cellular immune responses induced in aCD20-MS patients (203 donors/350 samples) compared to a healthy control group (43/146) after initial exposure to CoV-2 spike antigen and subsequent re-challenges. Low rates of seroconversion and RBD-hACE2 blocking activity were observed in aCD20-MS patients, even after multiple exposures (responders after 1st exposure = 17.5%; 2nd exposure = 29.3%). Regarding cellular immunity, an increase in the number of spike-specific monofunctional IFNγ+-, IL-2+-, and polyfunctional IFNγ+/IL-2+-secreting T-cells after 2nd exposure was found most noticeably in healthy controls. Nevertheless, a persistently higher T-cell response was detected in aCD20-MS patients compared to control individuals before and after re-exposure (mean fold increase in spike-specific IFNγ+-, IL-2+-, and IFNγ+/IL-2+-T cells before re-exposure = 3.9X, 3.6X, 3.5X/P< 0.001; after = 3.2X, 1.4X, 2.2X/P = 0.002, P = 0.05, P = 0.004). Moreover, cellular responses against sublineage BA.2 of the currently circulating omicron variant were maintained, to a similar degree, in both groups (15-30% T-cell response drop compared to ancestral). Overall, these results highlight the potential for a severely impaired humoral response in aCD20-MS patients even after multiple exposures, while still generating a strong T-cell response. Evaluating both humoral and cellular responses in vaccinated or infected MS patients on B-cell depletion therapy is essential to better assess individual correlations of immune protection and has implications for the design of future vaccines and healthcare strategies.

SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition.

Background: Following SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic. Methods: The study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA. Results: The frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups. Conclusions: The antibody responses to the spike protein, but not the anti-RBD B cell responses diverge between convalescent males and females who develop PCC. Our findings also suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens.

Humoral Immune Response Diversity to Different COVID-19 Vaccines: Implications for the "Green Pass" Policy.

In the COVID-19 pandemic year 2021, several countries have implemented a vaccine certificate policy, the "Green Pass Policy" (GPP), to reduce virus spread and to allow safe relaxation of COVID-19 restrictions and reopening of social and economic activities. The rationale for the GPP is based on the assumption that vaccinated people should maintain a certain degree of immunity to SARS-CoV-2. Here we describe and compare, for the first time, the humoral immune response to mRNA-1273, BNT162b2, Ad26.COV2.S, and ChAdOx1 nCoV-19 vaccines in terms of antibody titer elicited, neutralizing activity, and epitope reactogenicity among 369 individuals aged 19 to 94 years. In parallel, we also considered the use of a rapid test for the determination of neutralizing antibodies as a tool to guide policymakers in defining booster vaccination strategies and eligibility for Green Pass. Our analysis demonstrates that the titer of antibodies directed towards the receptor-binding domain (RBD) of SARS-CoV-2 Spike is significantly associated with age and vaccine type. Moreover, natural COVID-19 infection combined with vaccination results, on average, in higher antibody titer and higher neutralizing activity as compared to fully vaccinated individuals without prior COVID-19. We also found that levels of anti-Spike RBD antibodies are not always strictly associated with the extent of inhibition of RBD-ACE2 binding, as we could observe different neutralizing activities in sera with similar anti-RBD concentrations. Finally, we evaluated the reactivity to four synthetic peptides derived from Spike protein on a randomly selected serum sample and observed that similar to SARS-CoV-2 infection, vaccination elicits a heterogeneous antibody response with qualitative individual features. On the basis of our results, the use of rapid devices to detect the presence of neutralizing antibodies, even on a large scale and repeatedly over time, appears helpful in determining the duration of the humoral protection elicited by vaccination. These aspects and their implications for the GPP are discussed.

Differential Dynamics of Humoral and Cell-Mediated Immunity with Three Doses of BNT162b2 SARS-CoV-2 Vaccine in Healthcare Workers in Japan: A Prospective Cohort Study.

Vaccines against SARS-CoV-2 with good efficacy are now available worldwide. However, gained immunity diminishes over time. Here, we investigate the course of both humoral and cell-mediated immunity in response to three doses of the Pfizer mRNA BNT162b2 SARS-CoV-2 vaccine in healthcare workers in Japan. SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies (total Ig, IgG), neutralizing antibodies (NAb), and ELISpot were measured in serum and whole blood samples collected after each vaccine dose. ELISpot numbers were higher than the cutoff values in most participants at all times. It was suggested that the difference in behavior between humoral immunity and cell-mediated immunity with age is complementary. Anti-RBD total Ig, IgG, and NAb indicated a high correlation at each time point after vaccine doses. Total Ig was retained long-term after the second dose and increased significantly faster by the booster dose than IgG. Nab levels of all subjects were ≤20% six months after the second dose, and the correlation coefficient was greatly reduced. These are due to the avidity of each antibody and differences among commercial kits, which may affect the evaluation of immunokinetics in previous COVID-19 studies. Therefore, it is necessary to harmonize reagents categorized by the same characteristics.

Comparison of the effectiveness and duration of anti-RBD SARS-CoV-2 IgG antibody response between different types of vaccines: Implications for vaccine strategies.

BACKGROUND AND OBJECTIVES: Little is known about the efficacy and durability of anti-RBD IgG antibodies induced by certain SARS-CoV-2 vaccines. It has been shown that neutralizing antibodies are associated with the protection against re-infection. This study aims to compare the mean titers, duration, and efficacy of generating protective anti-RBD IgG antibody response among recipients of Pfizer/BioNTech, AstraZeneca, Sputnik V, Johnson & Johnson, Moderna, and Sinopharm COVID-19 vaccines. In addition, we aimed to compare the susceptibility of getting COVID-19 breakthrough infections after various types of vaccines. MATERIALS AND METHODS: Samples from 2065 blood bank donors and healthcare workers at King Hussein Cancer Center (KHCC) were collected between February and September 2021. Anti-Spike/RBD IgG levels were measured using Chemiluminescent microparticle-immunoassay (CMIA) (ARCHITECT IgG II Quant test, Abbott, USA). RESULTS: The mean titer of anti-RBD IgG levels was significantly diverse among different types of vaccines. The highest titer level was seen in participants who took a third booster vaccine shot, followed by Pfizer/BioNTech, AstraZeneca, and Sinopharm vaccine. The mean titer levels of anti-RBD IgG antibodies in the Pfizer vaccinated group was the highest after vaccination but started to drop after 60 days from vaccination unlike AstraZeneca and Sinopharm vaccine-induced antibodies where the mean titers continued to be stable until 120 days but their levels were significantly lower. Most of the breakthrough infections were among the Sinopharm vaccinated group and these breakthroughs happened at random times for the three main types of vaccines. CONCLUSIONS: Our data demonstrate that the mean-titer of anti-RBD IgG levels drop after four months which is the best time to take the additional booster shot from a more potent vaccine type such as mRNA vaccines that might be needed in Jordan and worldwide.

Kinetics of anti-SARS-CoV-2 antibody titer in healthy adults up to 6 months after BNT162b2 vaccination measured by two immunoassays: A prospective cohort study in Japan.

BACKGROUND: Although several assays are used to measure anti-receptor-binding domain (RBD) antibodies induced after severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination, the assays are not fully comparable in practice. This study evaluated the immunogenicity of the BNT162b2 mRNA vaccine in healthy adults using two immunoassays. METHODS: This prospective cohort study included SARS-CoV-2-naïve adults, predominantly healthcare workers, aged 20-64 years, who received two BNT162b2 vaccine doses between March and May 2021. Blood samples were collected before the first vaccination (S0), before the second vaccination (S1), 4 weeks after the second vaccination (S2), and 6 months after the second vaccination (S3). anti-RBD antibodies were measured using the Architect SARS-CoV-2 IgG II Quant (Abbott Laboratory) and Elecsys anti-SARS-CoV-2 S (Roche Diagnostics) assays. RESULTS: Among the 385 participants, the geometric mean antibody titers (GMTs) on the Architect assay (AU/mL) were 7.5, 693, 7007, and 1030 for S0, S1, S2, and S3, respectively. The corresponding GMTs on the Elecsys assay (U/mL) were 0.40, 24, 928, and 659, respectively. The GMT ratio (S3/S2) was 0.15 on the Architect and 0.71 on the Elecsys assay. The correlation between antibody titers measured with the two assays were strong at all time points after vaccination (Spearman's correlation coefficient: 0.74 to 0.86, P < 0.01 for all). GMT was significantly lower in the older age group after vaccination (P < 0.01), with no significant differences according to sex. Seroprotection (≥5458 AU/mL on the Architect assay and ≥ 753 U/mL on the Elecsys) at each time point was 0 %, 1 %, 67 %, and 1 % on the Architect assay and 0 %, 1 %, 62 %, and 43 % on the Elecsys, respectively. CONCLUSIONS: Two BNT162b2 vaccine doses resulted in adequate anti-RBD antibody response, which varied by age. As the two assays showed different kinetics, the results of single immunoassays should be interpreted with caution.

Pulling-Force Spinning Top for Serum Separation Combined with Paper-Based Microfluidic Devices in COVID-19 ELISA Diagnosis.

The spread of Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), resulting in a global pandemic with around four million deaths. Although there are a variety of nucleic acid-based tests for detecting SARS-CoV-2, these methods have a relatively high cost and require expensive supporting equipment. To overcome these limitations and improve the efficiency of SARS-CoV-2 diagnosis, we developed a microfluidic platform that collected serum by a pulling-force spinning top and paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) for quantitative IgA/IgM/IgG measurements in an instrument-free way. We further validated the paper-based microfluidic ELISA analysis of SARS-CoV-2 receptor-binding domain (RBD)-specific IgA/IgM/IgG antibodies from human blood samples as a good measurement with higher sensitivity compared with traditional IgM/IgG detection (99.7% vs 95.6%) for early illness onset patients. In conclusion, we provide an alternative solution for the diagnosis of SARS-CoV-2 in a portable manner by this smart integration of pulling-force spinning top and paper-based microfluidic immunoassay.