Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

Trajectory and Demographic Correlates of Antibodies to SARS-CoV-2 Nucleocapsid in Recently Infected Blood Donors, United States.

We evaluated antibodies to the nucleocapsid protein of SARS-CoV-2 in a large cohort of blood donors in the United States who were recently infected with the virus. Antibodies to the nucleocapsid protein of SARS-CoV-2 indicate previous infection but are subject to waning, potentially affecting epidemiologic studies. We longitudinally evaluated a cohort of 19,323 blood donors who had evidence of recent infection by using a widely available serologic test to determine the dynamics of such waning. We analyzed overall signal-to-cutoff values for 48,330 donations (average 2.5 donations/person) that had an average observation period of 102 days. The observed peak signal-to-cutoff value varied widely, but the waning rate was consistent across the range, with a half-life of 122 days. Within the cohort, only 0.75% of persons became seronegative. Factors predictive of higher peak values and longer time to seroreversion included increasing age, male sex, higher body mass index, and non-Caucasian race.

Immunogenicity and safety of SARS-CoV-2 mRNA vaccine in patients with nephrotic syndrome receiving immunosuppressive agents.

BACKGROUND: As there are no large-scale reports of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) mRNA vaccination in patients with nephrotic syndrome using immunosuppressive agents, we conducted the prospective study. METHODS: SARS-CoV-2 mRNA vaccines were administered to patients with nephrotic syndrome receiving immunosuppressive agents. The titers of SARS-CoV-2 spike protein receptor-binding domain antibodies were measured before and after vaccination. We evaluated factors associated with antibody titers after vaccination and analyzed adverse events. RESULTS: We enrolled 40 patients and evaluated vaccine immunogenicity in 35 of them. Seroconversion (> 0.8 U/mL) was achieved in all patients, and the median antibody titer was 598 U/mL (interquartile range, 89-1380 U/mL). Patients using mycophenolate mofetil (MMF) showed lower antibody titers than those who were not (median: 272 U/mL vs. 2660 U/mL, p = 0.0002), and serum immunoglobulin G (IgG) levels showed a weak linear relationship with antibody titers (R2 = 0.16). No breakthrough infections were noted. Three patients (7.5%) suffered from a relapse of nephrotic syndrome (2 and 3 days, respectively, after the first dose and 8 days after the second dose), two of whom had a history of relapse within 6 months before the vaccination. CONCLUSIONS: The SARS-CoV-2 mRNA vaccine was immunogenic in patients with nephrotic syndrome using immunosuppressive agents, although the use of MMF and low levels of serum IgG were associated with lower antibody titers after vaccination. Patients with high disease activity may experience a relapse of nephrotic syndrome after vaccination. A higher resolution version of the Graphical abstract is available as Supplementary information.

Analysis of Antibody Responses After COVID-19 Vaccination in Liver Transplant Recipients: A Single-Center Study.

BACKGROUND: Liver transplant (LT) recipients were considered a vulnerable population during the coronavirus disease 2019 (COVID-19) pandemic. The clinical efficacy of the COVID-19 vaccine is unknown in immunocompromised patients. The purpose of this study was to provide evidence of antibody responses after COVID-19 vaccination in LT recipients. METHODS: This study enrolled 46 patients who underwent LT at Samsung Medical Center (Seoul, Korea) before implementation of the one-dose vaccine in Korea. Those who completed the two-dose COVID-19 vaccine between August 2021 and September 2021 were included and followed through December 2021. Semiquantitative anti-spike serologic testing was performed using the Roche Elecsys anti-SARS-CoV-2 S enzyme immunoassay (Roche Diagnostics, Rotkereuz, Switzerland) with a positive cutoff of at least 0.8 U/mL. RESULTS: Among all 46 participants, 40 (87%) demonstrated an antibody response after the second dose of a COVID-19 vaccine, while six (13%) had no antibody response after the second dose. Upon univariate analysis, patients with higher antibody titer had longer years since LT (2.3 ± 2.8 vs. 9.4 ± 5.0, P < 0.001). A lower median tacrolimus (TAC) level before vaccination and after the second dose of COVID-19 vaccine indicated a significantly higher antibody response (2.3 [1.6-3.2] vs. 7.0 [3.7-7.8], P = 0.006, 2.5 [1.6-3.3] vs. 5.7 [4.2-7.2], P = 0.003). Period between 2nd vaccination and serologic testing was significantly higher in the antibody-response group compared to the no-antibody-response group (30.2 ± 24.0 vs. 65.9 ± 35.0, P = 0.012). A multivariate analysis of antibody responses revealed TAC level before vaccination as a statistically significant factor. CONCLUSION: A higher TAC level before vaccination resulted in less effective vaccination in LT patients. Booster vaccinations are required, especially for patients in the early stage after LT who have compromised immune function.

Anti-S Antibodies Against SARS-CoV-2 Infection Among Four Types of Vaccines in Malaysia.

Monitoring SARS-CoV-2 antibody levels can provide insights into a person's immunity to COVID-19 and inform decisions about vaccination and public health measures. Anti-S may be useful as an indicator of an effective immune response. Thus, we conducted this study that aimed to determine the immune response of anti-S antibodies against SARS-CoV-2 for all the vaccine types over time among adult recipients in Malaysia and to determine the associated factors. This study was a cohort that recruited 2513 respondents aged 18 years and above from June to December 2021. Each participant was followed-up for 1-year period from the initial vaccine dose (baseline). We found that the anti-S antibody generally increased for all vaccine types and peaked at two weeks after the second dose vaccination, with Pfizer recipients having the highest median of 100 (100.00-100.00). During the third-month follow-up, the seropositivity of anti-S antibody and the median level decreased for all vaccines. We found that type of vaccines, comorbid status, infection, and booster status were significantly associated with the anti-S antibody level after one year.

Performance of Three Anti-SARS-CoV-2 Anti-S and One Anti-N Immunoassays for the Monitoring of Immune Status and Vaccine Response.

This study aimed to evaluate and compare the performance of three anti-S and one anti-N assays that were available to the project in detecting antibody levels after three commonly used SARS-CoV-2 vaccines (Pfizer, Moderna, and Johnson & Johnson). It also aimed to assess the association of age, sex, race, ethnicity, vaccine timing, and vaccine side effects on antibody levels in a cohort of 827 individuals. In September 2021, 698 vaccinated individuals donated blood samples as part of the Association for Diagnostics & Laboratory Medicine (ADLM) COVID-19 Immunity Study. These individuals also participated in a comprehensive survey covering demographic information, vaccination status, and associated side effects. Additionally, 305 age- and gender-matched samples were obtained from the ADLM 2015 sample bank as pre-COVID-19-negative samples. All these samples underwent antibody level analysis using three anti-S assays, namely Beckman Access SARS-CoV-2 IgG (Beckman assay), Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG (Ortho assay), Siemens ADVIA Centaur SARS-CoV-2 IgG (Siemens assay), and one anti-N antibody assay: Bio-Rad Platelia SARS-CoV-2 Total Ab assay (BioRad assay). A total of 827 samples (580 COVID-19 samples and 247 pre-COVID-19 samples) received results for all four assays and underwent further analysis. Beckman, Ortho, and Siemens anti-S assays showed an overall sensitivity of 99.5%, 97.6%, and 96.9%, and specificity of 90%, 100%, and 99.6%, respectively. All three assays indicated 100% sensitivity for individuals who received the Moderna vaccine and boosters, and over 99% sensitivity for the Pfizer vaccine. Sensitivities varied from 70.4% (Siemens), 81.5% (Ortho), and 96.3% (Beckman) for individuals who received the Johnson & Johnson vaccine. BioRad anti-N assays demonstrated 46.2% sensitivity and 99.25% specificity based on results from individuals with self-reported infection. The highest median anti-S antibody levels were measured in individuals who received the Moderna vaccine, followed by Pfizer and then Johnson & Johnson vaccines. Higher anti-S antibody levels were significantly associated with younger age and closer proximity to the last vaccine dose but were not associated with gender, race, or ethnicity. Participants with higher anti-S levels experienced significantly more side effects as well as more severe side effects (e.g., muscle pain, chills, fever, and moderate limitations) (p < 0.05). Anti-N antibody levels only indicated a significant correlation with headache. This study indicated performance variations among different anti-S assays, both among themselves and when analyzing individuals with different SARS-CoV-2 vaccines. Caution should be exercised when conducting large-scale studies to ensure that the same platform and/or assays are used for the most effective interpretation of the data.

Post-Vaccination Detection of SARS-CoV-2 Antibody Response with Magnetic Nanoparticle-Based Electrochemical Biosensor System.

Here, we report magnetic nanoparticle-based biosensor platforms for the rapid detection of SARS-CoV-2 antibody responses in human serum. The use of the proposed system enabled the detection of anti-SARS-CoV-2 spike (S) and nucleocapsid (N) proteins at a concentration of ng/mL in both buffer and real serum samples. In particular, the protocol, which is considered an indicator of innate immunity after vaccination or post-infection, could be useful for the evaluation of antibody response. We included a total of 48 volunteers who either had COVID-19 but were not vaccinated or who had COVID-19 and were vaccinated with CoronoVac or Biontech. Briefly, in this study, which was planned as a cohort, serum samples were examined 3, 6, and 12 months from the time the volunteers' showed symptoms of COVID-19 with respect to antibody response in the proposed system. Anti-S Ab and anti-N Ab were detected with a limit of detection of 0.98 and 0.89 ng/mL, respectively. These data were confirmed with the corresponding commercial an electrochemiluminescence immunoassay (ECLIA) assays. Compared with ECLIA, more stable data were obtained, especially for samples collected over 6 months. After this period, a drop in the antibody responses was observed. Our findings showed that it could be a useful platform for exploring the dynamics of the immune response, and the proposed system has translational use potential for the clinic. In conclusion, the MNP-based biosensor platform proposed in this study, together with its counterparts in previous studies, is a candidate for determining natural immunity and post-vaccination antibody response, as well as reducing the workload of medical personnel and paving the way for screening studies on vaccine efficacy.

Short Research Communication Anti-Spike Antibody Response to COVISHIELD™ (SII-ChAdOx1 nCoV-19) Vaccine in Patients with B-Cell and Plasma Cell Malignancies and Hematopoietic Cell Transplantation Recipients.

Introduction: There is limited data on the serologic antibody responses after the ChAdOx1 vaccine in patients with hematological malignancies and hematopoietic cell transplantation recipients. There is no data on the safety and efficacy of the Indian COVISHIELD™ vaccine in this population. Methods: This study reports the anti-S antibody response to the COVISHIELD™ vaccine in a prospective cohort of patients with B-cell and plasma cell malignancies and HCT recipients at a single center. The quantitative antibodies to the SARS-CoV-2 S protein receptor-binding domain in human plasma were determined by the validated Roche Elecsys Anti-SARS-CoV-2 S kit. Results: A total of 118 patients were included over the study period from April 2021 to August 2021. The seropositivity rate at baseline and after the first and second dose of the vaccine was 39%, 66%, and 79%, respectively (p < 0.0001). The seronegative cohort had a higher median age (65 vs. 60 years, p = 0.03), were more likely to be males (81% vs. 42%, p = 0.009), had a diagnosis of B-CLPD (100% vs. 42%, p < 0.001) and were more likely to be on ibrutinib therapy (56% vs. 15%, p = 0.001). Conclusions: This study confirms the safety and efficacy of the COVISHIELD™ vaccine in patients with hematological malignancies.

Treatment with Sotrovimab and Casirivimab/Imdevimab Enhances Serum SARS-CoV-2 S Antibody Levels in Patients Infected with the SARS-CoV-2 Delta, Omicron BA.1, and BA.5 Variants.

Background: The neutralizing ability of sotrovimab and casirivimab/imdevimab against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is attenuated in the subvariant BA.5. However, the efficacy of sotrovimab in the clinical setting remains to be investigated. Methods: Patients admitted to Kishiwada City Hospital with COVID-19 delta, omicron BA.1, or BA.5 subvariants were evaluated retrospectively for serum SARS-CoV-2 S and N antibody levels using the Elecsys Anti-SARS-CoV-2 assay. Results: In patients with COVID-19 during the BA.5 wave of the COVID-19 pandemic, anti-SARS-CoV-2 S antibody titers (median [interquartile range]) increased from 2154.0 (864.0−6669.3) U/mL on day 0 to 21,371.0 (19,656.3−32,225.0) U/mL on day 3 in the group treated with sotrovimab (N = 40) and were significantly higher than in the group treated with remdesivir plus dexamethasone plus baricitinib (p < 0.001). Conclusion: Treatment with sotrovimab could prevent severe disease in high-risk patients infected with SARS-CoV-2 subvariant BA.5.

Immunogenicity of Third-dose BNT162b2 mRNA Vaccine Following Two Doses of ChAdOx1 in Health Care Workers: A Prospective Longitudinal Study.

Following the original severe acute respiratory syndrome coronavirus 2 strain (Wuhan-Hu-1) in December 2019, the Delta variant in May 2021 and the Omicron variant in December 2021 were classified as variants of concern. The pandemic has been ongoing for more than two years, and the three-dose vaccination rate has reached approximately 50% in Korea. We analyzed anti-S antibodies (Abs) and neutralizing Abs (NAbs) in 32 healthcare workers at a university hospital, focusing on the first to third doses of ChAdOx1-ChAdOx1-BNT162b2, which is the most common vaccination regimen in Korea. Antibodies were analyzed at eight time points according to the vaccine regimen. The first to third doses of ChAdOx1-ChAdOx1-BNT162b2 produced high Ab concentrations; NAb concentrations after the third dose were predicted to remain high for a longer period than those after the first and second doses. The effectiveness of a second dose of ChAdOx1 in the real world was demonstrated by analyzing samples collected during an outbreak that occurred in the study period, 4-5 months after the second dose. The relative risk ratio was 88.0%, and the efficacy of the second ChAdOx1 dose was 12.0% (P<0.05). Therefore, maintaining appropriate Ab concentrations through regular vaccination will help protect against coronavirus disease-19.

High Anti-CoV2S Antibody Levels at Hospitalization Are Associated with Improved Survival in Patients with COVID-19 Vaccine Breakthrough Infection.

BACKGROUND: Although vaccination against COVID-19 is highly effective, breakthrough infections occur, often leading to severe courses and death. The extent of protection provided by individual antibody levels in breakthrough infections is still unknown and cut-off levels have yet to be determined. METHODS: In 80 consecutive fully vaccinated patients hospitalized between August and December 2021 with COVID-19 breakthrough infection (Delta variant), anti-CoV2S antibody levels were analyzed for the endpoint of death. RESULTS: Ten out of the 12 patients who died (83.3%) had antibody levels < 600 U/mL; 5 (41.7%) of these had antibody levels < 200 U/mL. Only 2 patients with a level of >600 U/mL died from vaccine breakthrough infection. Correction for the number of comorbidities and age revealed that anti-CoV2S antibody levels at the time of hospitalization were a significant predictor for reduced risk of death (OR = 0.402 for every 1000 U/mL, p = 0.018). CONCLUSIONS: In this retrospective data analysis, we show that almost all patients who died from COVID-19 vaccine breakthrough infection had antibody levels < 600 U/mL, most of them below 200 U/mL. In logistic regression corrected for the number of comorbidities and age, anti-CoV2S antibody levels at the time of hospitalization proved to be a significantly protective predictor against death.

Benefits of Switching Mycophenolic Acid to Sirolimus on Serological Response after a SARS-CoV-2 Booster Dose among Kidney Transplant Recipients: A Pilot Study.

Kidney transplant recipients (KTRs) have a suboptimal immune response to COVID-19 vaccination due to the effects of immunosuppression, mostly mycophenolic acid (MPA). This study investigated the benefits of switching from the standard immunosuppressive regimen (tacrolimus (TAC), MPA, and prednisolone) to a regimen of mammalian target of rapamycin inhibitor (mTORi), TAC and prednisolone two weeks pre- and two weeks post-BNT162b2 booster vaccination. A single-center, opened-label pilot study was conducted in KTRs, who received two doses of ChAdOx-1 and a single dose of BNT162b2. The participants were randomly assigned to continue the standard regimen (control group, n = 14) or switched to a sirolimus (an mTORi), TAC, and prednisolone (switching group, n = 14) regimen two weeks before and two weeks after receiving a booster dose of BNT162b2. The anti-SARS-CoV-2 S antibody level after vaccination in the switching group was significantly greater than the control group (4051.0 [IQR 3142.0-6466.0] BAU/mL vs. 2081.0 [IQR 1077.0-3960.0] BAU/mL, respectively; p = 0.01). One participant who was initially seronegative in the control group remained seronegative after the booster dose. These findings suggest humoral immune response benefits of switching the standard immunosuppressive regimen to the regimen of mTORi, TAC, and prednisolone in KTRs during vaccination.

Differences in SARS-CoV-2-Specific Antibody Responses After the First, Second, and Third Doses of BNT162b2 in Naïve and Previously Infected Individuals: A 1-Year Observational Study in Healthcare Professionals.

Background: Safe and effective vaccines against COVID-19 are critical for preventing the spread of SARS-CoV-2, but little is known about the humoral immune response more than 9 months after vaccination. We aimed to assess the humoral immune response after the first, second, and third (booster) doses of BNT162b2 vaccine in SARS-CoV-2 naïve and previously infected healthcare professionals (HCP) and the humoral immune response after infection in vaccinated HCP. Methods: We measured anti-spike (anti-S) and anti-nucleocapsid antibodies at different time points up to 12 months in the sera of 300 HCP who had received two or three doses of BNT162b2 vaccine. Mixed-model analyses were used to assess anti-S antibody dynamics and to determine their predictors (age, sex, BMI, and previous infection). Results: Naïve individuals had statistically lower anti-S antibody concentrations after the first dose (median 253 BAU/ml) than previously infected individuals (median 3648 BAU/ml). After the second dose, anti-S antibody concentrations increased in naïve individuals (median 3216 BAU/ml), whereas the second dose did not significantly increase concentrations in previously infected individuals (median 4503 BAU/ml). The third dose resulted in an additional increase in concentrations (median 4844 BAU/ml in naïve and median 5845 BAU/ml in previously infected individuals). Anti-S antibody concentrations steadily decreased after the second dose and after the third dose in naïve and previously infected individuals. In addition, we found that age had an effect on the humoral immune response. Younger individuals had higher anti-S antibody concentrations after the first and second doses. After infection with the new variant Omicron, a further increase in anti-S antibody concentrations to a median value of 4794 BAU/ml was observed in three times vaccinated HCP whose anti-S antibody concentrations were relatively high before infection (median 2141 BAU/ml). Our study also showed that individuals with systemic adverse events achieved higher anti-S antibody concentrations. Conclusion: In this study, significant differences in humoral immune responses to BNT162b2 vaccine were observed between naïve and previously infected individuals, with age playing an important role, suggesting that a modified vaccination schedule should be practiced in previously infected individuals. In addition, we showed that the high anti-S antibodies were not protective against new variants of SARS-CoV-2.

Anti-human protein S antibody induces tissue factor expression through a direct interaction with platelet phosphofructokinase.

INTRODUCTION: Autoantibodies including anti-human protein S antibody (anti-hPS Ab) and anti-human protein C antibody (anti-hPC Ab) can be detected in patients with autoimmune diseases with hypercoagulability. The objective of the present study was to determine the effects and molecular pathways of these autoantibodies on tissue factor (TF) expression in human coronary artery endothelial cells (HCAECs). MATERIALS AND METHODS: HCAECs were treated with anti-hPS Ab or anti-hPC Ab for 3 hours. TF expression was measured by real-time PCR and Western blot. TF-mediated procoagulant activity was determined by a commercial kit. MAPK phosphorylation was analyzed by Bio-Plex luminex immunoassay and Western blot. The potential proteins interacting with anti-hPS Ab were studied by immunoprecipitation, mass spectrometry and in vitro pull-down assay. RESULTS: Anti-hPS Ab, but not anti-hPC Ab, specifically induced TF expression and TF-mediated procoagulant activity in HCAECs in a concentration-dependent manner. This effect was confirmed in human umbilical endothelial cells (HUVECs). ERK1/2 phosphorylation was induced by anti-hPS Ab treatment, while inhibition of ERK1/2 by U0216 partially blocked anti-hPS Ab-induced TF upregulation (P<0.05). In addition, anti-hPS Ab specifically cross-interacted with platelet phosphofructokinase (PFKP) in HCAECs. Anti-hPS Ab was able to directly inhibit PFKP activities in HCAECs. Furthermore, silencing of PFKP by PFKP shRNA resulted in TF upregulation in HCAECs, while activation of PFKP by fructose-6-phosphate partially blocked the effect of anti-hPS Ab on TF upregulation (P<0.05). CONCLUSIONS: Anti-hPS Ab induces TF expression through a direct interaction with PFKP and ERK1/2 activation in HCAECs. Anti-hPS Ab may directly contribute to vascular thrombosis in the patient with autoimmune disorders.