Association between IgG responses against the nucleocapsid proteins of alphacoronaviruses and COVID-19 severity.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Comparable cellular and humoral immunity upon homologous and heterologous COVID-19 vaccination regimens in kidney transplant recipients.

Background: Kidney transplant recipients (KTRs) are at high risk for a severe course of coronavirus disease 2019 (COVID-19); thus, effective vaccination is critical. However, the achievement of protective immunogenicity is hampered by immunosuppressive therapies. We assessed cellular and humoral immunity and breakthrough infection rates in KTRs vaccinated with homologous and heterologous COVID-19 vaccination regimens. Method: We performed a comparative in-depth analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses using multiplex Fluorospot assays and SARS-CoV-2-specific neutralizing antibodies (NAbs) between three-times homologously (n = 18) and heterologously (n = 8) vaccinated KTRs. Results: We detected SARS-CoV-2-reactive T cells in 100% of KTRs upon third vaccination, with comparable frequencies, T-cell expression profiles, and relative interferon γ and interleukin 2 production per single cell between homologously and heterologously vaccinated KTRs. SARS-CoV-2-specific NAb positivity rates were significantly higher in heterologously (87.5%) compared to homologously vaccinated (50.0%) KTRs (P < 0.0001), whereas the magnitudes of NAb titers were comparable between both subcohorts after third vaccination. SARS-CoV-2 breakthrough infections occurred in equal numbers in homologously (38.9%) and heterologously (37.5%) vaccinated KTRs with mild-to-moderate courses of COVID-19. Conclusion: Our data support a more comprehensive assessment of not only humoral but also cellular SARS-CoV-2-specific immunity in KTRs to provide an in-depth understanding about the COVID-19 vaccine-induced immune response in a transplant setting.

RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers.

The ability of antibodies to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important correlate of protection. For routine evaluation of protection, however, a simple and cost-efficient anti-SARS-CoV-2 serological assay predictive of serum neutralizing activity is needed. We analyzed clinical epidemiological data and blood samples from two cohorts of health care workers in Barcelona and Munich to compare several immunological readouts for evaluating antibody levels that could be surrogates of neutralizing activity. We measured IgG levels against SARS-CoV-2 spike protein (S), its S2 subunit, the S1 receptor binding domain (RBD), and the full length and C terminus of nucleocapsid (N) protein by Luminex, and against RBD by enzyme-linked immunosorbent assay (ELISA), and assessed those as predictors of plasma surrogate-neutralizing activity measured by a flow cytometry assay. In addition, we determined the clinical and demographic factors affecting plasma surrogate-neutralizing capacity. Both cohorts showed a high positive correlation between IgG levels to S antigen, especially to RBD, and the levels of plasma surrogate-neutralizing activity, suggesting RBD IgG as a good correlate of plasma neutralizing activity. Symptomatic infection, with symptoms such as loss of taste, dyspnea, rigors, fever and fatigue, was positively associated with anti-RBD IgG positivity by ELISA and Luminex, and with plasma surrogate-neutralizing activity. Our serological assays allow for the prediction of serum neutralization activity without the cost, hazards, time, and expertise needed for surrogate or conventional neutralization assays. Once a cutoff is established, these relatively simple high-throughput antibody assays will provide a fast and cost-effective method of assessing levels of protection from SARS-CoV-2 infection. IMPORTANCE Neutralizing antibody titers are the best correlate of protection against SARS-CoV-2. However, current tests to measure plasma or serum neutralizing activity do not allow high-throughput screening at the population level. Serological tests could be an alternative if they are proved to be good predictors of plasma neutralizing activity. In this study, we analyzed the SARS-CoV-2 serological profiles of two cohorts of health care workers by applying Luminex and ELISA in-house serological assays. Correlations of both serological tests were assessed between them and with a flow cytometry assay to determine plasma surrogate-neutralizing activity. Both assays showed a high positive correlation between IgG levels to S antigens, especially RBD, and the levels of plasma surrogate-neutralizing activity. This result suggests IgG to RBD as a good correlate of plasma surrogate-neutralizing activity and indicates that serology of IgG to RBD could be used to assess levels of protection from SARS-CoV-2 infection.

Dynamics of humoral and cellular immune responses after homologous and heterologous SARS-CoV-2 vaccination with ChAdOx1 nCoV-19 and BNT162b2.

BACKGROUND: Vaccines are an important means to overcome the SARS-CoV-2 pandemic. They induce specific antibody and T-cell responses but it remains open how well vaccine-induced immunity is preserved over time following homologous and heterologous immunization regimens. Here, we compared the dynamics of humoral and cellular immune responses up to 180 days after homologous or heterologous vaccination with either ChAdOx1-nCoV-19 (ChAd) or BNT162b2 (BNT) or both. METHODS: Various tests were used to determine the humoral and cellular immune response. To quantify the antibody levels, we used the surrogate neutralization (sVNT) assay from YHLO, which we augmented with pseudo- and real virus neutralization tests (pVNT and rVNT). Antibody avidity was measured by a modified ELISA. To determine cellular reactivity, we used an IFN-γ Elispot, IFN-γ/IL Flurospot, and intracellular cytokine staining. FINDINGS: Antibody responses significantly waned after vaccination, irrespective of the regimen. The capacity to neutralize SARS-CoV-2 - including variants of concern such as Delta or Omicron - was superior after heterologous compared to homologous BNT vaccination, both of which resulted in longer-lasting humoral immunity than homologous ChAd immunization. All vaccination regimens induced stable, polyfunctional T-cell responses. INTERPRETATION: These findings demonstrate that heterologous vaccination with ChAd and BNT is a potent alternative to induce humoral and cellular immune protection in comparison to the homologous vaccination regimens. FUNDING: The study was funded by the German Centre for Infection Research (DZIF), the European Union's "Horizon 2020 Research and Innovation Programme" under grant agreement No. 101037867 (VACCELERATE), the "Bayerisches Staatsministerium für Wissenschaft und Kunst" for the CoVaKo-2021 and the For-COVID projects and the Helmholtz Association via the collaborative research program "CoViPa". Further support was obtained from the Federal Ministry of Education and Science (BMBF) through the "Netzwerk Universitätsmedizin", project "B-Fast" and "Cov-Immune". KS is supported by the German Federal Ministry of Education and Research (BMBF, 01KI2013) and the Else Kröner-Stiftung (2020_EKEA.127).

Association between IgG responses against the nucleocapsid proteins of alphacoronaviruses and COVID-19 severity

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Infection Control Measures and Prevalence of SARS-CoV-2 IgG among 4,554 University Hospital Employees, Munich, Germany.

Hospital staff are at high risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the coronavirus disease (COVID-19) pandemic. This cross-sectional study aimed to determine the prevalence of SARS-CoV-2 infection in hospital staff at the University Hospital rechts der Isar in Munich, Germany, and identify modulating factors. Overall seroprevalence of SARS-CoV-2-IgG in 4,554 participants was 2.4%. Staff engaged in direct patient care, including those working in COVID-19 units, had a similar probability of being seropositive as non-patient-facing staff. Increased probability of infection was observed in staff reporting interactions with SARS-CoV-2‒infected coworkers or private contacts or exposure to COVID-19 patients without appropriate personal protective equipment. Analysis of spatiotemporal trajectories identified that distinct hotspots for SARS-CoV-2‒positive staff and patients only partially overlap. Patient-facing work in a healthcare facility during the SARS-CoV-2 pandemic might be safe as long as adequate personal protective equipment is used and infection prevention practices are followed inside and outside the hospital.

Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern.

Infection-neutralizing antibody responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 vaccination are an essential component of antiviral immunity. Antibody-mediated protection is challenged by the emergence of SARS-CoV-2 variants of concern (VoCs) with immune escape properties, such as omicron (B.1.1.529), which is rapidly spreading worldwide. Here we report neutralizing antibody dynamics in a longitudinal cohort of coronavirus disease 2019 convalescent and infection-naive individuals vaccinated with mRNA BNT162b2 by quantifying SARS-CoV-2 spike protein antibodies and determining their avidity and neutralization capacity in serum. Using live-virus neutralization assays, we show that a superior infection-neutralizing capacity against all VoCs, including omicron, developed after either two vaccinations in convalescents or a third vaccination or breakthrough infection of twice-vaccinated, naive individuals. These three consecutive spike antigen exposures resulted in an increasing neutralization capacity per anti-spike antibody unit and were paralleled by stepwise increases in antibody avidity. We conclude that an infection-plus-vaccination-induced hybrid immunity or a triple immunization can induce high-quality antibodies with superior neutralization capacity against VoCs, including omicron.

Dynamics of spike-and nucleocapsid specific immunity during long-term follow-up and vaccination of SARS-CoV-2 convalescents.

Anti-viral immunity continuously declines over time after SARS-CoV-2 infection. Here, we characterize the dynamics of anti-viral immunity during long-term follow-up and after BNT162b2 mRNA-vaccination in convalescents after asymptomatic or mild SARS-CoV-2 infection. Virus-specific and virus-neutralizing antibody titers rapidly declined in convalescents over 9 months after infection, whereas virus-specific cytokine-producing polyfunctional T cells persisted, among which IL-2-producing T cells correlated with virus-neutralizing antibody titers. Among convalescents, 5% of individuals failed to mount long-lasting immunity after infection and showed a delayed response to vaccination compared to 1% of naïve vaccinees, but successfully responded to prime/boost vaccination. During the follow-up period, 8% of convalescents showed a selective increase in virus-neutralizing antibody titers without accompanying increased frequencies of circulating SARS-CoV-2-specific T cells. The same convalescents, however, responded to vaccination with simultaneous increase in antibody and T cell immunity revealing the strength of mRNA-vaccination to increase virus-specific immunity in convalescents.

Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection.

T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs-classic features of protective immunity-are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.

Longitudinal Assessment of Health and Quality of Life of COVID-19 Patients Requiring Intensive Care-An Observational Study.

Long-term health consequences in survivors of severe COVID-19 remain unclear. Eighteen COVID-19 patients admitted to the intensive care unit at the University Hospital Rechts der Isar, Munich, Germany, between 14 March and 23 June 2020, were prospectively followed-up at a median of 36, 75.5, 122 and 222 days after discharge. The health-related quality of life (HrQoL) (36-item Short Form Health Survey and St. George's Respiratory Questionnaire, SGRQ), cardiopulmonary function, laboratory parameters and chest imaging were assessed longitudinally. The HrQoL assessment revealed a reduced physical functioning, as well as increased SGRQ impact and symptoms scores that all improved over time but remained markedly impaired compared to the reference groups. The median radiological severity scores significantly declined; persistent abnormalities were found in 33.3% of the patients on follow-up. A reduced diffusion capacity was the most common abnormal pulmonary function parameter. The length of hospitalization correlated with role limitations due to physical problems, the SGRQ symptom and the impact score. In conclusion, in survivors of severe COVID-19, the pulmonary function and symptoms improve over time, but impairments in their physical function and diffusion capacity can persist over months. Longer follow-up studies with larger cohorts will be necessary to comprehensively characterize long-term sequelae upon severe COVID-19 and to identify patients at risk.

Phantosmia, Parosmia, and Dysgeusia Are Prolonged and Late-Onset Symptoms of COVID-19.

Deficiencies in smell and taste are common symptoms of COVID-19. Quantitative losses are well surveyed. This study focuses on qualitative changes such as phantosmia (hallucination of smell), parosmia (alteration of smell), and dysgeusia (alteration of taste) and possible connections with the adaptive immune system. Subjective experience of deficiency in taste and smell was assessed by two different questionnaires after a median of 100 and 244 days after first positive RT-PCR test. SARS-CoV-2-specific antibody levels were measured with the iFlash-SARS-CoV-2 assay. After 100 days a psychophysical screening test for olfactory and gustatory dysfunction was administered. 30 of 44 (68.2%) participants reported a chemosensory dysfunction (14 quantitative, 6 qualitative, 10 quantitative, and qualitative) during COVID-19, eleven (25.0%) participants (1 quantitative, 7 qualitative, 3 quantitative, and quantity) after 100 days, and 14 (31.8%) participants (1 quantitative, 10 qualitative, 3 quantitative and qualitative) after 244 days. Four (9.1%) participants, who were symptom-free after 100 days reported now recently arisen qualitative changes. Serological and T-cell analysis showed no correlation with impairment of taste and smell. In conclusion, qualitative changes can persist for several months and occur as late-onset symptoms months after full recovery from COVID-19-induced quantitative losses in taste and smell.

Convalescent plasma therapy in B-cell-depleted and B-cell sufficient patients with life-threatening COVID-19 - A case series.

OBJECTIVE: To investigate the effect of convalescent plasma therapy (CPT) on clinical courses of B-cell-sufficient and B-cell-depleted patients with life-threatening COVID-19. PATIENTS AND METHODS: In this case series, we retrospectively analysed clinical, laboratory and cardiopulmonary parameters of six patients with life-threatening COVID-19 receiving convalescent plasma (CP) as rescue therapy between April 11, 2020 to October 10, 2020. Clinical and laboratory parameters before and after transfusion were compared in two B-cell-depleted patients and four B-cell sufficient patients (control group). RESULTS: Both B-cell-depleted patients cleared SARS-CoV-2 virus and survived, while all other patients died within 14 days from intervention despite maximal therapeutic efforts. D-dimer levels increased in both cohorts subsequent to CPT. In control patients, mean Interleukin-6 increased and platelet levels decreased as opposed to decreasing and stable levels in B-cell-depleted patients, respectively. Control patients required increased doses of vasopressor compared to decreasing doses in B-cell depleted patients subsequent to CPT. PO2/FiO2 decrease was more pronounced and respiratory deterioration required postinterventional extracorporeal membrane oxygenation in two control patients. Transpulmonary thermodilution revealed a further increase of the Extravascular Lung Water Index upon CPT in control patients. CONCLUSION: Use of CP in late stages of life-threatening COVID-19 should be used with caution but may be beneficial in B-cell-depleted patients. Further studies are necessary to assess factors predicting potential therapeutic benefits as well as possible hazards.

SARS-CoV-2 serology increases diagnostic accuracy in CT-suspected, PCR-negative COVID-19 patients during pandemic.

BACKGROUND: In the absence of PCR detection of SARS-CoV-2 RNA, accurate diagnosis of COVID-19 is challenging. Low-dose computed tomography (CT) detects pulmonary infiltrates with high sensitivity, but findings may be non-specific. This study assesses the diagnostic value of SARS-CoV-2 serology for patients with distinct CT features but negative PCR. METHODS: IgM/IgG chemiluminescent immunoassay was performed for 107 patients with confirmed (group A: PCR + ; CT ±) and 46 patients with suspected (group B: repetitive PCR-; CT +) COVID-19, admitted to a German university hospital during the pandemic's first wave. A standardized, in-house CT classification of radiological signs of a viral pneumonia was used to assess the probability of COVID-19. RESULTS: Seroconversion rates (SR) determined on day 5, 10, 15, 20 and 25 after symptom onset (SO) were 8%, 25%, 65%, 76% and 91% for group A, and 0%, 10%, 19%, 37% and 46% for group B, respectively; (p < 0.01). Compared to hospitalized patients with a non-complicated course (non-ICU patients), seroconversion tended to occur at lower frequency and delayed in patients on intensive care units. SR of patients with CT findings classified as high certainty for COVID-19 were 8%, 22%, 68%, 79% and 93% in group A, compared with 0%, 15%, 28%, 50% and 50% in group B (p < 0.01). SARS-CoV-2 serology established a definite diagnosis in 12/46 group B patients. In 88% (8/9) of patients with negative serology > 14 days after symptom onset (group B), clinico-radiological consensus reassessment revealed probable diagnoses other than COVID-19. Sensitivity of SARS-CoV-2 serology was superior to PCR > 17d after symptom onset. CONCLUSIONS: Approximately one-third of patients with distinct COVID-19 CT findings are tested negative for SARS-CoV-2 RNA by PCR rendering correct diagnosis difficult. Implementation of SARS-CoV-2 serology testing alongside current CT/PCR-based diagnostic algorithms improves discrimination between COVID-19-related and non-related pulmonary infiltrates in PCR negative patients. However, sensitivity of SARS-CoV-2 serology strongly depends on the time of testing and becomes superior to PCR after the 2nd week following symptom onset.