Nucleocapsid-specific T cell responses associate with control of SARS-CoV-2 in the upper airways before seroconversion.

Despite intensive research since the emergence of SARS-CoV-2, it has remained unclear precisely which components of the early immune response protect against the development of severe COVID-19. Here, we perform a comprehensive immunogenetic and virologic analysis of nasopharyngeal and peripheral blood samples obtained during the acute phase of infection with SARS-CoV-2. We find that soluble and transcriptional markers of systemic inflammation peak during the first week after symptom onset and correlate directly with upper airways viral loads (UA-VLs), whereas the contemporaneous frequencies of circulating viral nucleocapsid (NC)-specific CD4+ and CD8+ T cells correlate inversely with various inflammatory markers and UA-VLs. In addition, we show that high frequencies of activated CD4+ and CD8+ T cells are present in acutely infected nasopharyngeal tissue, many of which express genes encoding various effector molecules, such as cytotoxic proteins and IFN-γ. The presence of IFNG mRNA-expressing CD4+ and CD8+ T cells in the infected epithelium is further linked with common patterns of gene expression among virus-susceptible target cells and better local control of SARS-CoV-2. Collectively, these results identify an immune correlate of protection against SARS-CoV-2, which could inform the development of more effective vaccines to combat the acute and chronic illnesses attributable to COVID-19.

Impact of Omicron Variant Infection on Assessment of Spike-Specific Immune Responses Using the EUROIMMUN Quan-T-Cell SARS-CoV-2 Assay and Roche Elecsys Anti-SARS-CoV-2-S.

The currently prevailing variants of SARS-CoV-2 are subvariants of the Omicron variant. The aim of this study was to analyze the effect of mutations in the Spike protein of Omicron on the results Quan-T-Cell SARS-CoV-2 assays and Roche Elecsys anti-SARS-CoV-2 anti-S1. Omicron infected subjects ((n = 37), vaccinated (n = 20) and unvaccinated (n = 17)) were recruited approximately 3 weeks after a positive PCR test. The Quan-T-Cell SARS-CoV-2 assays (EUROIMMUN) using Wuhan and the Omicron adapted antigen assay and a serological test (Roche Elecsys anti-SARS-CoV-2 anti-S1) were performed. Using the original Wuhan SARS-CoV-2 IGRA TUBE, in 19 of 21 tested Omicron infected subjects, a positive IFNy response was detected, while 2 non-vaccinated but infected subjects did not respond. The Omicron adapted antigen tube resulted in comparable results. In contrast, the serological assay detected a factor 100-fold lower median Spike-specific RBD antibody concentration in non-vaccinated Omicron infected patients (n = 12) compared to patients from the pre Omicron era (n = 12) at matched time points, and eight individuals remained below the detection threshold for positivity. For vaccinated subjects, the Roche assay detected antibodies in all subjects and showed a 400 times higher median specific antibody concentration compared to non-vaccinated infected subjects in the pre-Omicron era. Our results suggest that Omicron antigen adapted IGRA stimulator tubes did not improve detection of SARS-CoV-2-specific T-cell responses in the Quant-T-Cell-SARS-CoV-2 assay. In non-vaccinated Omicron infected individuals, the Wuhan based Elecsys anti-SARS-CoV-2 anti-S1 serological assay results in many negative results at 3 weeks after diagnosis.

Enhanced Spike-specific, but attenuated Nucleocapsid-specific T cell responses upon SARS-CoV-2 breakthrough versus non-breakthrough infections.

SARS-CoV-2 vaccine breakthrough infections frequently occurred even before the emergence of Omicron variants. Yet, relatively little is known about the impact of vaccination on SARS-CoV-2-specific T cell and antibody response dynamics upon breakthrough infection. We have therefore studied the dynamics of CD4 and CD8 T cells targeting the vaccine-encoded Spike and the non-encoded Nucleocapsid antigens during breakthrough infections (BTI, n=24) and in unvaccinated control infections (non-BTI, n=30). Subjects with vaccine breakthrough infection had significantly higher CD4 and CD8 T cell responses targeting the vaccine-encoded Spike during the first and third/fourth week after PCR diagnosis compared to non-vaccinated controls, respectively. In contrast, CD4 T cells targeting the non-vaccine encoded Nucleocapsid antigen were of significantly lower magnitude in BTI as compared to non-BTI. Hence, previous vaccination was linked to enhanced T cell responses targeting the vaccine-encoded Spike antigen, while responses against the non-vaccine encoded Nucleocapsid antigen were significantly attenuated.

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection.

The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remains unclear. Here, we combine a multi-omics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype associated with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies.

The interplay of viral loads, clinical presentation, and serological responses in SARS-CoV-2 - Results from a prospective cohort of outpatient COVID-19 cases.

Risk factors for disease progression and severity of SARS-CoV-2 infections require an understanding of acute and long-term virological and immunological dynamics. Fifty-one RT-PCR positive COVID-19 outpatients were recruited between May and December 2020 in Munich, Germany, and followed up at multiple defined timepoints for up to one year. RT-PCR and viral culture were performed and seroresponses measured. Participants were classified applying the WHO clinical progression scale. Short symptom to test time (median 5.0 days; p = 0.0016) and high viral loads (VL; median maximum VL: 3∙108 copies/mL; p = 0.0015) were indicative for viral culture positivity. Participants with WHO grade 3 at baseline had significantly higher VLs compared to those with WHO 1 and 2 (p = 0.01). VLs dropped fast within 1 week of symptom onset. Maximum VLs were positively correlated with the magnitude of Ro-N-Ig seroresponse (p = 0.022). Our results describe the dynamics of VLs and antibodies to SARS-CoV-2 in mild to moderate cases that can support public health measures during the ongoing global pandemic.

Community SARS-CoV-2 seroprevalence before and after the second wave of SARS-CoV-2 infection in Harare, Zimbabwe.

BACKGROUND: By the end of July 2021 Zimbabwe, has reported over 100,000 SARS-CoV-2 infections. The true number of SARS-CoV-2 infections is likely to be much higher. We conducted a seroprevalence survey to estimate the prevalence of past SARS-CoV-2 in three high-density communities in Harare, Zimbabwe before and after the second wave of SARS-CoV-2. METHODS: Between November 2020 and April 2021 we conducted a cross-sectional study of randomly selected households in three high-density communities (Budiriro, Highfield and Mbare) in Harare. Consenting participants answered a questionnaire and a dried blood spot sample was taken. Samples were tested for anti-SARS-CoV-2 nucleocapsid antibodies using the Roche e801 platform. FINDINGS: A total of 2340 individuals participated in the study. SARS-CoV-2 antibody results were available for 70·1% (620/885) and 73·1% (1530/2093) of eligible participants in 2020 and 2021. The median age was 22 (IQR 10-37) years and 978 (45·5%) were men. SARS-CoV-2 seroprevalence was 19·0% (95% CI 15·1-23·5%) in 2020 and 53·0% (95% CI 49·6-56·4) in 2021. The prevalence ratio was 2·47 (95% CI 1·94-3·15) comparing 2020 with 2021 after adjusting for age, sex, and community. Almost half of all participants who tested positive reported no symptoms in the preceding six months. INTERPRETATION: Following the second wave, one in two people had been infected with SARS-CoV-2 suggesting high levels of community transmission. Our results suggest that 184,800 (172,900-196,700) SARS-CoV-2 infections occurred in these three communities alone, greatly exceeding the reported number of cases for the whole city. Further seroprevalence surveys are needed to understand transmission during the current third wave despite high prevalence of past infections. FUNDING: GCRF, Government of Canada, Wellcome Trust, Bavarian State Ministry of Sciences, Research, and the Arts.

Following Pediatric and Adult IBD Patients through the COVID-19 Pandemic: Changes in Psychosocial Burden and Perception of Infection Risk and Harm over Time.

BACKGROUND: COVID-19-associated restrictions impact societies. We investigated the impact in a large cohort of inflammatory bowel disease (IBD) patients. METHODS: Pediatric (pIBD) and adult patients and pIBD parents completed validated questionnaires for self-perceived stress (Perceived Stress Questionnaire, PSQ) and quality of life from July to October 2020 (1st survey) and March to April 2021 (2nd survey). Analyses were stratified by age groups (6-20, >20-40, >40-60, >60 years). Perceived risk of infection and harm from COVID-19 were rated on a 1-7 scale. An index for severe outcome (SIRSCO) was calculated. Multivariable logistic regression analysis was performed. RESULTS: Of 820 invited patients, 504 (62%, 6-85 years) patients and 86 pIBD parents completed the 1st, thereof 403 (80.4%) the 2nd survey. COVID-19 restrictions resulted in cancelled doctoral appointments (26.7%), decreased physical activity, increased food intake, unintended weight gain and sleep disturbance. PSQ increased with disease activity. Elderly males rated lower compared to females or younger adults. PSQ in pIBD mothers were comparable to moderate/severe IBD adults. Infection risk and harm were perceived high in 36% and 75.4%. Multivariable logistic models revealed associations of higher perceived risk with >3 household members, job conditions and female gender, and of perceived harm with higher SIRSCO, unintended weight change, but not with gender or age. Cancelled clinic-visits were associated with both. SARS-CoV-2 antibodies prior 2nd infection wave were positive in 2/472 (0.4%). CONCLUSIONS: IBD patients report a high degree of stress and self-perceived risk of complications from COVID-19 with major differences related to gender and age. Low seroprevalence may indicate altered immune response.

From first to second wave: follow-up of the prospective COVID-19 cohort (KoCo19) in Munich (Germany).

BACKGROUND: In the 2nd year of the COVID-19 pandemic, knowledge about the dynamics of the infection in the general population is still limited. Such information is essential for health planners, as many of those infected show no or only mild symptoms and thus, escape the surveillance system. We therefore aimed to describe the course of the pandemic in the Munich general population living in private households from April 2020 to January 2021. METHODS: The KoCo19 baseline study took place from April to June 2020 including 5313 participants (age 14 years and above). From November 2020 to January 2021, we could again measure SARS-CoV-2 antibody status in 4433 of the baseline participants (response 83%). Participants were offered a self-sampling kit to take a capillary blood sample (dry blood spot; DBS). Blood was analysed using the Elecsys® Anti-SARS-CoV-2 assay (Roche). Questionnaire information on socio-demographics and potential risk factors assessed at baseline was available for all participants. In addition, follow-up information on health-risk taking behaviour and number of personal contacts outside the household (N = 2768) as well as leisure time activities (N = 1263) were collected in summer 2020. RESULTS: Weighted and adjusted (for specificity and sensitivity) SARS-CoV-2 sero-prevalence at follow-up was 3.6% (95% CI 2.9-4.3%) as compared to 1.8% (95% CI 1.3-3.4%) at baseline. 91% of those tested positive at baseline were also antibody-positive at follow-up. While sero-prevalence increased from early November 2020 to January 2021, no indication of geospatial clustering across the city of Munich was found, although cases clustered within households. Taking baseline result and time to follow-up into account, men and participants in the age group 20-34 years were at the highest risk of sero-positivity. In the sensitivity analyses, differences in health-risk taking behaviour, number of personal contacts and leisure time activities partly explained these differences. CONCLUSION: The number of citizens in Munich with SARS-CoV-2 antibodies was still below 5% during the 2nd wave of the pandemic. Antibodies remained present in the majority of SARS-CoV-2 sero-positive baseline participants. Besides age and sex, potentially confounded by differences in behaviour, no major risk factors could be identified. Non-pharmaceutical public health measures are thus still important.

A dried blood spot protocol for high throughput analysis of SARS-CoV-2 serology based on the Roche Elecsys anti-N assay.

BACKGROUND: Since 2020 SARS-CoV-2 spreads pandemically, infecting more than 119 million people, causing >2·6 million fatalities. Symptoms of SARS-CoV-2 infection vary greatly, ranging from asymptomatic to fatal. Different populations react differently to the disease, making it very hard to track the spread of the infection in a population. Measuring specific anti-SARS-CoV-2 antibodies is an important tool to assess the spread of the infection or successful vaccinations. To achieve sufficient sample numbers, alternatives to venous blood sampling are needed not requiring medical personnel or cold-chains. Dried-blood-spots (DBS) on filter-cards have been used for different studies, but not routinely for serology. METHODS: We developed a semi-automated protocol using self-sampled DBS for SARS-CoV-2 serology. It was validated in a cohort of matched DBS and venous-blood samples (n = 1710). Feasibility is demonstrated with two large serosurveys with 10247 company employees and a population cohort of 4465 participants. FINDINGS: Sensitivity and specificity reached 99·20% and 98·65%, respectively. Providing written instructions and video tutorials, 99·87% (4465/4471) of the unsupervised home sampling DBS cards could be analysed. INTERPRETATION: DBS-sampling is a valid and highly reliable tool for large scale serosurveys. We demonstrate feasibility and accuracy with a large validation cohort including unsupervised home sampling. This protocol might be of big importance for surveillance in resource-limited settings, providing low-cost highly accurate serology data. FUNDING: Provided by Bavarian State Ministry of Science and the Arts, LMU University-Hospital; Helmholtz-Centre-Munich, German Ministry for Education and Research (project01KI20271); University of Bonn; University of Bielefeld; the Medical Biodefense Research Program of Bundeswehr-Medical-Service; Euroimmun, RocheDiagnostics provided discounted kits and machines.

Spatially resolved qualified sewage spot sampling to track SARS-CoV-2 dynamics in Munich - One year of experience.

Wastewater-based epidemiology (WBE) is a tool now increasingly proposed to monitor the SARS-CoV-2 burden in populations without the need for individual mass testing. It is especially interesting in metropolitan areas where spread can be very fast, and proper sewage systems are available for sampling with short flow times and thus little decay of the virus. We started in March 2020 to set up a once-a-week qualified spot sampling protocol in six different locations in Munich carefully chosen to contain primarily wastewater of permanent residential areas, rather than industry or hospitals. We used RT-PCR and sequencing to track the spread of SARS-CoV-2 in the Munich population with temporo-spatial resolution. The study became fully operational in mid-April 2020 and has been tracking SARS-CoV-2 RNA load weekly for one year. Sequencing of the isolated viral RNA was performed to obtain information about the presence and abundance of variants of concern in the Munich area over time. We demonstrate that the evolution of SARS-CoV-2 RNA loads (between <7.5 and 3874/ml) in these different areas within Munich correlates well with official seven day incidence notification data (between 0.0 and 327 per 100,000) obtained from the authorities within the respective region. Wastewater viral loads predicted the dynamic of SARS-CoV-2 local incidence about 3 weeks in advance of data based on respiratory swab analyses. Aligning with multiple different point-mutations characteristic for certain variants of concern, we could demonstrate the gradual increase of variant of concern B.1.1.7 in the Munich population beginning in January 2021, weeks before it became apparent in sequencing results of swabs samples taken from patients living in Munich. Overall, the study highlights the potential of WBE to monitor the SARS-CoV-2 pandemic, including the introduction of variants of concern in a local population.

In Search of the SARS-CoV-2 Protection Correlate: Head-to-Head Comparison of Two Quantitative S1 Assays in Pre-characterized Oligo-/Asymptomatic Patients.

BACKGROUND: Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in oligo-/asymptomatic individuals from a population-based cohort. METHODS: In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction [RT-PCR]-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay [EI-S1-IgG-quant]) and Roche Elecsys® Anti-SARS-CoV-2 S [Ro-RBD-Ig-quant]), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript®cPass™. Square roots R of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons. RESULTS: Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases. CONCLUSIONS: Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days.

Broad T Cell Targeting of Structural Proteins After SARS-CoV-2 Infection: High Throughput Assessment of T Cell Reactivity Using an Automated Interferon Gamma Release Assay.

Background: Adaptive immune responses to structural proteins of the virion play a crucial role in protection against coronavirus disease 2019 (COVID-19). We therefore studied T cell responses against multiple SARS-CoV-2 structural proteins in a large cohort using a simple, fast, and high-throughput approach. Methods: An automated interferon gamma release assay (IGRA) for the Nucleocapsid (NC)-, Membrane (M)-, Spike-C-terminus (SCT)-, and N-terminus-protein (SNT)-specific T cell responses was performed using fresh whole blood from study subjects with convalescent, confirmed COVID-19 (n = 177, more than 200 days post infection), exposed household members (n = 145), and unexposed controls (n = 85). SARS-CoV-2-specific antibodies were assessed using Elecsys® Anti-SARS-CoV-2 (Ro-N-Ig) and Anti-SARS-CoV-2-ELISA (IgG) (EI-S1-IgG). Results: 156 of 177 (88%) previously PCR confirmed cases were still positive by Ro-N-Ig more than 200 days after infection. In T cells, most frequently the M-protein was targeted by 88% seropositive, PCR confirmed cases, followed by SCT (85%), NC (82%), and SNT (73%), whereas each of these antigens was recognized by less than 14% of non-exposed control subjects. Broad targeting of these structural virion proteins was characteristic of convalescent SARS-CoV-2 infection; 68% of all seropositive individuals targeted all four tested antigens. Indeed, anti-NC antibody titer correlated loosely, but significantly with the magnitude and breadth of the SARS-CoV-2-specific T cell response. Age, sex, and body mass index were comparable between the different groups. Conclusion: SARS-CoV-2 seropositivity correlates with broad T cell reactivity of the structural virus proteins at 200 days after infection and beyond. The SARS-CoV-2-IGRA can facilitate large scale determination of SARS-CoV-2-specific T cell responses with high accuracy against multiple targets.