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Additional file 3: Figure S3. Proximity cluster analysis at Follow-ups 2 to 4. The grey points and curves show the distribution of mean within-cluster variances for 10,000 random permutations of cluster assignments. The horizontal lines show the observed values. Cluster variables are households, buildings, and geospatial clusters of different sizes. Household membership was left invariant when considering buildings and geospatial clusters. p-values indicate the one-sided probability of observing smaller than observed values under random cluster assignments. Results indicate within-household clustering and suggest neighbourhood transmission only in the cluster with 500m.

Additional file 1: Figure S1. Missing pattern in the baseline questionnaire. Bottom middle: variable analysed for missing information. Bottom left: bar chart depicting numbers of missing information for that variable. Bottom right: description of intersection pattern between variables (all possible combinations of the variables for which a missing information was given, from left to right e.g. only income information missing, income & living & household type information missing, all variables missing, etc.). Top: bar chart depicting the numbers of participants that did not give information for that intersection pattern.

Additional file 2: Figure S2. Cohort description based on current lab result (in contrast to ever-positivity as in Figure 2). Change of serological status of participants: only infected (anti-N positive and stated to be non-vaccinated in the questionnaire), naïve (anti-N and anti-S negative), vaccinated (only anti-S positive), infected & vaccinated (anti-N positive and in previous round only anti-S positive, or anti-N positive and stated to be vaccinated in the questionnaire), infected without information on vaccination status (infected, undefined vaccination) and non-responders/missing.

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.

BackgroundThe duration of immune response to COVID-19 vaccination is of major interest. Our aim was to analyze the determinants of anti-SARS-CoV-2 IgG titer at 6 months after 2-dose vaccination in an international cohort of vaccinated healthcare workers (HCWs).MethodsWe analyzed data on levels of anti-SARS-CoV-2 Spike antibodies and sociodemographic and clinical characteristics of 6,327 vaccinated HCWs from 8 centers from Germany, Italy, Romania and Slovakia. Time between 1st dose and serology ranged 150-210 days. Serological levels were log-transformed to account for the skewness of the distribution and normalized by dividing them by center-specific standard errors, obtaining standardized values. We fitted center-specific multivariate regression models to estimate the cohort-specific relative risks (RR) of an increase of 1 standard deviation of log antibody level and corresponding 95% confidence interval (CI), and finally combined them in random-effects meta-analyses.ResultsA 6-month serological response was detected in 99.6% of HCWs. Female sex (RR 1.10, 95%CI 1.00-1.21), past infection (RR 2.26, 95%CI 1.73-2.95) and two vaccine doses (RR 1.50, 95%CI 1.22-1.84) predicted higher IgG titer, contrary to interval since last dose (RR for 10-day increase 0.94, 95%CI 0.91-0.97) and age (RR for 10-year increase 0.87, 95%CI 0.83-0.92). M-RNA-based vaccines (pConclusionsFemale gender, young age, past infection, two vaccine doses, and m-RNA and heterologous vaccination predicted higher antibody level at 6 months. These results corroborate previous findings and offer valuable data for comparison with trends observed with longer follow-ups.

Additional file 1: Figure S1. For sensitivity and specificity adjusted (left) and unadjusted (right) SARS-CoV-2 sero-prevalence over the follow-up period excluding DBS intermediates. The 95% confidence intervals for the weekly sero-prevalence are based on the 2.5 and 97.5 percentiles from 5,000 repetitions of a cluster bootstrap that accounts for within household clustering. The estimates do not account for sample weights. Figure S2. Comparison of the sero-positivity over the follow-up period accounting for sampling weights. Figure S3. Sero-prevalence estimates across the different boroughs of Munich using CAR model priors with a single level of spatial autocorrelation as random effects. Figure S4. Sero-prevalence estimates across the different boroughs of Munich with CAR model priors through the dissimilarity of the population density in neighbouring boroughs for spatial autocorrelation. Figure S5. Distribution of mean within-cluster variance of test results under 10,000 random permutation of cluster assignments, with clusters being households, buildings, and geospatial clusters of different sizes. Household membership left invariant for building and geospatial clusters. Left: Value distribution. Right: 50%, 95%, 99% CIs. Black lines (left) and dots (right) indicate the observed values. Table S1. Comparison of the follow-up plasma results, using DBS or Venous Blood Samples. Table S2. Comparison of the estimates for the covariate of population density at the constituency level using the Poisson and the negative binomial model. Table S3. Course of SARS-CoV-2 antibody status within the KoCo19 follow-up participants. Table S4. Summary across age group and sex for the response to the behaviour questionnaires stratified by sex. p-values are from Pearson’s Chi-squared test with simulated p-value (based on 10000 replicates). Table S5. Summary across age group and sex for the response to the behaviour questionnaires stratified by age group. p- values are from Pearson’s Chi-squared test with simulated p-value (based on 10000 replicates).

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, Graphical abstract Unlabelled Image

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. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-021-06589-4.

Durante las últimas décadas ha crecido el interés en cuestiones como qué nos hace humanos o cómo difiere a nivel molecular el cerebro humano del de nuestros parientes más cercanos. Se han podido identificar cientos de genes con diferencias de expresión entre el ser humano y otros primates no humanos. Sin embargo, es importante estudiar más en detalle estos genes para comprobar si realmente están involucrados en las características específicas de nuestro cerebro. Las trombospondinas son glicoproteínas extracelulares multiméricas que modulan las interacciones entre células y con la matriz extracelular y que se han implicado en la sinaptogénesis. Dentro de la familia de las trombospondinas, los genes de la trombospondina-2 (THBS2) y trombospondina-4 (THBS4) se expresan, respectivamente, alrededor de 2 y 6 veces más en la corteza cerebral humana en comparación con la de chimpancés o macacos. Para conocer las causas de estas diferencias de expresión, hemos llevado a cabo un análisis comparativo y funcional de la región promotora de THBS4 en humanos y en chimpancés. Hemos identificado y validado un sitio de inicio de transcripción (TSS) alternativo para THBS4 que se encuentra 44 kb aguas arriba del TSS de referencia y que genera una nueva isoforma de ARNm que podría haber aparecido más tarde durante la evolución. Para comparar los niveles de expresión de ambos transcritos se realizó RT-PCR cuantitativa en diferentes tejidos humanos y en muestras de corteza cerebral de 11 humanos, 11 chimpancés y 8 macacos. Curiosamente, la nueva isoforma de THBS4 se expresa principalmente en los tejidos cerebrales. Por otra parte, la diferencia de expresión entre humanos y primates no humanos para la isoforma alternativa son consistentes con la encontrada al analizar la expresión total de THBS4. Por tanto, el aumento de la expresión de THBS4 en el cerebro humano parece estar relacionado con una mayor transcripción a partir del promotor alternativo. Para evaluar la actividad de ambas secuencias promotoras en humanos y en chimpancés, hemos llevado a cabo ensayos con un gen indicador en diferentes líneas celulares humanas. Se han encontrado diferencias significativas entre los dos promotores, aunque en las líneas de neuroblastoma que utilizamos no existen diferencias significativas entre especies. Este resultado es consistente con la búsqueda de sitios de unión de factores de transcripción en la región del promotor alternativo, ya que sólo se detectaron tres posibles sitios de unión diferentes entre ambas especies. Se ha comparado también la metilación del ADN en una isla CpG situada aguas arriba de la nueva isoforma de THBS4 en 5 humanos y en 5 chimpancés, detectando niveles bajos de metilación en ambas especies. Basándonos en las predicciones informáticas disponibles y en experimentos piloto de ChIP-Seq, hemos buscado posibles enhancers que estén controlando el promotor alternativo de THBS4, pero no hemos encontrado ningún candidato fiable. Por último, en humanos, se ha visto que hay dos haplotipos de THBS4 diferentes que se encuentran mantenidos mediante selección equilibradora. Sin embargo, la comparación experimental de ambos no muestra ningún efecto del genotipo sobre la expresión de THBS4. Aunque no se ha conseguido identificar la causa concreta del incremento en los niveles de THBS4 en humanos, en base a nuestros resultados sugerimos que la expresión diferencial del gen podría estar relacionada con una secuencia potenciadora específica del cerebro que no hemos conseguido localizar. Comprender como se encuentra regulada esta posible secuencia potenciadora podría ser relevante para entender cuales son las consecuencias funcionales de las diferencias de expresión de THBS4 sobre la evolución del cerebro y, en última instancia, darnos pistas sobre como nos convertimos en humanos., The last decades have seen a growing interest in what makes us humans and how the human brain differs from that of our closest relatives at the molecular level. Hundreds of genes with expression differences between human and non-human primates have been identified. However, it is important to study these genes in more detail to see if they are really involved in human brain characteristics. Thrombospondins are multimeric extracellular glycoproteins that modulate cell-cell and extracellular matrix interactions and have been implicated in synaptogenesis. Within the thrombospondin family, thrombospondin-2 (THBS2) and thrombospondin-4 (THBS4) show, respectively, a ~2-fold and ~6-fold upregulation in human cerebral cortex compared to chimpanzees and macaques. To analyze the causes of these expression differences, we have carried out a comparative and functional analysis of the THBS4 promoter region in humans and chimpanzees. We have identified and validated an alternative transcription start site (TSS) for THBS4 that is located ~44 kb upstream from the known TSS and generates a new mRNA isoform that might have appeared later that the reference one during evolution. To compare expression levels of both mRNAs, we performed quantitative RT-PCR in different human tissues and cortical regions of 11 humans, 11 chimpanzees and 8 macaques. Interestingly, the new isoform of THBS4 is expressed mainly in brain tissues. Moreover, expression differences between human and non-human primate cortex for this alternative isoform are consistent with those shown for total THBS4 expression. Increased THBS4 expression in the human brain therefore appears to be related to higher transcription from the alternative promoter. To evaluate the activity of both THBS4 promoter sequences we performed reporter assays from humans and chimpanzees in different human cell lines. We have found significant differences between both promoters, but not between species, in the neuroblastoma cell lines assayed. This result is consistent with the search for transcription factor binding sites (TFBS) in the alternative promoter region, which only detected three putative TFBS differentially predicted between both species. We also compared the DNA methylation in a CpG island upstream the new isoform in 5 humans and 5 chimpanzees detecting similar low methylation levels in all of them. Based in the computational predictions available online and ChIP-Seq pilot experiments, we searched for a putative enhancer region controlling the THBS4 alternative promoter without finding any reliable candidate. Finally, in humans, THBS4 has been associated to two different haplotypes that are maintained by balancing selection, but experimental analysis did not show any effect of the genotype over THBS4 gene expression. Although we have not been able to identify the ultimate cause of the increased THBS4 levels in humans, based on all our results we suggest that the differential gene expression might be related to a brain-specific enhancer sequence that has so far escaped our scrutiny. Understanding its regulation could be relevant to the functional consequences of THBS4 expression differences during human brain evolution and ultimately could give us clues of how we became humans.

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 ElecsysAnti-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. Copyright © 2021 Brand, Gilberg, Bruger, Gari, Wieser, Eser, Frese, Ahmed, Rubio-Acero, Guggenbuehl Noller, Castelletti, Diekmannshemke, Thiesbrummel, Huynh, Winter, Kroidl, Fuchs, Hoelscher, Roider, Kobold, Pritsch and Geldmacher.