Estimates of protection levels against SARS-CoV-2 infection and severe COVID-19 in Germany before the 2022/2023 winter season: the IMMUNEBRIDGE project.

PURPOSE: Despite the need to generate valid and reliable estimates of protection levels against SARS-CoV-2 infection and severe course of COVID-19 for the German population in summer 2022, there was a lack of systematically collected population-based data allowing for the assessment of the protection level in real time. METHODS: In the IMMUNEBRIDGE project, we harmonised data and biosamples for nine population-/hospital-based studies (total number of participants n = 33,637) to provide estimates for protection levels against SARS-CoV-2 infection and severe COVID-19 between June and November 2022. Based on evidence synthesis, we formed a combined endpoint of protection levels based on the number of self-reported infections/vaccinations in combination with nucleocapsid/spike antibody responses ("confirmed exposures"). Four confirmed exposures represented the highest protection level, and no exposure represented the lowest. RESULTS: Most participants were seropositive against the spike antigen; 37% of the participants ≥ 79 years had less than four confirmed exposures (highest level of protection) and 5% less than three. In the subgroup of participants with comorbidities, 46-56% had less than four confirmed exposures. We found major heterogeneity across federal states, with 4-28% of participants having less than three confirmed exposures. CONCLUSION: Using serological analyses, literature synthesis and infection dynamics during the survey period, we observed moderate to high levels of protection against severe COVID-19, whereas the protection against SARS-CoV-2 infection was low across all age groups. We found relevant protection gaps in the oldest age group and amongst individuals with comorbidities, indicating a need for additional protective measures in these groups.

Bridging the gap - estimation of 2022/2023 SARS-CoV-2 healthcare burden in Germany based on multidimensional data from a rapid epidemic panel.

OBJECTIVES: Throughout the SARS-CoV-2 pandemic, Germany like other countries lacked adaptive population-based panels to monitor the spread of epidemic diseases. METHODS: To fill a gap in population-based estimates needed for winter 2022/23 we resampled in the German SARS-CoV-2 cohort study MuSPAD in mid-2022, including characterization of systemic cellular and humoral immune responses by interferon-γ-release assay (IGRA) and CLIA/IVN assay. We were able to confirm categorization of our study population into four groups with differing protection levels against severe COVID-19 courses based on literature synthesis. Using these estimates, we assessed potential healthcare burden for winter 2022/23 in different scenarios with varying assumptions on transmissibility, pathogenicity, new variants, and vaccine booster campaigns in ordinary differential equation models. RESULTS: We included 9921 participants from eight German regions. While 85% of individuals were located in one of the two highest protection categories, hospitalization estimates from scenario modeling were highly dependent on viral variant characteristics ranging from 30-300% compared to the 02/2021 peak. Our results were openly communicated and published to an epidemic panel network and a newly established modeling network. CONCLUSIONS: We demonstrate feasibility of a rapid epidemic panel to provide complex immune protection levels for inclusion in dynamic disease burden modeling scenarios.

Suitability and user acceptance of the eResearch system "Prospective Monitoring and Management App (PIA)"-The example of an epidemiological study on infectious diseases.

BACKGROUND: The eResearch system "Prospective Monitoring and Management App (PIA)" allows researchers to implement questionnaires on any topic and to manage biosamples. Currently, we use PIA in the longitudinal study ZIFCO (Integrated DZIF Infection Cohort within the German National Cohort) in Hannover (Germany) to investigate e.g. associations of risk factors and infectious diseases. Our aim was to assess user acceptance and compliance to determine suitability of PIA for epidemiological research on transient infectious diseases. METHODS: ZIFCO participants used PIA to answer weekly questionnaires on health status and report spontaneous onset of symptoms. In case of symptoms of a respiratory infection, the app requested participants to self-sample a nasal swab for viral analysis. To assess user acceptance, we implemented the System Usability Scale (SUS) and fitted a linear regression model on the resulting score. For investigation of compliance with submitting the weekly health questionnaires, we used a logistic regression model with binomial response. RESULTS: We analyzed data of 313 participants (median age 52.5 years, 52.4% women). An average SUS of 72.0 reveals good acceptance of PIA. Participants with a higher technology readiness score at the beginning of study participation also reported higher user acceptance. Overall compliance with submitting the weekly health questionnaires showed a median of 55.7%. Being female, of younger age and being enrolled for a longer time decreased the odds to respond. However, women over 60 had a higher chance to respond than women under 60, while men under 40 had the highest chance to respond. Compliance with nasal swab self-sampling was 77.2%. DISCUSSION: Our findings show that PIA is suitable for the use in epidemiologic studies with regular short questionnaires. Still, we will focus on user engagement and gamification for the further development of PIA to help incentivize regular and long-term participation.

Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies.

BACKGROUND: Lyme borreliosis (LB) is the most common tick-borne infectious disease in the northern hemisphere. The diagnosis of LB is usually made by clinical symptoms and subsequently supported by serology. In Europe, a two-step testing consisting of an enzyme-linked immunosorbent assay (ELISA) and an immunoblot is recommended. However, due to the low sensitivity of the currently available tests, antibody detection is sometimes inaccurate, especially in the early phase of infection, leading to underdiagnoses. METHODS: To improve upon Borrelia diagnostics, we developed a multiplex Borrelia immunoassay (Borrelia multiplex), which utilizes the new INTELLIFLEX platform, enabling the simultaneous dual detection of IgG and IgM antibodies, saving further time and reducing the biosample material requirement. In order to enable correct classification, the Borrelia multiplex contains eight antigens from the five human pathogenic Borrelia species known in Europe. Six antigens are known to mainly induce an IgG response and two antigens are predominant for an IgM response. RESULTS: To validate the assay, we compared the Borrelia multiplex to a commercial bead-based immunoassay resulting in an overall assay sensitivity of 93.7% (95% CI 84.8-97.5%) and a specificity of 96.5% (95%CI 93.5-98.1%). To confirm the calculated sensitivity and specificity, a comparison with a conventional 2-step diagnostics was performed. With this comparison, we obtained a sensitivity of 95.2% (95% CI 84.2-99.2%) and a specificity of 93.0% (95% CI 90.6-94.7%). CONCLUSION: Borrelia multiplex is a highly reproducible cost- and time-effective assay that enables the profiling of antibodies against several individual antigens simultaneously.

Diminished neutralization responses towards SARS-CoV-2 Omicron VoC after mRNA or vector-based COVID-19 vaccinations.

SARS-CoV-2 variants accumulating immune escape mutations provide a significant risk to vaccine-induced protection against infection. The novel variant of concern (VoC) Omicron BA.1 and its sub-lineages have the largest number of amino acid alterations in its Spike protein to date. Thus, they may efficiently escape recognition by neutralizing antibodies, allowing breakthrough infections in convalescent and vaccinated individuals in particular in those who have only received a primary immunization scheme. We analyzed neutralization activity of sera from individuals after vaccination with all mRNA-, vector- or heterologous immunization schemes currently available in Europe by in vitro neutralization assay at peak response towards SARS-CoV-2 B.1, Omicron sub-lineages BA.1, BA.2, BA.2.12.1, BA.3, BA.4/5, Beta and Delta pseudotypes and also provide longitudinal follow-up data from BNT162b2 vaccinees. All vaccines apart from Ad26.CoV2.S showed high levels of responder rates (96-100%) towards the SARS-CoV-2 B.1 isolate, and minor to moderate reductions in neutralizing Beta and Delta VoC pseudotypes. The novel Omicron variant and its sub-lineages had the biggest impact, both in terms of response rates and neutralization titers. Only mRNA-1273 showed a 100% response rate to Omicron BA.1 and induced the highest level of neutralizing antibody titers, followed by heterologous prime-boost approaches. Homologous BNT162b2 vaccination, vector-based AZD1222 and Ad26.CoV2.S performed less well with peak responder rates of 48%, 56% and 9%, respectively. However, Omicron responder rates in BNT162b2 recipients were maintained in our six month longitudinal follow-up indicating that individuals with cross-protection against Omicron maintain it over time. Overall, our data strongly argue for booster doses in individuals who were previously vaccinated with BNT162b2, or a vector-based primary immunization scheme.

Comparative Magnitude and Persistence of Humoral SARS-CoV-2 Vaccination Responses in the Adult Population in Germany.

Recent increases in SARS-CoV-2 infections have led to questions about duration and quality of vaccine-induced immune protection. While numerous studies have been published on immune responses triggered by vaccination, these often focus on studying the impact of one or two immunisation schemes within subpopulations such as immunocompromised individuals or healthcare workers. To provide information on the duration and quality of vaccine-induced immune responses against SARS-CoV-2, we analyzed antibody titres against various SARS-CoV-2 antigens and ACE2 binding inhibition against SARS-CoV-2 wild-type and variants of concern in samples from a large German population-based seroprevalence study (MuSPAD) who had received all currently available immunisation schemes. We found that homologous mRNA-based or heterologous prime-boost vaccination produced significantly higher antibody responses than vector-based homologous vaccination. Ad26.CoV2S.2 performance was particularly concerning with reduced titres and 91.7% of samples classified as non-responsive for ACE2 binding inhibition, suggesting that recipients require a booster mRNA vaccination. While mRNA vaccination induced a higher ratio of RBD- and S1-targeting antibodies, vector-based vaccines resulted in an increased proportion of S2-targeting antibodies. Given the role of RBD- and S1-specific antibodies in neutralizing SARS-CoV-2, their relative over-representation after mRNA vaccination may explain why these vaccines have increased efficacy compared to vector-based formulations. Previously infected individuals had a robust immune response once vaccinated, regardless of which vaccine they received, which could aid future dose allocation should shortages arise for certain manufacturers. Overall, both titres and ACE2 binding inhibition peaked approximately 28 days post-second vaccination and then decreased.

Photomotor response data analysis approach to assess chemical neurotoxicity with the zebrafish embryo.

The photomotor response (PMR) of zebrafish embryos, a light pulse-triggered undirected movement, is known to be altered by neuroactive chemicals. Here, we developed an approach for data analysis of the distribution of PMR movement activities along the time axis; differences between treatment and respective controls are expressed by an aggregated value integrating the time-resolved density of the movement parameter as a measure for a chemically elicited PMR effect. Logistic concentration-PMR effect relationships were modeled for neuroactive test compounds with different modes of action (acetylcholinesterase inhibition, activation and inhibition of voltage-gated sodium channels); 50% effect concentrations (EC50) were in the low to medium µM range (EC50 < 10 µM for flucythrinate, esfenvalerate, azinphos-methyl, propoxur; EC50 > 10 µM for tricaine). Modulation of movement activities in different phases of the PMR (i.e., "fingerprint") by neuroactive test compounds varied across concentrations, showing that mode of action-specific PMR fingerprints are also concentration-dependent. Above concentrations causing 10% lethality (LC10; 48 h), 3,4-dichlo­roaniline caused movement inhibition. This substance presumably is not neuroactive; its effect on the PMR therefore is considered a secondary toxic effect. Quantitative morphological examinations of chemically exposed embryos showed that malformations occurred only above PMR effect concentrations, indicating that changes in the PMR were not due to such indirect effects. The PMR assay will provide a useful measure in ecotoxicological risk assessment of neuroactive chemicals with zebrafish embryos and could potentially be used to infer acute fish toxicity levels from PMR effect concen­trations of neurotoxic compounds.

SARS-CoV-2 Seroprevalence in Germany.

BACKGROUND: Until now, information on the spread of SARS-CoV-2 infections in Germany has been based mainly on data from the public health offices. It may be assumed that these data do not include many cases of asymptomatic and mild infection. METHODS: We determined seroprevalence over the course of the pandemic in a sequential, multilocal seroprevalence study (MuSPAD). Study participants were recruited at random in seven administrative districts (Kreise) in Germany from July 2020 onward; each participant was tested at two different times 3-5 months apart. Test findings on blood samples were used to determine the missed-case rate of reported infections, the infection fatality rate (IFR), and the association between seropositivity and demographic, socio-economic, and health-related factors, as well as to evaluate the self-reported results of PCR and antigenic tests. The registration number of this study is DRKS00022335. RESULTS: Among non-vaccinated persons, the seroprevalence from July to December 2020 was 1.3-2.8% and rose between February and May 2021 to 4.1-13.1%. In July 2021, 35% of tested persons in Chemnitz were not vaccinated, and the seroprevalence among these persons was 32.4% (07/2021). The surveillance detection ratio (SDR), i.e., the ratio between the true number of infections estimated from seroprevalence and the actual number or reported infections, varied among the districts included in the study from 2.2 to 5.1 up to December 2020 and from 1.3 to 2.9 up to June 2021, and subsequently declined. The IFR was in the range of 0.8% to 2.4% in all regions except Magdeburg, where a value of 0.3% was calculated for November 2020. A lower educational level was associated with a higher seropositivity rate, smoking with a lower seropositivity rate. On average, 1 person was infected for every 8.5 persons in quarantine. CONCLUSION: Seroprevalence was low after the first wave of the pandemic but rose markedly during the second and third waves. The missed-case rate trended downward over the course of the pandemic.

A KNIME-Based Analysis of the Zebrafish Photomotor Response Clusters the Phenotypes of 14 Classes of Neuroactive Molecules.

Recently, the photomotor response (PMR) of zebrafish embryos was reported as a robust behavior that is useful for high-throughput neuroactive drug discovery and mechanism prediction. Given the complexity of the PMR, there is a need for rapid and easy analysis of the behavioral data. In this study, we developed an automated analysis workflow using the KNIME Analytics Platform and made it freely accessible. This workflow allows us to simultaneously calculate a behavioral fingerprint for all analyzed compounds and to further process the data. Furthermore, to further characterize the potential of PMR for mechanism prediction, we performed PMR analysis of 767 neuroactive compounds covering 14 different receptor classes using the KNIME workflow. We observed a true positive rate of 25% and a false negative rate of 75% in our screening conditions. Among the true positives, all receptor classes were represented, thereby confirming the utility of the PMR assay to identify a broad range of neuroactive molecules. By hierarchical clustering of the behavioral fingerprints, different phenotypical clusters were observed that suggest the utility of PMR for mechanism prediction for adrenergics, dopaminergics, serotonergics, metabotropic glutamatergics, opioids, and ion channel ligands.

Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos.

Craniofacial malformations, reduced locomotion and induction of genes encoding for enzymes involved in thyroid hormone synthesis were assessed using methimazole and N-phenylthiourea in zebrafish embryos. Gene expression, the most sensitive endpoint (EC50_MMI=372-765µM, EC50_PTU=7.6-8.6µM), was analysed in wild-type and in a transgenic strain, tg(tg:mCherry), expressing mCherry fluorescence protein under the control of the thyroglobulin gene. Reduction of locomotion and craniofacial malformations were observed at one or two orders of magnitude above concentrations affecting gene expression, respectively. Both effects could be linked to the malformations caused by reduced thyroxin levels. Our results show that due to the presence of the autoregulatory loop of the hypothalamus-pituitary-thyroid axis, various molecular initiating events of thyroid disruption are amenable for the zebrafish embryo. We propose the tg(tg:mCherry) bioassay as a sensitive tool in medium scale screening of goitrogens, given the minimal effort for sample preparation and analysis of gene expression.

Fish embryo toxicity test: identification of compounds with weak toxicity and analysis of behavioral effects to improve prediction of acute toxicity for neurotoxic compounds.

The fish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, but concerns have been raised for its predictivity given that a few compounds have been shown to exhibit a weak acute toxicity in the fish embryo. In order to better define the applicability domain and improve the predictive capacity of the fish embryo test, we performed a systematic analysis of existing fish embryo and acute fish toxicity data. A correlation analysis of a total of 153 compounds identified 28 compounds with a weaker or no toxicity in the fish embryo test. Eleven of these compounds exhibited a neurotoxic mode of action. We selected a subset of eight compounds with weaker or no embryo toxicity (cyanazine, picloram, aldicarb, azinphos-methyl, dieldrin, diquat dibromide, endosulfan, and esfenvalerate) to study toxicokinetics and a neurotoxic mode of action as potential reasons for the deviating fish embryo toxicity. Published fish embryo LC50 values were confirmed by experimental analysis of zebrafish embryo LC50 according to OECD guideline 236. Except for diquat dibromide, internal concentration analysis did not indicate a potential relation of the low sensitivity of fish embryos to a limited uptake of the compounds. Analysis of locomotor activity of diquat dibromide and the neurotoxic compounds in 98 hpf embryos (exposed for 96 h) indicated a specific effect on behavior (embryonic movement) for the neurotoxic compounds. The EC50s of behavior for neurotoxic compounds were close to the acute fish toxicity LC50. Our data provided the first evidence that the applicability domain of the fish embryo test (LC50s determination) may exclude neurotoxic compounds. However, neurotoxic compounds could be identified by changes in embryonic locomotion. Although a quantitative prediction of acute fish toxicity LC50 using behavioral assays in fish embryos may not yet be possible, the identification of neurotoxicity could trigger the conduction of a conventional fish acute toxicity test or application of assessment factors while considering the very good fish embryo-acute fish toxicity correlation for other compounds.

Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays.

Cyanobacteria contain various types of bioactive compounds, which could cause adverse effects on organisms. They are released into surface waters during cyanobacterial blooms, but there is little information on their potential relevance for effects in vivo. In this study presence of bioactive compounds was characterized in cyanobacteria Microcystis aeruginosa (Chroococcales), Planktothrix agardhii (Oscillatoriales) and Aphanizomenon gracile (Nostocales) with selected in vitro assays. The in vivo relevance of detected bioactivities was analysed using transgenic zebrafish embryos tg(cyp19a1b-GFP). Teratogenic potency was assessed by analysis of developmental disorders and effects on functions of the neuromuscular system by video tracking of locomotion. Estrogenicity in vitro corresponded to 0.95-54.6 ng estradiol equivalent(g dry weight (dw))(-1). In zebrafish embryos, estrogenic effects could not be detected potentially because they were masked by high toxicity. There was no detectable (anti)androgenic/glucocorticoid activity in any sample. Retinoid-like activity was determined at 1-1.3 µg all-trans-retinoic acid equivalent(g dw)(-1). Corresponding to the retinoid-like activity A. gracile extract also caused teratogenic effects in zebrafish embryos. Furthermore, exposure to biomass extracts at 0.3 gd wL(-1) caused increase of body length in embryos. There were minor effects on locomotion caused by 0.3 gd wL(-1)M. aeruginosa and P. agardhii extracts. The traditionally measured cyanotoxins microcystins did not seem to play significant role in observed effects. This indicates importance of other cyanobacterial compounds at least towards some species or their developmental phases. More attention should be paid to activity of retinoids, estrogens and other bioactive substances in phytoplankton using in vitro and in vivo bioassays.

Extensive review of fish embryo acute toxicities for the prediction of GHS acute systemic toxicity categories.

Distribution and marketing of chemicals require appropriate labelling of health, physical and environmental hazards according to the United Nations global harmonisation system (GHS). Labelling for (human) acute toxicity categories is based on experimental findings usually obtained by oral, dermal or inhalative exposure of rodents. There is a strong societal demand for replacing animal experiments conducted for safety assessment of chemicals. Fish embryos are considered as alternative to animal testing and are proposed as predictive model both for environmental and human health effects. Therefore, we tested whether LC50s of the fish embryo acute toxicity test would allow effectively predicting of acute mammalian toxicity categories. A database of published fish embryo LC50 containing 641 compounds was established. For these compounds corresponding rat oral LD50 were identified resulting in 364 compounds for which both fish embryo LC50 and rat LD50 was available. Only a weak correlation of fish embryo LC50 and rat oral LD50 was obtained. Fish embryos were also not able to effectively predict GHS oral acute toxicity categories. We concluded that due to fundamental exposure protocol differences (single oral dose versus water-borne exposure) a reverse dosimetry approach is needed to explore the predictive capacity of fish embryos.

Transient overexpression of adh8a increases allyl alcohol toxicity in zebrafish embryos.

Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization.