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

From June to the end of November 2022, new survey rounds on vaccination status and previous infections were performed, blood samples were collected, and antibodies against SARS-Cov-2 were measured in four existing population-based cohort studies: the ELISA-Study [7, 13], MuSPAD [4], the German National Cohort (NAKO) [8], and STAAB [5]. Four new cross-sectional studies were set up, one to provide regional depth (GUIDE Study; DRKS00029693 [6]) and three to provide relevant data on children (studies of the University Hospitals in Dresden (adolescents; DRKS00022549), Bochum (school children) [2], and Würzburg (pre-school children; Wü-Kita-CoV) [3, 14]). Studies were included if they were able to quickly survey and sample in the general population regionally or supra-regionally, and provide the MDS. For efficiency reasons, the GUIDE study used a self-sampling method in which capillary blood was collected by the participants themselves and put on dried blood cards. For all other studies, venous blood samples were collected on-site. The principle desirable characteristics of epidemic panels such as speed, adaptability, and linkage to other sources were realised differently in the participating studies. If some characteristics were not available in a particular study, the network format of the project ensured that these aims could be reached for the overall project. For example, not all studies were able to collect samples in June 2022, but this was compensated by the capacity of other studies or study sites to do this. Further, as not all studies were able to provide regional depth, other studies were built up for this purpose (Online Resource 1 Table S1).

In addition to the population-based studies, a prospective hospital-based study called IMMUNEBRIDGE_ED was conducted by the Central Emergency Department of the University Medical Center Göttingen to provide information on defined risk groups not covered in sufficient depth by the available population-based studies. Inclusion criteria for IMMUNEBRIDGE_ED comprised geriatric patients as well as patients with pre-existing immunodeficiency or severe pre-existing diseases. We followed STROBE as a reporting guideline for observational studies [15]. Further information on the participating studies, such as eligibility criteria, can be found in cohort profile publications of these studies [2‐7, 16] (Online Resource 1 Table S1).

For all participating studies, local ethics committees` votes were obtained prior to the start of the IMMUNEBRIDGE project. Participants and patients were included only after written informed consent in each clinical study.

The serum samples obtained in the STAAB, MuSPAD, ELISA, all children cohorts and IMMUNEBRIDGE_ED by on-site blood collection were analysed in the Institute of Clinical Chemistry and Laboratory Medicine in Oldenburg. Serum samples from NAKO were analysed in the Institute of Clinical Chemistry and Laboratory Medicine in Greifswald. The Elecsys® Anti-SARS-CoV-2 S and Elecsys® Anti-SARS-CoV-2 NC (both Roche Diagnostics, Mannheim, Germany) were used to determine the quantitative antibody response to the S- and qualitative antibody response to the N-antigen of SARS-CoV-2. All antibody measurements were provided as BAU/ml (BAU: Binding Antibody Units; S-antigen) or a ratio (N-antigen). Seropositivity to the S-antigen was defined as ≥ 0.80 BAU/ml, seropositivity to the N-antigen as being above the cut-off index of 1.0.

For GUIDE, capillary blood was collected by the participants themselves, put on dried blood cards (Ahlstrom-Munksjö TFN 460), and sent by mail to the laboratory MVZ Labor Krone GbR (Bad Salzuflen). To analyse the dried blood spot (DBS) samples, the Anti-SARS-CoV-2-QuantiVac ELISA (IgG) was used for the S-antigen and the Anti-SARS-CoV-2-NCP ELISA (IgG) for the N-antigen. Both assays have been validated for use with sample material from DBS cards by both the manufacturer and the performing laboratory. Seropositivity against the S-antigen was defined as ≥ 35.2 BAU/ml, against the N-antigen as a ratio of > 1.0. In the main analysis, DBS samples with borderline findings (ratio ≥ 0.8 to ≤ 1.0 for the N-antigen and ≥25.6 to <35.2 BAU/ml for the S-antigen) were defined as seropositive. This represents a conservative estimate of the waning function with the aim not to underestimate true protection in the population (as a positive antibody response served only as a confirmation of a self-reported infection in the main analysis).

Data pooling took place using the Serohub (www.​serohub.​net) based on the MDS previously agreed on with the participating studies. The Serohub was designed during the SARS-CoV-2 pandemic as a digital tool able to link and collect individual participant data using common data sharing and MDS documents. In the MDS, variables regarding demographic, socioeconomic, and medical characteristics of participants were agreed on, and reporting categories of these variables were defined. Furthermore, variables regarding test characteristics, assay results, and reporting categories were fixed (Online Resource 1 Table S2).

As a proxy for protection levels against SARS-CoV-2 infection and severe course of disease, we formed four categories of protection levels based on a targeted literature review and associated them with graded levels of protection against (re-)infection and severe COVID-19 (Table 1). These four categories were based on a combination of the number of self-reported previous infections and vaccinations and the current antibody status against the N- and S-antigen; as the categories are based on a combination of infection, vaccination and antibody status, we will refer to this categorisation as the “combined endpoint” from here onwards. The number of previous self-reported infections and vaccinations will be denoted as “exposures” from hereafter. We defined a confirmed exposure as a self-reported infection or vaccination with a corresponding humoral immune correlate (antibody to N-antigen for infection, antibody to S-antigen for vaccination).

The four combined endpoint categories are as follows (highest to lowest protection levels): 3 + 1 confirmed exposures (with at least one confirmed exposure in 2022), corresponding to the highest level of protection; 3 confirmed exposures (regardless of the timing of the exposures); 1– < 3 confirmed exposures (regardless of the timing of the exposures); and 0 exposures, indicating no protection (Table 1).

The combined endpoint was reported stratified by age groups, self-reported comorbidities (hypertension, diabetes, lung diseases, cardiovascular diseases, cancers, diseases leading to immunosuppression), and self-reported sex. All estimates represent results that were age standardised according to the 2021 population status update of the 2011 German census of the Federal Statistical Office using the survey package in R [17, 18]. Results that were not age standardised can be found in Online Resource 1. In addition, the combined endpoint was presented stratified according to Nomenclature des Unités territoriales statistiques (NUTS) 2 regions as well as federal states (NUTS 1), using the R package eurostat [19]. NUTS divides areas of the European Union into three levels, allowing for cross-border comparisons [20].

In the analyses comparing vulnerable groups from the population-based studies with the IMMUNEBRIDGE_ED cohort, the results of the population-based cohorts were age standardised according to the age distribution in the IMMUNEBRIDGE_ED comorbidity groups.

In the sensitivity analyses, we assessed the combined endpoint if only self-reported information on infection and vaccination and no humoral immunity correlates for confirmation were included. Additionally, we evaluated the combined endpoint and seropositivity if DBS samples with borderline N-antigen findings (ratio ≥ 0.8 to ≤ 1.0) were treated as seronegative to provide a lower bound of protection levels compatible with the antibody analysis.

All analyses were carried out in R version 4.1 and 4.2 [21].

A total of 33,637 participants from 9 studies surveyed between June and November 2022 were included. Online Resource 1, Table S1 describes the different participating studies, and Table 2 shows the baseline characteristics of the participants in these studies.

In the adult age groups, between 5% (of those over 79 years old) and 16% (of those 30–39 years old) of individuals had less than three confirmed exposures (Table 3, Fig. 1A). Among persons over 79 years of age, 37% had less than four antibody-confirmed exposures (Table 3, Fig. 1A). Among children and adolescents, 80% had less than three confirmed exposures, and 13% had neither a reported exposure nor an immunocorrelate. We found no relevant differences in the results when treating borderline antibody findings of the GUIDE study as seronegative (Online Resource 1 Figure S6).

Among adults with self-reported comorbidities, 46–56% (depending on the pre-existing comorbidity) had less than four confirmed exposures and 5–10% had less than three confirmed exposures (Table 3).

When ignoring antibody results and building categories based on reported exposures only, the proportion of those with less than three exposures was lower in all age groups. However, the proportion of those with no reported exposure was higher, in particular in the age group under 18 years (Fig. 1B). While seropositivity against the N-antigen decreased with older age (Fig. 1D), this was not the case for seropositivity against the S-antigen, where a difference is only seen between children (80%) and adults (> 95%; Fig. 1C).

Figure 2A–F visualises the relative frequencies of the levels of the combined endpoint and seropositivity estimates on maps of Germany stratified by NUTS 2 units (usually German administrative districts). The combined endpoint is shown over time from June to September in Online Resource 1 Figure S1, over NUTS-2 regions in Online Resource 1 Figure S2, and over federal states in Online Resource 1 Figure S3. The combined endpoint could not be formed for 12,185 participants due to missing information from the participants from NAKO (n = 10,595) and for an additional 1,590 participants from the other cohorts. This was largely due to missing results from antibody tests due to difficulties in collecting blood for the DBS samples by the participants themselves and the resulting insufficient amount of sample material in the GUIDE study. Overall, between 0 and 12% of participants in all participating studies had missing laboratory results. In the GUIDE study, a higher proportion of missing lab results was seen among people in the older and very old age groups because of self-sampling. For between 0 and 13% of the study participants in the participating studies, information about vaccination and infection was missing.

Across all age groups, 95% of the study participants had antibodies against the S-antigen. In the age group 1–17 years, this proportion was lowest with 80% (Fig. 1C; Table 3).

Across all age groups, 52% of the study participants had antibodies against the N-antigen. This proportion was highest in the age group 1–17 at 68% and lowest in people over 79 years of age at 28%; overall, there was a trend of decreasing proportions of antibodies against the N-antigen with increasing age (Fig. 1D; Table 3). A lower proportion of older people (over 64 years of age) and people with comorbidities had antibodies against the N-antigen (Table 3). An antibody response against the N-antigen was present in 71% of participants who reported a first infection in 2020, in 76% of those who reported a first infection in 2021, and in 84% of those with a first reported infection in 2022 (Table 3). 25% of participants who reported no infection showed antibodies against the N-antigen (Table 3).

In participants reporting the first infection after being vaccinated at least two times, seropositivity for antibodies against the N-antigen was 89% up to 5 months after infection and dropped to 68% in those with reported infections longer than 5 months ago. This was similar albeit slightly less pronounced in persons without vaccination (91% up to 5 months and 76% after 5 months). This corresponded to markedly lower titre levels against the N-antigen in those participants of the GUIDE study with infections after at least two vaccinations compared to those with infections without vaccination and a higher decrease in antibody titres after 5 months amongst vaccinated persons.

In relation to NUTS 2, the proportion of people with less than three confirmed exposures was highest in Dresden (28%) and lowest in Schleswig–Holstein (4%; Fig. 2, Online Resource 1 Figure S2). The proportion of people who had antibodies against the N-antigen ranged from 37% in Lüneburg to 60% in Leipzig (Fig. 2).

In the hospital-based study IMMUNEBRIDGE_ED, the proportion of study participants who had less than three confirmed exposures (between 11 and 14%) was higher than that in the analogous strata in the population-based studies (between 4 and 7%; Fig. 3).

Compared to the population-based studies, there were lower or similar proportions of study participants with antibodies against the S-antigen and against the N-antigen in IMMUNEBRIDGE_ED in the higher age groups and defined comorbidity groups (Fig. 3).