Discussion
The study was performed to unveil the group of serological non-responders in PCR+ individuals of the first pandemic wave with Wuhan type SARS-CoV-2 in spring 2020 6 to 9 months after diagnosis and to evaluate their specific cellular immune response towards spike molecule compared to two groups of PCR- and of not PCR-tested (NT) household contact persons. The present study revealed a large group of serological non-responders (NR) in PCR+ persons after 6 to 9 months post infection, and in contrast, determined a SARS-CoV-2-specific serologically positive and cellular reactive group of individuals in PCR- contact persons and in household contact persons who were not PCR-tested.
Our results are in concordance with previous studies showing that SARS-CoV-2 infected persons are able to develop a robust antibody response particularly in symptomatic persons (Zhang, Li et al. 2020, Sonnleitner, Prelog et al. 2021). However, we could refer to a group of serological non-responders of 35.3% in the PCR+ group, which concurs to previous studies that showed that donors, especially with milder symptoms, middle-aged, and without comorbidities failed to have or had low levels of IgG-anti-SARS-CoV-2 antibodies (Baron, Risch et al. 2020, Marklund, Leach et al. 2020).
Interestingly, in the majority of IgG positive persons and particularly in those who were tested PCR+, positive serum IgA concentrations were still present over 6 months post diagnosis. The IgA concentrations correlated well with the IgG concentrations, and underline the observation that those individuals who develop a significant IgG response likely maintain positive serum IgA levels. IgA is one of the first line of defense against infection and has an important role mediating adaptive humoral response protecting mucosal surfaces (Devito, Hinkula et al. 2000, Planque, Salas et al. 2010). Recently, a group demonstrated that early response to SARS-CoV-2 is dominated by IgA antibodies with higher neutralizing capacity than IgG in the first weeks post infection. Moreover, the study also showed that IgA serum concentrations are diminished after one month of infection onset (Sterlin, Mathian et al. 2021). Undetectable IgA concentrations in some donors from the PCR- and NT group with positive IgG may be explained by possibly shortened periods of viral shedding (Callow 1985). The fact that IgA has a production peak approximately 10 days after mucosal antigen contact (Moldoveanu, Clements et al. 1995) suggest that time of collection of the samples for the analysis of the IgA response in the cohort studied is an important factor to be considered. However, our results suggest that IgA can persist even several months after primary SARS-CoV-2 infection.
A surrogate marker for the binding capacity was the assessment of IgG avidity in IgG positive individuals. As known from serological measures against other infectious pathogens, the IgG avidity matures within the first 3 to 5 months after primary infection due to clonal hypermutation of memory B cells (Prelog, Almanzar et al. 2013, Prelog, Schonlaub et al. 2013). Moderate to high RAI was developed in most of serologically responding PCR+ individuals. Although the neutralizing ability of the IgG was not tested in this study, this finding suggests a high binding strength of the detected IgG in those individuals with potentially neutralizing activity. A subgroup of the cohort was tested by neutralization assays in a former study at 5 months post positive PCR and 29 of 34 (85.3%) showed positive results in an enzyme-linked neutralization assays previously published (Sonnleitner, Prelog et al. 2021). In addition, the IgG ELISA assay used to analyze the concentration of IgG towards SARS-CoV-2 showed a highly significant correlation with neutralizing antibodies (Stromer, Rose et al. 2020).
Inflammation may occur during virus infection resulting in recruitment of CD4+ and CD8+ T cells and the production of pro-inflammatory cytokines such as IFNγ or TNFα. The subsequent generation of virus-specific T cells that are in charge of the elimination of the infection is as important as the generation of specific antibodies for the control of the infection and long-term neutralizing immunity. The present study identifies serological non-responders who were able to mount a cellular immune response, although to a significant lower level than found in individuals with positive IgG concentrations. Also, individuals of the PCR- and NT group developed positive cellular responses. These findings support the results by others who showed that unexposed or serologically negative donors have a mixed SARS-CoV-2-specific IFNγ response to nucleoprotein or to non-structural protein (NSP) 7 and NSP13 (Grifoni, Weiskopf et al. 2020, Le Bert, Tan et al. 2020).
One of the most astonishing results was that serological and cellular reactive samples could be identified in the PCR- and in the non-tested household contact groups. Although IgG responses in the PCR- and in the NT groups were lower compared to PCR+ group, avidities were fairly good and positive IgA levels could be found. The cellular responses were equal to PCR+ individuals. Thus, the present study underlines the impact of undetected SARS-CoV-2 during the first pandemic wave in spring 2020 by Wuhan type.
This cohort study is limited by its cross-sectional character, which does not allow to describe antibody dynamics over time and the relatively small sample size and the geographic locations. Study participants were not investigated regarding the disease severity, underlying diseases, COVID-19 risk factors or specific treatments and are highly heterogeneous regarding their age. However, no correlations with age could be demonstrated regarding humoral and cellular immune parameters. As the initial antibody and cellular response shortly after the infection is not known in our cohort, it is difficult to allow prognosis on the further dynamics of the humoral and cellular immune response in the study participants. A subgroup of our study participants showed positive IgG antibodies against S1/S2 domain with a CLIA assay in 28 of 34 individuals (82.4%) after about one month post infection and 27 (81.8%) after 5 months (Sonnleitner, Prelog et al. 2021). Several studies showed that most individuals with positive antibody reactions shortly after infection maintain their antibodies; however, a sharp decrease is reported after 6 months (Jaaskelainen, Ahava et al. 2021, Pradenas, Trinite et al. 2021), which may explain the relatively large number of serological non-responders in our cohort. These results underline the need for immunological booster after at least 6 months post infection. A booster by vaccination could be shown in reconvalescent individuals by several groups (Tretyn, Szczepanek et al. 2021) .
In conclusion, our findings showed that individuals who were tested negative for SARS-CoV-2 in PCR and individuals who were not tested (NT) although they were household contact persons to PCR+ persons developed SARS-CoV-2-specific humoral and cellular immune responses. The relatively large proportion of serological non-responders but also the proportion of cellular non-responders within the group of IgG positive individuals after PCR+ SARS-CoV-2 infection at least 6 months ago underlines the need for COVID-19 vaccinations in the reconvalescent group. Thus, the present study provides further evidence for the recommendation to vaccinate reconvalescent individuals from the first pandemic wave after at least 6 months post positive PCR to boost their humoral and cellular immune response.