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.