The serological responses towards severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein, receptor-binding domain (RBD), and spike protein S1 are characterized by incomplete avidity maturation. Analysis with varying concentrations of urea allows to determine distinct differences in avidity maturation, though the total process remains at an unusually low level. Despite incomplete avidity maturation, this approach allows to define early and late stages of infection. It therefore can compensate for the recently described irregular kinetic patterns of immunoglobulin M and immunoglobulin G (IgG) directed towards SARS-CoV-2 antigens. The serological responses towards seasonal coronaviruses neither have a negative nor positive impact on SARS-CoV-2 serology in general. Avidity determination in combination with measurement of antibody titers and complexity of the immune response allows to clearly differentiate between IgG responses towards seasonal coronaviruses and SARS-CoV-2.Cross-reactions seem to occur with very low probability. They can be recognized by their pattern of response and through differential treatment with urea. As high avidity has been shown to be essential in several virus systems for the protective effect of neutralizing antibodies, it should be clarified whether high avidity of IgG directed towards RBD indicates protective immunity. If this is the case, monitoring of avidity should be part of the optimization of vaccination programs.

Avidity is defined as the binding strength of immunoglobulin G (IgG) toward its target epitope. Avidity is directly related to affinity, as both processes are determined by the best fit of IgG to epitopes. We confirm and extend data on incomplete avidity maturation of IgG toward severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleoprotein (NP), spike protein‐1 (S1), and its receptor‐binding domain (RBD) in coronavirus disease 2019 (COVID‐19) patients. In SARS‐CoV‐2‐infected individuals, an initial rise in avidity maturation was ending abruptly, leading to IgG of persistently low or intermediate avidity. Incomplete avidity maturation might facilitate secondary SARS‐CoV‐2 infections and thus prevent the establishment of herd immunity. Incomplete avidity maturation after infection with SARS‐CoV‐2 (with only 11.8% of cases showing finally IgG of high avidity, that is, an avidity index > 0.6) was contrasted by regular and rapid establishment of high avidity in SARS‐CoV‐2 naïve individuals after two vaccination steps with the BioNTech messenger RNA (mRNA) Vaccine (78% of cases with high avidity). One vaccination step was not sufficient for induction of complete avidity maturation in vaccinated SARS‐CoV‐2 naïve individuals, as it induced high avidity only in 2.9% of cases within 3 weeks. However, one vaccination step was sufficient to induce high avidity in individuals with previous SARS‐CoV‐2 infection.

The humoral immune systems controls viral infections through recognition of critical viral target structures, selective proliferation stimulation of IgM-presenting B cells, class switch to IgG-generating B cells and subsequent affinity maturation of IgG. Affinity maturation is achieved through proliferation, hypermutation and clonal selection of IgG-generating B cells (1-4). The establishment of high affinity IgG is essential for sustained protective antiviral effects (5-10). While analyzing the maturation of functional affinity (avidity) of IgG towards SARS CoV-2 nucleoprotein, surface protein and its receptor-binding domain, we realized that avidity maturation was frequently not completed after infection. This finding gives insight into the biological strategy of SARS CoV-2 and is important for serodiagnosis. Incomplete avidity maturation might explain the observed decline of the humoral response (11-14), allow for secondary SARS CoV-2 infections and prevent the establishment of herd immunity. Therefore, future immunization strategies should achieve the goal to induce neutralizing IgG of high avidity.

In classical viral infections, the avidity of immunoglobulin G (IgG) is low during acute infection and high a few months later. As recently reported, SARS‐CoV‐2 infections are not following this scheme, but they are rather characterized by incomplete avidity maturation. This study was performed to clarify whether infection with seasonal coronaviruses also leads to incomplete avidity maturation. The avidity of IgG toward the nucleoprotein (NP) of the seasonal coronaviruses 229E, NL63, OC43, HKU1 and of SARS‐CoV‐2 was determined in the sera from 88 healthy, SARS‐CoV‐2‐negative subjects and in the sera from 70 COVID‐19 outpatients, using the recomLine SARS‐CoV‐2 assay with recombinant antigens. In the sera from SARS‐CoV‐2‐negative subjects, incomplete avidity maturation (persistent low and intermediate avidity indices) was the lowest for infections with the alpha‐coronaviruses 229E (33.3%) and NL63 (61.3%), and the highest for the beta‐coronaviruses OC43 (77.5%) and HKU1 (71.4%). In the sera from COVID‐19 patients, the degree of incomplete avidity maturation of IgG toward NP of 223E, OC43, and HKU1 was not significantly different from that found in SARS‐CoV‐2‐negative subjects, but a significant increase in avidity was observed for IgG toward NP of NL63. Though there was no cross‐reaction between SARS‐CoV‐2 and seasonal coronaviruses, higher concentrations of IgG directed toward seasonal coronaviruses seemed to indirectly increase avidity maturation of IgG directed toward SARS‐CoV‐2. Our data show that incomplete IgG avidity maturation represents a characteristic consequence of coronavirus infections. This raises problems for the serological differentiation between acute and past infections and may be important for the biology of coronaviruses.

The avidity (binding strength) of IgG directed towards the receptor‐binding domain (RBD) of spike protein has been recognized as a central marker in severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) serology. It seems to be linked to increased infection‐neutralization potential and therefore might indicate protective immunity. Using a prototype line assay based on the established recomLine SARS‐CoV‐2 assay, supplemented with RBD of the delta and the omicron variant, differential avidity determination of IgG directed towards RBD of wild‐type (WT) SARS‐CoV‐2 and distinct variants was possible within one assay. Our data confirm that natural SARS‐CoV‐2 infection or one vaccination step lead to low avidity IgG, whereas further vaccination steps gradually increase avidity to high values. High avidity is not reached by infection alone. After infection with WT SARS‐CoV‐2 or vaccination based on mRNA WT, the avidity of cross‐reacting IgG directed towards RBD of the delta variant only showed marginal differences compared to IgG directed towards RBD WT. In contrast, the avidity of IgG cross‐reacting with RBD of the omicron variant was always much lower than for IgG RBD WT, except after the third vaccination step. Therefore, parallel avidity testing of RBD WT and omicron seems to be mandatory for a significant assessment of protective immunity towards SARS‐CoV‐2.