key: cord-0907720-zsurmz5f
authors: Struck, Friedhelm; Schreiner, Patrick; Staschik, Eva; Wochinz‐Richter, Karin; Schulz, Sarah; Soutschek, Erwin; Motz, Manfred; Bauer, Georg
title: Vaccination versus infection with SARS‐CoV‐2: Establishment of a high avidity IgG response versus incomplete avidity maturation
date: 2021-08-20
journal: J Med Virol
DOI: 10.1002/jmv.27270
sha: c5d5bb7bd40d3cc466787fc176596d263ab26402
doc_id: 907720
cord_uid: zsurmz5f

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.

differential determination of SARS-CoV-2-specific IgM and IgG. 6 Also, the humoral response toward SARS-CoV-2 seemed to decline after an initial rise. [7] [8] [9] [10] [11] Avidity of IgG is defined as the strength of IgG binding to its specific epitope. This strength can be easily determined by the degree of removal of bound IgG through treatment with chaotropic agents like urea. 6, 12 Avidity maturation is strictly correlated with affinity maturation, as both characteristics depend on the best fit between IgG and epitope. 6, [13] [14] [15] [16] The avidity of neutralizing IgG is also crucial for protective immunity, 5,6 as will be discussed in detail under Introduction Section in Supporting Information Material. An initial analysis revealed that avidity maturation after SARS-CoV-2 infection remained at an untypical low level in most cases. 12 This is in line with the reports by other groups. 9, 10, [17] [18] [19] [20] The mechanism(s) underlying incomplete avidity maturation after SARS-CoV-2 infections have not been clarified so far. Restricted availability of SARS-CoV-2 antigen for the immune system, either with respect to concentration or time, might hypothetically be the cause for incomplete avidity maturation after the first contact between SARS-CoV-2 antigens and the immune system. It has also been reported that SARS-CoV-2 infection/COVID-19 can strongly impair the function of germinal centers in secondary lymphoid organs. 21 As germinal centers are the site of affinity/avidity maturation, 22 SARS-CoV-2 infections might as well contribute to incomplete avidity maturation through this mechanism.

The importance of high avidity IgG for the immunological defense toward SARS-CoV-2 is obvious, as the interaction between the SARS-CoV-2 RDB and the cellular angiotensin-converting enzyme 2 (ACE2) receptor is driven by high affinity. 23 Therefore, protective immunity toward SARS-CoV-2 infection depends on neutralizing antibodies of high affinity/avidity. 5, 23 It was therefore suggested that (a) natural SARS-CoV-2 infection would not lead to sustained protection toward SARS-CoV-2 and COVID-19, and (b) that the goal of immunization should be the induction of neutralizing antibodies of high avidity. 5 Here we confirm and extend our previous findings on incomplete avidity maturation after natural SARS-CoV-2 infection by increasing (a) the number of patients studied and (b) the time of observation. We also show that two vaccination steps with the BioNTech vaccine lead to an IgG response that is different from the humoral response after SARS-CoV-2 infection both in quantitative and qualitative terms.

Whereas avidity maturation after infection with SARS-CoV-2 remained at low or intermediate avidity levels, two vaccination steps allowed for complete avidity maturation of IgG directed toward RBD/S1 in most vaccinated subjects. 2.2 | Statements of ethics approval and of performance according to relevant guidelines and regulations are expressed after discussion Sera were stored at -20°C until they were tested in the immunoassays.

A. Production of recomLine SARS-CoV-2 IgG nitrocellulose strips:

Individual concentrations of purified recombinant antigens NP, RBD, S1 of SARS-CoV-2, as well as NP of 229E, NL63, OC43, HKU1 were applied directly onto nitrocellulose membranes in separate lanes.

Production was standardized and the resultant strips were evaluated (see below for details), resulting in product # 7374 of Mikrogen GmbH. The assay has been CE-marked.

B. Procedure of the line immunoassay: The reactivity of 1: 100 dilutions of serum antibodies against the recombinant antigens was detected with peroxidase-labeled anti-human IgG antibody and the use of precipitating tetramethylbenzidine. The first incubation of serum and test strips was for 1 h, followed by three washing steps with buffer. The incubation of the strips with peroxidase-labeled antihuman IgG antibody was for 45 min, followed by three washing steps.

Treatment with tetramethylbenzidine was for 8 min.

The line immunoassays were carried out in a semi-automatic processor Dynablot (Dynex Technologies GmbH) with manual serum pipetting according to the instruction manual provided by Mikrogen

GmbH. An Epson J371A scanner (Epson) and recomScan software (Mikrogen GmbH) were used according to the instruction manuals.

C. Avidity determination: sera were incubated for 1 h with the recomLine SARS-CoV-2 IgG test strips in duplicate; then both replicates were incubated for 5 min with wash buffer, and one assay was incubated in the wash solution, while the parallel assay replicate was treated with 7 M urea for 3 min; after three additional washing steps both assay replicates were processed with anti-human IgG antibody labeled with peroxidase and detected as outlined above to describe the line immunoassay procedure. The gray intensity area output by recomScan on the urea-treated test strip was divided by the gray intensity of the parallel assay replicate to determine the avidity index arithmetically. Standard urea treatment for a sharp distinction between IgGs of low and high avidity was 7 M. Where indicated, other concentrations of urea were used during our analysis.

The precision and reproducibility of the immunoassay have been previously described. 12 The data analysis by G. Bauer was performed on the basis of raw data.

The Yates continuity corrected χ 2 test (two-sided) was used for the statistical determination of significances (p < 0.01 = significant; p < 0.001 = highly significant).

3.1 | Infection with SARS-CoV-2 leads to incomplete avidity maturation of IgG directed toward SARS-CoV-2 nucleoprotein (NP), spike protein-1 (S1), and the receptor-binding domain (RBD) of S1

The performance of the recomLine SARS-CoV-2 IgG immuno-test with highly purified recombinant SARS-CoV-2 NP, S1, and RBD has been recently described. 12 Its analytical power was further evaluated (Figures S1 and S2). The long-term kinetics of the IgG responses toward NP, S1, and RBD after symptomatic SARS-CoV-2 infections showed an initial rapid increase in the IgG concentration, followed by a continuous decline ( Figure 1A −H). This is in line with the reports by several other groups. [8] [9] [10] [11] As the IgG was efficiently removed by 7 M urea in the assays described in Figure 1A −G, most of these antibodies seemed to exhibit relatively low or intermediate avidity. The avidity indices showed a striking initial increase, but then remained at a plateau of low or intermediate values ( Figure 1A −G). These findings confirm that the avidity maturation of the IgGs toward SARS-CoV-2 NP, S1, and RBD has not been completed. The curves obtained for IgG directed toward SARS-CoV-2 NP were gradually different from those obtained for IgG directed toward SARS-CoV-2 RBD or S1, whereas the values for IgG toward RBD and S1 were similar.

Avidity maturation of IgG directed toward SARS-CoV-2 RBD and S1 in the case described in Figure 1H Even in those cases that reached higher avidity values, the process of avidity maturation seemed to be rather slow and discontinuous. High avidity indices of more than 0.6 were the exception. They were only found in rare cases and later than 50 days after the onset of disease. In general, avidity indices of IgG toward RBD and S1 seemed to be higher than those for IgG toward NP, though they remained also at an untypical range of low avidity even long times after the onset of disease (see Figures S4−S9 for more details). These findings are in contrast to the findings for infection with other viruses, where low avidity of IgG is characteristic only for the early phase of infection, and then is regularly followed by the establishment of high avidity at later time points 6 (more details under Introduction section in Supporting Information Material). Avidity indices for IgG toward S1

and RBD of SARS-CoV-2 were nearly identical in most samples, in contrast to marked differences in avidity between IgG toward one of these two markers and NP ( Figures S7−S9) . A more refined analysis of avidity maturation, 12 using variable urea concentrations, illustrates the kinetics of incomplete avidity maturation, characterized by an initial onset of maturation, followed by an arrest at low avidity ( Figure S10 ).

The comparison of the antibody concentrations and avidity indices of sera from COVID-19 patients without and with the requirement for hospitalization showed that there was a trend for higher gray intensities and a marked increase in avidity in the hospitalized patients STRUCK ET AL. The responses of IgG directed toward NP (A) or RBD (B) in 16 cases of COVID-19 cases were followed for about 12 months, and the corresponding avidity indices were calculated (IgG NP: C; IgG RBD: D). The results for the IgG levels described under A and B show a rapid increase in IgG toward NP or RBD, followed by a decline over time, characterized by a high degree of variability. Avidity indices (C, D) indicate that avidity maturation is not completed. In the case of IgG directed toward SARS-CoV-2 NP, only one serum reached high avidity (avidity index > 0.6). In the case of RBD, one 5/16 sera reached high avidity, whereas 11/16 sera remained at a plateau of intermediate avidity. COVID-19, coronavirus disease 2019; IgG, immunoglobulin G; NP, nucleoprotein; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 F I G U R E 1 Kinetics of the serological responses to SARS-CoV-2 nucleoprotein (NP), receptor-binding domain (RBD), and spike protein S1. Sera from four patients with COVID-19 confirmed by positive RT-PCR and clinical symptoms were tested for IgG toward SARS-CoV-2 NP (A, C, E, G) or RBD and S1 (B, D, F, H) using a line immunoassay with purified recombinant antigens, as described under Methods. The immunoassays were performed without ("Control") and with urea treatment (7 M) for the determination of avidity. The determined gray intensity values (black) and the calculated avidity indices (red) are plotted toward the days after the onset of the disease. These findings demonstrate that, after a rapid initial increase, the concentrations of IgG directed toward NP, RBD, and S1 are declining. Incomplete avidity maturation seems to be characteristic for the IgG response toward SARS-CoV-2 NP, RBD, and S1, as the avidity indices remain at plateaus at low (avidity index < 0.4) or intermediate avidity (avidity index between 0.4 and 0.6). The avidities of IgG toward RBD and S1 under H are exceptional, showing an initial plateau of avidity after 50 days, followed by a secondary increase in avidity that is finalized at the second plateau of higher avidity. This pattern is indicative of secondary infection with SARS-CoV-2.COVID-19, coronavirus disease 2019; IgG, immunoglobulin G; RT-PCR, real-time polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 ( Figure S11 ). These data show that at least in a low percentage of patients, one of the outcomes of severe illness due to COVID-19 is the final establishment of anti-SARS-CoV-2 IgG of higher avidity.

Vaccination with the BioNTech/Pfizer vaccine caused efficient induction of IgG directed toward SARS-CoV-2 RBD ( Figure 4A ). Despite a marked variability with regard to the onset of IgG generation after the first vaccination, an additional strong increase in the IgG concentration was seen after the second vaccination in all sera. This strong induction of IgG after vaccination was in contrast to the less pronounced IgG response after infection, which was also characterized by a subsequent decline.

The first vaccination step essentially induced IgG of low avidity, whereas the second vaccination step caused rapid and nearly uniform maturation to high avidity ( Figure 4B ). At 19 days after the second vaccination, in most of the sera, high avidity IgG directed toward SARS-CoV-2 RBD (AI > 0.6) had been reached. This impressive effect is contrasted by the delayed and incomplete avidity maturation after natural infection, which resulted in a plateau within the lower avidity range. Figure S20 summarizes and further F I G U R E 3 (See caption on next page) strengthens these findings by presenting the respective median values. Figure 5 summarizes some special cases of IgG responses after vaccination. Cases a-d had been infected with SARS-CoV-2 up to 300 days before vaccination, as confirmed by positive RT-PCR and antibody titers directed toward SARS-CoV-2 NP, RBD, and S1. These titers had been declining over time, thereby maintaining low avidity (see Figures S14−S17 for detailed kinetics). Except for case "a," antibody titers toward RBD were at a level of 100 gray intensity units and less, shortly before vaccination. The first vaccination step was then sufficient to induce an immediate rise to high concentrations of RBD-specific IgG ( Figure 5A ) and to the highest possible level of avidity ( Figure 5B ). This finding shows that a preceding SARS-CoV-2 infection, even after a strong decline of the IgG concentration directed toward SARS-CoV-2, provides strong and immediate support for highly efficient avidity maturation and strong IgG production already after one vaccination step. These findings are extended in Case g represents the exemption within all cases studied by us so far. The concentration of IgG induced by two vaccination steps and determined after the second vaccination was of unusually low concentration and was completely removed by urea treatment. This example seems to represent a rare case of completely failing avidity maturation. This rare case is of specific individual importance, as it is questionable whether the vaccinated subject has established protective immunity.

A summary of our data shows that vaccination with the BioNTech/Pfizer vaccine caused rapid induction of IgG directed toward SARS-CoV-2 RBD (Figure 6A,B) . Within less than 23 days after the first vaccination, 46.1% of sera showed more than 200 gray intensity units. Intensities ranged between 0 and 650 units (median: 168 gray intensity units). Thus 13-30 days after the second vaccination, 97.6% of sera were in the range of more than 400 gray intensity units (median: 613). With one exception, avidity was low after the first vaccination ( Figure 6C ). After the second vaccination ( Figure 6D) , 78% of the sera reached high avidity, whereas only 17.1% remained at intermediate avidity. Avidity maturation seemed to have completely failed in one serum. This failure has been confirmed in a follow-up test. Another serum showed an avidity index at the border between low and intermediate avidity.

A follow-up study performed after the first submission of our manuscript confirmed these findings for a substantially increased number of subjects ( Figure S20 ).

The response toward SARS-CoV-2 S1 corresponded very well to the response toward RBD, whereas there was no detectable response toward SARS-CoV-2 NP, as expected ( Figures S12, S13) . The result shows a broad distribution of gray scales, which was similar at all time points. The percentages of sera showing values in the ranges 0-250, 250-500, and more than 500 gray intensity were not significantly different when sera were taken before or after 50 days after the onset of the disease were compared, with the exception of IgG toward NP at less than 250 units. Importantly, all sera that gave a positive result showed positivity toward all three antigens tested. In 93.5% of the sera with positive IgG toward S1, IgG toward RBD showed a value that was less than 20 gray intensity units different from that obtained for IgG S1, whereas, in 6.5% of the sera, IgG values toward S1 and RBD were different more than 20 gray intensity units (p < 0.001). In contrast, only 15% of sera showed IgG toward NP at a value that differed less than 20 gray intensity units from that of the respective IgG S1 value, and 84.9% showed a higher difference (p < 0.001). (B) Avidity indices. Whereas the gray intensity values (indicative of the respective concentrations of IgG) were broadly similar between sera taken within 50 days or after 50 days after the onset of the disease; the avidity of the IgG was increasing between these groups. However, no complete avidity maturation was reached. A total of 91.9% of the sera taken before Day 50 showed an avidity index below 0.4, whereas only 65.4% of the sera taken after 50 days showed similar low avidity (p < 0.001). As an indication of partial avidity maturation, 8.1% of sera taken before 50 days showed an avidity index between 0.4 and 0.6, in contrast to 22.0% of sera in the group where sera were taken after 50 days (p < 0.01). Avidity indices above 0.6 were only found at 12.6% in the group where sera had been taken later than 50 days after the onset of the disease (p < 0.001). In summary, an overall low degree of avidity maturation was seen, with a minority of samples of high avidity at late time points. These findings confirm incomplete avidity maturation after SARS-CoV-2 infection. COVID-19, coronavirus disease 2019; IgG, immunoglobulin G; RT-PCR, real-time polymerase chain reaction; NP, nucleoprotein; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 4 | DISCUSSION 4.1 | Infection with SARS-CoV-2 leads to immature avidity maturation of IgG directed toward NP, RBD, and S1

Our data confirm that specific IgG responses after SARS-CoV-2 infections are characterized by an initial rapid increase in concentration, followed by a continuous decline. This finding, obtained for IgG toward NP, RBD, and spike protein S1, is in line with the reports by other groups. 7-11 Importantly, after SARS-CoV-2 infection, a limited rapid initial increase in avidity was followed by a continuous plateau As suggested by Khatri et al., 23 high avidity IgG induced by vaccination seems to be essential for interference with the highaffinity interaction between ACE2 and viral spike protein. This concept is in line with the suggestion that protective immunity toward SARS-CoV-2 infection and COVID-19 should require high avidity neutralizing antibodies. 5 These considerations are also in perfect agreement with the highly protective effect of the BioNTech/ Pfizer vaccine in Phase III studies that preceded its approval.

Our findings therefore define S1 and RBD as excellent antigens for the performance of avidity testing, based on the application of the chaotropic agent urea. The observed span between avidity indices F I G U R E 5 Analysis of special cases of vaccination. The figure shows the increase in gray intensity units (A) or avidity index (B) of special cases of vaccinated individuals. Cases a−e represent individuals with natural coronavirus infections several months before vaccination. Before vaccination, in four out of the five cases, the antibody titers derived from primary infection had declined to very low levels. One vaccination step was sufficient to induce a strong IgG response with extremely high avidity in all five cases. Case f represents a case without known preceding SARS-CoV-2 infection. However, the high response with high avidity already after the first vaccination step and the detection of IgG directed toward NP (not shown in the figure) indicates that this case is analogous to cases a-e, that is, it seems to represent a vaccinated individual with (clinical inapparent) SARS-CoV-2 infection. Finally, Case g represents the only case seen so far in our study, which was characterized by a complete failure in avidity maturation after vaccination, paralleled by a low IgG response. V1 and V2 indicate the time point of the first and second vaccination. IgG, immunoglobulin G; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 STRUCK ET AL. shown that lowering the antigenic load through treatment of HIV or tuberculosis infections was leading to the prevalence of low avidity IgG directed toward the respective agents. 28, 29 The impairment of germinal centers in secondary lymphoid organs by SARS-CoV-2, as reported by Kaneko et al., 21 would allow a straightforward explanation for incomplete avidity maturation after SARS-CoV-2 infection. However, as the important findings by Kaneko et al. 21 have been established in lymph nodes derived from patients who had died from COVID-19, it is uncertain whether this mechanism also applies to patients with milder disease. Furthermore, the relative increase in avidity in patients, who required hospitalization compared with outpatients, as shown in Figure S11 This allows the assumption that a higher viral load associated with the more severe disease might increase avidity maturation, but that the failure to reach higher avidity increases the risk of a more severe outcome of the disease. The latter finding strengthens the role of high avidity for protective immunity. 5 The SARS-CoV-2 infection has been shown to lead to the generation of neutralizing antibodies. 32, 33 It was demonstrated that the parallel determination of the IgG response toward RBD and spike protein of SARS-CoV-2 correlated very well with the presence of neutralizing antibodies. 29 Only one out of 5882 individuals positive for both IgG toward RBD and spike protein did not show neutralizing antibodies. This is in line with findings by other groups. [9] [10] [11] However, classical neutralization tests are performed by preincubation of virus and serum, before the mixture is applied to test cells. Therefore, neutralization tests determine the concentration of IgG with specific binding to epitopes that are actually involved in the attachment of the virus to the cellular receptor. However, as the test systems for neutralization do not involve direct competition between antibodies and cellular receptors for viral spike proteins, they do not allow to determine the role of IgG avidity, which might be critical for virus neutralization in vivo. 23 This deficiency can be compensated by direct avidity determination of IgG directed toward SARS-CoV-2 RBD. The importance of neutralization, as well as avidity for antibody functionality, has also been explicitly pointed out by Gaspar and De Gaspari. 34 Protective immunity can be predicted to depend on a sufficiently high concentration of neutralizing antibodies, ensuring optimal load of the virus with antibody. In addition, a sufficiently high affinity/ avidity of these antibodies is most likely required, to cope with the competition of ACE2 for viral spike protein. The determination of avidity of IgG directed toward SARS-CoV-2 RBD in the small percentage of patients who developed COVID-19 despite vaccination might allow to clarify the predictive value of avidity determination for the determination of individual protective immunity.

F I G U R E 6 IgG responses after vaccination and natural infection with SARS-CoV-2. Thirty-nine sera taken between 9 and 22 days after first vaccination with the BioNTech/Pfizer vaccine (A) and 41 sera taken between 6 and 25 days after second vaccination with the BioNTech/Pfizer vaccine (B) were tested for IgG directed toward SARS-CoV-2 RBD. The determined gray intensity units are shown under A and B, whereas the avidity indices, determined by parallel treatment with 7 M urea after incubation of serum with test antigens, are shown under C (sera obtained after the first vaccination) and D (sera obtained after the second vaccination). For comparison, 37 sera were taken from COVID-19 outpatients between 17 and 30 days after the onset of the disease (E), 42 sera from COVID-19 outpatients taken between 31 and 60 days after the onset of the disease (F) and 34 sera from COVID-19 outpatients taken between 61 and 140 days after the onset of the disease were tested for IgG directed toward RBD (gray intensity units). The avidity indices of these sera were determined and are shown (H−J). The figure shows a variable induction of IgG after the first vaccination (A), which extends to uniformly high values after the second vaccination (B) (p < 0.001). This is paralleled by low avidity indices after the first vaccination (with one exception) and high avidity in 78.0% of the samples taken after the second vaccination (p < 0.001). Though natural infection with SARS-CoV-2 can also induce IgG concentrations of higher levels, a broad range of intensities is maintained over long times without statistically significant changes (E−G). Though a certain degree of avidity maturation in the low and intermediate range was seen with time for sera from patients with natural SARS-CoV-2 infection, only a minority of sera (11.8%) reached high avidity values (>0.6) (H−J) at 61−140 days after natural infection, whereas 78% of sera taken from vaccinated individuals had reached avidity indices above 0.6 between 6 and 25 days after the second vaccination (p < 0.001). The median values calculated from the data presented in Figure 6 are summarized in Figure S20 . The confirmation of the data presented in Figure 6A −D through the increase in the number of vaccinated subjects are presented in Figure S18 . COVID-19, coronavirus disease 2019; IgG, immunoglobulin G; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 4.4 | Induction of low avidity IgG: Potential advantage for coronaviruses?

As seasonal coronavirus infections are also frequently characterized by subsequent incomplete avidity maturation, 12, 26 it may be speculated that maintaining low avidity of the IgG response, either through the suboptimal supply of the immune system with viral antigens or through impairment of avidity maturation, is part of the biology of coronaviruses in general. This might ensure the observed repeated waves of reinfection over time. [35] [36] [37] From this perspective, the establishment of high avidity IgG directed toward SARS-CoV-2 RBD through vaccination seems to be a rational approach to break the dynamics of SARS-CoV-2 infections and reinfections. The determination of avidity of IgG directed toward RBD might thereby play an essential role in the determination of individual protective immunity.

The authors thank Dr. P. Luppa (Technical University Munich) and 

All raw data used for this study are presented within the study as "Gray intensity units". Additional data are presented under Supplementary Materials.

http://orcid.org/0000-0003-3134-3885

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