key: cord-0867011-v8unyiik
authors: Markewitz, Robert; Pauli, Daniela; Dargvainiene, Justina; Steinhagen, Katja; Engel, Sarah; Herbst, Victor; Zapf, Dorinja; Krüger, Christina; Sharifzadeh, Shahpour; Schomburg, Benjamin; Leypoldt, Frank; Rupp, Jan; Görg, Siegfried; Junker, Ralf; Wandinger, Klaus-Peter
title: B-cell-responses to vaccination with BNT162b2 and mRNA-1273 six months after second dose
date: 2022-03-05
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2022.02.028
sha: 9b5620e951043f7fb4339ba324063a7d13eddbc0
doc_id: 867011
cord_uid: v8unyiik

OBJECTIVES: To examine the state of B-cell immunity six months after the second vaccination against SARS-CoV-2 in comparison to the state observed two weeks after it. METHODS: Sera of 439 participants, whose immune responses to two doses of an mRNA-based vaccine (BNT162b2 or mRNA-1273) has been previously characterized, was examined for anti-S1 IgG and IgA, anti-NCP IgG and neutralizing antibodies (nAb), as well as antinuclear antibodies (ANA). RESULTS: Levels of all examined markers decreased significantly from two weeks to six months after the second vaccination (anti-S1 IgG: 3744±2571.4 vs. 253±144 BAU/ml; anti-S1 IgA: 12±0 vs. 1.98±1.75 OD ratio; nAb: 100±0 vs. 82±19.3 %), the vast majority of participants retaining reactive levels of anti-S1 IgG (436/439) and anti-S1 IgA (334/439) at six months. Immune responses were stronger for mRNA-1273 compared with BNT162b2 (anti-S1 IgG: 429±289 vs. 243±143 BAU/ml; anti-S1 IgA: 5.38±3.91 vs. 1.89±1.53 OD ratio; nAb: 90.5±12.6 vs. 81±19.3 %). There was no meaningful influence of sex and age on the examined markers. There was a strong correlation between anti-S1 IgG and the SNA (rho = 0.91, p < 0.0001), but not for for IgA and the SNA (rho = 0.52, p < 0.0001). There was a ceiling effect for the association between anti-S1 IgG titers and the inhibition of the binding between S1 and ACE2. ANA prevalence was unchanged from two to six months after the second vaccination (87/498 vs. 77/435), as were the median, ANA titers (1:160 vs. 1:160)). CONCLUSIONS: While the clinical consequences of decreasing anti-SARS-CoV-2 antibody titers cannot be estimated with certainty, a lowered degree of clinical protection against SARS-CoV-2 is possible. Persistently stronger responses to mRNA-1273 suggest that it might confer greater protection than BNT162b2, even six months after the second vaccination. Both examined vaccinations do not induce ANA within the examined time frame.

Vaccinations against the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been rolled out on a worldwide basis since their approval by the CDC and the EMA (among others) in late 2020. Since then, multiple studies have been conducted on the immune system's response to different vaccines as well as the resulting clinical efficacy (1-6). We, among others, found that vaccination with BNT162b2 or mRNA-1273 leads to high titers of specific IgG antibodies against the spike protein of SARS-CoV-2, with stronger reactions for mRNA-1273 (7) . In the short term, there was no induction of antinuclear antibodies (ANA) as a correlate for autoimmunity caused by the vaccination. Contrary to findings by others (8, 9) , we could not find a pronounced influence of age on the B-and T-cell responses two weeks after the second dose of the vaccination.

In the meantime, there is evidence for the waning of antibody titers over time after vaccination against SARS-CoV-2 (10) . In this current study, we examined whether we could find a similar waning of antibodies against SARS-CoV-2 for our cohort six months after the second vaccination; and if yes, how large was the reduction of different markers of B-cell immunity against SARS-CoV-2. Also, we examined associations between B-cell immunity and age, sex, J o u r n a l P r e -p r o o f the vaccine received (BNT162b2 vs. mRNA-1273), as well as correlations between the different markers. Lastly, we examined the possible induction of ANA by the vaccine. We hypothesized that -Levels of markers of B-cell immunity after vaccination against SARS-CoV-2 would decrease significantly, -Reactions would remain stronger for recipients of mRNA-1273 compared to those of BNT162b2, -There would be no relevant influence of sex and age (within the examined age span) on the measured immune responses and -There would be no induction of ANA six months after the second vaccination.

All 531 participants of the previous study were asked to participate in a follow-up examination of their B-cell immunity against SARS-CoV-2. The initial recruitment of this cohort as well as details on the vaccination program they underwent is detailed elsewhere (7) . In short, all participants were health-care professionals from a German university hospital who received either BNT1662b2 or mRNA-1273 with a five-week inter-dose interval between.

All participants provided written informed consent for this study. The study was approved by the University of Kiel institutional review board (AZ: D642/20). The study was conducted in accordance with the Declaration of Helsinki (11).

Antibodies of the classes IgG and IgA against the S1 subunit of the spike protein of SARS-CoV-2 (anti-S1) as well as IgG against the nucleocapsid protein of SARS-CoV-2 (anti-NCP) were measured using the respective ELISA test kits by EUROIMMUN (Lübeck Germany). S1 was chosen as target antigen to monitor the response to the vaccination, as the vaccination induces the production of S1 within the body as immunogenic target, and NCP was chosen in order to identify possible infections with SARS-CoV-2 within the cohort, as anti-NCP is only present after infection with SARS-CoV-2 and not after vaccination. More information on the assays can be found in the supplement.

Neutralizing antibodies were examined via a surrogate neutralization assay (SNA) (NeutraLISA; EUROIMMUN, Lübeck, Germany) according to the manufacturers' instructions. In brief, this is a competitive ELISA in which anti-S1 antibodies within the examined serum competes with ACE2 contained in a buffer for binding at S1 bound to a solid phase. The amount of bound ACE2 that can be detected via enzymatic reaction after a washing step is inversely proportional to the level of inhibition of SARS-CoV-2 achieved via anti-SARS-CoV-2 antibodies. More information on the SNA can be found in the supplement.

Differences in continuous variables between groups were examined via analyses of variance (ANOVAs), with a Benjamini-Yekutieli correction in case of multiple comparisons (12) . Posthoc testing was performed via Tukey's honest significant differences (Tukey's HSD).

Differences between two groups were tested via student's t-test. Differences in the distribution of categorically scaled variables between groups was tested via Pearson's Chi-squared test. For associations between two continuous variables, correlations using Spearman's rho were calculated. Statistical significance was assumed for p<0.05. Average values with a measure of dispersion are reported as median and median absolute deviation (MAD), unless otherwise stated. All statistical analyses were performed using the open-source software for statistical computing and graphics R (version 4.1.0) with the integrated development environment RStudio (Version 1.4.1717) (13) .

Of the 531 participants who donated a serum sample during the previous study, 439 (82.7 %) did so again during the current study. Of these, 322 (73.3 %) were female and 403 (91.8 %) received BNT162b2. Their median age was 45±14.8 (range: 20-66) years old. There was no statistically significant difference in age between the two sexes (df = 1, F = 3.729, p = 0.054) or between recipients of the different vaccines (df = 2, F = 2.379, p = 0.124). Pearson's chisquared test revealed no statistically significantly different distribution of sexes between recipients of the two vaccines (chi 2 = 0.444, df = 1, p = 0.505).

Samples were donated at a median of 181 (±1.48) days after the second dose of the vaccination against SARS-CoV-2.

Seven participants had a known history of COVID-19, five of them before the administration of the first dose, one who was tested positive (via PCR) in between both doses and one who tested positive (via PCR) after having received both doses of the vaccine.

Levels of all examined markers decreased significantly from 14 days to six months after the second vaccination (all p-values <0.0001, see Figure 1 , panels A-C). Two-way ANOVAs with the factors sex and vaccine revealed a statistically significant main effect of the vaccine administered for all markers (with higher levels for recipients of mRNA-1273; all p-values <0.001, see Figure 1 , panels G-I), but no significant main effect of sex or interaction effect of sex and the vaccine administered (all p values >0.1; see Figure S1 , panels A-C). Notably, only three participants, who exhibited reactive anti-S1 IgG titers two weeks after the second dose, showed levels of anti-S1 IgG below the cutoff for reactivity. However, their anti-S1 IgG titers two weeks after the second dose had already been below the 1 st percentile of the cohort. 334 out of 439 participants (76.1 %) were anti-S1 IgA reactive after six months.

Correlations between all examined markers and age were of small to negligible effect size (albeit statistically significant: anti-S1 IgG and age: rho = -0.23, p < 0.0001; anti-S1 IgA and age: rho = -0.16, p < 0.001; inhibition via SNA and age: rho = -0.25, p < 0.0001; see Figure 1 , panels D-F). Effect sizes were not significantly greater when these correlations were examined separately for recipients of the two different vaccines, for which a significant main effect on the levels of examined markers was shown (see Figure S1 , panels D-I).

Among the examined markers, there was a strong correlation between anti-S1 IgG and inhibition via SNA (rho = 0.91, p < 0.0001) and correlations of medium effect size between anti-S1 IgA and inhibition via SNA (rho = 0.53, p < 0.0001) and between anti-S1 IgG and anti-S1 IgA (rho = 0.52, p < 0.0001; see Figure 2 ).

Of the 439 participants of the current study, seven tested positive for anti-NCP IgG. Of these seven, one had a known history of COVID-19, five were deemed false positives and for one a possible asymptomatic infection could not be ruled out. Four participants with a history of COVID-19 who were anti-NCP positive in the previous study were negative in the current study and two participants never developed anti-NCP-IgG at any time point despite a history of COVID-19.More detailed data of all previously infected participants, as well as all participants who tested anti-NCP IgG positive after six months can be found in the supplement.

ANA testing via indirect immunofluorescence testing revealed that six months after the administration of the second dose of the vaccine, there was no change in qualitative ANA results (McNemar's chi-squared = 2.5574, df = 3, p = 0.465), nor in the semiquantitative titers of those individuals who tested ANA positive at any time point (Wilcoxon signed-rank test: V = 46, p = 0.606; see Figure S2 of the supplement).

In the current study, we found that levels of markers of B-cell immunity against SARS-CoV-2, such as anti-S1 IgG and IgA, as well as in vitro inhibition via SNA decrease significantly over the course of six months since the administration of the second dose of the vaccination against SARS-CoV-2.

We again found that recipients of mRNA-1273 had significantly higher titers of all examined markers compared to recipients of BNT162b2. This difference, found by others as well (9, 14) , manifests itself as early as 14 days after the first dose of the vaccination (7) and apparently persists long-term, despite the overall decrease of antibody levels. The fact that vaccination with mRNA-1273 leads to higher levels of all examined markers than vaccination with BNT162b2 is most likely explained by the a greater amount of mRNA contained in the former (100 µg) compared to the latter (30 µg)(2,3), although it is surprising that this difference is persistent. Granted that higher levels of anti-S1 IgG convey a higher degree and longer duration of protection from SARS-CoV-2, mRNA-1273 may be preferable to BNT162b2 in this particular respect.

Nearly all participants still exhibited anti-S1 IgG reactive titers six months after the second dose of the vaccination. Furthermore, those participants who did not exhibit reactivity at six months after the second dose already exhibited low levels of anti-S1 IgG two weeks after the second dose. Therefore, the exact consequences of waning titers for the protection against SARS-CoV-2 are difficult to estimate, especially in the absence of a clearly established cutoff value for anti-S1 IgG above which protection can be assumed.

Notably, anti-S1 IgG correlates strongly with inhibition via SNA but anti-S1 IgA does not (the correlation of medium effect size between anti-S1 IgA and the SNA is likely explained by a correlation of the same effect size between anti-S1 IgA and IgG). This is surprising because in theory, anti-S1 IgA competes with ACE2 for binding at the S1 bound on the solid phase in the same extent as anti-S1 IgG. Our findings, however, suggest that, at least in vitro, serum anti-J o u r n a l P r e -p r o o f S1 contributes markedly less to the neutralization of SARS-CoV-2 compared to anti-S1 IgG. On the other hand, it is also surprising that a high proportion of participants (76.1 %) still exhibit anti-S1 IgA six months after the second dose of the vaccination, as it is usually considered an early marker of humoral immunity against SARS-CoV-2 with a tendency to wane faster than anti-S1 IgG (15) . Conceivably, anti-S1 IgA remains an important part of the mucosal first line of defense against SARS-CoV-2 even in the long term, which might explain its persistence in serum.

The correlation between anti-S1 IgG and the SNA reveals another insight: levels of inhibition sharply increase with increasing anti-S1 IgG only until anti-S1 IgG titers of about 350 BAU/ml. After this point a ceiling effect is reached with inhibition levels reaching levels close to 100 %. As a consequence of the assay's design, apparently all S1 on the solid phase is bound by anti-S1, which, probably through a combination of higher affinity and concentration, vastly outcompetes the ACE2 in the buffer for binding at S1. On one hand, this demonstrates the limitations of this SNA, on the other hand, as the SNA was modeled after "actual" plaque reduction neutralization tests (PRNT) (16) , it is conceivable that a similar constellation of anti-S1, S1 and ACE2 is reached in vivo at titers of 350-400 BAU/ml. The resulting hypothesis that titers above these limits might confer a relatively certain degree of immunity is highly speculative would have to be tested in a clinical setting however.

Almost all participants, who had developed anti-NCP IgG in the course of the previous study as a result of a previous infection with SARS-CoV-2 returned a negative result during the current study, suggesting that anti-NCP IgG had waned in the meantime. On the other hand, the majority of positive anti-NCP IgG results were likely false positives. This is most likely explained by the low prevalence of COVID-19 in our cohort and the resulting low pre-test probability. In a low-prevalence population, such as our own, even a test with excellent sensitivity and specificity can have a low positive predictive value, leading to a relatively high proportion of false positives. In our experience, previously infected individuals are most reliably identified by the particular dynamics of their antibody response to the vaccination, with significantly higher titers of anti-S1 IgG already after the first dose of the vaccination. Oligo-or asymptomatic cases of COVID-19 that may lack development of anti-NCP IgG (as was the case for one participant in our cohort) are difficult to detect serologically and may go undetected without PCR testing.

Finally, we did not find any induction of ANA by the vaccination against SARS-CoV-2 six months after the second dose, nor did we see increases in ANA titers in participants with a preexisting ANA. Therefore, we could not establish any link between anti-SARS-CoV-2 vaccination and autoimmunity (as detected via ANA).

Our study had several limitations: There were obvious imbalances within the cohort with females and recipients of BNT162b2 being overrepresented. But as the distribution of age groups between the sexes, as well as the distribution of age groups and sexes between the recipients of the different vaccines was not significantly different, it is fair to assume that the effects we found (e.g. stronger reactions for mRNA-1273) are representative and not caused by skewed distributions. Also, there was no routine PCR testing for SARS-CoV-2 within the cohort. Oligo-or asymptomatic cases of SARS-CoV-2 without development of anti-NCP IgG during or after the vaccination program might therefore have been overlooked. Also, while ANA testing via IIF is a relatively broad screening for autoantibodies within the serum, it is possible that the induction of autoantibodies not covered by this assay may have gone unnoticed.

In conclusion, we found that markers of B-cell immunity against SARS-CoV-2 decrease significantly within six months after the second dose of the vaccination against SARS-CoV-2, with little to no discernible influence of age or sex in our cohort. Despite this, recipients of mRNA-1273 still exhibit higher levels of all examined markers than recipients of BNT162b2 at this time point, continuing a phenomenon that can already be detected after the first dose of the vaccination. At least in vitro, anti-S1 IgG seems to contribute significantly more to the inhibition of binding between S1 and ACE2 than serum anti-S1 IgA. Lastly, we did not see any induction of autoimmunity (as examined via ANA IIF) via the vaccination against SARS-CoV-2 six months after the second dose. Fig. 1 : Panels A-C show the decrease of measured levels from 14 days to six months after the second dose of the vaccination for anti-S1 IgG (panel A), anti-S1 IgA (panel B) and inhibition via SNA (panel C); panels D-F depict the correlation of these markers at six months after the second dose with age (the blue line representing the conditional smoothed mean, calculated via locally estimated scatterplot smoothing (LOESS), with a gray 95 % confidence band around it); panels G-I show the comparison of levels of these markers between recipients of mRNA-1273 and BNT162b2 at six months after the second dose. The individual measurements, plotted as dots, are color-coded according to the vaccination received (lilac: mRNA-1273; orange: BNT162b2).

Panels A-C depict the correlations found between inhibition (via SNA) and anti-S1-IgG (panel A), inhibition and anti-S1 IgA (panel B) and anti-S1 IgA and anti-S1 IgG (panel C). The blue lines in panels A-C again represent the smoothed means (via LOESS) with a 95 % confidence band. Panel D provides a correlation matrix for all examined markers of immunity against SARS-CoV-2 as well as age. The individual measurements, plotted as dots, are colorcoded according to the vaccination received (lilac: mRNA-1273; orange: BNT162b2). Table 1 : Average values (reported via medians ± median absolute deviation) for all markers measured at six months after the second vaccination for the whole cohort and the following subgroups: women, men, participants previously infected with SARS-CoV-2, participants having received BNT162b2, and participants having received mRNA-1273. Shown are data for the time points two weeks after the second vaccination (data already published elsewhere) and six months after the second vaccination (data newly accumulated).

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass Vaccination Setting

Interim Estimates of Vaccine Effectiveness of BNT162b2 and mRNA-1273 COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Health Care Personnel, First Responders, and Other Essential and Frontline Workers -Eight

Early Evidence of the Effect of SARS-CoV-2 Vaccine at One Medical Center

The temporal course of T-and B-cell-responses to vaccination with BNT162b2 and mRNA-1273

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2

Comparison of SARS-CoV-2 Antibody Response by Age Among Recipients of the BNT162b2 vs the mRNA-1273 Vaccine

Waning Immune Humoral Response to BNT162b2 Covid-19 Vaccine over 6 Months

World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects

The control of the false discovery rate in multiple testing under dependency

R: A Language and Environment for Statistical Computing

Comparison of SARS-CoV-2 Antibody Response Following Vaccination With BNT162b2 and mRNA-1273

IgA dominates the early neutralizing antibody response to SARS-CoV-2

Virological assessment of hospitalized patients with COVID-2019

The authors want to express their gratitude towards all colleagues and coworkers who have made this study possible, special thanks go out to: Mrs Taylor 

R.M. declares support from the EUROIMMUN AG for attending the 15 th Dresden Symposium on autoantibodies 2021. K.S., V.H., D.Z., and C.K. currently are employees of the EUROIMMUN AG (Lübeck, Germany). They furthermore declare a planned patent for a method and reagents for the detection of an immune response to SARS-CoV-2. All other coauthors have no conflict of interest to declare.

No external funding was received for this study. Anti-S1 IgG (BAU/ml) Neutral. antibodies (%)Anti-S1 IgA (OD ratio)Anti-S1 IgG (BAU/ml)