key: cord-0718330-9qm9lbyc
authors: Terpos, Evangelos; Stellas, Dimitris; Rosati, Margherita; Sergentanis, Theodoros N.; Hu, Xintao; Politou, Marianna; Pappa, Vassiliki; Ntanasis-Stathopoulos, Ioannis; Karaliota, Sevasti; Bear, Jenifer; Donohue, Duncan; Pagoni, Maria; Grouzi, Elisavet; Korompoki, Eleni; Pavlakis, George N.; Felber, Barbara K.; Dimopoulos, Meletios A.
title: SARS-CoV-2 Antibody Kinetics Eight Months from COVID-19 Οnset: Persistence of Spike Antibodies but Loss of Neutralizing Antibodies in 24% of Convalescent Plasma Donors
date: 2021-05-18
journal: Eur J Intern Med
DOI: 10.1016/j.ejim.2021.05.010
sha: 3245d8d0a19279d362665d7528840b70817ffca9
doc_id: 718330
cord_uid: 9qm9lbyc

Elucidating the characteristics of human immune response against SARS-CoV-2 is of high priority and relevant for determining vaccine strategies. We report the results of a follow-up evaluation of anti-SARS-CoV-2 antibodies in 148 convalescent plasma donors who participated in a phase 2 study at a median of 8.3 months (range 6.8-10.5 months) post first symptom onset. Monitoring responses over time, we found contraction of antibody responses for all four antigens tested, with Spike antibodies showing higher persistence than Nucleocapsid antibodies. A piecewise linear random-effects multivariate regression analysis showed a bi-phasic antibody decay with a more pronounced decrease during the first 6 months post symptoms onset by analysis of two intervals. Interestingly, antibodies to Spike showed better longevity whereas their neutralization ability contracted faster. As a result, neutralizing antibodies were detected in only 76% of patients at the last time point. In a multivariate analysis, older age and hospitalization were independently associated with higher Spike, Spike-RBD, Nucleocapsid, N-RBD antibodies and neutralizing antibody levels. Results on persistence and neutralizing ability of anti-SARS-CoV-2 antibodies, especially against Spike and Spike-RBD, should be considered in the design of future vaccination strategies.

showing higher persistence than Nucleocapsid antibodies. A piecewise linear 48 random-effects multivariate regression analysis showed a bi-phasic antibody decay 49 with a more pronounced decrease during the first 6 months post symptoms onset by 50 analysis of two intervals. Interestingly, antibodies to Spike showed better longevity 51 whereas their neutralization ability contracted faster. As a result, neutralizing 52 antibodies were detected in only 76% of patients at the last time point. In a 53 multivariate analysis, older age and hospitalization were independently associated 54 with higher Spike, Spike-RBD, Nucleocapsid, N-RBD antibodies and neutralizing 55 antibody levels. Results on persistence and neutralizing ability of anti-SARS-CoV-2 56

The duration of antibody responses against severe acute respiratory syndrome 63 response with anti-SARS-CoV-2 antibodies; (v) two negative SARS-CoV-2 PCR 111 results (nasal and/or pharyngeal swab); the time interval between the two negative 112 tests should be at least 7 days. defined as the last dilution value being higher than the cut-off. A sample below the 140 threshold at 1:200 dilution is defined as background of the assay (dilution 1:50) and 141 is entered as 50. A modelfit approach was conducted in R to model the curve to 142 more accurately define endpoint titers. Antibody levels were also expressed as area-143 under-the curve (AUC) values covering serial serum dilutions (log10 transformed) 144 above the endpoint titer cut-off. If a sample has an endpoint titer of 1:50, it is 145 considered below threshold of the assay and the value is entered as 0.1. 146

Comparison of AUC and modelfit measurments show significant correlations 147 (p<0.0001) between these analysis for all the assays with Spearman r values from 148 0.933 to 0.972 (N=249 to 375 data points for the 4 assays). 149

We employed ELISA assays to measure antibodies binding to the trimeric Spike and 150 Nucleocapsid as well as to Spike-RBD and N-RBD. The rationale of this evaluation 151 was based on our goal to (i) monitor the durability of two key SARS-CoV-2 152 antibodies (Spike and Nucleocapsid) and (ii) to compare antibody responses to the 153 complete protein to those recognizing key functional regions (Spike-RBD, N-RBD). 154 For the modelfit determination of the endpoint titers, the right side of the sigmoid 171 dilution curve (all points after the largest drop in measured value or the highest four 172 dilution points, which ever was longer) was fit to a self-starting asymptotic regression 173 model (R functions SSasymp() and nls() from the "stats" R package) used to 174 determine the nonlinear least-squares estimate of the model parameters 175 (or from the first positive PCR assay (PCR+) for those with 191 asymptomatic disease). The characteristics of the patients are summarized in Table  192 1. The follow-up measurement (n=135) was performed at a median of 5.9 months 193 (range 2.9-7.2 months) post symptom onset, and termed "6-month follow-up" in this 194 report. All these patients had recovered from symptomatic COVID-19. A subset of 195 patients (n=94) was tested again at a median of 8.3 months (range 6.8-10.5 months) 196 and termed "8-month follow-up" in this report. The long-term follow-up was 197 performed on patients (N=81) analyzed at all three timepoints (2, 6 and 8 months). 198

The median age was 50 years (range: 18-65). Ninety-one patients did not need Nucleocapsid and N-RBD, except 1 of 148 donors, who did not have measurable 208 antibodies to N-RBD (Table 2) . At the 6-month follow-up, positive responses were found to Spike (100%), whereas 1/135 (0.74%), 1/82 (1.2%) and 8/135 (5.9%) of 210 donors showed values below the threshold of the respective assays (Table 2) . At the 211 8-month follow-up, all donors (N=92) had positive responses to Spike (100%) and 212

Spike-RBD (100%), whereas 2/81 (2.4%) and 2/92 (2.2%) had responses below the 213 threshold of the assay to Nucleocapsid and N-RBD, respectively (Table 2 ). These 214 data show better persistence of antibodies to Spike than to Nucleocapsid (see 215 below). 216

Monitoring responses over time, we found a significant contraction of antibody 217 levels to all four antigens (paired t test, Table 3 ). A significant correlation of Spike 218 antibody levels was found between the values at screening and the 6-month follow-219 up ( Figure 2A ); this correlation became even stronger when comparing antibody 220 levels from the 6-month and the 8-month follow-up ( Figure 2B ). Similar observations 221

were made for the S-RBD, Nucleocapsid and N-RBD measurements ( Figure 2C) . 222

The stronger correlation may be the result of rapid antibody changes during the first 223 period (early after infection) compared to the later stages, that reflected the activity of 224 long-lasting antibody producing cells. 225

We further analyzed the relation between antibodies to and between antibodies to Nucleocapsid and N-RBD over time. We previously 227 reported significant correlations of these measurements at screening using a smaller 228 cohort [9]. Here, we report that this significant correlation was maintained for Spike 229 and Spike-RBD at the 6-month ( Figure 2D ) and at the 8-month ( Figure 2E ) 230 evaluation. Similar significant correlations were maintained for Nucleocapsid and N-231 RBD ( Figure 2F ). Together, our data indicate better persistence of antibodies to 232

Spike than to Nucleocapsid (see below) and that the Spike and Nucleocapsid 233 antibody relations were maintained over time.

of the antibody responses to Spike and Nucleocapsid in more detail. We calculated 236 the level of antibody persistence for the four different assays at the 8-month follow-237 up in relation to their respective screening measurements (Figure 3 ). This analysis 238

showed that Spike antibodies have better persistence than Nucleocapsid antibodies 239 ( Figure 3A , p <0.0001). In addition, antibodies recognizing only Spike-RBD persisted 240 less than the mixture of antibodies recognizing the complete Spike protein ( Figure  241 3B, p <0.0001). These data indicate that Spike-RBD antibodies are less durable than 242 other specificities within the mixture of antibodies recognizing the complete Spike. 243 No difference was found between antibodies recognizing N-RBD and the complete 244

Nucleocapsid protein in this comparison ( Figure 3C ; p=0.0656). 245

The antibody measurements were also used to determine their half-life over 246 the entire period. We employed the piecewise, linear random-effects generalized 247 least squares multivariate regression analysis to evaluate decay in two sequential 248 time intervals. This analysis showed that time emerged as an independent factor 249 inversely associated with antibody levels (Table 4 ). Up to 6 months post symptom 250 onset, the estimated half-time for Spike, Spike-RBD, Nucleocapsid, and N-RBD was 251 97, 62, 47 and 47 days, respectively. These data support a significant difference 252 between Spike and the Nucleocapsid antibody half-life, in agreement with data 253 shown in Figure 3A 

In summary, measuring the decline of CoV-2 Spike antibodies in these 286 sequentially collected sera, we found that the 100% positivity at the screening was 287 reduced to 76% at 6 to 8-month follow-up. Of note, 7% of the negative samples are 288 below the detection threshold of the assay and 17% show very low positivity but 289 below the level of quantification. These data show lack of long-term durability of NAb 290

in CoV-2 infected persons despite the better persistence of Spike binding antibodies. 291

The data from the sequentially analyzed antibodies ( Cross-sectional analysis showed that donors with age >50 years showed 301 higher antibodies levels for Spike, Spike-RBD, Nucleocapsid and N-RBD from the 302 screening to 8-month follow-up ( Figure 5B ). Similar data were obtained for 303

Nucleocapsid and N-RBD antibodies (Table 5) . Using the piecewise, multivariate 304 regression analysis, we found that older age (>50) was independently associated 305 with higher log10-transformed antibody levels (Spike, p=0.023; Spike-RBD, p=0.002; 306 significantly higher antibodies to all four antigens ( Figure  5D  309 ). Hospitalization correlated with higher antibody values 310 (Spike, p<0.001; Spike-RBD, p<0.001; Nucleocapsid, p=0.003; N-RBD, p=0.024) 311 (Table 6) . 312

The univariate analysis showed further that there is a significant association 313 between antibody levels and gender against Spike and Nucleocapsid at the 314 screening time point ( Figure 5F ). Anti-SARS-CoV-2 antibody levels were higher in 315 male donors, however, this association did not persist over time in this cohort ( Figure  316 5F, Table 5 ). Gender differences in anti-Spike antibodies were also observed by 317

We also show the NAb data measured overtime, separated by age, 319 hospitalization and gender ( Figure 5B , 5D, 5F, right panels). These data mirror the 320 results found for the binding antibodies measured by ELISA showing higher levels in 321 male, in donors <50 years of age and in donors with previous hospitalization. 322

In a sub-cohort of 86 patients, age (p<0.001) and hospitalization (p=0.003) 323 correlated with higher neutralizing antibody titers (Table 5) . 

Plots show the median 648 with box and whiskers at the 10-90 percentile

Figure 4. Persistence of Neutralizing Antibodies (NAb) responses

Neutralizing antibodies were measured using the pseudotype SARS-CoV-2 virus 652 inhibition assay in a sub-cohort of patients (N=86). The log serum dilution inhibiting 653 virus by 50% (ID50) values are plotted over time

AUC) measured at the matching 655 time points at screen time (top panels), at the 6-month follow-up (middle panels), and 656 at the 8-month follow-up (bottom panels). The NAb ID50 threshold of quantification 657 (0.5 log) and threshold of detection (0.1 log)

Figure 5. Associations of antibodies levels with time and clinical 660 characteristics

Spike antibody measurements as shown in Figure 1 are analyzed for different clinical 662 characteristics and plotted over time post symptom onset. Donors were grouped by 663 (A, B) by age with >50 and <50 years

) Cross-sectional comparisons of 665 endpoint antibody levels to Spike, Spike-RBD, Nucleocapsid and N-RBD and of NAb 666 are shown. p values (Mann Whitney t test) are given and the 3 time points of 667 analysis are shown