High Initial Titres of Anti-Spike Antibodies following SARS-CoV-2 Infection is Associated with Faster Decay Rates at Four Months Follow-Up

SARS-CoV-2 Infection, anti-spike antibody, decay rate, COVID-19 14 15 Running Title: Decay Rates following SARS-CoV-2 Infection 16 17 Word count: 3396 18 19 *Corresponding Author: 20 Vidya Menon, MD, FACP 21 NYC HHC/Lincoln 22 234 E 149th Street 23 The Bronx, New York 10451 24 Email: [email protected] 25 Phone: 718-579-5000 ext. 3485 26 27 Key Points 28 Question: What is the decay rate of neutralizing antibodies among SARS-CoV-2 infected 29 healthcare workers? 30 31 Findings: In this cohort study that included 178 healthcare workers, over a 4-month period 32 following the COVID-19 pandemic, participants had an initial rise in anti-nucleocapsid (N) and 33 anti-spike (S) antibodies, which was followed by decay and stabilization of the titres. Significant 34 correlation with rate of decay was noted with the symptomatic participants. 35 Meaning: A strong correlation is observed in the decay of anti-S antibody titres based on 36 symptomology, thus eluding to the fact that continued recommendations for infection protection 37 and COVID-19 vaccine campaigns are necessary. 38 39 40 41 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2

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43 Dynamics of humoral immune responses to SARS-CoV-2 antigens following infection suggests 44 an initial decay of antibody followed by subsequent stabilization. We aim to understand the 45 longitudinal humoral responses to SARS-CoV-2 nucleocapsid (N) protein and spike (S) protein 46 and to evaluate their correlation to clinical symptoms among healthcare workers (HCW). 47

48 In this cross-sectional longitudinal cohort study done in two phases over four months, HCW 49 underwent serial qualitative serology testing for anti-N antibody, quantitative MSH-ELISA to 50 detect Receptor Binding Domain and full-length S reactive antibodies and completed online 51 surveys about COVID-19 related symptoms and healthcare/community exposure. 52

53 Anti-N antibody positivity was 27% and anti-S positivity was 28% in Phase 1. In Phase 2 anti-S 54 titres were higher in symptomatic than in asymptomatic positive subjects in Phase 1. Marginally 55 higher titers were seen in asymptomatic compared to the symptomatic positive subgroup in 56 Phase 2. A positive correlation was noted between age, number and duration of symptoms, and 57 Phase 1 anti-S antibody titre. A strong correlation was observed between Phase 1 titers and decay 58 of anti-S antibody titres between the two phases. Significant correlation with rate of decay was 59 also noted with fever, GI symptoms, and total number and duration of COVID-19 symptoms. 60

61 Higher initial anti-S antibody titres were associated with larger number and longer duration of 62 symptoms as well as faster decay during the two time points. 63 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 3

64 In light of the unprecedented coronavirus disease 2019 (COVID-19) pandemic, understanding 65 the role of the immune system in countering the viral infection is critical not just to design 66 effective antiviral strategies but also to aid us in taking appropriate public health decisions. The 67 early publication of the viral genome led to a rapid development of many nucleic acid based 68 diagnostic assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. 69 While nucleic acid-based tests are widely employed in the diagnosis of acute (current) SARS-70 CoV-2 infections, they are often limited in their clinical utility in identifying past infections or 71 assess the level of immunity to SARS-CoV-2 within the communities. Evaluation of antibody 72 responses is the other well-known modality used in a clinical setting that can detect both current, 73 and past infections and is the preferred approach for surveillance to determine the true 74 prevalence of infections. The currently available serological assays for SARS-CoV-2 target 75 either the viral nucleoprotein (N) or the spike su rface protein (S) antigens. The S-protein, which 76 contains the receptor binding domain (RBD), binds to host cells via the angiotensin converting-77 enzyme-2 (ACE2) receptor, followed by membrane fusion 1,2. The spike is the target of most 78 neutralizing antibodies 3-5, while the N plays an important role in transcription enhancement and 79 viral assembly 6. Studies have demonstrated that antibodies against the N and S appeared around 80 the same time - between day 8 and day 14 after the onset of symptoms with antibodies to the N 81 being more sensitive than anti S antibodies for detecting early infection7. Neutralizing antibodies 82 confer protective immunity and can be detected in most infected individuals 10-15 days 83 following the onset of COVID-19 symptoms and remain elevated following initial viral 84 clearance 8-12. There is compelling evidence suggesting that serological assays for anti-S 85 antibodies predict neutralizing activity, in contrast to N based assays11,13. 86 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 4 87 The detailed characterization of the dynamics of humoral immune responses to the SARS-CoV-2 88 viral antigens following infection is still ongoing and early evidences suggest an initial decay of 89 antibody followed by stabilization at a certain level 11,14-18. These dynamics are likely driven by 90 an initial expansion of plasmablasts which produce large amounts of antibody but die off quickly 91 followed by a slower decay of antibody titres (the half-life of IgG is approximately three weeks) 92 which then transitions into a steady state level of antibody produced by long-lived plasma cells19. 93 However, it is currently unknown, if the magnitude of the initial expansion of plasmablast and 94 the associated antibody titres are correlated with the steady state level of serum antibody 95 produced by long-lived plasma cells. This is an important question since steady state antibody 96 levels may provide superior protection from re-infection20,21. 97 98 Specifically, there is currently a paucity of information on the kinetics of antibody decay among 99 health care workers (HCW). It is suspected that SARS-CoV-2 infections among HCW are 100 usually asymptomatic or mildly symptomatic and frequently associated with either 101 underreporting of symptoms or heterogenous PCR and/or serologic diagnostics leading to most 102 of them going undetected or unrecognized 22. A large cohort study of HCWs in the greater New 103 York City (NYC) area showed a seroprevalence of SARS-CoV-2 antibodies of 13.7% 23. Our 104 own data of anti N antibody screening among HCW at a New York City public hospital in the 105 Bronx following the first “surge” of COVID-19 in May 2020, found that SARS-CoV-2 106 seroprevalence was at 27%24. Understanding the longitudinal kinetics of SARS-CoV-2 antibody 107 response and the effectiveness of commercial antibody measurement assays is crucial to 108 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 5 correctly determine infection rates, sero-prevalence and true sero-reversion rates in both infected 109 and vaccinated individuals – and to better understand protection associated with sero-positivity. 110 111 In this study, we aimed to investigate the longitudinal humoral responses to viral N and the spike 112 and to evaluate their correlation to clinical symptoms and baseline characteristics in our HCW 113 study cohort. Importantly, having access to samples during the initial antibody peak and several 114 months out, we also aimed to determine if initial high antibody levels correlated with high 115 antibody titers at steady state. 116 117

118 Study setting and population 119 The study was a cross sectional cohort study done in two phases after receiving Institutional 120 Review Board approval (IRB # 20-009, Lincoln Medical Center, Office of the Institutional 121 Review Board approved as per 45 CFR 46 & 21 CFR50,56 under a full board committee and 122 gave its approval on 4/28/2020). The Phase 1 was conducted in May/June, 2020 and the Phase 2 123 was completed August/September 2020. The cohort included HCWs who worked at the New 124 York City Public Hospital in the South Bronx and were willing to participate in both phases of 125 the study. In the Phase 1 of the study, after informed consent, participants underwent qualitative 126 serology testing (Abbott Architect SARS-CoV-2 IgG Assay, Abbott Park, IL 60064 USA) 25 and 127 a nasopharyngeal swab for SARS-CoV-2 (Bio-Reference Laboratories, Inc., Elmwood Park, NJ, 128 USA). They also completed an initial online survey on demographics, symptoms of COVID-19, 129 healthcare/community exposure etc. An extra sample was collected and stored at -80°C for 130 subsequent analysis. These samples were processed using a quantitative enzyme-linked 131 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 6 immunosorbent assay (ELISA) that correlates well with virus neutralization, developed by 132 Mount Sinai Health System (MSH ELISA) 26,27, to detect RBD and full-length spike (S) reactive 133 antibodies. Participants from Phase 1 who agreed to return for follow up serology testing (Abbott 134 and MSH ELISA) and completion of a follow-up online survey were part of Phase 2 of the study. 135 136 Antibody assays 137 The Abbott Architect assay uses a qualitative chemiluminescent microparticle immunoassay 138 technology targeting the N antigen of the virus with a reported sensitivity of 100% (CI 95.8–139 100%) and specificity of 99.6% (CI 99–99.9%)25. The MSH ELISA consists of an initial ELISA 140 using serum or plasma to detect specific IgG against the RBD of SARS-CoV-2 at a single 141 dilution, followed by quantitative titrations of presumptive positives in a confirmatory ELISA 142 against full length SARS-CoV-2 spike protein (S)28. The positive result from the spike ELISA is 143 reported as antibody at a titre of 1:80 or higher. Test performance assessment revealed that PCR+ 144 samples were 94 % positive and all negative samples returned a negative result for 100% 145 negative agreement29. 146 147 Survey 148 The online survey was accessed by a unique identification number assigned to each participant, 149 blinded to the research team to ensure confidentiality. The survey requested information on age, 150 race/ethnicity, comorbidities, domestic/international travel and healthcare and community 151 exposure details during and prior to both phases. The first phase collected information about 152 symptoms of COVID-19 including their timing and duration in the preceding weeks of the blood 153 draw24. The Phase 2 survey requested information on new comorbidities, persistent COVID-19 154 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 7 symptoms (cough, shortness of breath, anosmia, ageusia, myalgia, nausea, and/or diarrhea), 155 interim testing via antibody and/or reverse transcription polymerase chain reaction (RT -PCR) (if 156 present) and their result (positive/negative), presence of positive SARS-CoV-2 PCR results in 157 the preceding months, interim domestic/international travel and continued use of personal 158 protective equipment (PPE). The risk of exposure in the healthcare setting and community 159 exposure was determined based on CDC guidelines30. 160 161 Statistical analysis 162 Descriptive statistics were used to summarize the baseline characteristics of the cohort and key 163 study outcome variables. Categorical variables were compared by the Chi–squared test, while 164 continuous variables were compared by a Student’s t-test. The spike antibody titres were 165 described as geometric means. Correlations were calculated using standard Pearson and 166 Spearmen correlation. Multiple linear regression was applied to determine the predictors of 167 log10 rate of decay from Phase 1 to Phase 2 of anti-spike antibodies. A p-value of <0.05 was 168 considered significant. All statistical analyses were performed using SPSS version 27 (IBM, 169 USA). 170 171

172 For Phase 1 of our study, 500 healthcare workers underwent both PCR and serology testing. Of 173 these, 137 were positive by for anti-N antibody (Abbott) and 142 were positive by the MSH 174 ELISA. For the second phase 178 participants from the initial cohort consented and underwent 175 evaluation with PCR, antibody assays (Abbott and MSH ELISA) and completed the online 176 follow up survey. The details of patient enrolment are described in Figure 1. While 46 of the 178 177 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 8 tested subjects remained positive for the anti-N antibody (Abbott), 70 were positive by the MSH 178 ELISA in the second phase. Anti -spike titres of the 5 subjects in the first phase were close to the 179 cut off for positivity. Twenty-two subjects who were negative for anti-N antibody in Phase 2 had 180 positive titres of anti-RBD and anti-spike antibodies, though lower than their Phase 1 levels. 181 Among the subjects who participated in the Phase 1 and Phase 2 study, 68 were positive in both 182 phases by the MSH ELISA, 110 were negative in both phases and 2 were positive only in Phase 183 2 with previously negative results in Phase 1. 184 185 The baseline characteristics of study participants who were positive by MSH ELISA in both 186 phases (n=68) and those who were negative in both phases (n=108) are shown in Table 1. The 187 mean age of the participants was 44.7±12.4 years, and 63.1% were female. Overall, 30.7% of the 188 HCWs were Latinx, 29.5% were Asian, 16.5% were Black and 17.6% were White. COVID-19 189 related symptoms were present in 83.8% (57) of the subjects who were positive in both phases, 190 while only 42.6% (46) of the subgroup who had negative antibodies in both phases admitted to 191 symptoms prior to Phase 1. The duration of symptoms prior to Phase 1 was longer among the 192 symptomatic positive group (48.3% for >14 days) in comparison to symptomatic negative group 193 (17.8% for >14 days). The mean duration of symptoms to Phase 1 testing in the symptomatic 194 positive sub cohort was 47.9 ±16.0 days. Persisting symptoms of COVID-19 were reported in 19 195 (27.9%) subjects from the cohort with positive antibodies in both phases. 196 197 Clinical characteristics and seropositivity to spike protein in both phases 198 Table 2 describes the characteristics of the symptomatic and asymptomatic subjects who were 199 positive for anti-spike antibody in both phases. Baseline characteristics were comparable 200 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 9 between the groups and no difference either in the healthcare or community exposure or in the 201 location of work (ED/Inpatient/intensive care unit, OR etc.) between the two groups was 202 observed. Titres of anti-spike antibodies (geometric mean area under the curve (AUC)) were 203 higher in symptomatic subjects than in asymptomatic positive subjects (6754 AUC vs. 5803 204 AUC) in Phase 1. However, in the Phase 2 analysis we observed marginally higher titres in the 205 asymptomatic subgroup compared to the symptomatic subgroup (2383 AUC vs. 2198 AUC). The 206 rate of decay was higher in the symptomatic subgroup (geometric mean 32.96 per day) compared 207 to the asymptomatic (geometric mean 23.42 per day) suggesting delayed antibody/kinetics in the 208 asymptomatic cohort. 209 210 Phase 1 anti-spike antibody titre and clinical correlations 211 A Pearson’s product and Spearman’s rank-order correlation was run to assess the relationship 212 between cohort characteristics including age, gender, comorbidities, number of symptoms of 213 COVID-19, healthcare exposure and Phase 1 anti-spike titres in our cohort ( Figure 2 ). One 214 hundred-forty-three subjects with a positive test in Phase 1 were included in the analysis. Scatter 215 plot analysis showed a monotonic relationship between the variables. A statistically significant 216 weak positive correlation was observed between age and Phase 1 anti-spike antibody titres 217 (R=0.269, p<0.005). Moderate positive correlation was present between presence of fever 218 (R=0.319, p<0.005), number of symptoms (R=0.310, p<0.005) and days of symptoms (R=0.434, 219 p<0.005) and anti-spike antibody titre; and weak positive correlation was observed with upper 220 respiratory symptoms (R=0.278, p<0.005) and gastrointestinal (GI) symptoms (R=0.204, 221 p<0.05) with anti-spike antibody titres. 222 223 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 10 Correlation of rate of decay of anti-spike antibody titres from Phase 1 to Phase 2 and 224 clinical characteristics 225

of Pearson correlation to assess the relationship between cohort characteristics including 226 Phase 1 anti-spike antibody titres, age, gender, comorbidities, symptoms of COVID-19, number 227 of symptoms of COVID-19, healthcare exposure and decay of anti-spike titres between the two 228 phases in our cohort is shown in Figure 3. A strong positive statistically significant correlation 229 was observed between Phase 1 titers and decay of anti-spike antibody titres between the two 230 phases (R=0.898, p<0.000). Medium positive correlation was observed between presence of 231 fever (R=0.428, p<0.001), GI symptoms (R=0.340, p<0.011), number of symptoms (R=0.357, 232 p<0.007), duration of symptoms (R=0.469, p<0.000) with decay of anti-spike antibody titres 233 between the two phases respectively. 234 235 A pairwise comparison was performed between rate of decay of anti-spike antibody titres and 236 patient characteristics ( Figure 4 ). Rate of decay by gender was comparable (male; 30.73 237 AUC/day vs. female;34.68 AUC/day, p=0.413). Asian (86.0 AUC/day) race showed higher rate 238 of decay compared with White (7.2 AUC/day) and Black (19.61 AUC/day) individuals; while 239 Latinx (47.28 AUC/day) race had higher rate of decay compared with White (7.2 AUC/day) 240 individuals. Subjects with fever had a higher rate of decay than those who did not report fever 241 (53.08 AUC/day vs.16.14 AUC/day, p=0.002). Similarly subjects with GI symptoms had a 242 higher rate of decay than those without (55.81 AUC/day vs.21.94 AUC/day, p=0.019). Subjects 243 with symptoms restricted to less than seven days demonstrated a lower decay rate compared with 244 symptomatic subjects over 7-14 days (13.60 AUC/day vs. 36.12 AUC/day, p=0.046) and when 245 compared with symptomatic subjects with more than 14 days (13.60 AUC/day vs. 59.72 246 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 11 AUC/day, p=0.001). This finding was statistically significant. No difference was found when 247 degree of exposure (High/Moderate: 28.18 AUC/day vs. Mild: 34.78 AUC/day, p=0.395) or job 248 role (physician: 29.57 AUC/day vs. nurse: 53.59 AUC/day vs. Other: 26.83 AUC/day; p=0.361) 249 was compared to rate of decay. 250 251 Predictors of rate of decay from Phase 1 to Phase 2 of anti-spike antibodies 252 Multiple linear regression analysis to predict the rate of decay with respect to age, Bacillus 253 Calmette Guerin vaccination, number of symptoms, and Phase 1 (log10) anti-spike antibody 254 titres is shown in Table 3. On the basis of a linear regression model that included the participants 255 age, history of BCG vaccination, total number of COVID-19 symptoms and the Phase 1 256 concentration of log 10 spike antibody titres, the estimated change (decay) was 23.6 AUC/day 257 when age was centred at median (42.6 years), there was positive history of BCG vaccination, the 258 total number of COVID-19 symptoms were centred at a median of 4, and the geometric mean of 259 the log10 spike antibody titre was 3.78. 260 261

262 With the COVID-19 pandemic showing no signs of abating, healthcare workers at the epicentre 263 are at risk of infection due to occupational exposure as well as community exposure. Sero-264 surveillance is the foundation for determining the scale and rate of exposures. With a multitude 265 of serological assays getting emergency use approval from FDA, interpretation of the results of 266 these assays and their clinical significance remains challenging. It is critical to understand the 267 timing of the antibody response for acute interpretation. Confidence in analytical specificity of 268 the assay is a critical requirement in measurement of the specific antibody responses. Recent 269 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 12 studies have confirmed that anti spike titres especially anti-RBD titres can serve as surrogates for 270 virus neutralization31,32. The Abbott SARS-CoV-2 IgG assay that targets antibodies to the 271 nucleoprotein has a reported specificity and sensitivity of greater than 99% at 14 days or more 272 following symptom onset and these measurements are not indicative or correlated to virus 273 neutralization titres33. In comparison, the MSH ELISA targets the full-length S protein including 274 RBD, a major target for neutralizing antibodies and has demonstrated excellent correlation to 275 virus neutralization11,26. Longitudinal measurements of antibody levels have revealed that anti-N 276 and anti S IgG antibodies continue to increase until the third week post symptoms and an 277 approach that combines the detection of both of these antibodies would precisely detect almost 278 100% of all infectious exposures34. In our study, the mean number of days after symptoms to 279 testing in Phase 1 was 47 days suggesting a higher likelihood of accuracy of the utilized assay. 280 281 Longitudinal blood sampling among HCWs working at a public hospital in the epicentre of the 282 pandemic in NYC allowed for analysis of kinetics of anti-S and anti-N antibody responses. At 283 two months after the first surge of infections, anti-N antibodies were detected in 27% and anti-S 284 antibodies in 28% of participating HCWs. After an interval of four months, it is not surprising to 285 note that among the participants who returned, 26% remained positive for anti N antibodies, 286 while 31% of the previously anti-N antibody positive subjects tested negative in phase 2. On the 287 other hand, a similar analysis of the anti-S antibodies levels, confirmed that all the previously 288 positive retested subjects continued to remain positive, albeit with lower titres. 289 290 COVID-19 related symptoms were significantly associated with positive anti-spike antibodies in 291 both phases, with a similar association with longer duration (>14 days) of symptoms. Previous 292 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 13 studies have demonstrated a lower level of IgG response among patients without symptoms or 293 with mild symptoms compared to those with severe and critical disease 35,36. A comparison of 294 symptomatic versus asymptomatic subjects who tested positive for anti-spike antibodies in both 295 phases, confirmed that the rate of decay of anti-spike antibody titres were faster in the 296 symptomatic cohort than the asymptomatic subjects, which was seen also in the anti-N antibody 297 kinetics. However, we observed a faster decay in this group with a lower titre of anti-spike 298 antibodies in Phase 2 compared to the asymptomatic cohort (though the difference was not 299 statistically significant). This could additionally be supported by the finding of fever and GI 300 symptoms contributing to faster decay. Similar results of decreasing neutralizing antibody titre in 301 symptomatic than asymptomatic patients were observed by Choe et al.37 302 303 Positive correlations for age, presence of fever, upper respiratory symptoms, GI symptoms, total 304 number and duration of symptoms was observed with increased levels of anti-spike titres at 305 Phase 1. Similar results of neutralizing antibody titres were also observed by 306 Boonyaratanakornkit et al. wherein they showed higher levels of neutralizing antibody titres 307 were significantly associated with male gender, older adults, higher disease severity and shorter 308 interval from recovery 38. Based on a linear regression model with age centred at median (42.6 309 years), positive history of BCG vaccination, the total number of COVID-19 symptoms centred at 310 a median of 4, and the geometric mean of the log10 anti-spike antibody titre at 3.78, we observed 311 that the rate of decay of these antibody titres was 23.6 AUC/day. Evaluation of other 312 characteristics with rate of decay between Phase 1 and Phase 2 showed a faster reduction in titres 313 in Asian participants and in those with fever and GI symptoms. A slower decrease was noted 314 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 14 among patients with shorter duration (<7 days) of symptoms, with no other significant 315 correlation noted with any other baseline demographics or clinical characteristics. 316 As described above, higher antibody titers are associated with a larger number of symptoms, 317 longer duration of symptoms and – as described by others as well – disease severity in general. 318 We also found that higher initial antibody titers were associated with faster antibody decay 319 during the two time points. Initial antibody responses are driven by short lived plasmablasts, 320 which decay after a few days after producing massive amounts of antibody. IgG has a relatively 321 long half-life of approximately three weeks, but decay is inevitable since the plasmablasts 322 initially producing it disappear. Usually, titers then drop until they reach relatively stable levels 323 of antibody which are maintained by long-lived plasma cells in the bone marrow19. The two time 324 points described in this study represent the initial peak response and likely the stable level after 325 the initial decay. We found that individuals with higher initial titers had a faster decay rate 326 during the observation period meaning the difference between peak and stable, long-lived 327 antibody levels were larger. This indicates that there is likely no direct correlation between the 328 magnitude of the initial expansion of plasmablasts and the number of long-lived plasma cells that 329 migrate to the bone marrow. 330 Our study has the following limitations: First being a single center study with a small 331 convenience sampling that included a smaller number of participants in Phase 2 of the study. 332 Following the pandemic, the HCWs who volunteered from around the country were transferred 333 back and lost to follow-up, which did decrease the overall sample size, but the rates of positive 334 and negative results remained proportional. Second, the likelihood of a recall bias in the 335 participant’s responses on the online survey may exist. Lastly, as a cross-sectional 336 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 15 seroprevalence study the findings can underestimate rates of prior infections based on timing of 337 the testing given that antibodies are only transiently detectable following infection. 338 In conclusion, findings from this study are similar to other studies that have reported that higher 339 magnitude of anti-spike titres may correlate with protection against reinfection, in spite of the 340 observed decay in the antibody levels20,21. Nevertheless, further studies to evaluate the longevity 341 of immunity, especially in context of widespread administration of spike-based vaccine among 342 HCWs would be important in predicting herd immunity to COVID-19 infections. 343 Funding 344 The authors received no specific funding for this work. 345 346

347 The authorship is structured in first-last-author-emphasis. We thank the COVID-19 Testing Tent 348 staff for their invaluable assistance with this study with testing and accommodation of study 349 participants for Phase 2: Patrick C McNeil PA-C MPAS; Aney D Patel PA-C MPAS; and Megan 350 Corley DNP ANP-BC. We thank the Nursing staff for their commitment and support with the 351 workflow, especially, Karen Philip and Kenisha Williamson with others; additionally, the 352 Registration (Lexus Gonzalez and Taj Washington) and Patient Care Associate (Eva Penn) that 353 assisted the research team. We also thank the Occupational Health Services for accommodating 354 the research team during project completion. The Clinical Laboratory staff played a part in 355 allocating resources for Abbott Architect usage: Dior Ndao and Ayman Elshamshery. We thank 356 the study participants, who were essential health care workers, who volunteered to follow-up in 357 this protocol. 358 359 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 16 Contributions 360 V.M., M.A.S. and U.V. designed the study. J.M.C., M.A.S., B.Y., V.P.G. analysed the data. E.V. 361 and M.G. assisted with participant follow-up and coordination with the assistance of A.P., U.V., 362 V.M., and M.A.S. The Mount Sinai Health System team, J.M.C. and F.K., performed the 363 measurements for anti-Spike and Anti-RBD antibodies. V.M., U.V. and A.P. were responsible 364 for the clinical care of the research participants and supervised the day-to-day operation and 365 coordination of the study by M.K., V.D., M.A.S., B.Y., V.P.G., M.G., E.V., and M.G. 366 V.M. and F.K wrote the manuscript and is the guarantor of this work and has full access to all 367 data in the study and takes responsibility for the integrity of the data and the accuracy of the data 368 analysis, with the assistance of J.C.Q., M.A.S., B.Y., V.P.G., and M.G. 369 All authors critically revised the draft and approved the final manuscript. 370 371 Potential conflicts of interest 372 F.K. is listed as a co-inventor on a patent application filed by The Icahn School of Medicine at Mount 373 Sinai relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines. Mount Sinai 374 has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for 375 Merck and Pfizer (before 2020), and is currently co nsulting for Seqirus and Avimex. F.K.’s Krammer 376 laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. 377 All other authors report no potential conflicts. All authors have submitted the ICMJE Form for 378 Disclosure of Potential Conflicts of Interest. 379 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 17

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(which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 20 Table 1: Broad characteristics among health care workers assessed for antibody reactivity to 481 spike SARS-CoV-2 protein in Phase 1 and Phase 2 482 483 Overall † Spike ELISA (AUC) positive in both phases Negative Reactivity to spike (AUC) in both phases p value N=176 n=68 n=108 Age, years 44.7+12.4 42.9+11.9 45.8+12.7 0.099 Female, Gender 111 (63.1%) 40 (58.8%) 71 (65.7%) 0.467 Race 0.666 Latinx 54 (30.7%) 21 (30.9%) 33 (30.6%) Asian 52 (29.5%) 18 (26.5%) 34 (31.5%) Black 29 (16.5%) 15 (22.1%) 14 (13.0%) White 31 (17.6%) 10 (14.7%) 21 (19.4%) Other 10 (5.7%) 4 (5.9%) 6 (5.9%) Comorbidities 54 (30.7%) 25 (36.8%) 29 (26.9%) 0.214 BCG vaccine received in childhood 87 (49.4%) 35 (51.5%) 52 (48.1%) 0.902 COVID-19 related symptoms prior to Phase 1 103 (58.5%) 57 (83.8%) 46 (42.6%) <.001 Duration of symptoms <.001 14 days 36 (35.0%) 28 (48.3%) 8 (17.8%) Time from symptom to positive result, days 45.7+19.9 47.9+16.0 42.9+24.1 0.062 RT-PCR positive result for SARS-CoV-2 prior to Phase 1 51 (29.0%) 49 (72.1%) 2 (1.9%) <.001 RT-PCR positive result for SARS-CoV-2 during Phase 1 14 (8.0%) 13 (19.1%) 1 (0.9%) <.001 Persisting symptoms from COVID-19 25 (14.2%) 19 (27.9%) 6 (5.6%) <.001 Nature of work 0.306 Physicians 81 (46.0%) 29 (42.6%) 52 (51.5%) Nurses 29 (16.5%) 15 (22.1%) 14 (13.0%) Others 64 (36.4%) 24 (35.3%) 40 (39.6%) Hospital areas worked in: Emergency department/Inpatient units 118 (67.0%) 50 (73.5%) 68 (63.0%) 0.141 Ambulatory care/Clinics 72 (40.9%) 27 (39.7%) 45 (41.7%) 0.631 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 21 Administration/Non-clinical care areas 24 (13.6%) 9 (13.2%) 15 (13.9%) 0.867 Community exposure 47 (26.7%) 19 (27.9%) 28 (25.9%) 0.591 Household exposure 39 (22.2%) 17 (25.0%) 22 (20.4%) 0.343 PPE use at work 173 (98.3%) 67 (98.5%) 106 (98.1%) 0.226 Use of facemask outside of the hospital 158 (89.8%) 58 (85.3%) 100 (92.6%) 0.062 Continuous variables are expressed as mean (SD), categorical variables as n (%). BCG, Bacillus Calmette–Guérin vaccine; PPE, personal protective equipment; RT-PCR, reverse transcription polymerase chain reaction. † Demographic data is missing for 2 participants from the overall cohort. 484 485 486 487 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 22 488 Table 2: Broad characteristics among health care workers with positive antibody reactivity to SARS-CoV-2 spike in both phases Overall Asymptomatic for SARS-CoV-2 infection Symptomatic for SARS-CoV-2 infection p value n=68 n=11 n=57 Age, Mean (±SD) 42.9 (±1.45) 44.5 (±3.8) 42.6 (±1.6) 0.557 Female, n (%) 40 (58.8%) 6 (54.5%) 34 (40.4%) 0.502 Race 0.753 Latinx 21 (30.9%) 3 (27.3%) 18 (31.6%) Asian 18 (26.5%) 3 (27.3%) 18 (26.3%) Black 15 (22.1%) 3 (27.3%) 12 (21.1%) White 10 (14.7%) 2 (18.2%) 8 (14.0%) Other 4 (5.8%) 0 (0%) 4 (7.0%) Comorbidities Hypertension 13 (19.1%) 2 (18.2%) 11 (19.3%) 0.650 Diabetes 6 (8.8%) 0 (0%) 6 (10.5%) 0.332 COPD and asthma 13 (19.1%) 1 (9.1%) 12 (21.1%) 0.326 Number of symptoms, median (IQR) - - 4.0 (2.0-5.0) Length of symptoms 14 days - - 26 (45.6%) Degree of HCW exposure 0.492 High and Moderate 16 (23.5%) 2 (18.2%) 14 (24.6%) Minor 52 (76.5%) 9 (81.8%) 43 (75.4%) Community exposure 19 (27.9%) 3 (27.3%) 16 (28.1%) 0.635 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 23 Household exposure 17 (25.4%) 3 (27.3%) 14 (24.6%) 0.557 Use of facemask outside of hospital 58 (85.3%) 9 (81.8%) 49 (86.0%) 0.722 Principal means of transportation 0.663 Public 33 (48.5%) 6 (54.5%) 27 (47.7%) Private 35 (51.5%) 5 (45.5%) 30 (52.6%) Nature of work 0.502 Physician 29 (42.6%) 4 (36.4%) 25 (43.9%) Nurse 15 (22.1%) 2 (18.2%) 13 (22.8%) Other 24 (35.3%) 5 (45.5%) 19 (33.3%) Hospital areas work in: 0.288 Emergency department/inpatient units 32 (47.1%) 6 (54.5%) 26 (45.6%) Ambulatory care/clinics 9 (13.2%) 2 (18.2%) 7 (12.3%) Impatient and outpatient setting 18 (26.5%) 3 (27.3%) 15 (26.3%) Administration/nonclinical care areas 9 (13.2%) 0 (0%) 9 (15.8%) Anti-spike reactivity (AUC) Reactivity in phase 1, G-Mean (IQR) 6590 (5165- 8410) 5803 (2825- 11920) 6754 (5177- 8812) 0.647 Days from symptoms to first test, Mean (±SD) - - 47.7 (±1.9) Reactivity in phase 2, G-Mean (IQR) 2226 (1824- 2718) 2382 (1494-3797) 2198 (1753- 2755) 0.980 Days from symptoms to second test 174.5 (±4.1) Rate of decay, G-Mean (IQR) 31.14 (22.11- 43.87) 23.42 (8.45-64.93) 32.96 (22.73- 47.82) 0.382 Continuous variables are expressed as mean (SD) or interquartile range (IQR), categorical . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 24 variables as n (%). AUC, area under the curve; COPD, Chronic obstructive pulmonary disease; HCW, health care worker 489 490 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 25 491 Table 3: Multiple linear regression analysis of rate of decay for anti-spike antibodies between Phase 1 and Phase 2 Rate of decay (log10) B 95.0% CI for B SE B ß R2 ▲ R2 LL UL Model 0.83 0.82 Constant -3.203** -3.647 -2.759 .222 Age (per 10-year change) .014 -.005 .007 .002 .030 BCG vaccination .131** .030 .310 .046 .121 Number of symptoms .013 -.029 .060 .012 .050 ELISA reactivity (Log10) 1.159** 1.050 1.419 .059 .916 B: Unstandardized regression coefficient; CI: confidence interval; LL: lower limit; UL: upper limit; SE B: standard error of the coefficient; ß: standardized coefficient; R2: coefficient of determination; ▲ R2: adjusted R2. **P<0.05 BCG, Bacillus Calmette–Guérin vaccine 492 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 2 6 Figure 1: Flow Chart of patient enrollment, follow up and analysis 493 494 495 496 497 498 499 500 501 6 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 27 Fig u re 2: S imple co rr elation analy sis o f H C W w ith po s itive r eac tivity fo r a n ti- spik e antib ody i n Pha se 1 with base l i n e 502 chara cteristics and sy mpto m s 503 504 505 506 507 508 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint 28 Fig u re 3: S imple co rr elation analy sis o f rate o f de ca y o f anti-sp ik e anti b od i es betwe e n both ph a s es with b a sel ine 509 chara cteristics and sy mpto m s 510 511 512 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint Figure 4: Paired comparison between rate of decay of anti-spike antibody titres and patient 513 characteristics 514 515 516 517 518 519 520 521 . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint . CC-BY-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 2, 2021. ; https://doi.org/10.1101/2021.03.02.21252362doi: medRxiv preprint