key: cord-0758377-5t5l5rrx
authors: Letizia, A. G.; Ge, Y.; Vangeti, S.; Goforth, C.; Weir, D. L.; Kuzmina, N. A.; Chen, H. W.; Ewing, D.; Soares-Schanoski, A.; George, M.-C.; Graham, W. D.; Jones, F.; Bharaj, P.; Lizewski, R. A.; Lizewski, S. A.; Marayag, J.; Marjanovic,; N., Miller; C., Mofsowitz; S., Nair; V. D., Nunez; E., Parent; D. M., Porter; C. K., Santa Ana; E., Schilling; M., Stadlbauer; D., Sugiharto; V., Termini; M. S., Sun; P., Tracy; R. P., Krammer; F., Bukreyev; A., Irene; R., Sealfon; S., C.
title: SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study
date: 2021-01-29
journal: nan
DOI: 10.1101/2021.01.26.21250535
sha: e60b4b5d55b37a7374e9d0cf3b564a8e946acb5a
doc_id: 758377
cord_uid: 5t5l5rrx

Background: The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subsequent infection among seropositive young adults was studied prospectively. Methods: The study population comprised 3,249 predominantly male, 18-20-year-old Marine recruits. Upon arrival at a Marine-supervised two-week quarantine, participants were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a 1:150 dilution or greater on receptor binding domain and full-length spike protein enzyme-linked immunosorbent (ELISA) assays. SARS-CoV-2 infection was assessed by PCR at initiation, middle and end of the quarantine. After appropriate exclusions, including participants with a positive PCR during quarantine, we performed three biweekly PCR tests in both seropositive and in seronegative groups once recruits left quarantine and entered basic training and baseline neutralizing antibody titers on all subsequently infected seropositive and selected seropositive uninfected participants. Findings: Among 189 seropositive participants, 19 (10.1%) had at least one positive PCR test for SARS-CoV-2 during the six-week follow-up (1.1 cases per person-year). In contrast, 1,079 (48.0%) of the 2,247 seronegative participants tested positive (6.2 cases per person-year). The incidence rate ratio was 0.18 (95% CI 0.11-0.28, p<0.00001). Among seropositive recruits, infection was associated with lower baseline full-length spike protein IgG titers (p<0.0001). Compared with seronegative recruits, seropositive recruits had about 10-fold lower viral loads (ORF1ab gene, p<0.005), and trended towards shorter duration of PCR positivity (p=0.18) and more frequent asymptomatic infections (p=0.13). Among seropositive participants, baseline neutralizing titers were detected in 45 of 54 (83.3%) uninfected and in 6 of 19 (31.6%) infected participants during the 6 weeks of observation (ID50 difference p<.0001). Interpretation: Seropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralization activity or immunity against subsequent infection. These findings may be relevant for optimization of mass vaccination strategies.

As of mid-December 2020, more than 72 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been diagnosed world-wide 1 . Serological surveys indicate that the actual Within 48 hours of arriving at the supervised quarantine location, recruits were offered the 117 opportunity to volunteer for CHARM. Recruits were eligible if they were ≥18 years of age. Since recruits 118 are a vulnerable population and at risk for coercion, special measures were undertaken including study 119 briefers, who are active-duty Navy personnel wore civilian clothes, did not disclose military ranks, did not 120 have members in the recruit's chain of command present, and ensured that participation would not affect 121 a recruit's medical care or influence the grading of a recruit's military performance by superiors.

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 number NMRC.2020.0006) in compliance with all applicable U.S. federal regulations governing the The presence and levels of IgG SARS-CoV-2-specific antibodies in serum were determined using an enzyme-linked immunosorbent assay (ELISA) as previously described 26 . Briefly, 384-well Immulon 4 (SinoBiological) or full-length spike protein (LakePharma) at a concentration of 2 µg/ml in phosphate-155 buffered saline (PBS). Plates were washed three times with 0.1% Tween-20 (Fisher Scientific) in PBS 156 (PBS-T) using an automated ELISA plate washer (Aquamax 4000, Molecular devices), and blocked for 1 157 h at room temperature (RT) with 3% milk (BioRad) PBS-T. Blocking solution was removed, and serum 158 samples diluted in 1% milk PBS-T were dispensed in the wells. At least 2 positive controls (sera with 159 known IgG presence), 8 negative controls (sera collected before July 2019) and 4 blanks (no serum) were 160 included in every assay. Plates were incubated for 2 h at room temperature, and then washed 3 times with 161 PBS-T. Next, peroxidase conjugated goat F(ab')2 Anti-Human IgG (Abcam) was added at a dilution 162 1:5,000-1:10,000 dilutions (determined after optimization for each antibody lot) in 1% milk PBS-T, and 163 plates were incubated for 1 h at room temperature. Plates were washed 6 times with PBS-T, developed 164 using o-Phenylenediamine (Sigma), and the reaction was stopped after 10 min with 3M HCl. Optical 165 density (OD) at 492 nm was measured using a microplate reader (SpectramaxM2, Molecular Devices).

All serum samples were screened at a 1:50 dilution with S-RBD. Those samples with an OD 492 nm 167 value higher than the average of the negative controls plus 3 times their standard deviation (SD) in the 168 screening assay underwent titration assay (6 serial 1:3 serum dilutions starting at 1:50) using both S-RBD CC-BY 4.0 International license It 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 January 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Two-fold serial dilutions of heat-inactivated serum at an initial dilution of 1:20, were prepared in serum is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10. 1101 /2021 The funders had no role in the design of the protocol, data collection, data management, data analysis, 204 data interpretation, or writing of the report. SCS, AGL, YG, CP and IR had access to all deidentified data.

SCS and AGL had final responsibility for the decision to submit for publication.

The flow of participants through the study is shown in Figure 1 . Two weeks before the initiation 209 of basic training and the start of the prospective cohort study period, a total of 3,249 out of 4,657 eligible 210 new Marine recruits (70%) enrolled in CHARM and underwent a supervised two-week quarantine.

Exclusions before the prospective study period included 73 participants who were SARS-CoV-2 PCR 212 positive on at least one of the three PCR tests performed during quarantine, 53 who lacked baseline 213 serology results and 47 who were lost to follow-up.

Of the remaining 3,076 participants, 225 were baseline seropositive, having SARS-CoV-2 IgG seropositive participants, 36 (16.0%) were excluded from analysis due either to being lost to follow up 218 (n=34), or to lacking valid PCR results during the study period (n=2). In the seronegative group, 604 219 (21.2%) participants were excluded due to either being lost to follow up (n=532), or lacking valid PCR 220 results (n=72). Participants were lost to follow up for specific reasons unknown to the study team,

including dropping out of the study, being separated from the Marines or being removed from the base for 222 medical or administrative reasons. Demographic characteristics of the two groups in the study cohort are 223 shown in Table 1 . Most participants were 18-20 years old and male. The two groups were well-balanced 224 with the exception of a higher proportion of participants who self-identified as Hispanic and as black in 225 the seropositive group.

A total of 19 out of 189 (10.1%, 1.1 cases per person-year) seropositive participants and 1,079 out 227 of 2,247 (48.0%, 6.2 cases per person-year) seronegative participants had at least one positive SARS-

CoV-2 PCR result during the six-week study period, representing an 0.18 (95% CI: 0.11 to 0.28, p<0.001)

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101/2021.01.26.21250535 doi: medRxiv preprint incidence of infection in seropositive and seronegative groups is shown in Figure 2A . After adjusting the baseline titers and the risk of infection. As shown in Figure 2B , supplementary Figure 2 and Table 3 , we 235 found a strong association between subsequent PCR positive infection and lower titers of IgG antibodies 236 directed to full-length spike protein (p<0.0001) as well as to S-RBD (p=0.0019). The detailed Cox 237 proportional hazard analysis in supplementary Table 1 gives a hazard ratio of 0.45 (95% CI 0.32-0.65, 238 p<0.001) and 0.67 (95% CI 0.47-0.96, p=0.028) respectively for the full-length spike protein titer and S-239 RBD titer, both log-transformed.

We examined baseline SARS-CoV-2 IgG neutralizing antibody activity in all seropositive 241 participants who became PCR positive during the observation period and in the first 54 participants who 242 were seropositive but remained PCR negative. Neutralizing activity was above the limit of detection in 45 243 of 54 (83.3%) seropositive participants who never became PCR positive and in 6 of 19 (31.6%) of 244 participants infected during the 6 weeks of observation. The neutralizing activity assessed as 50% 245 inhibitory dose (ID50) was significantly higher in the participants who did not become PCR positive 246 during the study (p<.0001, Cochran-Armitage test, Table 3 , supplementary Figure 2 ).

We also compared virus loads estimated by PCR Ct values between the seronegative and 248 seropositive PCR infected groups and found that seronegative individuals had on average 4, 2.6 and 3.3 249 lower cycle values for ORF1ab gene (p=0.004, 95% CI 1.23 to 6.67), S gene (p=0.11, 95% CI -0.58 to 250 5.77) and N gene (p=0.033, 95% CI 0.27 to 6.33), respectively, than seropositive individuals. The lower

Ct values suggest an approximately 10-fold higher virus load in the samples from seronegative 252 participants (Table 4 ). In addition, seronegative participants tended to have a longer duration of PCR 253 positivity than seropositive individuals (p=0.18). The proportion of asymptomatic participants was 84.2%

(16 of 19) and 67.8% (732 of 1079) in seropositive and seronegative infected individuals, respectively, a 255 difference that did not reach significance (p=0.13).

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101/2021.01.26.21250535 doi: medRxiv preprint Discussion seropositive group, the participants that became infected had lower antibody titers than those that were 262 uninfected, and were more likely to lack detectable baseline neutralizing antibody activity. Our results

indicate that although antibodies induced by infection are largely protective, they do not guarantee 264 effective immunity against subsequent infection.

This study leveraged a two-week USMC-mandated quarantine period during which baseline 

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101/2021.01.26.21250535 doi: medRxiv preprint prevalence in self-identified Hispanic and non-Hispanic black participants in the seropositive group. The 284 seropositive group was almost 50% Hispanic and 22% non-Hispanic black participants compared to 22% 285 and 12%, respectively in the seronegative group. This is likely due to minority populations having higher 286 seroprevalence rates during the COVID-19 pandemic in general and among young adults specifically 27 .

Rates of infection and the risk reduction provided by seropositivity are important for 288 understanding transmission dynamics for COVID-19, for epidemiologic modeling, and for estimating and 

The crowded living conditions, demanding regimen and requirement for personal contact during 299 basic training despite the pandemic leads not only to an increased risk for respiratory epidemics 29 , but also 300 potentially to higher exposure levels. The close quarters and constant contact among recruits that are 301 needed for team building allows a viral infection to rapidly proliferate within a unit. The physically and 302 mentally demanding training environment may also suppress immunity. These conditions may contribute 303 to the high infection rate we observed during the six-week study period. These factors are not typically 304 present in the civilian community. Therefore, the study setting limits the generalizability of our findings 305 to other settings where the frequency and intensity of exposure and the susceptibility of the host might CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 were separated from the Marine Corps or were removed from the base for medical or administrative 395 reasons. The study team did not know the reason for participants missing study visits. 396 397 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10. 1101 /2021 Overall cumulative incidence for testing PCR positive in the baseline seropositive and seronegative 399 groups. B. Cumulative incidence for testing PCR positive in the seropositive group at different baseline CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 29, 2021. 

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10.1101/2021.01.26.21250535 doi: medRxiv preprint Figure 1 : Flow chart of the study design and outcomes. The baseline analyses were performed within 515 two days of enrollment and two weeks before the prospective study period. Nearly all participants were 516 tested at scheduled biweekly intervals during the prospective study period. A few participants were 517 diagnosed by the Marine Corps Recruit Depot Parris Island clinic, and were tested by the study team at 518 times not corresponding to the regularly scheduled longitudinal follow ups. Participants lost to follow up 519 either dropped out of the study, were separated from the Marine Corps or were removed from the base for 520 medical or administrative reasons. The study team did not know the reason for participants missing study 521 visits.

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10. 1101 /2021 Overall cumulative incidence for testing PCR positive in the baseline seropositive and seronegative 527 groups. B. Cumulative incidence for testing PCR positive in the seropositive group at different baseline 528 full-length spike protein IgG titers which ranged from 1:150 to 1:12150. The cumulative incidence rate is 529 computed by the Kaplan-Meier method, and the cumulated censored participants are listed.

A B . CC-BY 4.0 International license It 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 January 29, 2021. ; https://doi.org/10. 1101 /2021 N seropositive group at different baseline RBD IgG titers, which ranged from 1:150 to 1:12150. The 549 numbers at risk and cumulated censored participants are listed below the table. . CC-BY 4.0 International license It 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)

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The copyright holder for this preprint this version posted January 29, 2021. ; https://doi.org/10. 1101 /2021 

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