key: cord-0906256-32ughvdh
authors: Gallichotte, Emily N; Nehring, Mary; Stromberg, Sophia; Young, Michael C; Snell, Ashley; Daniels, Josh; Pabilonia, Kristy L; VandeWoude, Sue; Ehrhart, Nicole; Ebel, Gregory D
title: Impact of prior infection on SARS-CoV-2 antibody responses in vaccinated long-term care facility staff
date: 2022-04-05
journal: bioRxiv
DOI: 10.1101/2022.04.04.487083
sha: 54059e1fe3c51c0da60215d1d7a03fca810ba919
doc_id: 906256
cord_uid: 32ughvdh

SARS-CoV-2 emerged in 2019 and has resulted in millions of deaths worldwide. Certain populations are at higher risk for infection, especially staff and residents at long term care facilities (LTCF), due to the congregant living setting, and residents with many comorbidities. Prior to vaccine availability, these populations represented a large fraction of total COVID-19 cases and deaths in the U.S. Due to the high-risk setting and outbreak potential, staff and residents were among the first groups to be vaccinated. To define the impact of prior infection on response to vaccination, we measured antibody responses in a cohort of staff members at a LTCF, many of whom were previously infected by SARS-CoV-2. We found that neutralizing, receptor-binding-domain (RBD) and nucleoprotein (NP) binding antibody levels were significantly higher post-full vaccination course in individuals that were previously infected, and NP antibody levels could discriminate individuals with prior infection from vaccinated individuals. While an anticipated antibody titer increase was observed after vaccine booster dose in naïve individuals, boost response was not observed in individuals with previous COVID-19 infection. We observed a strong relationship between neutralizing antibodies and RBD-binding antibodies post-vaccination across all groups, suggesting RBD-binding antibodies may be used as a correlate of neutralization. One individual with high levels of neutralizing and binding antibodies experienced a breakthrough infection (prior to the introduction of Omicron), demonstrating that the presence of antibodies is not always sufficient for complete protection against infection. These results highlight that history of COVID-19 exposure significantly increases SARS-CoV-2 antibody responses following vaccination. Importance Long-term care facilities (LTCFs) have been disproportionately impacted by COVID-19, due to their communal nature, high-risk profile of residents and vulnerability to respiratory pathogens. In this study, we analyzed the role of prior natural immunity to SARS-CoV-2 on post-vaccination antibody responses. The LTCF in our cohort experienced a large outbreak with almost 40% of staff becoming infected. We found that individuals that were infected prior to vaccination, had higher levels of neutralizing and binding antibodies post-vaccination. Importantly, the second vaccine dose significantly boosted antibody levels in those that were immunologically naïve prior to vaccination, but not those that had prior immunity. Regardless of pre-vaccination immune status, levels of binding and neutralizing antibodies were highly correlated. The presence of NP-binding antibodies can be used to identify individuals that were previously infected when pre-vaccination immune status is not known. Our results reveal that vaccination antibody responses differ depending on prior natural immunity.

pre-immune with either a documented prior infection, or serological evidence of prior infection (seropositive). 73

Early work examined the role of pre-existing immunity on the level of binding antibodies up to four weeks 74 following a single dose of an mRNA vaccine (both Pfizer and Moderna) and found that levels were higher in 75 those that were seropositive [12] . Additional work has evaluated longer term responses after two vaccine 76 doses, and similarly found that those with prior infections generated higher levels of binding antibodies [13] [14] [15] . 77

Most of these studies do not measure polyclonal antibody neutralization of live SARS-CoV-2 virus, and instead 78 use pseudotyped virus, or receptor blocking assays as surrogates of true neutralization. 79 80 Staff at a local long-term care facility (LTCF), in parallel with their weekly SARS-CoV-2 nasal surveillance 81 qPCR testing, provided blood samples for antibody analyses [8] . This facility experienced a SARS-CoV-2 82 outbreak in September 2020 prior to vaccine availability, resulting in infection and seroconversion of almost 83 35% of the staff members [8] . In January 2021, a Pfizer vaccine clinic was provided at their workplace, with the 84 second dose provided three weeks later in early February. Vaccines were not required at this time, though 85 vaccination is now required with rare exceptions [16] . As of January 30, 2022, 96% of staff and 97% of 86 residents at this facility were fully vaccinated, slightly higher than Colorado statewide averages (92% and 93% 87 of staff and residents respectively) [2] . We collected and analyzed sera from staff at this facility from August to 88

December 2020 [8] . We found that during an outbreak at the facility, many staff (~30%) became infected and 89 subsequently seroconverted, generating neutralizing, spike-and RBD-binding antibodies. Here we report 90 seroantibody levels detected in samples collected from February through September 2021 to examine humoral 91 immune response duration. We characterized antibody neutralization, binding to receptor-binding domain 92 (RBD, contained within the spike protein component of the vaccine), and nucleocapsid (NP, not present within 93 the mRNA vaccines). We found that individuals with a prior SARS-CoV-2 infection, have higher post-94 vaccination neutralizing, RBD and NP binding antibodies than those that were seronegative prior to 95 vaccination, and individuals that were never infected with SARS-CoV-2 did not harbor anti-NP seroreactivity. 96 97

Human specimens. This study was approved by the Colorado State University Institutional Review Board 99 under protocol number 20-10057H. Participation in providing blood samples was voluntary. Participants were 00 consented and enrolled and informed of test results. Staff represented a range of job classifications, including 01 those in direct patient care roles (e.g. nurses) and non-direct patient care roles (e.g. administrative). 02 03 Serum collection. Whole blood was collected in BD Vacutainer blood collection tubes and allowed to clot at 04 room temperature for at least 30 minutes. Tubes were spun at 1,300xg for 10 minutes to separate sera from 05 the blood clot. Sera was aliquoted, heat inactivated at 56°C for 30 minutes, and stored at 4°C. 06 Viruses and cells. Vero cells (ATCC-81) were maintained in DMEM with 10% fetal bovine serum (FBS), and 08 1% antibiotic/antimycotic at 37°C and 5% CO 2 . SARS-CoV-2 virus (2019-nCoV/USA-WA1/2020 strain) was 09 used to infect Vero cells for 3 days, supernatant was harvested, centrifuged at maximum speed for 10 minutes 10 to pellet cell debris, aliquoted into single-use aliquots, and stored at -80°C until use. 11 12 Neutralization assay. Standard plaque reduction neutralization test (PRNT) was performed as previously 13 described [8] . Briefly, diluted sera were mixed with virus, incubated for one hour at 37°C, added to a Vero cell 14 monolayer, incubated an additional hour at 37°C, then overlaid with tragacanth media and incubated for two 15 days. Cells were fixed and stained with ethanol and crystal violet, and plaques counted manually. 16 17 RBD and NP ELISA. Binding assays were performed as described previously [8] . Briefly, 96-well plates were 18 coated with SARS-CoV-2 protein (RBD and NP from Sino Biological), blocked with non-fat dried milk, and 19 diluted sera was added. Plates were washed and a secondary anti-human IgG-horseradish peroxidase 20 conjugated secondary antibody was added. Plates were developed and read at 490nm on a 21 spectrophotometer. 22

23 Surveillance qPCR testing. Surveillance testing was performed as previously described [8, 9] . Briefly, nasal 24 swabs were collected, processed, viral RNA extracted, and quantitative reverse transcriptase PCR (qPCR) 25 was performed using the Thermo Fisher Scientific TaqPath Samples without neutralization detected are plotted at half the limit of detection (10). 48 49

Receptor binding domain (RBD) and nucleoprotein (NP) antibody levels after vaccination. We next 50 measured RBD and NP antibody binding levels following vaccination in our cohort participants. RBD levels 51 reached their maximum level in all individuals by day 70 post-vaccination and gradually decreased over the 52 next 6 months (Figure 2A) . Seropositive individuals had slightly higher RBD absorbance values than those 53 that were immunologically naïve prior to vaccination, though this enhancement was not as marked as 54 neutralizing antibody levels ( Figure 2B) . Participants in our cohort received either the Pfizer or Moderna 55 mRNA vaccines, which encode the viral spike protein (which contains the RBD). Therefore, as expected, only 56 participants with NP-reactive antibodies ( Figure 2C ) were previously infected with SARS-CoV-2 ( Figure 2D) . 57 

subset of the cohort with known serostatus prior to vaccination provided blood samples following both their first 82 and second vaccine doses. We compared levels of neutralizing, RBD binding, and NP binding across these 83 two time points and cohorts and looked at relative changes in antibody levels (Figure 4) . In immunologically 84 naïve individuals prior to vaccination, neutralizing and RBD binding levels significantly increased between first 85 and second doses (p<0.001) (Figure 4A & B) . Importantly, some individuals did not have detectable 86 neutralizing antibodies until after their second dose. In contrast, in previously infected individuals, neutralizing 87 and RBD binding antibody levels did not significantly increase following their second dose (Figure 4A & B) . 88

Additionally, vaccination did not alter NP antibody binding levels regardless of pre-vaccination immune status 89 ( Figure 4C) . In seronegative individuals, following the second vaccine dose, neutralizing and RBD binding 90 levels increased significantly (average of 17-fold and 1.5-fold respectively) (Figure 4D & E) . Conversely, in 91 pre-vaccination seropositive individuals, on average, neutralizing, RBD binding, and NP binding antibody levels 92 did not change following the second vaccine dose (0.7-, 1-and 0.9-fold changes respectively) ( Figure 4D , E & 93 F). 94

Neutralization titers, B) RBD binding and C) NP binding of serum from individuals following their first and 97 second vaccine doses (3 weeks after the first dose, and 7 weeks after the second dose), stratified by pre-98 vaccination immune status. Fold change between D) neutralization, E) RBD binding and F) NP binding relative 99 to levels following their first vaccine dose. A) dashed lines represent the limit of detection. Samples without 00 neutralization detected are plotted at half the limit of detection (10). B, C) Dashed line represents the 01 background level for each assay. ***p<0.001, ****p<0.0001 by Mann-Whitney test. 02 03

Relationship between neutralizing and binding antibodies in vaccinated individuals. We next compared 04 the relationship between neutralizing and binding (both RBD and NP) antibodies in vaccinated individuals 05 (including samples collected after just the first dose), stratified by pre-vaccination immune status. We saw a 06 strong relationship (r > 0.75) between neutralizing titer and RBD binding antibody absorbance regardless of 07 immune status ( Figure 5A) . Because NP antibodies are only found in individuals that experienced a natural 08 SARS-CoV-2 infection, the relationships with NP antibodies (both PRNT 50 vs NP and RBD vs NP) were poorly 09 correlated (r < 0.45) in post-vaccination sera samples (Figure 4B & C) . 10 Figure 6A) . There was no evidence that 24 antibody levels had waned prior to infection (Figure 6B & C) . Neutralizing antibody levels rapidly increased 25 following infection ( Figure 6B ) whereas their RBD binding antibodies did not ( Figure 6C) . Detection of anti-NP 26 antibodies confirmed the breakthrough infection ( Figure 6D) . Early following SARS-CoV-2 vaccine approval, it was unclear if both doses of the mRNA vaccine would be 37 necessary for individuals that had previously been infected, to achieve full protection [18] . It was predicted the 38 first dose would boost humoral immunity acquired from a natural infection. Multiple studies have demonstrated 39 that in previously infected, seropositive individuals, a single vaccine dose is sufficient to generate robust 40 immune responses (both humoral and cellular), often to levels higher than in naïve individuals that received 41 two vaccine doses [19] [20] [21] . Our data confirm that individuals with a prior infection generate a robust, 42 neutralizing antibody response that is not further increased upon a second dose. These results have led to 43 calls for a single-dose vaccine regimen in previously infected individuals to stretch vaccine supplies, improve 44 worldwide vaccine access, and increase vaccine uptake among hesitant . 45 46 Conversely, in seronegative individuals, antibody levels significantly increased following a second vaccine 47 dose [19] [20] [21] . Three individuals in our cohort did not generate neutralizing antibodies until after the second 48 vaccine dose, and one individual never seroconverted following vaccination. It is therefore critical that 49 individuals without prior infection receive the full vaccination course to ensure maximum immune response 50 [26] . 51 52 Neutralizing and binding antibody levels are being developed as correlates of protection, as they are highly 53 correlated with vaccine efficacy across diverse cohorts and vaccine platforms [27] [28] [29] . There are reports 54 describing breakthrough infections post-vaccination, likely due to reduced/waning antibody levels and timing 55 post-vaccination [30] [31] [32] [33] . The breakthrough infection that occurred in our cohort, was in an individual with high 56 neutralizing antibody levels similar to other recent reports [34, 35] . These data suggest that while antibody 57 levels may be broadly predictive of vaccine efficacy, they are not sufficient as a singular correlate of protection 58 in all individuals. 59 60 Our work, along with others [36] [37] [38] , describes the use of nucleoprotein antibody detection as a tool to identify 61 natural infection using serum collected post-vaccination. This assay could be used to further define and refine 62 correlates of protection, or generate a better predictor of breakthrough risk, by stratifying post-vaccination 63 serum into those that had and had not been previously infected. Importantly, this strategy is only effective in 64 individuals that received a vaccine without a nucleocapsid component (Pfizer, Moderna, etc.) as opposed to 65 inactivated whole virus vaccines (or other similar vaccine platforms) containing nucleocapsid, such as Sinovac. 66 67 There remain many unknowns regarding the immune response following vaccination, 68 booster, . Boosters, which have been widely accessible in the U.S., combat waning 69 immunity by boosting pre-existing adaptive immunity (both humoral and cellular), furthering protection against severe disease [42] . There is relatively high booster uptake among staff and residents of LTCFs in Colorado 71 (76% and 40% of residents and staff with boosters, respectively), with slightly higher rates in the facility 72 described in this paper (80% residents, 44% staff) [2] . Despite high vaccination and booster rates, the Omicron 73 variant seems to efficiently evade vaccine-elicited immunity [43, 44] . These results suggest additional 74 boosters, and variant-specific boosters might be required to maintain long-term immunity against SARS-CoV-2 75 Systems Center for Health Aging, and the CSU One Health Institute. We thank the members of the CSU 82 Veterinary Diagnostic Lab for their assistance with surveillance testing and diagnostic support. Additionally, we 83 thank the ongoing support and participation of the LTCF staff who participated in the study. 84 85

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