key: cord-1011300-o6xmys26 authors: Haymond, Amanda; Damluji, Abdulla A; Narayanan, Aarthi; Mueller, Claudius; Reeder, Alex; Alem, Farhang; Maxwell, G Larry; Petricoin, Emanuel F; Liotta, Lance; deFilippi, Christopher R title: Viral Neutralization is Durable in Asymptomatic COVID-19 for at least 60 Days date: 2021-03-15 journal: J Infect Dis DOI: 10.1093/infdis/jiab140 sha: fe9609ed9708b75c16d21635e86ad4a13c7833ec doc_id: 1011300 cord_uid: o6xmys26 A cohort consisting of asymptomatic health care workers (HCW) donated temporal serum samples following infection with SARS-CoV-2. Analysis shows that all asymptomatic HCW had neutralizing antibodies, that these antibodies persist for at least 60 days, and that anti-spike RBD IgG levels were correspondingly durable over the same time period. A c c e p t e d M a n u s c r i p t A cohort consisting of asymptomatic health care workers (HCW) donated temporal serum samples following infection with SARS-CoV-2. Analysis shows that all asymptomatic HCW had neutralizing antibodies, that these antibodies persist for at least 60 days, and that anti-spike RBD IgG levels were correspondingly durable over the same time period. A c c e p t e d M a n u s c r i p t The reported decay of anti-spike antibodies [1] and the temporal reduction in viral neutralization titers [2] have led to debate on the longevity of protection following SARS-CoV-2 infection. To address this question, we report a study enrolling asymptomatic health care workers (HCW) without prior COVID-19 symptoms, but detectable SARS-CoV-2 antibodies, and mildly symptomatic patients. Current evidence in animal models and natural experiments suggests that the presence of neutralizing antibodies predicts protection from SARS-CoV-2 [3] [4] [5] . While correlates of protection in COVID-19 are as of yet unknown, including how cellular responses may provide protection apart from, or in conjunction with, antibody responses [6, 7] , the prophylactic protection provided by administered neutralizing antibodies supports the hypothesis that neutralizing antibodies can prevent infection [8] . Therefore, analyzing the antibody immune response of asymptomatic or mildly symptomatic patients may shed light on protection from reinfection. All participants were consented and samples collected under IRB approval from the appropriate institution. Asymptomatic HCW cohort participants were identified as follows: HCW previously infected with to SARS-CoV-2 were identified via an Ortho Diagnostics Vitros anti-S1 total Ig ELISA serology screening program during April/May 2020 at Inova Hospital in Northern Virginia. Each HCW was asked whether they had developed symptoms prior to or during enrollment. During followup, each participant had to answer the same set of questions of whether they developed symptoms including, but not limited to, fever, cough, chest tightness or shortness of breath, throat pain, loss of taste, loss of smell, or other respiratory symptoms related to COVID-19 disease. The follow-up questionnaire was performed at 2-and 6-months intervals. Individuals identified as positive via the Ortho Diagnostics assay were consented and donated sera samples, which are denoted as baseline. The same individuals returned at 60 days past the initial donation and provided a second temporal sera sample, denoted as 60 days. Of n=17 matched samples, two sera samples (one baseline sample and Symptomatic participants were not screened via the Ortho Diagnostics anti-S1 total antibody assay. interviews with the participants (n=12). As shown in Figure 1A , the presence of anti-spike RBD IgG was predictive of viral neutralizing activity in a standard PRNT 90 assay in both symptomatic and asymptomatic cohorts. For the symptomatic cohort, those without confirmatory PCR were in general non-neutralizing, indicating symptoms were likely due to an illness other than COVID-19. However, as shown in Figure 1B , we do not see a strong correlation between anti-RBD titers and viral neutralization titers in our cohort, even in a subgroup analysis separating symptomatic patients from asymptomatic patients, though this correlation is reported by some other groups [9, 10] . For the asymptomatic cohort, we were able to obtain an additional serum samples 60 days following their initial sample. We determined the longitudinal PRNT 90 values for the asymptomatic participants (n=15, two samples unavailable, see methods) to evaluate the persistence of neutralizing antibodies for this cohort. As shown in Figure 1C , it is clear that all asymptomatic HCW made detectable neutralizing antibodies at baseline. Furthermore, these titers are largely unchanged at 60 days post-baseline. Given that it is unknown how long after infection an asymptomatic participant's baseline sample was obtained, protection could easily last longer than 60 days. Because baseline samples were acquired in April/May 2020, and the first confirmed SARS-CoV-2 case in the state of Virginia was reported on March 7 th [11] , it is unlikely any participant had been infected more than a two months prior to donation of the baseline sample. Notably, consistent neutralizing titers over 60 days are mirrored by consistent positivity via our anti-RBD ELISA over the same time period, as A c c e p t e d M a n u s c r i p t shown in Figure 1D . One participant showed very low anti-RBD titers at baseline that rose significantly by 60 days, suggesting this participant may have provided a baseline sample very early in the convalescent phase. Due to the unknown timeline of exposure or infection prior to donation at baseline, it is unclear if the titers are -stable‖ over the two month window observed here as peak antibody concentrations may have been missed; however, the durability of such a response over the time frame is clear. We conclude that anti-RBD ELISAs, while excellent to determine previous infection and predict presence of neutralizing antibodies, may be a poor surrogate for the level of viral neutralization activity depending on the composition and size of the cohort based on the results reported in Figure 1B . Our cohort of asymptomatic HCW may be different immunologically than primarily symptomatic cohorts [9, 10] in which the strong correlation between anti-RBD antibody titers and neutralizing antibody titers is reported. Recent work suggests that mildly affected cohorts, such as children, make distinct antibody responses[12], and one report has shown reduced anti-spike IgG generation specifically in an asymptomatic cohort, as well as reduced correlation between anti-RBD titers and neutralizing titers, as we report here. [13] It is worth noting that the lack of correlation between anti-RBD IgG and neutralization antibody could be explained in part by the fact that neutralization titers in this study population of asymptomatic participants are at the low end of the spectrum, which could make their association less linear. Furthermore, our PRNT 90 assay has differences from the assays used by some other groups [9, 10] . Our measured endpoint is a plaques, rather than quantification of virus present through RT-PCR, luminesce assays, or anti-N antibody staining. While anti-RBD responses may be predictive of previous infection, further research into additional epitopes targeted by neutralizing antibodies in this cohort may shed light on other epitopes relevant to protection in asymptomatic or mildly affected cohorts. We eagerly report that asymptomatic HCW mount viral-neutralizing responses that are durable over at least 60 days. Previous reports have suggested that asymptomatic patient mount neutralizing titers at reduced intensity as compared to symptomatic cohorts [13] , which is a finding we A c c e p t e d M a n u s c r i p t replicate here with median PRNT 90 titers of 320 and 80 in the symptomatic and asymptomatic cohorts respectively ( Figure 1B) . We however do not find marked reduction in neutralizing titers in short periods of time in asymptomatic patients, as feared following reports of reductions in IgG titers in asymptomatic patients by others [14] . While we cannot rule out the possibility of subsequent asymptomatic reinfection of subjects during the 60 days, data suggests this is unlikely [15] , and particularly unlikely that would apply to significant numbers of cohort participants. Sustained viral neutralization titers are an independent metric indicating protection of asymptomatic patients regardless of whether anti-RBD IgG decays or persists. The durability of neutralizing responses even in asymptomatic participants as reported here has clinical implications on public health and society as a whole as vaccination or other immune therapy for COVID-19 disease becomes available in clinical practice and serology assays are used to guide treatment [16] . While there is data to support potentially giving only a single mRNA vaccine dose for symptomatic, previously-infected COVID-19 patients [17] , it is unclear if this research is translatable to those who remain asymptomatic. Our data suggests some level of durable protection even in asymptomatic infections, suggesting that single dose mRNA vaccinations constitute a worthy area of further study in asymptomatic individuals. Additional follow-up for durability of neutralizing antibodies beyond 60 days in asymptomatic, but infected individuals is sorely needed. 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Signal Transduction and Targeted Therapy Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections Association of SARS-CoV-2 Seropositive Antibody Test With Risk of Future Infection Clinical Application of Serologic Testing for Coronavirus Disease 2019 in Contemporary Cardiovascular Practice 19: People who have had infection might only need one dose of mRNA vaccine A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Figure 1 : Mild and asymptomatic patients produce neutralizing antibodies, which for asymptomatic patients are shown to be durable over the course of 60 days in viral neutralization assays with authentic SARS-CoV-2 yet not strongly correlated with anti-RBD IgG titers. A) Anti-spike RBD ELISA OD450 values at 1:20 dilution are predictive of the presence of viral neutralizing antibodies in patient serum samples. Differences between medians were evaluated by unpaired t-test; medians were significantly different (p < 0.0001). Asymptomatic patient data shown at baseline. B) All patients with neutralizing antibody titers (n = 34) were evaluated for correlation between neutralizing titers and signal intensity in the anti-spike RBD ELISA. Spearman's r for both cohorts is 0.4297 (p = 0.011). Subgroup analysis with neutralizers from the symptomatic cohort (n = 18) give a poorer correlation where Spearman's r for this subgroup is 0.232 (p=0.354). Subgroup analysis with neutralizers from the asymptomatic cohort show an improved but still weak correlation where Spearman's r for this subgroup is 0.591 (p = 0.018). C) Differences between PRNT 90 values at baseline and at 60 days for patients in the asymptomatic HCW cohort were analyzed via paired t-test; differences in PRNT 90 value were not significant (p > 0.99). D) Differences between OD450 values at 1:20 dilution in anti-spike RBD ELISA values at baseline and at 60 days for patients in the asymptomatic HCW cohort were analyzed via paired t-test; differences in PRNT 90 value were not significant (p = 0.1793).