Persistence and baseline determinants of seropositivity in health care workers up to nine months after COVID-19


 We determined the duration and baseline determinants of antibody responses to SARS-CoV-2 up to nine months after COVID-19 symptoms onset in 173 primary health care worker patients from Spain. Seropositivity to SARS-CoV-2 spike and RBD antigens was 92.49% (60.69% IgM, 76.3% IgA, 90.17% IgG), with four suspected reinfection cases. Antibody levels significantly correlated with fever, hospitalization, anosmia/hypogeusia, allergies, smoking and occupation, and persisted 149-270 days in this cohort of patients


Main Text
One of the key questions to understand the evolution of the COVID-19 pandemic is the duration of the immune response generated upon infection with SARS-CoV-2. Studies have shown that most patients induce a robust humoral and cellular response 1 but with high heterogeneity and a percentage of non-responders. Diversity in epitope speci city, quality and functional capacity of antibodies will likely affect the e cacy of the immunity mediated. Although a number of cases of SARS-CoV-2 reinfection have been reported, antibodies were recently associated with protective immunity over a 30-week follow up 2 . The spike (S) protein on the virus surface is considered the main target of protective antibodies, and the component of leading vaccine candidates 3 .
Functional neutralizing antibodies highly correlate with IgG levels to the receptor binding domain (RBD) of S 1 , but IgA and IgM also have neutralizing properties 4 , and non-neutralizing antibodies with Fc receptor-mediated functions are likely important for protective immunity.
Despite an increasing understanding of the nature of antibody responses, their longevity remains to be de ned as the pandemic evolves. This is a critical question to predict whether the frequency of reinfection may increase if immunity wanes. As massive global immunization campaigns advance, this knowledge will give clues as to how long vaccine immunity may last and how preexisting antibodies and other baseline variables could affect vaccine effectiveness. Although some initial reports indicated a marked decline in antibodies after 3 months 5 , subsequent studies have shown relatively stable antibody levels, mostly IgG, over a period of up to 6-8 months in small sets of patients 1,6-10 .
We aimed to evaluate the seroprevalence and levels of antibodies 149-270 days after the onset of symptoms in a cohort of 173 primary health care workers (HCW) from 5 counties in Barcelona province, Spain, with COVID-19 at the rst peak of the pandemic (March-April 2020) and being followed up for 1.5 years. HCW are at the forefront of this medical emergency and thus considered a high-risk population. The study protocol was approved by the IRB Comitè Ètic d'Investigació Clínica IDIAP Jordi Gol (codes 20/094-PCV and 20/162-PCV) and written informed consent was obtained from participants.
Demographic and clinical data were collected to characterize the factors associated with disease presentation, presence of sequelae, long COVID-19 and reinfection in this cohort. Most cases were mild-moderate COVID-19, with 24 hospitalized, and 64 presenting with sequelae. Median age was 49 years (IQR 41-58) and 137 were females. Clinical information included history of previous allergies and smoking (13 smokers, 31 ex-smokers).
Three cross-sectional surveys were done between September and November 2020 to obtain venous blood for assessing maintenance of anti-SARS-CoV-2 seropositivity and analyze baseline factors associated with antibody levels. Levels of IgM, IgA and IgG to RBD and S recombinant proteins expressed from plasmids donated by F.
Krammer (Mount Sinai, NY) were quanti ed in plasma by Luminex, as described 11 . The cutoff for seropositivity was calculated with 55 prepandemic samples as 10 to the mean plus 3 standard deviations of log 10 -transformed mean uorescence intensity values.
We did not detect a signi cant decline in antibody levels as a function of time since symptoms onset ( Figure 1).
The percentage of seropositivity 149-270 days after symptoms onset combining RBD and S antigens was 60.69% for IgM, 76.30% for IgA, and 90.17% for IgG, consistent with the expected longer duration of the latter isotype. Unexpectedly, seropositivity was considerably sustained also for IgM and IgA, considered of shorter duration.
Computing all immunoglobulin isotypes, seroprevalence 5-9 months after the initial COVID-19 episode was as high as 92.49%, indicating highly stable persistence of responses.
There were four suspected reinfections (Table S1). Before the second positive PCR, two symptomatic cases were seronegative, one asymptomatic was seropositive with low antibodies, and one had unknown serostatus.
Unfortunately, viral RNA was not available from the rst episodes for genome sequencing and demonstration of different strains.
The baseline factors most consistently and signi cantly associated with higher levels of antibodies 5-9 months later in stepwise multivariable regression analyses were having been admitted to hospital, presenting with fever (n=131), anosmia and/or hypogeusia (n=106), and previous allergies (n=24) ( Table 1). Speci cally, for anti-S IgG, HCW with fever had 2.5 times higher levels, patients with anosmia and/or hypogeusia had 2.6 times higher levels, and those with allergies had 1.9 times higher levels, than patients without those conditions. Baseline factors associated with lower levels of IgA and IgG included being a nurse (n=68) or a physician (n=70) compared to other occupations (n=35), and smoking. For anti-S IgA, physicians had 34.84% and nurses 45.67% lower levels than the other jobs, and smokers had 46.17% less than non-smokers (Table 1). Other factors were associated with only certain isotypes. Presenting with sputum and/or hemoptysis (n=13) was associated with higher IgM levels, and shivers (n=86) were associated with higher IgAs. Of note, hospitalized patients had 2.1 times higher IgM levels to RBD than non-hospitalized. Age correlated positively with IgGs, having 1.39% higher antibody levels to RBD with each year of age older (Table 1). Higher IgGs (and IgAs less strongly) positively correlated with duration of symptoms (median 24 days, IQR 13-36; S rho=0.229 P=0.002; RBD rho=0.246, P=0.001) and number of symptoms (median 10, IQR 6-12; S rho=0.351 P<0.001; RBD rho=0.364, P<0.001). All other variables, symptoms, or sequelae, were either not statistically signi cantly associated with antibody levels or weakly in univariable models.
Previous acute phase studies showed that COVID-19 severity was associated with higher antibody responses.
Here, hospitalization was associated with higher immunoglobulin levels many months after convalescence, suggesting that severity does not affect stability of memory B cell and plasma cells producing antibodies 12 .
Common symptoms like fever and highly speci c symptoms like alteration in smell and taste were also associated with higher antibodies. Interestingly, having previous allergies also correlated positively with higher antibody levels, which to our knowledge has not been reported. This could be related to disease exacerbation and increased risk of respiratory infections associated with some allergies 13 although this relationship remains unclear. Lower antibody levels in nurses and physicians than other HCW could indicate a lower exposure due to PPE use and higher awareness of risks 14 . Smoking had previously been associated with lower antibody responses 15 and we show that this effect persists after several months primarily affecting IgA, the main mucosal antibody.
In conclusion, despite the large heterogeneity in antibody levels induced by SARS-CoV-2 infection, the majority of HCW patients remained seropositive for anti-S antibodies up to nine months after COVID-19. The ndings that after PCR reversion, 2 out of 13 seronegatives had another symptomatic episode, and that one low responder had a second (asymptomatic) infection, are consistent with a protective role for antibodies 2 . Data suggest that immune memory induced by rst generation vaccines could also be long-lasting, therefore reducing the probability that periodical boosters might be required to sustain protective immunity. Date of start and end of the rst and last symptoms. All dates are 2020 unless otherwise indicated.

Declarations
2 PCR was done prior to a surgical procedure and not as part of a routine screening, the participant had no symptoms. 3 Low level antibody responses above the seropositivity threshold. Antibody levels by days since COVID-19 symptoms onset.