key: cord-0952426-et6oehx1
authors: Bastug, Aliye; Bodur, Hurrem; Aydos, Omer; Filazi, Nazlican; Oksuz, Ergun; Ozkul, Aykut
title: The changing dynamics of neutralizing antibody response within 10 months of SARS‐CoV‐2 infections
date: 2022-01-07
journal: J Med Virol
DOI: 10.1002/jmv.27544
sha: 10f22ca77454837653f3a41afcbb386e4f0d3d73
doc_id: 952426
cord_uid: et6oehx1

There are limited data on how long neutralizing antibody (NAb) response elicited via primary SARS‐CoV‐2 infection will last. Eighty‐four serum samples were obtained from a prospective cohort of 42 laboratory‐confirmed COVID‐19 inpatients at the time of discharge from the hospital and in the late convalescent phase. A virus neutralization assay was performed to determine the presence and titers of NAbs with authentic SARS‐CoV‐2. Long‐term dynamics of NAbs and factors that may have an impact on humoral immunity were investigated. Mild and moderate/severe patients were compared. The mean sampling time was 11.12 ± 5.02 days (4–28) for the discharge test and 268.12 ± 11.65 days (247–296) for the follow‐up test. NAb response was present in 83.3% of the patients about 10 months after infection. The detectable long‐term NAb rate was significantly higher in mild patients when compared to moderate/severe patients (95.7% vs. 68.4%, p = 0.025). In the follow‐up, NAb‐positive and ‐negative patients were compared to determine the predictors of the presence of long‐term humoral immunity. The only significant factor was disease severity. Patients with mild infections have more chance to have NAbs for a longer time. Age, gender, and comorbidity did not affect long‐term NAb response. NAb titers decreased significantly over time, with an average rank of 24.0 versus 19.1 (p = 0.002). Multivariate generalized estimating equation analysis revealed that no parameter has an impact on the change of NAb titers over time. The majority of the late convalescent patients still had detectable low levels of neutralizing antibodies. The protective effect of these titers of NAbs from re‐infections needs further studies.

1 h for neutralization. Then, the mixture of virus and serum was inoculated into 90% confluent Vero E6 cells grown in 96-well plates. Virus control cells were prepared with 100TCID 50 dilution test virus. Therefore, 100% cytopathogenic effect occurrence in control wells was accepted for the best time for test assessment. Reciprocals of serum dilutions that neutralize a minimum of 50% of 100TCID 50 virus infection were accepted as virus-neutralizing antibody (NAb) titer of each serum sample.

Mean and standard deviation, minimum, maximum, median, and interquartile range were used for continuous variables. Number and percentage were used for categorical variables. The Kruskal-Wallis and Mann-Whitney U-tests were used for the data that did not have a normal distribution. The categorical data were compared using the χ2 test or Fisher's exact test. The Wilcoxon signed-rank test was used to determine baseline and follow-up antibody test titers. p-values were calculated using the Wilcoxon rank-sum test to compare titer values between groups of different categories of disease severity and sampling days at a given time point. p ≤ 0.05 was considered significant. The association between the variation of neutralizing antibody titers and potential factors such as gender, age, clinical disease severity, and time from onset of symptoms was calculated with the generalized estimating equation (GEE) model, which takes into account the correlation between repeated measurements. In the analyses, hypothesis testing was performed bi-directionally with an α value of 0.05. All analyzes were performed using SPSS 24.0 (IBM Corp) software.

A total of 42 laboratory-confirmed COVID-19 inpatients with a mean age of 40 ± 10.2 years were enrolled. Of the patients, 83.3% was PCR (+) on admission. The remaining patients had SARS CoV IgM and or IgG positivity at the time of hospital discharge in the pre-vaccine era. It was found that 57.1% were male and 21.4% had comorbidity. Twenty-three patients (54.7%) were in the mild group, 14 (33.3%) were moderate, and the remaining 5 (11.9%) were severe. Hypertension was the most frequent comorbidity with a ratio of 11.9% and it was statistically more frequent in the moderate/severe group (p = 0.015). Cough, fever, tachypnea, and myalgia were the most frequent symptoms. The mean length of time after symptom onset was 11.12 ± 5.02 days (4-28 days) for the sampling of the first NAb tests and 268.12 ± 11.65 days (247-296 days) for the follow-up NAb tests.

The presence of NAb response was observed in 50% of the patients at the time of hospital discharge, then it reached a level of 83.3% on the follow-up sera. In none of the patients, re-infection occurred after discharge. The detectable NAb rate was lower on discharge as 52.4% of the patients were tested 4-9 days after symptom onset at the time of discharge from the hospital. In other words, the reason for this result may be that 10-14 days required for antibody development has not passed yet at the test time of discharge NAbs.

In the follow-up test samples, the detectable NAb rate was significantly higher in mild patients compared to moderate/severe patients (95.7% vs. 68.4, p = 0.025). The follow-up NAb positive and negative patients were compared to determine the predictors of the presence of long-term humoral immunity. The only significant factor was determined as the degree of disease severity. Age, gender, and comorbidity had no effect on long-term NAb response.

A total of seven patients had negative follow-up NAb titers. Discharge NAb titers were also negative in three of them. The other four patients had positive discharge NAb titers (1:1250, 1:125, 1:25, and 1:7.5 respectively) which became negative on the follow-up test (Figure 1 ).

Mild patients constituted 54.7% of the study population. When the mild and moderate/severe groups were compared, the median NAb titers on discharge was higher in the moderate/severe group (median, 7.5; range, 0-1250 vs. median, 0.0; range 0-125) whilst it did not reach statistical significance at the Wilcoxon rank analysis ( Table 1) Wilcoxon signed-rank test shows that the observed difference between both measurements is significant (p = 0.002). There is also a significant decrease in the NAb titers of mild patients on the followup (average rank of 14.7 vs. average rank of 11.6, p = 0.004). However, the decrease of the NAb titers in the moderate/severe group was not statistically significant (average rank of 9.5 vs. average rank of 8.2, p = 0.119). In the comparative analysis according to the postsymptom day groups from which the first NAb test was obtained, follow up NAb titers were significantly higher in the mild 4-9 days group (median, 30; range, 0-40) when compared with moderate/ severe group (median, 7.5; range, 0-30), p = 0.019 ( Figure 2B ). | 1985 significant in the group of 15-28 days (average rank of 2.5 vs. 6.0, p = 0.498). Univariate GEE analyses revealed that age, gender, and disease severity had no effect on the change of antibody titers over time.

The only significant factor between the change of antibody titers over time was the post-onset sampling time of the discharge test. In other words, when the sampling time of the discharge NAb test was compared (4-9 days vs. 15-28 days post-onset), there was a significant increase in the long term NAb response compared to the baseline level in days 4-9 group in univariate analyses (p = 0.015). Multivariate analyses revealed that no significant parameter had an impact on the difference between baseline and follow-up NAb titers ( Table 3 ). The distribution of the number of patients for the discharge and follow-up NAb titers according to time post-onset is given in Figure 3A ,B.

NAbs have been expected as predictors of antibody-mediated immunity. 8 It is supposed that it will either eliminate the risk of re-infection or in case of re-infection, at least NAbs may decrease the severity of infection.

However, it is difficult to present the presence of NAbs as conclusive evidence for preventing reinfection. 9 Although the exact threshold for the NAb titer that will be more probable to prevent reinfection is unknown, higher titers are more likely to have a chance for prevention. Another important issue that needs to be clarified is how long antibody-mediated immunity is elicited after natural infection will last. Its importance derives from its contribution to the development of herd immunity and on the decision of the length of time for using "immunity passports." 10, 11 This is also necessary for determining whether a booster dose of vaccination is necessary for recovered patients and if so when it is supposed to be administered. 8, 12 Humoral immunity via primary infection of other coronaviruses lasts for several months although antibody titers wane over time. [13] [14] [15] In terms of SARS-CoV-2 infection, there are several studies conducted in acute and early convalescence periods and they have reported that NAb titers vary depending on the severity of the primary infection and the length of time from onset. 2, 5, 12 In addition, Legros et al. 16 and strong NAb response (NAb titers of 1:1250 and, 1:125) in the acute phase of infection, the antibody titers became negative within the 10 months after onset. These findings agree with previous reports suggesting that the severe infection leads to a strong reaction in the antibody response via short-lived plasmablast, and then wanes more rapidly. 5 However, further studies with large cohorts are needed to define this issue more precisely.

In the present study, milder infections were found as the only predictor of long-term detectable NAb response. Other than this, age, gender, and comorbidity did not have an effect. In contrast, the severe infection was reported to lead to a higher antibody response in the early phase of the disease. 8 

This study did not receive a specific grant.

The authors declare that there are no conflict of interests. 

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID Aliye Bastug http://orcid.org/0000-0002-8831-4877

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