Unveiling a New Perspective on Distinguishing Omicron Breakthrough Cases and Postimmune COVID-19-Naive Individuals: Insights from Antibody Profiles

ABSTRACT In the situation of mass vaccination against COVID-19, few studies have reported on the early kinetics of specific antibodies (IgG/IgM/IgA) of vaccine breakthrough cases. There is still a lack of epidemiological evidence about the value of serological indicators in the auxiliary diagnosis of COVID-19 infection, especially when the nucleic acid results were undetectable. Omicron breakthrough cases post-inactivated vaccination (n = 456) and COVID-19-naive individuals with two doses of inactivated vaccination (n = 693) were enrolled. Blood samples were collected and tested for SARS-CoV-2 antibody levels based on the magnetic chemiluminescence enzyme immunoassay. Among Omicron breakthrough cases, the serum IgG antibody level was 36.34 Sample/CutOff (S/CO) (95% confidence interval [CI], 31.89 to 40.79) in the acute phase and 88.45 S/CO (95% CI, 82.79 to 94.12) in the recovery phase. Serum IgA can be detected in the first week post-symptom onset (PSO) and showed an almost linear increase within 5 weeks PSO. Compared with those of breakthrough cases, IgG and IgA titers of the postimmune group were much lower (4.70 S/CO and 0.46 S/CO, respectively). Multivariate regression showed that serum IgG and IgA levels in Omicron breakthrough cases were mainly affected by the weeks PSO (P < 0.001). Receiver operating characteristic ROC0 curve analysis showed that the area under the curve (AUC) was 0.744 and 0.806 when the cutoff values of IgA and IgG were 1 S/CO and 15 S/CO, respectively. Omicron breakthrough infection can lead to a further increase in IgG and IgA levels relative to those of the immunized population. When nucleic acid real-time PCR was negative, we would use the kinetics of IgG and IgA levels to distinguish the breakthrough cases from the immunized population. IMPORTANCE This study fills a gap in the epidemiological evidence by investigating the value of serological indicators, particularly IgG and IgA levels, in the auxiliary diagnosis of COVID-19 infections when nucleic acid results are undetectable. The findings reveal that among Omicron breakthrough cases, both IgG and IgA antibody levels exhibit significant changes. Serum IgG levels increase during the acute phase and rise further in the recovery phase. Serum IgA can be detected as early as the first week post-symptom onset (PSO), showing a consistent linear increase within 5 weeks PSO. Furthermore, receiver operating characteristic (ROC) curve analysis demonstrates the potential of IgG and IgA cutoff values as diagnostic markers. The study’s conclusion underscores the importance of monitoring IgG and IgA kinetics in distinguishing Omicron breakthrough cases from vaccinated individuals. These findings contribute to the development of more accurate diagnostic approaches and help inform public health strategies during the ongoing COVID-19 pandemic.

improvement. The claimed diagnostic role is difficult to implement when two serum/plasma samples collected at different time points from the same individual are unavailable.

Reviewer #2 (Comments for the Author):
This article by Shihan Zhang et al. analyzed the kinetic changes of specific IgG, IgM, and IgA antibodies to SARS-CoV-2 in breakthrough cases after inactivated vaccination and in COVID-19 naive individuals who were vaccinated with 2 doses of inactivated vaccine over time. The results of the study showed that Omicron breakthrough cases had significantly higher levels of IgG and IgA antibodies than the immunized population. It gives us a glimpse of the current status of SARS-CoV-2 reinfection after mass population vaccination, especially after a pandemic of Omicron variant strains. However, the findings of this report require some modifications before publication. Major comments: 1. The name of article does not match the content enough, it is better to be changed. 2. Please clarify the case definition of the breakthrough cases in this study. 3. It is interesting to find in the article that the threshold values for IgA and IgG can be set to 1 and 15 S/CO for trying to distinguish the different between breakthrough cases and post-immunization populations, but the authors do not do a good job of explaining and analyzing this phenomenon in the discussion and whether it can be used for clinical diagnosis in the future. In particular, the duration of use of this threshold considers that antibody titers will decline over time. 4. Author calculated the proportion of IgG-recovery/IgG-acute ratios of four or more in the breakthrough cases with two blood collections in the acute and recovery phases and found that the proportion of ratios greater than four decreased as the level of IgG in the acute phase increased. However, considering that IgG antibodies in breakthrough cases rise much faster than in firsttime infected patients, using 4-fold as a criterion may not be appropriate. 5. In the article the authors tried to use the level of IgG or IgA antibody values to infer the different infection stages infected cases were in, but this inference could lead to errors due to the big individual differences. Minor comments: 1. In line 52, please explain whether the antibody level is the arithmetic mean or median? 2. In line 174, how many post-immune individuals received at least one booster immunization and what is the impact on the result.

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Reviewer #1:
Identifying breakthrough infection in inactivated virus vaccinees is very challenging when the nucleic acid-based diagnosis was found to be negative. The present study is a novel study since it adds more evidence to the role of the level and kinetics of binding antibodies in identifying breakthrough infection in individuals vaccinated with inactivated virus vaccine in whom. This study also provides the role of the ratio of the level of Recovery IgG titer to the level of Acute IgG titer not only identification of breakthrough infection but also in identifying acute infection. The major strength of this study is the inclusion of large cohorts (acute and recovery breakthrough cases and SARS-CoV-2 infection native vaccines). However, the manuscript still needs improvement. The claimed diagnostic role is difficult to implement when two serum/plasma samples collected at different time points from the same individual are unavailable.

Response:
We appreciate you very much for your positive and constructive comments and suggestions on our manuscript. In response to your mentioned implementability issue, in the prevention and control of the pandemic, we generally collect samples twice from suspected subjects, with a general interval of 1~2 weeks between samples. When the first sampling is finished, timely testing is done to apply quantitative values of IgG and IgA to roughly determine whether the host has a history of infection. When the antibody results of the second sampling are available, further verification will be done.

Reviewer #2:
This article by Shihan Zhang et al. analyzed the kinetic changes of specific IgG, IgM, and IgA antibodies to SARS-CoV-2 in breakthrough cases after inactivated vaccination and in COVID-19 naive individuals who were vaccinated with 2 doses of inactivated vaccine over time. The results of the study showed that Omicron breakthrough cases had significantly higher levels of IgG and IgA antibodies than the immunized population. It gives us a glimpse of the current status of SARS-CoV-2 reinfection after mass population vaccination, especially after a pandemic of Omicron variant strains. However, the findings of this report require some modifications before publication.
Major comments: 1. The name of article does not match the content enough, it is better to be changed.
Response: Thank you for your suggestion. We have revised the title to: "Unveiling a New Perspective on Distinguishing Omicron Breakthrough Cases and Post-immune COVID-19 Naïve Individuals: Insights from Antibody Profiles".

Please clarify the case definition of the breakthrough cases in this study.
Response: Thank you for your suggestions, and we sincerely apologize for our oversight during the writing process. We have now added a precise definition of breakthrough cases as the detection of SARS-CoV-2 on RT-PCR assay performed 14 or more days after vaccination of a second dose of CoronaVac/BBIBP in lines 117-119 of the manuscript.
3. It is interesting to find in the article that the threshold values for IgA and IgG can be set to 1 and 15 S/CO for trying to distinguish the different between breakthrough cases and post-immunization populations, but the authors do not do a good job of explaining and analyzing this phenomenon in the discussion and whether it can be used for clinical diagnosis in the future. In particular, the duration of use of this threshold considers that antibody titers will decline over time.
Response: Thank you for your careful review. When the threshold values for IgA and IgG were set to 1 and 15 S/CO, the AUC to distinguish the different between breakthrough cases and post-immunization populations were 0.744 and 0.806, respectively. Especially, the specificity were 93.36% and 93.22%, which means that it was of high accuracy to exclude one as an uninfected person when the IgA and IgG was less than 1 and 15 S/CO. However, the sensitivity were relatively low to confirm one as an infected person when the IgA and IgG was more than 1 and 15 S/CO. These immunological tests were mainly used in the course of epidemiological investigation and were not necessarily suitable for clinical diagnosis. Figure 2A showed that the IgG and IgA level had an upward trend in the fourth week of PSO in those breakthrough cases. Figure 2B showed that the overall trend of IgG levels decreased, and IgA antibody levels fluctuated at low levels throughout the study period in the post-immune population. Therefore, our results were relatively stable when extrapolating infection history from a month ago, and this time is valuable and important for tracing the source in the epidemiological investigation.
We have now modified it in lines 319-331 of the manuscript.
4. Author calculated the proportion of IgG-recovery/IgG-acute ratios of four or more in the breakthrough cases with two blood collections in the acute and recovery phases and found that the proportion of ratios greater than four decreased as the level of IgG in the acute phase increased. However, considering that IgG antibodies in breakthrough cases rise much faster than in first-time infected patients, using 4-fold as a criterion may not be appropriate.
Response: Thank you for the suggestion. When the population were with no history of SARS-CoV-2 immunization, a 4-fold or higher increase in IgG antibody titers in the recovery phase compared to the acute phase was a criterion based on the Diagnostic and Treatment Guidelines for Novel Coronavirus Infection (Trial Tenth Edition). It is worth investigating whether this general rule holds true after a history of inactivated vaccines. Therefore, this study validated the criterion by collecting serum samples from 46 individuals who had samples from both the acute and recovery phases, and found that 50% of the subjects met the guideline's criterion. Of course, it would make more sense to work out a more accurate fold as a criterion for the inactivated vaccine population. This will require more samples and more in-depth research.
We have now modified it in lines 332-344 of the manuscript.
5. In the article the authors tried to use the level of IgG or IgA antibody values to infer the different infection stages infected cases were in, but this inference could lead to errors due to the big individual differences.
Response: Thank you for your question. In our study, by univariate and multivariate regression analysis, based on factors such as age, sex and vaccination history, we found that IgG and IgA antibody levels in Omicron breakthrough cases were mainly affected by the weeks of symptom onset. These were analyzed from the perspective of population, and caution should be exercised when applied to specific individuals due to the individual differences. We have now modified it in lines 353-355 of the manuscript.
Minor comments: 1. In line 52, please explain whether the antibody level is the arithmetic mean or median?
Response: After verification, the mentioned antibody levels in the manuscript refer to arithmetic means.
2. In line 174, how many post-immune individuals received at least one booster immunization and what is the impact on the result. Table 1, all post-immune naïve individuals received two doses of inactivated vaccine, and no booster immunizations were administered.