Efficacy of the neutralizing antibodies after the booster dose on SARS-CoV-2 Omicron variant and a two-year longitudinal antibody study on Wild Type convalescents

Objectives Waning vaccine-induced immunity and emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants which may lead to immune escape, pose a major threat to the COVID-19 pandemic. Currently, enhanced efficacy of the neutralization antibodies (NAb) produced after the booster dose of vaccinations against the Omicron variant is the main focus of vaccine strategy research. In this study we have analyzed the potency of the NAbs and IgGs produced after the third vaccine dose in patients infected with Omicron variant and wild-type (WT) SARS-CoV-2. Methods We enrolled 75 patients with Omicron variant breakthrough infections, and 87 patients with WT infections. We recorded the clinical characteristics and vaccination information of all patients and measured the NAb and anti-S1 (spike protein) + N (nucleocapsid protein) IgG-binding antibodies against SARS-CoV-2 in serum samples of Omicron variant-infected patients at admission, and patients with WT COVID-19 infection from the time of admission and discharge, and one-year to two-years follow-ups. Results Our results demonstrated higher NAb levels, fewer clinical symptoms, and faster viral shedding in Omicron variant infected patients vaccinated with the booster dose. Hybrid immunity (natural infection plus vaccination) induces higher NAb levels than vaccine-only immunity. NAb and IgG levels decreased significantly at one-year follow-up in WT convalescents with natural infection. The NAb and IgG levels in booster-vaccinated COVID-19 patients were higher than those in two-dose-vaccinated patients. Conclusion Our results suggest that booster vaccinations are required to improve the level of protective NAbs. Moreover, our data provide important evidence for vaccination strategies based on existing vaccines.

Currently, vaccination remains the only effective way to control SARS-CoV-2 transmission and the adverse effects of COVID-19 [6]. However, whether vaccination can maintain the immune memory to protect healthy individuals from severe diseases caused by the new SARS-CoV-2 variants is has become an important center of scientific discussion. Several studies have indicated that vaccination can only confer 6-8 months of protective immunity against severe disease and death [7,8]. Numerous studies on COVID-19 vaccines and NAb activity have shown that vaccine-induced NAb titers against Omicron are low and decrease over time. A major cause of breakthrough infections may be prolonged duration between vaccine doses especially when antibody titers are waning and reduced recognition efficacy of vaccine-induced NAb due to the Omicron antigenic mutations [9]. The Omicron variant has been reported to exhibits significant immune evasion compared to other variants, but antibody neutralization is largely restored by vaccine booster doses, protecting against severe COVID-19. Therefore, measures to induce higher levels of NAb are of great significance.
In this study, we assessed the efficacy of booster vaccination against the SARS-CoV-2 Omicron variant and a two-year longitudinal antibody study against wild-type (WT) convalescents, providing important evidence for vaccination strategies based on existing vaccines.

Participants
We enrolled 301 patients with confirmed COVID-19 based on SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) test of nasopharyngeal, oropharyngeal, or sputum specimens, who were hospitalized at Taizhou Public Health Medical Center from January 17, 2020 to March 10, 2020, and from March 29, 2022 to April 18, 2022.
Out of these, we excluded 139 patients (82/157 Omicron variant and 57/144 wt cohorts, respectively) with incomplete data. Finally, 162 patients (75 with the omicron variant and 87 with the WT) were included in this study (Fig. 1).
We collected patients' information about demographics, clinical symptoms, comorbidities, radiologic characteristics, laboratory data, days since first symptom onset to admission and discharge, vaccination time, vaccination doses, manufacturer, and sampling time (Fig. 2). All patients with WT and Omicron variant had received two doses (28 days apart) Sinovac CoronaVac COVID-19 vaccine or the Sinopharm's Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV) of 4 μg dose of β-propiolactone-inactivated, aluminum hydroxideadjuvanted COVID-19 vaccine, followed by a third booster dose 7 months after the two initial doses. The vaccine is a 0.5 mL dose containing 600 SU of inactivated SARS-CoV-2 virus. To prepare the vaccine, SARS-CoV-2 (CN02 strain) was propagated in African green monkey kidney cells (WHO Vero 10-87 Cells).
Moreover, 73 and 58 patients with WT participated in the one-year and two-year follow-ups respectively. Patients with Omicron were infected for the first time and uninfected with other SARS-CoV-2 strains. Similarly, patients with WT were uninfected with other SARS-CoV-2 strains during the two-year follow-up period. The onset date was defined as the day on which the patient developed any symptoms. This study has been approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province (K20220133).

Antibody analyses
All patients were assessed using a surrogate virus neutralization test (sVNT) with a receptor-binding domain (RBD)-neutralizing antibody competition assay. The neutralizing capacity was estimated by performing the sVNT, which allows the indirect detection of potential SARS-CoV-2 NAb in the serum by blocking the binding of SARS-CoV-2 RBD (of the WT strain) to the human host receptor angiotensinconverting enzyme 2 (ACE2). NAb was measured with CLIA kits (Caris 200, Wantai, Xiamen, China) using the competitive chemiluminescence method on a Caris 200 automated magnetic CLIA analyzer from Wantai Biotech Co., ltd (Wantai, Xiamen, China). The NAb in the sample and biotinylated SARS-CoV-2 specific antibody competed with the acridine ester-labeled S protein. Streptavidin-coated magnetic particles were then added to interact with biotin, forming a complex consisting of magnetic particles coated with streptavidin, biotinylated SARS-CoV-2 specific antibody, and acridine ester-labeled S protein; the complex obtained after washing and removing substances that did not bind to the magnetic particles. The NAb concentration in the sample was inversely proportional to the instrumentally detected Relative Luminescence Unit (RLU). The cutoff value of NAb was 0.1 μg/mL. According to the assay specifications from the Wantai manufacturer, it shows excellent sensitivity (100 %), specificity (99.33 %), and correlation (R = 0.9042) compared to the pseudovirus neutralization test (pVNT) ( Figure S1).
Serum anti-S1 (spike protein) + N (nucleocapsid protein) IgGbinding antibodies against SARS-CoV-2 were measured using a chemiluminescence immunoassay (CLIA) kit (iFLASH3000, YHLO, Shenzhen, China). The recombinant N and S1 proteins of SARS-CoV-2 were coated on magnetic beads with an acridine ester-labeled mouse anti-human IgG antibody as the detection antibody. The concentration of anti-S1 + N IgG antibodies positively correlated with the RLU. The cut-off value for anti-S1 + N IgG antibodies was 10 AU/mL. According to the assay specifications from the manufacturer, the sensitivity and specificity of anti-S1 + N IgG-binding antibodies were 98.5 % and 100 %, respectively.

Flow cytometry analysis
The lymphocyte subsets and cytokines (interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ) were determined using the flow cytometer (FACS Canto TM II, BD, New Jersey, USA). Lymphocyte subset measurement: Fluorescent-labeled monoclonal antibodies bound to lymphocyte surface antigens and lymphocyte subsets were distinguished by flow cytometry based on the expression of cluster of differentiation (CD) molecules on the surface. The percentage of lymphocyte subsets was computed using BD FACSCanto Clinical Software and BD FACSDiva Software.
Cytokine detection: There were six types of captured microspheres in the kit mixture (Nuode, Jiangxi, China). Microspheres coated with cytokine-specific antibodies explicitly bound to cytokines in the patient samples. Phycoerythrin (PE) then marked the antibody, captured the microspheres and cytokines in the sample, forming a double antibody sandwich composite whose fluorescence intensity was analyzed to measure the cytokine concentration in the sample.

Statistical analysis
Categorical variables were described as frequency and percentage, and continuous variables were shown as mean and standard deviation or median and interquartile range (IQR), as appropriate. The categorical variables were compared using the χ 2 test and Fisher's exact test as appropriate. To compare continuous variables among data from different groups, the Mann-Whitney U test was performed when the data were non-normally distributed; otherwise, an independent t-test was used when the data were normally distributed. The Spearman's correlation coefficient was used. Statistical analyses were performed using GraphPad Prism (version 8.0) and R software (version 3.6.0). Statistical significance was set at P < 0.05, P < 0.01, and P < 0.001, as indicated by asterisks.

Clinical characteristics of patients with Omicron variant and WT SARS-CoV-2
In Omicron variant cohort, the median age was 33.0 (range, 27.5-39.0) years and 56.0 % were male. Regarding vaccination status of the patients, two (2.7 %) were not vaccinated, two (2.7 %) were vaccinated with one dose, 27 (36.0 %) were vaccinated with two doses, and 44 (58.7 %) had received booster vaccination (Table S1). Among the patients previously infected with WT, the median age was 47.0 (range, 38.5-55.5) years and 51.7 % were male. At the two-year follow-up, nine (10.3 %) patients were unvaccinated, 12 (13.8 %) were vaccinated with one dose, 57 (65.5 %) were vaccinated with two doses, and only nine (10.3 %) had received a booster dose (Table S1). Compared to patients with WT COVID-19, we observed a higher incidence of sore throat (42.7 vs 9.2 %) and nasal congestion (22.7 vs 4.6 %) in patients infected with Omicron-variant COVID-19. Patients infected with WT exhibited a higher incidence of fever (77.0 vs 50.7 %), patchy and ground-glass opacity (93.1 vs 27.9 %) compared to the Omicron-variant infected patients. Upon admission, the lymphocyte subsets CD3 + T cells and CD8 + T cells in the Omicron variant cohort were higher than those in the WT cohort, whereas cytokine levels were significantly lower. Our data showed that lung computed tomography (CT) abnormalities were rare (27.9 %) in the patients with Omicron-variant COVID-19 (Table 1).

The NAb levels are influenced by the number of clinical symptoms and duration of viral shedding in patients with Omicron variant COVID-19
We used chemiluminescence immunoassays (CLIAs) to assess the expression levels of NAb and IgG, and acquired electronic hospital records (EHRs) of the enrolled patients. The Omicron variant cohort was divided into two groups based on median symptom = 3, at the time of admission, with 0-2 symptoms in the "Few group" and 3-7 symptoms in the "More group". NAb and IgG levels (all P < 0.05) were higher in the Few group than in the More group. To investigate the differences between the two groups, we compared immune cells on admission. We found that the numbers of CD3 + , CD4 + and CD8 + T cells (all P < 0.05) were higher in the few-symptom group. Among these, CD19, CD16, and CD56 cells showed no statistical significance (Fig. 3A). Thus, the NAb and IgG levels positively correlated with lymphocyte subsets and negatively correlated with cytokine levels (Fig. 3B). The median duration of viral shedding for the Omicron variant was 14 days. We also observed that among the 44 Omicron variant patients who were vaccinated with three doses, NAb (P = 0.003) and IgG (P = 0.009) levels were higher in patients with a viral shedding duration of < 14 days (Fig. 3C). However, there was no difference between NAb and IgG levels and viral shedding duration in the 27 Omicron variant patients (P greater than 0.05) who were vaccinated with two doses (Fig. 3D). Furthermore, the duration of viral shedding was negatively correlated with NAb (P = 0.004) and IgG (P = 0.005) levels in 44 Omicron variant patients vaccinated with the three doses (Fig. 3E). Nevertheless, in patients infected with the Omicron variant, who were vaccinated with the two doses, there was no correlation between NAb and IgG levels, and the duration of viral shedding.
In summary, Omicron variant patients with higher NAb and IgG levels had mild clinical symptoms and those who received a booster vaccination had fast viral shedding. These results suggest that booster vaccinations can reduce symptoms and promote rapid viral clearance in patients harboring the Omicron variant.

The levels of NAb induced by inactivated vaccines in patients infected with Omicron variant and WT SARS-CoV-2
We measured the level of NAb in 75 Omicron variant COVID-19 patients at admission, which was induced by vaccination. In addition, we tested NAb and lymphocyte subsets among WT COVID-19 patients: 87 patients at admission and discharge, 73 patients at one-year followup, and 58 patients at two-year follow-up. Patients with Omicron-  variant were vaccinated with an inactivated vaccine against the WT in advance, which induced cross-protection against the Omicron variant. Compared with Omicron variant COVID-19 patients at admission, convalescent WT COVID-19 at two-year follow-up exhibited higher NAb levels. However, the numbers of CD3 + , CD4 + and CD8 + T cells were not significantly different (Fig. 4A). Fifty-seven (65.5 %) WT patients had received two doses of the vaccine between one-year follow-up and two-year follow-up, while 27 (36.0 %) Omicron variant patients had received two doses before admission. To analyze the effect of vaccine dose on NAb levels, we included 27 Omicron variants and 57 wt patients who had received two doses of the vaccine. The results showed that patients with Omicronvariant COVID-19 had lower NAb levels at admission than those with WT COVID-19 at two-year follow-up. Similarly, there were no significant differences in CD3 + , CD4 + and CD8 + T cells between Omicronvariant patients and WT convalescent patients (Fig. 4B). In addition, We compared the levels of NAb after same dose and same time from last vaccination in Omicron variant and WT infected COVID-19 patients. The NAb levels at 6 to 8 months after two doses of vaccine were lower in 8 patients with Omicron variant infection at admission than in 21 patients with WT infection at two-year follow-up (Fig. S2C). Similarly, the levels of NAb at 2 months after three doses of vaccine in 8 patients with Omicron variant infection at admission were lower than in 5 patients with WT infection at two-year follow-up (Fig. S2D).

A
Overall, hybrid immunity (natural infection plus vaccination) in WT convalescent patients induced higher NAb levels than vaccine-only immunity in Omicron-variant patients, which may be due to the existence of an immune memory response in WT patients.

Booster vaccination enhances NAb levels in patients with Omicron variant and WT COVID-19
We assessed the impact of booster vaccination on NAb and IgG levels, and found that the 44 Omicron variant patients vaccinated with three doses had higher NAb and IgG levels than the 27 Omicron-variant patients who received two doses of vaccination (Fig. 5A).
For patients with WT after natural infection, NAb and IgG levels peaked at discharge and decreased significantly at one-year follow-up. At the two-year follow-up, nine individuals were unvaccinated, 57 had received two doses, and nine had received a booster vaccination. For the unvaccinated WT individuals at two-year follow-up, their NAb levels were further reduced to below the reference lower limit, whereas their IgG levels remained close to the reference lower limit. NAb and IgG levels were higher in patients who received a booster vaccination than that for those who received two doses at two-year follow-up (Fig. 5B).
In WT patients with natural infection, NAb and IgG levels decreased significantly at one-year follow-up. Moreover, the levels of NAb and IgG in those who received three doses of the vaccination were much higher than those in the two-dose vaccinated population. For those suitable for vaccination, booster vaccinations are recommended to maintain a longterm immune memory response.

Discussion
This study analyzed the NAb dynamics of patients infected with WT virus after natural infection and with vaccination, from hospital admission and discharge, and one year to two years of follow-up and showed that the hybrid immunity produced by natural infection plus booster vaccination provided stronger protection. In addition, booster vaccinations can reduce symptoms and promote rapid viral clearance in patients harboring the Omicron variant. Therefore, booster vaccination is needed to improve the protective NAb levels. Our data provide important evidence for vaccination strategies based on the existing vaccines.
According to the present study, the higher the levels of NAb and IgG in Omicron variant patients, the fewer the clinical symptoms and faster the viral shedding. This is related to the protective effects of the booster vaccination. Our findings are in line with the results of a multicenter cohort study in Switzerland, which reported that hybrid immunity (infection plus vaccination) and receipt of booster vaccination resulted in fewer symptoms (median, 3) than unvaccinated participants (median, 4) [10]. It has been reported that lower NAb levels are associated with an increased risk of SARS-CoV-2 infection and a higher disease burden [11]. The results of a Brazilian real-world study of over 2.67 million subjects showed that the effectiveness of booster vaccination in preventing severe illness increased to 66.9 % compared with those who received only two doses of the CoronaVac vaccine [12]. This is because the distribution frequency of receptor-binding domain (RBD)-specific memory B cells increases significantly after booster vaccination and new memory B cell clones are induced [13]. A study also showed that three doses of the vaccine significantly reduced the viral load after Omicron variant infection [14]. This is the protective efficacy of booster vaccination, which shortens the length of hospitalization and quarantine and reduces the pressure on the country's medical resources and economy.
In the present study, the longitudinal part of the observations with a two-year follow-up of WT COVID-19 patients, showed that hybrid immunity (natural infection plus vaccination) in WT convalescents induced higher NAb levels than vaccine-only immunity in Omicronvariant COVID-19 patients, which may be due to the existence of an immune memory response in WT patients. This is consistent with the results of Vacharathi et al., who showed that the NAb levels of the CoronaVac vaccine in Omicron variant patients were lower than those of WT COVID-19 convalescents [15]. A history of infection was associated with high NAb levels in the vaccinated individuals [16]. A study by Université Paris Cité found that only 30 % of the IgG secreted by SARS-CoV-2-specific memory B cells had affinity for the Omicron variant RBD, but the protection against the variant was not completely lost [17]. In addition, we found that T cell levels in Omicron-variant patients were similar to those in WT convalescent patients. T cells, which are the arms of the adaptive immune response, can augment protection against Omicron infection. T cell responses to Omicron are preserved in most infected and vaccinated individuals and are enhanced shortly after booster vaccination [18]. Therefore, for those suitable for vaccination, an entire course of vaccine and booster vaccinations should be advocated.
Our data showed that NAb and IgG levels were significantly reduced at one-year follow-up in WT patients with natural infection. A modeling study also predicts that with the decline in NAb levels, immune protection from the infection may weaken over time and that booster vaccination may be required within a year [11]. However, it is encouraging to know that more and more evidence exhibited that three doses of heterologous or homologous booster vaccination had a 25-100fold increase in NAb titers compared to two doses of vaccination [16,19]. Liu et al. also confirmed that three doses of the vaccine provided good protection against the Omicron variant, a strain with strong immune escape, compared to two doses [20]. Our study also showed that the NAb level after the booster vaccination was higher than that of the two doses of the vaccine in Omicron variant patients and WT convalescents. Our results are comparable to those of studies with the same inactivated vaccine; a booster inactivated vaccine elevated NAb titers and provided increased protection against Omicron [21]. Nussenzweig revealed that antibodies secreted by B cells after the third dose of mRNA vaccine results in increase in, and evolution of, anti-receptor binding domain specific memory B cells. This significantly increased potency and breadth of the antibodies is especially due to the newly induced B cell clones following the booster vaccination, and the titer and activity of NAb secreted by these B cells increased significantly. After booster vaccination, more than 50 % of the NAb secreted by the memory B cells neutralized Omicron [13]. This explains why booster vaccinations can provide protection against Omicron variant infections.
This study has several limitations. This was a small-sample singlecenter cohort study, and in the near future, more multicenter clinical studies are needed to evaluate the immune protection efficacy of the vaccine against Omicron variant. Further analysis of the vaccine protection mechanism against the Omicron variant is required.

Conclusions
Taken together, our data demonstrates that a third dose of booster vaccination is necessary to improve protective levels of NAbs and provide an important basis for the immunization strategy of existing vaccines.

Data availability
Data will be made available on request.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.