Immunogenicity Following Two Doses of the BBIBP-CorV Vaccine and a Third Booster Dose with a Viral Vector and mRNA COVID-19 Vaccines against Delta and Omicron Variants in Prime Immunized Adults with Two Doses of the BBIBP-CorV Vaccine

Coronavirus disease 2019 (COVID-19) booster vaccination is being comprehensively evaluated globally due to waning immunity and the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Therefore, this study aimed to evaluate antibody responses in individuals vaccinated with two doses of the BBIBP-CorV vaccine and to explore the boosting effect of the different vaccine platforms in BBIBP-CorV-primed healthy adults, including a viral vector vaccine (AZD122) and mRNA vaccines (BNT162b2 and mRNA-1273). The results showed that in the BBIBP-CorV prime group, the total receptor-binding domain (RBD) immunoglobulin (Ig) and anti-RBD IgG levels waned significantly at three months after receiving the second dose. However, after the booster, RBD-specific binding antibody levels increased. Neutralizing antibody measured by a surrogate neutralization test showed inhibition over 90% against the SARS-CoV-2 delta variant but less than 70% against the omicron variant after the third dose on day 28. All booster vaccines could induce the total IFN-ɣ T-cell response. The reactogenicity was acceptable and well-tolerated without serious adverse events. This study supports the administration of the third dose with either a viral vector or mRNA vaccine for BBIBP-CorV-primed individuals to stimulate antibody and T-cell responses.

The participants were informed of the name of the vaccine and were observed adverse events for at least 30 min after vaccination. All participants were continuou monitored for adverse events after immunization (AEFI) for seven days after receiv the booster dose using self-assessment records via online or paper questionnaires. adverse events included system reactions and local reactions. System reactions inclu fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding dom immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminesce Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the che luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland) spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-R IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum sam from the third-booster group at all time points was also evaluated for neutralizing acti against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sV based on antibody-mediated blockage of the interaction between the viral receptor bi ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furt more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, cataway, NJ, USA) was used for all strains following the manufacturer's instructions. combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (contain N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum inhibit binding between RBD and ACE2 was calculated as a percentage as follows: (average OD of the sample/average OD of the negative control) × 100. A value ≥30% considered positive, indicating the presence of neutralizing antibodies. The lower lim detection was established as 0% inhibition.

IFNɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-boo group was taken at all time points on days 0, 14, 28, and 90 to assess the specific Tresponse. The heparinized whole blood sample was collected in a blood collection t from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) follow the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then cen fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stim lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combinatio specific SARS-CoV-2 antigens covering the S protein, followed by measurement in plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube contain CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube c taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by us the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated us IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ leve ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2

Introduction
The coronavirus disease 2019  pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It can result in mild to severe illness leading to hospitalization and death [1]. Currently available COVID-19 vaccines have been effective for the control and prevention of COVID-19 outbreaks. During this pandemic, many COVID-19 vaccines have been approved by the World Health Organization (WHO)
The virion is produced in genetically modified HEK293 cells. One dose (0.5 mL) contains 5 × 10 10 infectious units [23]. BNT162b2 (Pfizer-BioNTech Inc., New York, NY, USA) and mRNA-1273 (ModernaTX Inc., Cambridge, MA, USA) are modified RNA containing lipid nanoparticles encoding the full-length spike of SARS-CoV-2, modified by two proline mutations to lock it in the prefusion conformation. One dose contains 30 and 100 µg for BNT162b2 and mRNA-1273, respectively [24,25]. After receiving the additional dose, all study participants were monitored for immunogenicity and safety.

Monitoring of Adverse Events
The participants were informed of the name of the vaccine and were observed for adverse events for at least 30 min after vaccination. All participants were continuously monitored for adverse events after immunization (AEFI) for seven days after receiving the booster dose using self-assessment records via online or paper questionnaires. The adverse events included system reactions and local reactions. System reactions included Vaccines 2022, 10, 1071 4 of 13 fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. Local reactions included pain at the injection site, swelling, and redness.

Laboratory Experiments 2.4.1. Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding domain immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminescence Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the chemiluminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland), respectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-RBD IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum samples from the third-booster group at all time points was also evaluated for neutralizing activity against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) and B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of the interaction between the viral receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFN-
The participants were informed of the name of the vaccine and were observed for adverse events for at least 30 min after vaccination. All participants were continuously monitored for adverse events after immunization (AEFI) for seven days after receiving the booster dose using self-assessment records via online or paper questionnaires. The adverse events included system reactions and local reactions. System reactions included fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. Local reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding domain immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminescence Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the chemiluminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland), respectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-RBD IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum samples from the third-booster group at all time points was also evaluated for neutralizing activity against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) and B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of the interaction between the viral receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFNɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centrifuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimulation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination of specific SARS-CoV-2 antigens covering the S protein, followed by measurement in the plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube containing CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube containing CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction was used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA and measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by using the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated using IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ level of ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2.

Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 • C for 21 to 24 h and was then centrifuged for 15 min at 3500× g to harvest the plasma. This test is based on the in vitro stimulation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination of specific SARS-CoV-2 antigens covering the S protein, followed by measurement in the plasma of IFN- The participants were informed of the name of the vaccine and were observed for adverse events for at least 30 min after vaccination. All participants were continuously monitored for adverse events after immunization (AEFI) for seven days after receiving the booster dose using self-assessment records via online or paper questionnaires. The adverse events included system reactions and local reactions. System reactions included fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. Local reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding domain immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminescence Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the chemiluminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland), respectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-RBD IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum samples from the third-booster group at all time points was also evaluated for neutralizing activity against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) and B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of the interaction between the viral receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centrifuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimulation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination of specific SARS-CoV-2 antigens covering the S protein, followed by measurement in the plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube containing CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube containing CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction was used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA and measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by using the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated using IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ level of ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2.

Statistical Analysis
production by ELISA. This assay consisted of the Ag1 tube containing CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube containing CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction was used to determine the concentration of IFN-Vaccines 2022, 10, x FOR PEER REVIEW The participants were informed of the name of the vaccine and were observ adverse events for at least 30 min after vaccination. All participants were continu monitored for adverse events after immunization (AEFI) for seven days after rece the booster dose using self-assessment records via online or paper questionnaires adverse events included system reactions and local reactions. System reactions inc fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizzines cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding do immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemilumines Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the c luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Irelan spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The ant IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum sa from the third-booster group at all time points was also evaluated for neutralizing ac against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (s based on antibody-mediated blockage of the interaction between the viral receptor ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Fu more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech cataway, NJ, USA) was used for all strains following the manufacturer's instruction combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (conta N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a ser inhibit binding between RBD and ACE2 was calculated as a percentage as follow (average OD of the sample/average OD of the negative control) × 100. A value ≥30% considered positive, indicating the presence of neutralizing antibodies. The lower li detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-b group was taken at all time points on days 0, 14, 28, and 90 to assess the specific response. The heparinized whole blood sample was collected in a blood collection from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) follo the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then c fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro s lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combinat specific SARS-CoV-2 antigens covering the S protein, followed by measurement plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube conta CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tub taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fractio used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ le ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV The participants were informed of the name of the vaccine and were obs adverse events for at least 30 min after vaccination. All participants were con monitored for adverse events after immunization (AEFI) for seven days after the booster dose using self-assessment records via online or paper questionn adverse events included system reactions and local reactions. System reactions fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizz cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor bindin immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemilum Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and t luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ire spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum from the third-booster group at all time points was also evaluated for neutralizin against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (d B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization te based on antibody-mediated blockage of the interaction between the viral rece ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27 more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Bio cataway, NJ, USA) was used for all strains following the manufacturer's instruc combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (c N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a inhibit binding between RBD and ACE2 was calculated as a percentage as fo (average OD of the sample/average OD of the negative control) × 100. A value considered positive, indicating the presence of neutralizing antibodies. The low detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the thir group was taken at all time points on days 0, 14, 28, and 90 to assess the spec response. The heparinized whole blood sample was collected in a blood colle from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was th fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vi lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a comb specific SARS-CoV-2 antigens covering the S protein, followed by measurem plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube c CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fra used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® E measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated the QuantiFERON R&D Analysis Software. The seropositivity rate was calcula IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-standard curve was calculated by using the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated using IFN- The subset of participants' w group was taken at all time point response. The heparinized whole from QuantiFERON (QFN) SARS-the manufacturer's instruction and fuged for 15 min at 3500 g to harv lation of CD4 + and CD8 + lymphoc specific SARS-CoV-2 antigens cov plasma of IFN-ɣ production by E CD4 + epitopes derived from the S1 taining CD4 + and CD8 + epitopes fr used to determine the concentrati measured the absorbance at 450 n the QuantiFERON R&D Analysis IFN-ɣ levels from the stimulated t ≥0.15 IU/mL and ≥25% of nil were levels from the stimulated tube minus the negative control tube. An IFN-Vaccines 2022, 10, x FOR PEER REVIEW The participants were informed of the nam adverse events for at least 30 min after vaccinat monitored for adverse events after immunizatio the booster dose using self-assessment records adverse events included system reactions and lo fever, headache, myalgia, nausea, vomiting, diar cal reactions included pain at the injection site, sw

Immunoglobulin Assays
All serum samples were analyzed using SA immunoglobulin (total RBD Ig) and anti-RBD IgG Immunoassay (ECLIA) (Roche Diagnostics Gmb luminescent microparticle immunoassay (CMIA spectively [26]. A total RBD Ig level of ≥0.8 U/mL IgG level with a value ≥7.1 BAU/mL was conside 2.4.2. Surrogate Virus Neutralization Test (sVNT To assess SARS-CoV-2 surrogate virus neu from the third-booster group at all time points wa against the SARS-CoV-2 variants of concern B.1.1.529 (omicron), using an ELISA-based sur based on antibody-mediated blockage of the inte ing domain (RBD) and the angiotensin-convertin more, a cPass TM SARS-CoV-2 neutralizing antibo cataway, NJ, USA) was used for all strains follow combinant RBDs from B.1.617.2 (containing L452 N501Y, E484A, K417N, and D614G) were also us inhibit binding between RBD and ACE2 was ca (average OD of the sample/average OD of the ne considered positive, indicating the presence of ne detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole hepariniz group was taken at all time points on days 0, 14 response. The heparinized whole blood sample from QuantiFERON (QFN) SARS-CoV-2 RUO kit the manufacturer's instruction and incubated at fuged for 15 min at 3500 g to harvest the plasma lation of CD4 + and CD8 + lymphocytes in whole h specific SARS-CoV-2 antigens covering the S pr plasma of IFN-ɣ production by ELISA. This ass CD4 + epitopes derived from the S1 subunit (RBD taining CD4 + and CD8 + epitopes from S1 and S2 o used to determine the concentration of IFN-ɣ (IU measured the absorbance at 450 nm. The IFN-ɣ the QuantiFERON R&D Analysis Software. The IFN-ɣ levels from the stimulated tube minus the level of ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2.

Statistical Analysis
G*power software version 3.1.9.6 was used for calculating the sample size. The Graph-Pad Prism version 7.0 for Microsoft Windows was used for performing the graphical presentation and statistical analyses. The chi-square test and Welch's ANOVA were performed for categorical analyses of age and sex. Antibody responses against SARS-CoV-2 were designated as geometric mean titers (GMT) with a 95% confidence interval (CI). Neutralization activities and SARS-CoV-2-stimulating IFN-Vaccines 2022, 10, x FOR PEER REVIEW The participants were informed of the name of th adverse events for at least 30 min after vaccination. A monitored for adverse events after immunization (AE the booster dose using self-assessment records via on adverse events included system reactions and local rea fever, headache, myalgia, nausea, vomiting, diarrhea, jo cal reactions included pain at the injection site, swelling

Immunoglobulin Assays
All serum samples were analyzed using SARS-Co immunoglobulin (total RBD Ig) and anti-RBD IgG by us Immunoassay (ECLIA) (Roche Diagnostics GmbH, Man luminescent microparticle immunoassay (CMIA) (Abb spectively [26]. A total RBD Ig level of ≥0.8 U/mL was c IgG level with a value ≥7.1 BAU/mL was considered po

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutraliz from the third-booster group at all time points was also against the SARS-CoV-2 variants of concern (VOC B.1.1.529 (omicron), using an ELISA-based surrogate based on antibody-mediated blockage of the interactio ing domain (RBD) and the angiotensin-converting enzy more, a cPass TM SARS-CoV-2 neutralizing antibody det cataway, NJ, USA) was used for all strains following th combinant RBDs from B.1.617.2 (containing L452R and N501Y, E484A, K417N, and D614G) were also used wit inhibit binding between RBD and ACE2 was calculate (average OD of the sample/average OD of the negative considered positive, indicating the presence of neutrali detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized bloo group was taken at all time points on days 0, 14, 28, a response. The heparinized whole blood sample was co from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAG the manufacturer's instruction and incubated at 37 °C f fuged for 15 min at 3500 g to harvest the plasma. This were presented as medians with interquartile ranges. The Kruskal-Wallis test or Wilcoxon signed rank test (nonparametric) Vaccines 2022, 10, 1071 5 of 13 multiple comparison adjustments were used for calculating the differences in antibody titers, percentage inhibition, and IU/mL minus nil between groups. A p-value < 0.05 was considered to reveal statistical significance.

Antibody Responses against SARS-CoV-2 in the BBIBP-CorV-Primed Group
The BBIBP-CorV-primed group consisted of 21 male participants and 30 female participants. The average and median ages were 37.7 and 35.0, respectively. Ten participants were lost to follow-up during the study. The enrollment flow diagram is shown in Figure 1.
ical presentation and statistical analyses. The chi-square test and Welch's ANOVA were performed for categorical analyses of age and sex. Antibody responses against SARS-CoV-2 were designated as geometric mean titers (GMT) with a 95% confidence interval (CI). Neutralization activities and SARS-CoV-2-stimulating IFN-ɣ were presented as medians with interquartile ranges. The Kruskal-Wallis test or Wilcoxon signed rank test (nonparametric) multiple comparison adjustments were used for calculating the differences in antibody titers, percentage inhibition, and IU/mL minus nil between groups. A p-value < 0.05 was considered to reveal statistical significance.

Antibody Responses against SARS-CoV-2 in the BBIBP-CorV-Primed Group
The BBIBP-CorV-primed group consisted of 21 male participants and 30 female participants. The average and median ages were 37.7 and 35.0, respectively. Ten participants were lost to follow-up during the study. The enrollment flow diagram is shown in Figure  1. Total levels of RBD Ig and anti-RBD IgG at 30 days after two-dose vaccination in the BBIBP-CorV-primed group showed seropositivity rates of 97.8% and 93.6%, respectively. The maximum level of total RBD Ig and anti-RBD IgG was detected in all participants after completing two doses of BBIBP-CorV at 30 days, with GMT 42.8 U/mL and 50.0 BAU/mL, respectively. The waning of antibody titers was observed after 90 days of completed vaccination, as shown in Figure 2 and Supplementary Table S1. Total levels of RBD Ig and anti-RBD IgG at 30 days after two-dose vaccination in the BBIBP-CorV-primed group showed seropositivity rates of 97.8% and 93.6%, respectively. The maximum level of total RBD Ig and anti-RBD IgG was detected in all participants after completing two doses of BBIBP-CorV at 30 days, with GMT 42.8 U/mL and 50.0 BAU/mL, respectively. The waning of antibody titers was observed after 90 days of completed vaccination, as shown in Figure 2 and Supplementary Table S1.

Antibody Responses against SARS-CoV-2 in the Third Dose with a Viral Vector or mRNA COVID-19 Vaccine Group
In the third-booster group, healthy participants who received two doses of BBIBP-CorV over 6 ± 1 months were separated into three groups to receive the third dose with a viral vector or mRNA COVID-19 vaccine depending on vaccine availability. The enrolled participants had no underlying disease or had well-controlled disease, were not immunocompromised, or were receiving treatment with ongoing immunosuppressive therapy. The demographic characteristics of all participants, namely those who received vaccines AZD1222 (n = 33, median age = 45.0 years), BNT162b2 (n = 56, median age = 40.0 years), and mRNA-1273 (n = 55, median age = 46.0 years) vaccines; the average age of the participants enrolled in each group was 42.27, 41.89, and 44.02 years, respectively. There were no significant differences in sex and age in each group. The enrollment flow diagram is shown in Figure 1.

Antibody Responses against SARS-CoV-2 in the third dose with a viral vector or mRNA COVID-19 vaccine group
In the third-booster group, healthy participants who received two doses of BBIBP-CorV over 6 ± 1 months were separated into three groups to receive the third dose with a viral vector or mRNA COVID-19 vaccine depending on vaccine availability. The enrolled participants had no underlying disease or had well-controlled disease, were not immunocompromised, or were receiving treatment with ongoing immunosuppressive therapy. The demographic characteristics of all participants, namely those who received vaccines AZD1222 (n = 33, median age = 45.0 years), BNT162b2 (n = 56, median age = 40.0 years), and mRNA-1273 (n = 55, median age = 46.0 years) vaccines; the average age of the participants enrolled in each group was 42.27, 41.89, and 44.02 years, respectively. There were no significant differences in sex and age in each group. The enrollment flow diagram is shown in Figure 1.
The most common symptom induced by the third booster dose of the COVID-19 vaccine in three groups was injection-site pain. The requested injection site and systemic adverse reactions were reported. The most common adverse effects at the injection site and systemic adverse reactions were myalgias, headache, and chilling. The incidence of total local and systemic symptoms in the AZD1222, BNT162b2, and mRNA-1273 groups are shown in Figure 3 and Supplementary Table S1. The third dose of AZD1222 had a significantly lower incidence of swelling than BNT162b2 and the mRNA-1273 vaccine did. Fever symptoms had a higher incidence, but the remaining differences were not significant. Any adverse reactions resolved within a few days. After vaccination, no severe adverse events (SAEs) were observed in all third-booster subgroups. The most common symptom induced by the third booster dose of the COVID-19 vaccine in three groups was injection-site pain. The requested injection site and systemic adverse reactions were reported. The most common adverse effects at the injection site and systemic adverse reactions were myalgias, headache, and chilling. The incidence of total local and systemic symptoms in the AZD1222, BNT162b2, and mRNA-1273 groups are shown in Figure 3 and Supplementary Table S1. The third dose of AZD1222 had a significantly lower incidence of swelling than BNT162b2 and the mRNA-1273 vaccine did. Fever symptoms had a higher incidence, but the remaining differences were not significant. Any adverse reactions resolved within a few days. After vaccination, no severe adverse events (SAEs) were observed in all third-booster subgroups.
The antibody level before the booster in individuals from all third-booster subgroups showed that the total RBD Ig and anti-RBD IgG levels at baseline were maintained at a low level, similar to those in the BBIBP-CorV-primed group. After receiving the third dose, there was a substantial increase in the highest titers on day 14 and a slight decrease up to day 90, as shown in Figure 4 and Supplementary Table S1. Compared with the third-booster group, AZD1222 achieved total RBD Ig and anti-RBD IgG levels that were significantly lower than those achieved with BNT162b2 or mRNA-1273.

Surrogate Virus Neutralization Test
The functionally binding antibody that may inhibit SARS-CoV-2 delta and omicron variants was evaluated by sVNT. According to Figure 5 and Supplementary Table S1, before the third dose vaccination, the median percentage of inhibition of participants who received BBIBP-CorV was less than 30.0% for the delta and omicron variants. In the comparison of neutralizing activities between the different third-dose platforms, the highest percentage of inhibition against delta and omicron variants was achieved after the booster on day 14. The median of neutralizing activities against the delta variant showed a 97.0% inhibition in the vaccination with the three vaccines studied. The median neutralizing activities against the omicron variant of the third dose with AZD1222, BNT162b2, and mRNA-1273 were 49.8%, 67.4%, and 53.7%, respectively. On day 28, the percentage of inhibition against omicron variants decreased slightly to less than 30.0% on day 90, while the percentage of inhibition against delta variants was still higher than 90%. The neutralizing capacity of sera against the omicron variant in all third-dose groups was lower than that of the delta variant. In addition, recipients of the third dose of the AZD1222 vaccine had no significant difference in neutralizing activity against the delta variant compared with that of BNT162b2 and The antibody level before the booster in individuals from all third-booster s

Surrogate Virus Neutralization Test
The functionally binding antibody that may inhibit SARS-CoV-2 delta and omicron variants was evaluated by sVNT. According to Figure 5 and Supplementary Table S1, before the third dose vaccination, the median percentage of inhibition of participants who received BBIBP-CorV was less than 30.0% for the delta and omicron variants. In the comparison of neutralizing activities between the different third-dose platforms, the highest percentage of inhibition against delta and omicron variants was achieved after the booster on day 14. The median of neutralizing activities against the delta variant showed a 97.0% inhibition in the vaccination with the three vaccines studied. The median neutralizing activities against the omicron variant of the third dose with AZD1222, BNT162b2, and mRNA-1273 were 49.8%, 67.4%, and 53.7%, respectively. On day 28, the percentage of inhibition against omicron variants decreased slightly to less than 30.0% on day 90, while the percentage of inhibition against delta variants was still higher than 90%. The neutralizing capacity of sera against the omicron variant in all third-dose groups was lower than that of the delta variant. In addition, recipients of the third dose of the AZD1222 vaccine had no significant difference in neutralizing activity against the delta variant compared with that of BNT162b2 and mRNA-1273 vaccine recipients but had significantly lower neutralizing activity against the omicron variant (p-value < 0.01).
A correlation was plotted between the percentage of inhibition against the SARS-CoV-2 variants, total-RBD Ig, and anti-RBD IgG. Nonlinear regression, a one-phase decay model, could predict immunogenicity titers to neutralize inhibition against delta and omicron variants in the third dose with the different platforms (Supplementary Figures S1 and S2).

SARS-CoV-2 Stimulating IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ T-cell Response
The IFN-ɣ responses to SARS-CoV-2 IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ were evaluated, as shown in Figure 6 and Supplementary Table S1. Interestingly, potential antiviral-induced IFN-ɣ stimulation of T-cells was notably observed. The marked individual difference in terms of IFN-ɣ release induced by IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ ranged from 0.0 to 10.0 IU/mL. In terms of the serological response, the seropositivity rates for the IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ levels observed in most participants after the booster dose 14 days were 67.9%/82.1% for AZD1222, 83.3%/93.3% for BNT162b2, and 86.2%/93.1% for mRNA-1273. The production of IFN-ɣ by T-cells was decreased on days 28 and 90, consistent with the total RBD Ig and anti-RBD IgG levels. All booster vaccines showed that humoral immunity also developed cellular immunity. In contrast, both A correlation was plotted between the percentage of inhibition against the SARS-CoV-2 variants, total-RBD Ig, and anti-RBD IgG. Nonlinear regression, a one-phase decay model, could predict immunogenicity titers to neutralize inhibition against delta and omicron variants in the third dose with the different platforms ( Supplementary Figures S1 and S2).

SARS-CoV-2 Stimulating IFN-
The participants were informed of the name of the vaccine and were o adverse events for at least 30 min after vaccination. All participants were c monitored for adverse events after immunization (AEFI) for seven days aft the booster dose using self-assessment records via online or paper questio adverse events included system reactions and local reactions. System reactio fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and d cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor bind immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemilu Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) an luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. T IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of ser from the third-booster group at all time points was also evaluated for neutrali against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization based on antibody-mediated blockage of the interaction between the viral re ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [ more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript cataway, NJ, USA) was used for all strains following the manufacturer's instr combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 N501Y, E484A, K417N, and D614G) were also used with this kit. The ability o inhibit binding between RBD and ACE2 was calculated as a percentage as (average OD of the sample/average OD of the negative control) × 100. A valu considered positive, indicating the presence of neutralizing antibodies. The lo detection was established as 0% inhibition.

IFNɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the t group was taken at all time points on days 0, 14, 28, and 90 to assess the sp response. The heparinized whole blood sample was collected in a blood co from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, German the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a com The participants were informed of the name of the vaccin adverse events for at least 30 min after vaccination. All partici monitored for adverse events after immunization (AEFI) for se the booster dose using self-assessment records via online or p adverse events included system reactions and local reactions. S fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, cal reactions included pain at the injection site, swelling, and red

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total immunoglobulin (total RBD Ig) and anti-RBD IgG by using the E Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, G luminescent microparticle immunoassay (CMIA) (Abbott Diagn spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considere IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a s from the third-booster group at all time points was also evaluate against the SARS-CoV-2 variants of concern (VOCs), name B.1.1.529 (omicron), using an ELISA-based surrogate virus ne based on antibody-mediated blockage of the interaction betwee ing domain (RBD) and the angiotensin-converting enzyme 2 (AC more, a cPass TM SARS-CoV-2 neutralizing antibody detection ki cataway, NJ, USA) was used for all strains following the manufa combinant RBDs from B.1.617.2 (containing L452R and T478K) N501Y, E484A, K417N, and D614G) were also used with this kit inhibit binding between RBD and ACE2 was calculated as a p (average OD of the sample/average OD of the negative control) considered positive, indicating the presence of neutralizing anti detection was established as 0% inhibition.

IFNɣ Releasing Assay
The subset of participants' whole heparinized blood sampl group was taken at all time points on days 0, 14, 28, and 90 to response. The heparinized whole blood sample was collected i from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hil the manufacturer's instruction and incubated at 37 °C for 21 to fuged for 15 min at 3500 g to harvest the plasma. This test is bas lation of CD4 + and CD8 + lymphocytes in whole heparinized blo The participants were informed of the name of the vaccine and were observed for adverse events for at least 30 min after vaccination. All participants were continuously monitored for adverse events after immunization (AEFI) for seven days after receiving the booster dose using self-assessment records via online or paper questionnaires. The adverse events included system reactions and local reactions. System reactions included fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. Local reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding domain immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminescence Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the chemiluminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland), respectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-RBD IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum samples from the third-booster group at all time points was also evaluated for neutralizing activity against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) and B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of the interaction between the viral receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centrifuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimu- The participants were informed of the name of the vac adverse events for at least 30 min after vaccination. All par monitored for adverse events after immunization (AEFI) fo the booster dose using self-assessment records via online o adverse events included system reactions and local reactions fever, headache, myalgia, nausea, vomiting, diarrhea, joint pa cal reactions included pain at the injection site, swelling, and

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 to immunoglobulin (total RBD Ig) and anti-RBD IgG by using th Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim luminescent microparticle immunoassay (CMIA) (Abbott Dia spectively [26]. A total RBD Ig level of ≥0.8 U/mL was consid IgG level with a value ≥7.1 BAU/mL was considered positive

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, from the third-booster group at all time points was also evalu against the SARS-CoV-2 variants of concern (VOCs), na B.1.1.529 (omicron), using an ELISA-based surrogate virus based on antibody-mediated blockage of the interaction betw ing domain (RBD) and the angiotensin-converting enzyme 2 ( more, a cPass TM SARS-CoV-2 neutralizing antibody detection cataway, NJ, USA) was used for all strains following the man combinant RBDs from B.1.617.2 (containing L452R and T478 N501Y, E484A, K417N, and D614G) were also used with this inhibit binding between RBD and ACE2 was calculated as a (average OD of the sample/average OD of the negative contr considered positive, indicating the presence of neutralizing a detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood sam group was taken at all time points on days 0, 14, 28, and 90 response. The heparinized whole blood sample was collecte from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, the manufacturer's instruction and incubated at 37 °C for 21 fuged for 15 min at 3500 g to harvest the plasma. This test is The participants were informed of the na adverse events for at least 30 min after vaccin monitored for adverse events after immuniza the booster dose using self-assessment record adverse events included system reactions and fever, headache, myalgia, nausea, vomiting, di cal reactions included pain at the injection site,

Immunoglobulin Assays
All serum samples were analyzed using S immunoglobulin (total RBD Ig) and anti-RBD I Immunoassay (ECLIA) (Roche Diagnostics Gm luminescent microparticle immunoassay (CMI spectively [26]. A total RBD Ig level of ≥0.8 U/m IgG level with a value ≥7.1 BAU/mL was consi

Surrogate Virus Neutralization Test (sVN
To assess SARS-CoV-2 surrogate virus n from the third-booster group at all time points w against the SARS-CoV-2 variants of concer B.1.1.529 (omicron), using an ELISA-based s based on antibody-mediated blockage of the in ing domain (RBD) and the angiotensin-convert more, a cPass TM SARS-CoV-2 neutralizing anti cataway, NJ, USA) was used for all strains follo combinant RBDs from B.1.617.2 (containing L4 N501Y, E484A, K417N, and D614G) were also u inhibit binding between RBD and ACE2 was (average OD of the sample/average OD of the considered positive, indicating the presence of detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparin group was taken at all time points on days 0, response. The heparinized whole blood samp from QuantiFERON (QFN) SARS-CoV-2 RUO the manufacturer's instruction and incubated a fuged for 15 min at 3500 g to harvest the plasm CD4+ and CD8+ were evaluated, as shown in Figure 6 and Supplementary Table S1. Interestingly, potential Vaccines 2022, 10, 1071 9 of 13 antiviral-induced IFN-cataway, NJ, USA) was used for all strains following the manufacturer's instructions. R combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containin N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 (average OD of the sample/average OD of the negative control) × 100. A value ≥30% wa considered positive, indicating the presence of neutralizing antibodies. The lower limit detection was established as 0% inhibition.

IFNɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-boost group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-ce response. The heparinized whole blood sample was collected in a blood collection tub from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) followin the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centr fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimu lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination specific SARS-CoV-2 antigens covering the S protein, followed by measurement in th plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube containin CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube con taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction wa used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA an measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by usin the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated usin IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ level ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2.

Statistical Analysis stimulation of T-cells was notably observed. The marked individual difference in terms of IFN-
more, a cPass SARS-CoV-2 neutralizing antibody detection kit (GenScript Bio cataway, NJ, USA) was used for all strains following the manufacturer's instruc combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (c N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a inhibit binding between RBD and ACE2 was calculated as a percentage as fo (average OD of the sample/average OD of the negative control) × 100. A value considered positive, indicating the presence of neutralizing antibodies. The low detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the thir group was taken at all time points on days 0, 14, 28, and 90 to assess the spec response. The heparinized whole blood sample was collected in a blood collec from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was th fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vi lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a comb specific SARS-CoV-2 antigens covering the S protein, followed by measurem plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube c CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fra used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® E measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated the QuantiFERON R&D Analysis Software. The seropositivity rate was calcula IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-

Statistical Analysis
release induced by IFN-more, a cPass SARS-CoV-2 neutralizing antibody d cataway, NJ, USA) was used for all strains following combinant RBDs from B.1.617.2 (containing L452R an N501Y, E484A, K417N, and D614G) were also used w inhibit binding between RBD and ACE2 was calcula (average OD of the sample/average OD of the negativ considered positive, indicating the presence of neutra detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized bl group was taken at all time points on days 0, 14, 28 response. The heparinized whole blood sample was from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIA the manufacturer's instruction and incubated at 37 °C fuged for 15 min at 3500 g to harvest the plasma. Thi lation of CD4 + and CD8 + lymphocytes in whole hepar specific SARS-CoV-2 antigens covering the S protein plasma of IFN-ɣ production by ELISA. This assay co CD4 + epitopes derived from the S1 subunit (RBD) of S taining CD4 + and CD8 + epitopes from S1 and S2 of SA used to determine the concentration of IFN-ɣ (IU/mL measured the absorbance at 450 nm. The IFN-ɣ stand the QuantiFERON R&D Analysis Software. The sero IFN-ɣ levels from the stimulated tube minus the nega ≥0.15 IU/mL and ≥25% of nil were defined as a positiv

IFN-ɣ Releasing Assay
The subset of participants' whole h group was taken at all time points on response. The heparinized whole blood from QuantiFERON (QFN) SARS-CoV-the manufacturer's instruction and incu fuged for 15 min at 3500 g to harvest th lation of CD4 + and CD8 + lymphocytes i specific SARS-CoV-2 antigens covering plasma of IFN-ɣ production by ELISA CD4 + epitopes derived from the S1 subu taining CD4 + and CD8 + epitopes from S used to determine the concentration of measured the absorbance at 450 nm. T the QuantiFERON R&D Analysis Softw IFN-ɣ levels from the stimulated tube m ≥0.15 IU/mL and ≥25% of nil were defin

Statistical Analysis
CD4+ and CD8+ ranged from 0.0 to 10.0 IU/mL. In terms of the serological response, the seropositivity rates for the IFN-ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centrifuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimulation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination of specific SARS-CoV-2 antigens covering the S protein, followed by measurement in the plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube containing CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube containing CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction was used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA and measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by using the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated using IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ level of ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2.

Statistical Analysis
CD4+/IFN-ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Fu more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech cataway, NJ, USA) was used for all strains following the manufacturer's instruction combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (conta N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a ser inhibit binding between RBD and ACE2 was calculated as a percentage as follow (average OD of the sample/average OD of the negative control) × 100. A value ≥30% considered positive, indicating the presence of neutralizing antibodies. The lower li detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-bo group was taken at all time points on days 0, 14, 28, and 90 to assess the specific response. The heparinized whole blood sample was collected in a blood collection from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) follo the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then c fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro s lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combinat specific SARS-CoV-2 antigens covering the S protein, followed by measurement plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube conta CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tub taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fractio used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ le ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized group was taken at all time points on days 0, 14, response. The heparinized whole blood sample w from QuantiFERON (QFN) SARS-CoV-2 RUO kit (Q the manufacturer's instruction and incubated at 37 fuged for 15 min at 3500 g to harvest the plasma. T lation of CD4 + and CD8 + lymphocytes in whole hep specific SARS-CoV-2 antigens covering the S prot plasma of IFN-ɣ production by ELISA. This assay CD4 + epitopes derived from the S1 subunit (RBD) o taining CD4 + and CD8 + epitopes from S1 and S2 of used to determine the concentration of IFN-ɣ (IU/ measured the absorbance at 450 nm. The IFN-ɣ st the QuantiFERON R&D Analysis Software. The se IFN-ɣ levels from the stimulated tube minus the n ≥0.15 IU/mL and ≥25% of nil were defined as a pos

Statistical Analysis
by T-cells was decreased on days 28 and 90, consistent with the total RBD Ig and anti-RBD IgG levels. All booster vaccines showed that humoral immunity also developed cellular immunity. In contrast, both BNT162b2 and mRNA-1273 vaccines could stimulate robust antigen-specific T-cell responses after 14 days with especially high levels of IFN-

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neut from the third-booster group at all time points was against the SARS-CoV-2 variants of concern ( B.1.1.529 (omicron), using an ELISA-based surro based on antibody-mediated blockage of the inter ing domain (RBD) and the angiotensin-converting more, a cPass TM SARS-CoV-2 neutralizing antibod cataway, NJ, USA) was used for all strains followi combinant RBDs from B.1.617.2 (containing L452R N501Y, E484A, K417N, and D614G) were also used inhibit binding between RBD and ACE2 was calc (average OD of the sample/average OD of the neg considered positive, indicating the presence of neu detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized group was taken at all time points on days 0, 14, response. The heparinized whole blood sample w from QuantiFERON (QFN) SARS-CoV-2 RUO kit ( the manufacturer's instruction and incubated at 3 fuged for 15 min at 3500 g to harvest the plasma. lation of CD4 + and CD8 + lymphocytes in whole he specific SARS-CoV-2 antigens covering the S pro plasma of IFN-ɣ production by ELISA. This assay CD4 + epitopes derived from the S1 subunit (RBD) taining CD4 + and CD8 + epitopes from S1 and S2 of used to determine the concentration of IFN-ɣ (IU measured the absorbance at 450 nm. The IFN-ɣ st the QuantiFERON R&D Analysis Software. The s IFN-ɣ levels from the stimulated tube minus the n ≥0.15 IU/mL and ≥25% of nil were defined as a pos

Statistical Analysis
. There was a good correlation between the results for IFN-

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of ser from the third-booster group at all time points was also evaluated for neutrali against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization based on antibody-mediated blockage of the interaction between the viral re ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [ more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript cataway, NJ, USA) was used for all strains following the manufacturer's inst combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 N501Y, E484A, K417N, and D614G) were also used with this kit. The ability o inhibit binding between RBD and ACE2 was calculated as a percentage as (average OD of the sample/average OD of the negative control) × 100. A valu considered positive, indicating the presence of neutralizing antibodies. The lo detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the t group was taken at all time points on days 0, 14, 28, and 90 to assess the sp response. The heparinized whole blood sample was collected in a blood co from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, German the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a com specific SARS-CoV-2 antigens covering the S protein, followed by measure plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tub CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the A taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON measured the absorbance at 450 nm. The IFN-ɣ standard curve was calcula the QuantiFERON R&D Analysis Software. The seropositivity rate was calc IFN-ɣ levels from the stimulated tube minus the negative control tube. An IF ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SAR

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a from the third-booster group at all time points was also evaluat against the SARS-CoV-2 variants of concern (VOCs), nam B.1.1.529 (omicron), using an ELISA-based surrogate virus n based on antibody-mediated blockage of the interaction betwe ing domain (RBD) and the angiotensin-converting enzyme 2 (A more, a cPass TM SARS-CoV-2 neutralizing antibody detection k cataway, NJ, USA) was used for all strains following the manu combinant RBDs from B.1.617.2 (containing L452R and T478K N501Y, E484A, K417N, and D614G) were also used with this k inhibit binding between RBD and ACE2 was calculated as a (average OD of the sample/average OD of the negative contro considered positive, indicating the presence of neutralizing ant detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samp group was taken at all time points on days 0, 14, 28, and 90 t response. The heparinized whole blood sample was collected from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, H the manufacturer's instruction and incubated at 37 °C for 21 to fuged for 15 min at 3500 g to harvest the plasma. This test is b lation of CD4 + and CD8 + lymphocytes in whole heparinized bl specific SARS-CoV-2 antigens covering the S protein, followe plasma of IFN-ɣ production by ELISA. This assay consisted o CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoVused to determine the concentration of IFN-ɣ (IU/mL) using Q measured the absorbance at 450 nm. The IFN-ɣ standard curv the QuantiFERON R&D Analysis Software. The seropositivity IFN-ɣ levels from the stimulated tube minus the negative cont ≥0.15 IU/mL and ≥25% of nil were defined as a positive respon

SARS-CoV-2 Stimulating IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ T-cell Response
The IFN-ɣ responses to SARS-CoV-2 IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ were evaluated, as shown in Figure 6 and Supplementary Table S1. Interestingly, potential antiviral-induced IFN-ɣ stimulation of T-cells was notably observed. The marked individual difference in terms of IFN-ɣ release induced by IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ ranged from 0.0 to 10.0 IU/mL. In terms of the serological response, the seropositivity rates for the IFN-ɣ CD4+/IFN-ɣ CD4+ and CD8+ levels observed in most participants after the booster dose 14 days were 67.9%/82.1% for AZD1222, 83.3%/93.3% for BNT162b2, and 86.2%/93.1% for mRNA-1273. The production of IFN-ɣ by T-cells was decreased on days 28 and 90, consistent with the total RBD Ig and anti-RBD IgG levels. All booster vaccines showed that humoral immunity also developed cellular immunity. In contrast, both BNT162b2 and mRNA-1273 vaccines could stimulate robust antigen-specific T-cell responses after 14 days with especially high levels of IFN-ɣ. There was a good correlation between the results for IFN-ɣ CD4+/IFN-ɣ CD4 + and CD8 + (Spearman correlation coefficient, p-value < 0.01). The participants were informed of the name of the vaccine and we adverse events for at least 30 min after vaccination. All participants we monitored for adverse events after immunization (AEFI) for seven days the booster dose using self-assessment records via online or paper ques adverse events included system reactions and local reactions. System rea fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, an cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor b immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochem Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sli spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positiv IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of from the third-booster group at all time points was also evaluated for neut against the SARS-CoV-2 variants of concern (VOCs), namely B.1.61 B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralizati based on antibody-mediated blockage of the interaction between the vira ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) prote more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScr cataway, NJ, USA) was used for all strains following the manufacturer's i combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1. N501Y, E484A, K417N, and D614G) were also used with this kit. The abili inhibit binding between RBD and ACE2 was calculated as a percentage (average OD of the sample/average OD of the negative control) × 100. A v considered positive, indicating the presence of neutralizing antibodies. Th detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from th group was taken at all time points on days 0, 14, 28, and 90 to assess th response. The heparinized whole blood sample was collected in a blood from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germ the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and w fuged for 15 min at 3500 g to harvest the plasma. This test is based on the lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a specific SARS-CoV-2 antigens covering the S protein, followed by meas plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and th taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasm used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERO measured the absorbance at 450 nm. The IFN-ɣ standard curve was calc the QuantiFERON R&D Analysis Software. The seropositivity rate was c IFN-ɣ levels from the stimulated tube minus the negative control tube. A ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against S

Statistical Analysis
assay. The subset of heparinized samples was obtained from a third-booster group. Participants who received the third dose of AZD1222 (green), the BNT162b2 (red), or the mRNA-1273 (blue) were stimulated by a CD4+ epitope derived from RBD (Ag1) and a CD4+/CD8+ epitope derived from S1 and S2 subunits (Ag2) and incubated in a QFN blood collection tube for at least 21 h. QFN IFN-Vaccines 2022, 10, x FOR PEER REVIEW The participants were informed of the name of the vaccine adverse events for at least 30 min after vaccination. All participa monitored for adverse events after immunization (AEFI) for sev the booster dose using self-assessment records via online or pap adverse events included system reactions and local reactions. Sys fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, c cal reactions included pain at the injection site, swelling, and redn

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total re immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Ele Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Ge luminescent microparticle immunoassay (CMIA) (Abbott Diagnos spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a su from the third-booster group at all time points was also evaluated against the SARS-CoV-2 variants of concern (VOCs), namely B.1.1.529 (omicron), using an ELISA-based surrogate virus neu based on antibody-mediated blockage of the interaction between ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit ( cataway, NJ, USA) was used for all strains following the manufac combinant RBDs from B.1.617.2 (containing L452R and T478K) an N501Y, E484A, K417N, and D614G) were also used with this kit. T inhibit binding between RBD and ACE2 was calculated as a per (average OD of the sample/average OD of the negative control) × considered positive, indicating the presence of neutralizing antibo detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples group was taken at all time points on days 0, 14, 28, and 90 to a response. The heparinized whole blood sample was collected in from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilde the manufacturer's instruction and incubated at 37 °C for 21 to 24 fuged for 15 min at 3500 g to harvest the plasma. This test is base lation of CD4 + and CD8 + lymphocytes in whole heparinized blood specific SARS-CoV-2 antigens covering the S protein, followed b plasma of IFN-ɣ production by ELISA. This assay consisted of th CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2 taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. T used to determine the concentration of IFN-ɣ (IU/mL) using Qua measured the absorbance at 450 nm. The IFN-ɣ standard curve w the QuantiFERON R&D Analysis Software. The seropositivity ra IFN-ɣ levels from the stimulated tube minus the negative control ≥0.15 IU/mL and ≥25% of nil were defined as a positive response a The participants were informed of the name of the vaccine and were observed adverse events for at least 30 min after vaccination. All participants were continuo monitored for adverse events after immunization (AEFI) for seven days after receiv the booster dose using self-assessment records via online or paper questionnaires. adverse events included system reactions and local reactions. System reactions inclu fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding dom immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminesce Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the che luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland) spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-R IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum sam from the third-booster group at all time points was also evaluated for neutralizing acti against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sV based on antibody-mediated blockage of the interaction between the viral receptor b ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furt more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, cataway, NJ, USA) was used for all strains following the manufacturer's instructions. combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (contain N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum inhibit binding between RBD and ACE2 was calculated as a percentage as follows: (average OD of the sample/average OD of the negative control) × 100. A value ≥30% considered positive, indicating the presence of neutralizing antibodies. The lower lim detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-boo group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T response. The heparinized whole blood sample was collected in a blood collection t from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) follow the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then ce fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro sti lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combinatio specific SARS-CoV-2 antigens covering the S protein, followed by measurement in plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube contain CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by u the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated u IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ lev ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2 The participants were informed of the adverse events for at least 30 min after vac monitored for adverse events after immuni the booster dose using self-assessment reco adverse events included system reactions an fever, headache, myalgia, nausea, vomiting, cal reactions included pain at the injection si

Immunoglobulin Assays
All serum samples were analyzed using immunoglobulin (total RBD Ig) and anti-RBD Immunoassay (ECLIA) (Roche Diagnostics G luminescent microparticle immunoassay (C spectively [26]. A total RBD Ig level of ≥0.8 U IgG level with a value ≥7.1 BAU/mL was con

Surrogate Virus Neutralization Test (s
To assess SARS-CoV-2 surrogate viru from the third-booster group at all time poin against the SARS-CoV-2 variants of conc B.1.1.529 (omicron), using an ELISA-based based on antibody-mediated blockage of the ing domain (RBD) and the angiotensin-conv more, a cPass TM SARS-CoV-2 neutralizing an cataway, NJ, USA) was used for all strains fo combinant RBDs from B.1.617.2 (containing N501Y, E484A, K417N, and D614G) were als inhibit binding between RBD and ACE2 w (average OD of the sample/average OD of th considered positive, indicating the presence detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole hepa group was taken at all time points on days response. The heparinized whole blood sam from QuantiFERON (QFN) SARS-CoV-2 RU the manufacturer's instruction and incubate fuged for 15 min at 3500 g to harvest the pla lation of CD4 + and CD8 + lymphocytes in wh specific SARS-CoV-2 antigens covering the plasma of IFN-ɣ production by ELISA. This CD4 + epitopes derived from the S1 subunit ( taining CD4 + and CD8 + epitopes from S1 and used to determine the concentration of IFN measured the absorbance at 450 nm. The IF the QuantiFERON R&D Analysis Software. IFN-ɣ levels from the stimulated tube minu ≥0.15 IU/mL and ≥25% of nil were defined a 2.5. Statistical Analysis produced by CD4+ and CD8+ T-cells. The lines represent medians with interquartile ranges (IQR). The dotted lines represent cutoff titers. Statistical analysis was performed using the Wilcoxon signed-rank test (two-tailed); p-value < 0.05 (*).

Discussion
The BBIBP-CorV vaccine is a highly valuable tool against COVID-19 due to its less stringent storage and shipment requirements, which differ from those of mRNA-based vaccines. The BBIBP-CorV vaccine has been approved for use in more than 45 countries around the world, and the interim results of the phase 3 trials indicated 78.1% efficacy in preventing the ancestral variant [28]. Our data showed that immunogenicity could be detected after two doses of the BBIBP-CorV vaccine at 30 days, and the seropositivity rates of total RBD Ig and anti-RBD IgG were 97.8% and 93.6%, respectively. The magnitudes of the total RBD Ig and anti-RBD IgG responses were the highest (GMT 42.8 U/mL, and 50.0 BAU/mL, respectively) in individuals 30 days after having completed two doses of the BBIBP-CorV vaccine and slightly decreased and maintained at a low level until 90 days at levels similar to those as the baseline of the third-dose group which had over 6 ± 1 months of two doses of the BBIBP-CorV vaccine. These results are in line with a previous study reporting that on day 18 after vaccination with the BBIBP-CorV vaccine, more than half of the participants showed negative results for the IgG anti-spike protein antibody test and indicates that the BBIBP-CorV vaccine does not produce a sufficient immune response in most vaccinated individuals [6].
With regard to the side effects of the third dose, previous studies [29,30] have shown that the most reported adverse effects of AZD1222 are pain and tenderness at the injection site, fatigue, and headache. Side effects started on the first day after vaccination and lasted one to three days. The most common side effects for BNT162b2 and mRNA-1273 were pain at the injection site, headaches, fever, flu-like symptoms, and fatigue. Although post-vaccination side effects are the same for all vaccines, the number and severity of these side effects differ significantly according to the type of vaccine. In this study, an incidence rate of injection site and systemic reactions was reported within seven days after boost vaccination, all adverse symptoms were mild in severity and mostly transient. The results suggest that a third booster vaccination in healthy adults aged over 18 years is safe, and these results may help support booster vaccination strategies to be administered in the future [31].
Studies of the third dose of COVID-19 vaccination reported the effectiveness of reducing risk in symptomatic infection and hospitalization rates compared to two doses of vaccination [15,19,22]. Therefore, for the general population, vaccination is necessary. Furthermore, heterologous booster doses (two prime doses with inactive or viral vector vaccine and a third dose with an mRNA vaccine) appear to induce higher levels of immune response than homologous booster doses do, suggesting that heterologous immunization could be considered an alternative to homologous booster doses for immunization programs [22,32,33]. To assess the immunogenicity of a booster vaccination, our study found that a third booster dose with heterologous booster immunization of AZD1222, BNT162b2, and mRNA-1273 showed a high titer of total RBD Ig and anti-RBD IgG compared to that before the third booster. Immunogenicity and neutralizing antibody titers increased significantly on day 14. After 28 days from the third booster dose, the immunogenicity, and neutralizing titers continuously decreased until day 90. In correlation with previous studies, the vaccine effectiveness against delta and omicron variants of a third booster shot of CoronaVac, AZD1222, and BNT162b2 at 14 days after previous priming vaccination with two doses of CoronaVac was 56-80%, 56-90%, and 56-93%, respectively [14,34]. Neutralizing antibodies are the most important parameter for measuring the immune response to prevent disease and reinfections. Although neutralizing antibodies are difficult to investigate in routine laboratories, the level of anti-SARS-CoV-2 IgG is indicative. Previous studies found that neutralizing antibodies and anti-SARS-CoV-2 IgG titers are compatible. The level of total anti-RBD antibodies was strongly correlated with the neutralizing antibodies' titers of SARS-CoV-2, especially against the ancestral strain [35,36]. This study showed that the titers of total RBD Ig and anti-RBD IgG were correlated with the neutralizing antibody titers against delta and omicron variants. Such results advocate the idea that a higher immune response would provide better neutralizing antibodies in a real-life situation.
For the IFN- The participants were informed of the name of the vaccine and were observed for adverse events for at least 30 min after vaccination. All participants were continuously monitored for adverse events after immunization (AEFI) for seven days after receiving the booster dose using self-assessment records via online or paper questionnaires. The adverse events included system reactions and local reactions. System reactions included fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. Local reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding domain immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminescence Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the chemiluminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland), respectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-RBD IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum samples from the third-booster group at all time points was also evaluated for neutralizing activity against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) and B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of the interaction between the viral receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFNɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centrifuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimulation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination of specific SARS-CoV-2 antigens covering the S protein, followed by measurement in the plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube containing CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube containing CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction was used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA and measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by using the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated using IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ level of ≥0.15 IU/mL and ≥25% of nil were defined as a positive response against SARS-CoV-2.

Statistical Analysis
response, previous studies have reported that the third dose of an inactivated SARS-CoV-2 vaccine could generate SARS-CoV-2-specific CD4 + and CD8 + T-cells and elicit B-cell responses by antibody evolution [37]. This study observed seropositivity rates for the IFN- The participants were informed of the name of the vaccine and were observed for adverse events for at least 30 min after vaccination. All participants were continuously monitored for adverse events after immunization (AEFI) for seven days after receiving the booster dose using self-assessment records via online or paper questionnaires. The adverse events included system reactions and local reactions. System reactions included fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and dizziness. Local reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor binding domain immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemiluminescence Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and the chemiluminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, Ireland), respectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. The anti-RBD IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of serum samples from the third-booster group at all time points was also evaluated for neutralizing activity against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 (delta) and B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of the interaction between the viral receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [27]. Furthermore, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript Biotech, Piscataway, NJ, USA) was used for all strains following the manufacturer's instructions. Recombinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 (containing N501Y, E484A, K417N, and D614G) were also used with this kit. The ability of a serum to inhibit binding between RBD and ACE2 was calculated as a percentage as follows: 1 − (average OD of the sample/average OD of the negative control) × 100. A value ≥30% was considered positive, indicating the presence of neutralizing antibodies. The lower limit of detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the third-booster group was taken at all time points on days 0, 14, 28, and 90 to assess the specific T-cell response. The heparinized whole blood sample was collected in a blood collection tube from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was then centrifuged for 15 min at 3500 g to harvest the plasma. This test is based on the in vitro stimulation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a combination of specific SARS-CoV-2 antigens covering the S protein, followed by measurement in the plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube containing CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag2 tube containing CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma fraction was used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® ELISA and measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculated by using the QuantiFERON R&D Analysis Software. The seropositivity rate was calculated using IFN-ɣ levels from the stimulated tube minus the negative control tube. An IFN-ɣ level of The participants were informed of the name of the vaccine and were o adverse events for at least 30 min after vaccination. All participants were co monitored for adverse events after immunization (AEFI) for seven days afte the booster dose using self-assessment records via online or paper question adverse events included system reactions and local reactions. System reactio fever, headache, myalgia, nausea, vomiting, diarrhea, joint pain, chills, and di cal reactions included pain at the injection site, swelling, and redness.

Immunoglobulin Assays
All serum samples were analyzed using SARS-CoV-2 total receptor bind immunoglobulin (total RBD Ig) and anti-RBD IgG by using the Electrochemilu Immunoassay (ECLIA) (Roche Diagnostics GmbH, Mannheim, Germany) and luminescent microparticle immunoassay (CMIA) (Abbott Diagnostics, Sligo, spectively [26]. A total RBD Ig level of ≥0.8 U/mL was considered positive. Th IgG level with a value ≥7.1 BAU/mL was considered positive.

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutralization, a subset of seru from the third-booster group at all time points was also evaluated for neutraliz against the SARS-CoV-2 variants of concern (VOCs), namely B.1.617.2 B.1.1.529 (omicron), using an ELISA-based surrogate virus neutralization based on antibody-mediated blockage of the interaction between the viral rec ing domain (RBD) and the angiotensin-converting enzyme 2 (ACE2) protein [2 more, a cPass TM SARS-CoV-2 neutralizing antibody detection kit (GenScript B cataway, NJ, USA) was used for all strains following the manufacturer's instr combinant RBDs from B.1.617.2 (containing L452R and T478K) and B.1.1.529 N501Y, E484A, K417N, and D614G) were also used with this kit. The ability o inhibit binding between RBD and ACE2 was calculated as a percentage as (average OD of the sample/average OD of the negative control) × 100. A valu considered positive, indicating the presence of neutralizing antibodies. The lo detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blood samples from the th group was taken at all time points on days 0, 14, 28, and 90 to assess the sp response. The heparinized whole blood sample was collected in a blood col from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIAGEN, Hilden, Germany the manufacturer's instruction and incubated at 37 °C for 21 to 24 h and was fuged for 15 min at 3500 g to harvest the plasma. This test is based on the in lation of CD4 + and CD8 + lymphocytes in whole heparinized blood with a com specific SARS-CoV-2 antigens covering the S protein, followed by measure plasma of IFN-ɣ production by ELISA. This assay consisted of the Ag1 tube CD4 + epitopes derived from the S1 subunit (RBD) of SARS-CoV-2, and the Ag taining CD4 + and CD8 + epitopes from S1 and S2 of SARS-CoV-2. The plasma f used to determine the concentration of IFN-ɣ (IU/mL) using QuantiFERON ® measured the absorbance at 450 nm. The IFN-ɣ standard curve was calculate the QuantiFERON R&D Analysis Software. The seropositivity rate was calcu IFN-ɣ levels from the stimulated tube minus the negative control tube. An IF CD4 + and CD8 + levels in most participants after the booster dose of 14 days and a significant decrease in IFN-Vaccines 2022, 10, x FOR PEER REVIEW The participants were informed of the name of t adverse events for at least 30 min after vaccination. A monitored for adverse events after immunization (AE the booster dose using self-assessment records via on adverse events included system reactions and local re fever, headache, myalgia, nausea, vomiting, diarrhea, j cal reactions included pain at the injection site, swellin

Immunoglobulin Assays
All serum samples were analyzed using SARS-Co immunoglobulin (total RBD Ig) and anti-RBD IgG by u Immunoassay (ECLIA) (Roche Diagnostics GmbH, Ma luminescent microparticle immunoassay (CMIA) (Abb spectively [26]. A total RBD Ig level of ≥0.8 U/mL was IgG level with a value ≥7.1 BAU/mL was considered p

Surrogate Virus Neutralization Test (sVNT)
To assess SARS-CoV-2 surrogate virus neutraliz from the third-booster group at all time points was also against the SARS-CoV-2 variants of concern (VOC B.1.1.529 (omicron), using an ELISA-based surrogate based on antibody-mediated blockage of the interactio ing domain (RBD) and the angiotensin-converting enzy more, a cPass TM SARS-CoV-2 neutralizing antibody de cataway, NJ, USA) was used for all strains following th combinant RBDs from B.1.617.2 (containing L452R and N501Y, E484A, K417N, and D614G) were also used wi inhibit binding between RBD and ACE2 was calculat (average OD of the sample/average OD of the negativ considered positive, indicating the presence of neutral detection was established as 0% inhibition.

IFN-ɣ Releasing Assay
The subset of participants' whole heparinized blo group was taken at all time points on days 0, 14, 28, response. The heparinized whole blood sample was c from QuantiFERON (QFN) SARS-CoV-2 RUO kit (QIA the manufacturer's instruction and incubated at 37 °C fuged for 15 min at 3500 g to harvest the plasma. This lation of CD4 + and CD8 + lymphocytes in whole hepari specific SARS-CoV-2 antigens covering the S protein plasma of IFN-ɣ production by ELISA. This assay con CD4 + epitopes derived from the S1 subunit (RBD) of S taining CD4 + and CD8 + epitopes from S1 and S2 of SAR used to determine the concentration of IFN-ɣ (IU/mL) measured the absorbance at 450 nm. The IFN-ɣ stand the QuantiFERON R&D Analysis Software. The serop production T-cells after boosting the dose for 14 days, limiting the efficacy of the vaccines. This result is similar to that of a previous report [37] and suggests that a second dose improves an effective antibody response, and further boosters may be needed to retain vaccine effectiveness in adults. Whether enhanced B-and T-cell responses could lead to better protection is one of the key issues concerning boosting strategies. As a result, a third-dose vaccination after six months, even a viral vector or an mRNA vaccine, is in the new guidelines that make it mandatory to get a booster. However, more information is needed for use in a larger population in the future. The limitations of the current study include the limit of detection of the sVNT assay. Most individuals receiving a booster dose achieved an elevation in antibody levels that was greater than the upper limit of detection. This method may not reflect the actual neutralizing antibody level. Furthermore, the participants lost to follow-up due to breakthrough infection or lost contact during the follow-up period may not reflect the actual values at each time point.
In conclusion, a third-dose heterologous regimen, two initial doses of BBIBP-CorV vaccines followed by a viral vector or mRNA vaccine, induced a robust immunological response. This study found that a two-dose schedule generated good immune memory. However, immunogenicity titers decreased to near or below the lower limit of the seropositivity rate over time. A third dose given 6 ± 1 months after the second dose of BBIBP-CorV was highly effective in recalling a specific SARS-CoV-2 immune response, leading to a significant rebound in antibody levels. This approach would make it possible to optimize the third available booster dose of vaccines without increasing the risk of infection and allowing a faster expansion of vaccination coverage.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vaccines10071071/s1, Figure S1: Correlation between the percentage of inhibition against the SARS-CoV-2 variants and the total RBD Ig levels. The subset of serum samples was obtained from a third-booster group. Participants who received the third dose of AZD1222 (green), BNT162b2 (red), or mRNA-1273 (blue) were plotted with a nonlinear regression curve. (a) AZD1222 against the delta variant, (b) AZD1222 against the omicron variant, (c) BNT162b2 against the delta variant, (d) BNT162b2 against the omicron variant, (e) mRNA-1273 against the delta variant, and (f) mRNA-1273 against the omicron variant. Figure S2 The correlation between the percentage of inhibition against SARS-CoV-2 variants and anti-RBD IgG levels. The subset of serum samples was obtained from a third-booster group. The participants who received the third dose of AZD1222 (green), BNT162b2 (red), or mRNA-1273 (blue) were plotted with a nonlinear regression curve.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.