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Effects of boosted mRNA and adenoviral‐vectored vaccines on immune responses to omicron BA.1 and BA.2 following the heterologous CoronaVac/AZD1222 vaccination

Abstract The coronavirus 2019 omicron variant has surged rapidly and raises concerns about immune evasion even in individuals with complete vaccination, because it harbors mutations. Here we examine the capability of booster vaccination following CoronaVac/AZD1222 prime to induce neutralizing antibodies (NAbs) against omicron (BA.1 and BA.2) and T‐cell responses. A total of 167 participants primed with heterologous CoronaVac/AZD1222 for 4–5 months were enrolled, to receive AZD1222, BNT162b2, or mRNA‐1273 as a third dose. Reactogenicity was recorded. Immunogenicity analyses of severe acute respiratory syndrome coronavirus 2‐binding antibodies were measured using enzyme‐linked immunosorbent assay. The NAb titers against omicron BA.1 and BA.2 were determined using the focus reduction neutralization test (FRNT50) and total interferon‐γ responses were measured to observe the T‐cell activation. A substantial loss in neutralizing potency to omicron variant was found at 4–5 months after receiving the heterologous CoronaVac/AZD1222. Following booster vaccination, a significant increase in binding antibodies and neutralizing activities toward delta and omicron variants was observed. Neutralization to omicron BA.1 and BA.2 were comparable, showing the highest titers after boosted mRNA‐1273 followed by BNT162b2 and AZD1222. In addition, individuals boosted with messenger RNA (mRNA) vaccines develop a T‐cell response to spike protein, whereas those boosted with AZD1222 did not. Reactogenicity was mild to moderate without serious adverse events. Our findings demonstrated that mRNA booster vaccination is able to overcome waning immunity to provide antibodies that neutralize omicron BA.1 and BA.2, as well as a T‐cell response.


| INTRODUCTION
As of November 2021, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron (B.1.1.529) variant quickly surged worldwide and raised concern about immune evasion. 1 Due to high transmissibility and the potential to evade immunity, the World

Health Organization Technical Advisory Group on SARS-CoV-2 Virus
Evolution declared the omicron as a variant of concern. 2 Omicron variant is now reported in more than 190 countries and causes the global case count of over 10 million weekly cases between December 2021 and March 2022. 3 During the omicron wave, the mortality rate was 0.27% over positive cases and 0.021% overpopulation as of February 2022. 4 The total number of estimated infections was about twofold higher for omicron and showed a twofold lower rate of mortality relative to delta. 5 The omicron variant is characterized by many mutations in the spike protein. Among these, 15 mutations located on the receptorbinding domain (RBD), which are responsible for interactions with the angiotensin-converting enzyme 2 (ACE2) receptor, and 8 amino acid changes are found at the N-terminal domain (NTD). 6 In a study of mutated RBD profiles, various omicron mutations capable of escaping human neutralizing antibodies (NAbs) were found to contain epitopes overlapping the ACE2-binding motif, indicating evasion of immunity and reduction in vaccine effectiveness. 7 Furthermore, mutations in NTD related to partial escape from the Nabs have been reported. 8 These findings are consistent with other clinical data demonstrating that the emergence of the omicron variant has led to an increase in the risk of reinfection. 9 During the ongoing viral evolution, the omicron variant has been divided into four subvariants, including BA.1, BA.1.1, BA.2, and BA.3. 10 Among them, BA.1 surged earlier and became the dominant subvariant circulating worldwide. However, the BA.2 subvariant has recently increased in multiple countries and appears to be more transmissible than BA.1. 11 Previous studies suggest that BA.1 and BA.2 are highly resistant to neutralization by monoclonal antibody therapy and vaccine-induced immunity. 12,13 Another concern is the waning immunity that occurs over time. A previous study indicates that the IgG antibodies declined a consistent rate at 6 months after second dose of the messenger RNA (mRNA) vaccination, whereas NAbs declined rapidly over the first 3 months followed by a relatively slower decrease after that point. 14 A reduction in NAbs is related to an increased risk of symptomatic SARS-CoV-2 infection and reduced vaccine effectiveness. 15 Moreover, omicron variants were poorly or not at all neutralized in the sera sampled 5 months after completing the two-dose BNT162b2 or AZD1222 vaccination courses. 16 Due to the emergence of omicron and waning immunity, a booster vaccination program has been implemented in many countries. 17,18 Thus, data on boosting immunity against omicron variant are needed.
Besides humoral immunity, cell-mediated immunity also plays an essential role in limiting the SARS-CoV-2 infection 19 and reducing disease severity in acute coronavirus 2019 (COVID-19) patients. 20 The SARS-CoV-2-specific T cells persist at least 6 months after receiving the two-dose regimen of either mRNA or adenoviralvectored vaccines, whereas the levels of NAb severely declined. 21 Furthermore, SARS-CoV-2-specific T cells induced by vaccination or previous infection are highly cross-reactive with the omicron variant and the omicron cross-reactive T cells exhibiting the polyfunctional profiles were not significantly different compared with ancestral strain and other SARS-CoV-2 variants. 22 These findings indicate that a few mutations in the spike can minimally affect T-cell recognition.
Seven vaccines have now been authorized in Thailand: (1) inactivated CoronaVac, (2) BBIBP-CorV, (3) adenoviral-vectored ChAdOx1-S/ AZD1222, (4) Ad26.COV2.S, (5) mRNA-based BNT162b2, (6) mRNA-1273, and (7) protein-based NVX-CoV2373 vaccines. 23 However, the vaccine effectiveness and capability of inducing immune responses differ with different types of vaccines and are affected by emerging variants. 24 Our previous study indicated that mRNA vaccine-or AZD1222-boosted individuals after a two-dose CoronaVac course elicited a higher immune response than in those receiving boosted inactivated vaccines. 25 In the COV-BOOST trial, the heterologous boost after either two-dose of AZD1222 or BNT162b2 prime showed an increased humoral and cellmediated immune response compared to homologous booster vaccination, although the reactogenicity was increasing in some heterologous boosted combination. 26 However, information about the effects of booster vaccinations on safety and immune responses against omicron variants following heterologous primed has been limited.
Due to the limited vaccine supply, Thailand has administered the heterologous CoronaVac followed by AZD1222 vaccination as an alternative regimen for combatting the delta variant. This regimen could induce a higher immune response than the homologous CoronaVac regimen. 27   This study was registered with the Thai Clinical Trials Registry (TCTR20211120002). All participants signed a written consent before being enrolled in this study.

| Reactogenicity assessment
Participants were observed adverse events (AEs) at least 30 min after vaccination. At the baseline visit, the participants were given the AEs report form to self-record within 7 days after immunization. AEs report forms recorded the solicited local and systemic AEs, and unsolicited and AEs of special interests. The solicited local and systemic AEs were classified as mild (no limitation on normal activity), moderate (some limitation of daily activity), and severe (unable to perform or prevented daily activity).

| Measurement binding antibody and neutralizing activity using enzyme-linked immunosorbent assay (ELISA)-based assay
All sera samples measured anti-RBD IgG and anti-nucleocapsid (N) IgG using ELISAs (Abbott Diagnostics). The anti-RBD IgG was expressed as a binding antibody unit (BAU/ml) and a value ≥ 7.1 was considered positive. Anti-N IgG reported as S/C ratio (optical density (OD) sample divided by calibrator) with a value ≥1.4 was considered positive. In addition, a subset of samples was randomly selected to perform surrogate virus neutralization assay (sVNT) against delta and omicron variants using a cPass™ SAR-CoV-2 NAb detection kit (GenScript Biotech) as previously described. 27 Neutralizing activity was reported as the percentage of inhibition (inhibition [%] = [1 − OD value of sample/average OD of negative control] × 100). A value ≥ 30% was defined as positive, indicating the presence of NAbs. The lower limit of detection was set as 0% inhibition.

| Focus reduction neutralization test (FRNT50)
Live SARS-CoV-2 NAb titers in a subset of serum samples were determined using a 50% focus reduction neutralization test (FRNT50) against omicron BA.1 (accession number: EPI_ISL_8547017) and BA.2 (EPI_ISL_11698090) subvariants. Briefly, heat-inactivated sera were used to prepare serial dilutions starting from 1:10 to 1:7290 and incubated with live virus for 1 h at 37°C. The virus-sera mixtures were transferred to monolayers of Vero cells in a 96-well plate and incubated for 2 h. Foci development evaluation and infected cell counting were performed as previously described. 31 The focus reduction percentage for an individual sample was calculated and the half-maximal inhibitory concentration was evaluated using PROBIT software. If no neutralization was observed, the FRNT50 was set as 10, which is one dilution step below the lower limit of detection (dilution 1:20).

| Quantification of interferon-γ (IFN-γ) response
The SARS-CoV-2-specific T-cell response was evaluated by using a commercially available IFN-γ release assay in whole blood according to the manufacturer's instructions (QuantiFERON, Qiagen). Heparinized whole blood was incubated with different antigens, including negative (Nil), positive (Mitogen), and two different SARS-CoV-2 antigens (Ag1 and Ag2). The Ag1 tube was coated with S1 subunit (RBD) peptides with CD4+ stimulation and Ag2 contained S1 + S2 peptides for CD4+ and CD8+ stimulation. After stimulation, total IFN-γ production was measured as previously described. 27 The results were calculated from SUNTRONWONG ET AL. | 3 a standard curve and expressed as IU/ml after subtraction of the Nil control. The total IFN-γ with a value ≥0.15 IU/ml and ≥25% of Nil were considered a positive response against SARS-CoV-2.   (Table 1). Additionally, no statistically significant differences were observed in the time intervals between the first and the second doses for any group.

| Statistical analysis
However, the median interval between the second and third dose in AZD1222 (125 days, IQR: 118-134.5) was slightly shorter (but not statistically significant) than BNT162b2 (130 days, IQR: 110-141) and mRNA-1273 (131 days, IQR: 102.3-133).  3.3 | Neutralizing activity to delta and omicron measured using sVNT Neutralizing activity to the delta and omicron variants was measured in sera at baseline and 28 days post-boost using the surrogate virus neutralization test. All boosted individuals could restore neutralizing activity to delta by more than 90% (Figure 2A

| Total IFN-γ response
Besides the NAbs, the T-cell response were assessed by measuring total IFN-γ responses in whole blood from AZD1222-, BNT162b2-, and mRNA-1273-boosted individuals after S1 (RBD) peptides for CD4+ stimulation or Ag1 ( Figure 4A) and S1+ S2 peptides for CD4+ F I G U R E 3 Neutralizing antibody (NAb) titers against omicron BA.1 and BA.2 measured using focus reduction neutralization test (FRNT50). A subset of samples from boosted individuals with AZD1222 (purple), BNT162b2 (green), and mRNA-1273 (yellow), which was randomly selected to test the FRNT50 included sera collected at baseline (n = 10/group) and sera collected at 28 days post-boost (n = 30/group). NAb titers against omicron BA.1 (A) and omicron BA.2 (B) were compared between baseline (Day 0) and 28 days post-booster vaccination with different vaccines. Numbers above the plot indicate the geometric mean titers (GMTs). Fold increases for each comparison are denoted. NAb titers against BA.1 and BA.2 at 28 days after booster vaccination were compared (C). Statistical analysis was done using Wilcoxon signed-rank test (two-tailed). The horizontal dotted line indicates the limit of detectable value of FRNT50. Values below the limit of detection (<20) were set at a titer of 10 before statistical analysis. ns, no significant difference.
F I G U R E 4 Comparison of total interferon-γ (IFN-γ)-releasing T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens. Heparinized whole blood samples from vaccinated individuals receiving heterologous CoronaVac/AZD1222 followed by a third booster with AZD1222 (n = 30, purple), BNT162b2 (n = 30, green), or mRNA-1273 (n = 30, yellow) were stimulated by Ag1, which is a CD4+ epitope derived from receptor-binding domain (RBD), minus negative control (Nil) (A), and Ag2 which is CD4+ and CD8+ epitopes derived from S1 and S2 subunits, minus negative control (Nil) (B). Horizontal bars indicate the median. The cut-off values were represented by horizontal dotted line. Levels of IFN-γ above cut-off values (0.15 IU/ml and ≥25% of Nil) indicate a reactive response. Statistical analysis was done using Wilcoxon signed-rank test (two-tailed). ns, no significant difference; **p < 0.01; ***p < 0.001. and CD8+ stimulation or Ag2 ( Figure 4B) immunity. 14 It has been well established that booster vaccinations could overcome the waning immunity. 18,25,26,32 Rapid elevation in binding antibodies after boosting with mRNA and adenoviralvectored vaccines were found. Similar results have been reported after receiving either a viral-vectored or an mRNA vaccine as a third dose, indicating that it was sufficient to recall the memory B cells. 32 NAb titers is a highly predict the immune protection against SARS-CoV-2 infection. Higher levels of NAbs correlated with a reduced risk of infection and severe disease. 15 Our study found a substantial loss of NAb titers against omicron variants at pre-booster vaccination. This effect might be related to harboring of mutations concentrated around the RBD. 7 However, the NAb titers against omicron were retained upon booster vaccination even though the current vaccines composition was based on the ancestral strain.
Consistent results were observed after receiving mRNA vaccine following either homologous inactivated, adenoviral-vectored, or mRNA-based vaccines prime. 18,25,26,33 Although the low number of omicron-neutralizing memory B cells produced after two-doses of mRNA vaccination has previously been reported, their coverages were improved through affinity maturation over time and might be sufficient to expand the coverage of neutralization against SARS-CoV-2 variants after receiving a booster dose. 34 Furthermore, a reduced neutralizing activity against omicron compared with delta variant was found. Similar results were found concerning neutralizing activity to omicron, which was reduced by 6-to 23-fold lower than delta after booster vaccination. 16 With the surge of omicron BA.