Neutralization against Omicron sublineages (BA.2/BA.5/BQ.1.1/XBB/XBB.1.5) in bivalent BNT162b2-vaccinated HCWs with or without risk factors, or following BT infection with Omicron

SARS-CoV-2-BA.4/5-adapted-bivalent-BNT162b2-vaccine (bvBNT), developed in response to the recent emergence of immune-evasive Omicron-variants, has been given to individuals who completed at least 2-doses of the monovalent-BNT162b2-vaccine (mvBNT). In the present cohort study, we evaluated neutralization-titers (NT50s) against Wuhan-strain (SCoV2Wuhan) and Omicron-sublineages including BA.2/BA.5/BQ.1.1/XBB/XBB.1.5, and vaccine-elicited S1-binding-IgG in sera from participants-vaccinated with 5th-bvBNT following 4th-mvBNT. The 5th-bvBNT-dose elicited good protective-activity against SCoV2Wuhan with geometric-mean (gMean)-NT50 of 1966–2091, higher than the peak-values post-4th-mvBNT with no statistical significance, and favorable neutralization-activity against not only BA.5 but also BA.2, with ~ 3.2-/~ 2.2-fold greater gMean-NT50 compared to the peak-values post-4th-mvBNT-dose, in participants with or without risk factors. However, neutralization-activity of sera post-5th-bvBNT-dose was low against BQ.1.1/XBB/XBB.1.5. Interestingly, participants receiving bvBNT following breakthrough (BT) infection during Omicron-wave had significantly enhanced neutralization-activity against SCoV2Wuhan/BA.2/BA.5 with ~ 4.6-/~ 6.3-/~ 8.1-fold greater gMean-NT50, respectively, compared to uninfected participants receiving bvBNT. Sera from BT-infected-participants receiving bvBNT had enhanced neutralization-activity against BQ.1.1/XBB/XBB.1.5 by ~ 3.8-fold compared to those from the same participants post-4th-mvBNT-dose, and had enhanced gMean-NT50 ~ 5.4-fold greater compared to those of uninfected-participants’ sera post-bvBNT. These results suggest that repeated stimulation brought about by exposure to BA.5’s-Spike elicit favorable cross-neutralization-activity against various SARS-CoV-2-variants.

From the initial stage of the global pandemic, massive efforts were made toward development of novel vaccines against SARS-CoV-2 around the world [5][6][7] Currently, more than 50 vaccines have been approved by at least one country (https:// covid 19.track vacci nes.org/ vacci nes/ appro ved/).The efficacy of vaccines against SARS-CoV-2 had been beyond expectation.As of February 2023, more than 13.3 billion of anti-SARS-CoV-2 vaccine doses have already been administered in the world 8 .Among various vaccines, two mRNA vaccines, BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna), have previously shown ~ 95% efficacy in preventing symptomatic COVID-19 in early-phase of pandemic [9][10][11] , and these mRNA vaccines accounted for 90 and 97% of total administrated doses of COVID-19 vaccine in U.S. and European Union respectively, up to present 8 .
According to the recent emergence of immune-evasive Omicron variants, novel bivalent mRNA booster vaccines were developed by targeting the Spike protein of SARS-CoV-2 Wuhan and Omicron BA.4/BA.5 sublineages and have been provided to individuals who had completed at least 2 doses of monovalent COVID-19 vaccination.Although the Morbidity and Mortality Weekly Report (MMWR) described that among immunocompetent adults, who were ≥ 65 years, a bivalent booster dose provided 73% additional protection against COVID-19 hospitalization compared with monovalent mRNA vaccination only 12 , there have been multiple results reporting antibody evasion profiles of new Omicron sublineages BQ.1.1 and XBB, posing further concerns on the efficacy of anti-SARS-CoV-2 vaccines 13 .

Results
Effects of Omicron BA.4/5-adapted BNT162b2 ( bv BNT) vaccination in sera obtained from health care workers (HCWs) with risk factors.
Firstly, we examined SARS-CoV-2 neutralization activity of sera post-bv BNT booster vaccine dose, obtained from 23 out of 225 HCWs in Kumamoto General Hospital, Japan (225 individuals were initially recruited in the primary clinical study 14 ), who were either of ≥ 60-years of age and/or had pre-existing diseases/risk factors (see demographic characteristics in Table 1).

Effects of bv BNT in sera from HCWs who had experienced symptomatic/asymptomatic breakthrough infection during Omicron wave
Among HCWs enrolled in the present study, 20 participants proved to be SARS-CoV-2 positive by swab PCR from April 2022 to August 2022, and 13 out of the 20 participants received the 5th-dose bv BNT vaccination after recovery.These 13 participants who had experienced symptomatic breakthrough (BT) infection and 5thdose of vaccine were termed as "BT-Sym#1-13" (symptoms of each participant were indicated in Supplemental Table S2).Other 17 participants who had neither tested nor received any positive results for swab-PCR or antigen tests but proved to be positive for anti-SARS-CoV-2 nucleocapsid-specific-IgG in their sera obtained from August 2022 to December 2022.These 17 participants all received 5th-dose bv BNT vaccine and were termed as "BT-Asym#1-17".Detailed information regarding infection date (PCR positivity/serum N-IgG positivity) and longitudinal changes of serum NT 50 values against Omicron BA.5 are summarized in Fig. 4

Discussion
In the present cohort study, we studied in detail the effectiveness of BA.4/5 adapted bivalent BNT162b2 ( bv BNT) vaccine using various infectious SARS-CoV-2 s, by examining the participants' sera obtained pre-/post-2nd-4thdoses of mv BNT, from participants with/without risk factors or who had experienced BT-infection during the Omicron-wave (January 2022 through December 2022) in Japan.
In our previous data with mv BNT vaccinations between post-3rd and 4th-doses, neutralization activity against SCoV2 Wuhan elicited by 4th-dose of mv BNT were not greater than those after 3rd-dose of mv BNT 17,18 (Supplemental Table S1).Similarly, the magnitudes of neutralizing activity against SCoV2 Wuhan after the 5th-bv BNT dose were not greater compared to the significantly boosted response elicited by the 3rd-mv BNT in participants with/ without risk factors (Supplemental Table S1).These limited restoration regarding neutralization activity against SCoV2 Wuhan by 5th-bv BNT seems to reflect the difference the amounts of mRNA containing against original SCoV2 Wuhan between 3rd-dose and 5th-dose (30 μg of mRNA for 3rd-dose of mv BNT, and 15 μg of mRNA for 5th-dose of mv BNT).However, 4th dose-mv BNT also contains 30 μg of mRNA against original SCoV2 Wuhan , but 5th-bv BNT elicited higher neutralization activity against SCoV2 Wuhan than those of 4th-mv BNT (Supplemental Table S1), indicating bv BNT may have different property from that of mv BNT against SCoV2 Wuhan .
We also evaluated sera post-5th-bv BNT dose against not only BA.5 but also BA.2.All the post-5th-dose sera examined in the current study demonstrated significantly more robust neutralization activity against BA.5 and showed favorable neutralization activity also against BA.2 (p < 0.0001 for both BA.5 and BA.2 in Figs. 2 and 3).
In the present study, we also focused on the groups of participants who experienced BT-infection during the Omicron wave period (Fig. 4 and 5).BT-infected participants showed significant enhancement of neutralization activity after bv BNT dose against SCoV2 Wuhan , BA.2, and BA.5, as well as BQ.1.1 and XBB.These results suggest that repeated stimulation caused by the exposure to Omicron's Spike protein elicited broad and stronger neutralization activity against multiple SARS-CoV-2 variants.If it is the case and if further infection waves by SARS-CoV-2 variants arrive, booster bv BNT doses may have to be considered, although further data on the range of neutralization elicited by bv BNT have to be carefully examined.

Figure 1 .
Figure 1.Schedule of 4-times mv BNT doses and once bv BNT dose in this study.Administration schedule of 4-times mv BNT doses (red arrows), once bv BNT dose (blue arrows), and blood collections (yellow arrows) in this study are shown.HCWs with risk factors received 4th dose of mv BNT on day477, while HCWs without risk factor received it on day537.HCWs with risk factors had 11th blood collection (10-weeks post 4th dose) on day550.

Figure 2 .
Figure 2. Effect of Omicron BA.4/5-adapted BNT162b2 ( bv BNT) vaccination in sera obtained from HCWs with risk factors.Temporal changes of neutralizing activity of sera obtained from HCWs with risk factors over 650 days post-1st dose of BNT162b2 are shown.4th monovalent ( mv BNT) and 5th bivalent BNT162b2 ( bv BNT) doses were administered on days 477 and 637, respectively (n = 23).(A) The 50% neutralization titers (NT 50 ) of participants' sera against infection by SARS-CoV-2 Wuhan strain (SCoV2 Wuhan ) were determined on days 470, 490, 550, 630, and 650 post-1st-dose using VeroE6 TMPRSS2 cell-based neutralization assay.Solid circles denote NT 50 titers of each participant's serum and filled bars denote average NT 50 titers of 23 participants' sera at each time point.Geometric mean NT 50 titers (gMean-NT 50 ) and ranges of NT 50 at each time point are shown at the bottom.(B) Temporal changes of neutralizing activity of participants' sera at days 470, 490, 550, 630, and 650 post-1st dose against Omicrons BA.2, BA.5, BQ.1.1,XBB, and XBB.1.5 are shown.Solid circles denote NT 50 titers of each participant's serum and filled bars denote average NT 50 titers of 23 participants' sera at each time point.The circles and lines in same color indicate that the data were obtained from same participant's sera.

Figure 3 .
Figure 3.Effect of bv BNT vaccination in sera obtained from HCWs without risk factor.Temporal changes of neutralizing activity of sera obtained from HCWs without risk factor over 650 days post-1st dose of mv BNT are shown.4th mv BNT and 5th bv BNT doses were administered on days 537 and 637, respectively (n = 90).NT 50 of participants' sera against infection by SCoV2 Wuhan and Omicrons BA.2, BA.5, BQ.1.1,XBB, and XBB.1.5were determined using VeroE6 TMPRSS2 cell-based neutralization assay.Solid circles denote NT 50 titers of each participant's serum and filled bars denote average NT 50 titers of 90 participants' sera at each time point.gMean-NT 50 and ranges of NT 50 at each time point are shown at the bottom.The circles and lines in same color indicate that the data were obtained from same participant's sera.

Figure 4 .
Figure 4. Neutralization activity against Omicron BA.5 of sera obtained from HCWs who had received booster dose of mv BNT and experienced breakthrough infection during Omicron wave.Detailed information of neutralization activity (NT 50 ) of sera obtained from BT-infection experienced participants against Omicron BA.5, dates of swab PCR-positive, and positive periods of serum SARS-CoV-2 nucleocapsid-specific IgG (red colored columns) are shown.(A) shows the results of participants with risk factors, and (B) shows the results of participants without risk factor.

Figure 5 .
Figure 5.Effect of bv BNT vaccination in sera from HCWs who had experienced breakthrough (BT) infection during Omicron wave.Temporal changes of neutralizing activity of sera obtained from HCWs who had experienced breakthrough (BT) infection over 650 days post-1st dose of BNT162b2 are shown (n = 30).NT 50 of participants' sera against infection by SCoV2 Wuhan and Omicrons BA.2, BA.5, BQ.1.1,XBB, and XBB.1.5were determined using VeroE6 TMPRSS2 cell-based neutralization assay.Solid circles denote NT 50 titers of each participant's serum and filled bars denote average NT 50 titers of 30 BT-infected participants' sera at each time point.gMean-NT 50 and ranges of NT 50 at each time point are shown at the bottom.The circles and lines in same color indicate that the data were obtained from same participant's sera.

Table 1 .
Demographic characteristics of the participants who received Omicron BA.5-adapted 5th-dose of BNT162b2 vaccination.*143 of 225 health care workers participated in the study.None of the participants were in immunodeficient states or were receiving immunosuppressants or steroids.Risk factors contain age (≧ 60 y.o) and following diseases/conditions; asthma, hypertension, diabetes, malignancy, obesity, and liver disease.