Neutralizing Antibody Response of the Wild-Type/Omicron BA.1 Bivalent Vaccine as the Second Booster Dose against Omicron BA.2 and BA.5

ABSTRACT In addition to the original monovalent vaccines available for SARS-CoV-2, bivalent vaccines covering wild-type (WT) and Omicron BA.1 are also available. However, there is a lack of real-world data on the immunogenicity of bivalent vaccines as second boosters against the dominant Omicron sublineages, including BA.2 and BA.5. Healthcare workers (n = 565) who received the first booster vaccination were followed for 2 weeks after the second booster dose of the monovalent mRNA-1273 (WT group, n = 168) and bivalent BNT162b2 (WT+BA.1 group, n = 23) vaccines. Participants with previous SARS-CoV-2 infections were excluded from the study. The anti-receptor binding domain (RBD) antibody levels after the second booster dose in the WT and WT+BA.1 group were similar (median [interquartile range], 26,262.0 [16,951.0 to 38,137.0] U/mL versus 24,840.0 [14,828.0 to 41,460.0] U/mL, respectively). Although the neutralization activities of the pooled sera were lower against BA.5 than against other variants in both groups, the activities against BA.2 and BA.5 in the WT+BA.1 group were higher than those of the WT group in both pseudotyped and live virus assays. Vaccine-related symptoms, including systemic and local symptoms, were strongly correlated with anti-RBD antibody levels and neutralizing titers. In conclusion, the second booster dose of the bivalent (WT/Omicron BA.1) vaccine induced higher neutralizing activity against BA.2 and BA.5 than that of the original monovalent vaccine. IMPORTANCE Although Omicron BA.1-containing bivalent vaccines have been authorized, real-world data validating their safety and antibody responses remain scarce. We conducted a prospective longitudinal study to assess the safety, immunogenicity, and reactogenicity of the second booster dose with the Omicron BA.1 bivalent vaccine in health care workers. Compared with the original monovalent vaccine, the bivalent (WT+BA.1) vaccine elicited higher levels of neutralizing antibodies against the Omicron BA.2 and BA.5 subvariants. The frequency of adverse events after the second booster dose was similar to that of the monovalent vaccine. BA.5-neutralizing antibodies induced by the bivalent Omicron BA.1-containing vaccine were expected to decline. A prospective longitudinal study should be performed to determine the persistence of the humoral immunity.

One major observation I have is that the authors have misinterpreted the data at the time they make the main conclusion, since VAX efficacy was not measured in the present study; it is all about immunogenicity. This overstatement has to be corrected all along the manuscript including the title. Line 1: "Efficacy of the wild-type/Omicron BA.1 bivalent vaccine as the second booster dose against Omicron BA.2 and BA.5" Line 60: "However, there is a lack of real-world data on the effectiveness of bivalent vaccines as second boosters on the dominant Omicron sublineages, including BA.2 and BA.5" this is true as introductory statement, but then DO NOT mix it with your conclusion, since not efficacy was assessed here. Again in line 111: "These bivalent vaccines have already been used, but not much information is available on their clinical efficacy." Here in line 113-115, you postulate both concepts in one sentence: "It is crucial to assess newly authorized vaccines for different communities and provide an accurate understanding of their efficacy and side effects. We conducted a prospective longitudinal study to assess the safety, immunogenicity, and reactogenicity ...". Lack of data on efficacy is true, but you did nothing to improve that in the present study, so focus on what you are solving assessing here. The first sentence of the discussion is closer to what I think is correct statement for this study: Line 203-204: "Although Omicron BA.1-containing bivalent vaccines have been authorized,real-world data validating their safety and antibody responses remain scarce." Other limitations of the present work are the absence of a later measurement after the 2nd booster and the lack of testing NEUT activity against novel Omicron circulating variants (i.e., XBB.1.5, CH.1.1., CA.3.1., etc). These could be mentioned in the discussion. Related to this last subject: discussion could be enriched with the incorporation of recent studies exploring the immunogenicity of monovalent vs bivalent boosting approaches. One example for this is the recent preprint by Shan-Lu Liu "Extraordinary evasion of Nab response by XBB.1.5, CH.1.1., CA.3.1 variants" doi: https://doi.org/10.1101/2023.01.16.524244. There are other references, including peer-reviewed ones, about this particular subject.
In the abstract: Line 68-70: "Although the neutralization activity of the pooled sera of the WT+BA.1 group was the lowest against BA.5, the activities against BA.2 and BA.5 were higher than those of the WT group in both pseudotyped and live virus assays." This sentence is confusing and I do not observe that neutralization activity of the pooled sera of the WT+BA.1 group was the lowest against BA.5 (FIGURE 3).

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Responses to reviewers' comments
We thank the reviewers for their incisive comments and suggestions. Below are our responses in red. The revised manuscript and our responses were checked for grammar and edited by professional English editing service before re-submission.

Reviewer 1
Line 230: Please specify the duration instead of just mentioning few months.

Response:
We agree with the reviewer's comment. We specified the duration and revised the following sentence as suggested: "In fact, vaccine effectiveness against infection waned remarkably within three months of administration in clinical settings (19,20)." (Line 239-241) Line 240-241: Please mention the percentage of participants instead of mentioning many participants.
Response: Thank you for your valuable suggestion. We specified the percentage and revised the following sentence as suggested: "Furthermore, over 66.7% of participants who experienced symptoms after the first booster dose showed the same symptoms after the second booster dose." (Line 250-251) Line 269: The authors have mentioned -were not randomly divided in two groups. Please rephrase the sentence.

Response:
We are grateful for the valuable comments. We rephrased and added the following sentences to explain why only a small group of individuals received the bivalent vaccine. "In the present study, participants who provided blood samples at least once after the third dose were eligible for analysis and were voluntarily divided into two groups: those who received the original mRNA-1273 vaccine and those who received the recently authorized Pfizer BNT162b2 bivalent Omicron BA.1-containing vaccine as the second booster (fourth dose). (Lines 283-287) "The second booster dose had been promoted in our hospital according to the national vaccine campaigns before the bivalent vaccine was authorized. Therefore, most healthcare workers (HCWs) had already received the second booster dose of the original monovalent vaccine, while those who had waited for the bivalent vaccines received the bivalent vaccine as a second booster dose later." (Lines 289-294) Line 95-96: Reference for the statement needs to be provided.

Response:
We agree with the reviewer's comment. We revised the following sentence and added the references, as suggested. "Vaccination programs against COVID-19 have been conducted worldwide; however, vaccine coverage varies per country based on the social circumstances of each country Response: Thank you for your valuable suggestions. We rephrased the following sentence as suggested: "These bivalent vaccines have already been used, but not much information is available on their immunogenicity and safety compared to the monovalent vaccines." (Lines 109-111) One of the other major limitation of the study is no data regarding the number of participants who became infected after receiving the second booster dose with the prevailing Omicron variants at that timepoints.

Response:
We are grateful for the valuable comment. As you and reviewer 2 thoughtfully and insightfully suggested, the vaccine efficacy was not assessed in the present study. Because the follow-up time after the second booster dose was limited in the present study, we should conduct a follow-up study to monitor the incidences of symptomatic and asymptomatic SARS-CoV-2 infections in both groups. We added the following sentences as one of the major limitations. "Fourth, the present study did not assess the vaccine efficacy and investigate the number of participants who infected after receiving the second booster dose because the The authors describe a couple of major limitation related to de present study (i.e., the small number of individuals receiving the bivalent vaccine, thee age range, the absence of background information as for example comorbidities, etc.). This is a prospective study and the continuation of previously published data: Effectiveness of the third dose of BNT162b2 vaccine on neutralizing Omicron variant in the Japanese population. J Infect Chemother 28:1273-8. The present study adds novel and important information as it is the immunogenicity of bivalent vaccine, a longer follow-up period after previous boost, etc. This manuscript is overall, well written and methodologically correct. The English could be improved in order to make it more readable, though.
One major observation I have is that the authors have misinterpreted the data at the time they make the main conclusion, since VAX efficacy was not measured in the present study; it is all about immunogenicity. This overstatement has to be corrected all along the manuscript including the title.
Response: Thank you for your valuable suggestions, and we sincerely apologize for our misleading conclusion. We rephased it throughout the manuscript as follows: Line 1: "Efficacy of the wild-type/Omicron BA.1 bivalent vaccine as the second booster dose against Omicron BA.2 and BA.5"

Response:
We are grateful for the valuable comments. We revised the title to "Neutralizing antibody response of the wild-type/Omicron BA.1 bivalent vaccine as the second booster dose against Omicron BA.2 and BA.5" (Lines 1-2) Line 60: "However, there is a lack of real-world data on the effectiveness of bivalent vaccines as second boosters on the dominant Omicron sublineages, including BA.2 and BA.5" this is true as introductory statement, but then DO NOT mix it with your conclusion, since not efficacy was assessed here.

Response:
We agree with the reviewer's comment. We revised the following sentence as suggested: "However, there is a lack of real-world data on the immunogenicity of bivalent vaccines as second boosters against the dominant Omicron sublineages, including BA.2 and BA.5." (Line 59-61) Again in line 111: "These bivalent vaccines have already been used, but not much information is available on their clinical efficacy." Response: Thank you for your suggestion. We revised the following sentence: "These bivalent vaccines have already been used, but not much information is available on their immunogenicity and safety compared to the monovalent vaccines." (Lines 109-111) Here in line 113-115, you postulate both concepts in one sentence: "It is crucial to assess newly authorized vaccines for different communities and provide an accurate understanding of their efficacy and side effects. We conducted a prospective longitudinal study to assess the safety, immunogenicity, and reactogenicity ...". Lack of data on efficacy is true, but you did nothing to improve that in the present study, so focus on what you are solving assessing here.

Response:
We are grateful for the valuable comments. We revised the following sentence to focus on what was assessed and evaluated in the present study. "It is crucial to assess newly authorized vaccines in real-world clinical settings and provide an accurate understanding of their humoral immune response to circulating variants and side effects." (Lines 112-114) The first sentence of the discussion is closer to what I think is correct statement for this study: Line 203-204: "Although Omicron BA.1-containing bivalent vaccines have been authorized, real-world data validating their safety and antibody responses remain scarce." Response: According to your thoughtful comments, we revised the following sentences and added the reference (14)  We also revised the following sentence in the Discussion section: "Similar to our previous studies about the immunogenicity and safety following primary and booster vaccinations (25, 29), a positive relationship was observed between higher antibody responses and some specific adverse reactions." (Lines 247-250) Other limitations of the present work are the absence of a later measurement after the 2nd booster and the lack of testing NEUT activity against novel Omicron circulating variants (i.e., XBB.1.5, CH.1.1., CA.3.1., etc). These could be mentioned in the discussion.
Related to this last subject: discussion could be enriched with the incorporation of recent studies exploring the immunogenicity of monovalent vs bivalent boosting approaches. One example for this is the recent preprint by Shan-Lu Liu "Extraordinary evasion of Nab response by XBB.  (24). It is, therefore, critical to monitor viral evolution and the impact on the immunogenicity and efficacy of the bivalent vaccines." (Lines 222-234) "Third, the long-term persistence of neutralizing antibody levels after the second booster dose was not assessed, and neutralization activity against novel Omicron circulating variants (BQ.