Original SARS-CoV-2 monovalent and Omicron BA.4/BA.5 bivalent COVID-19 mRNA vaccines: phase 2/3 trial interim results

This ongoing, open-label, phase 2/3 trial compared the safety and immunogenicity of the Omicron BA.4/BA.5-containing bivalent mRNA-1273.222 vaccine with the ancestral Wuhan-Hu-1 mRNA-1273 as booster doses. Two groups of adults who previously received mRNA-1273 as primary vaccination series and booster doses were enrolled in a sequential, nonrandomized manner and received single-second boosters of mRNA-1273 (n = 376) or bivalent mRNA-1273.222 (n = 511). Primary objectives were safety and the noninferiority or superiority of neutralizing antibody (nAb) responses against Omicron BA.4/BA.5 and ancestral SARS-CoV-2 with the D614G mutation (ancestral SARS-CoV-2 (D614G)), 28 days post boost. Superiority and noninferiority were based on prespecified success criteria (lower bounds of 95% CI > 1 and < 0.677, respectively) of the mRNA-1273.222:mRNA-1273 geometric mean ratios. Bivalent Omicron BA.4/BA.5-containing mRNA-1273.222 elicited superior nAb responses against BA.4/BA.5 versus mRNA-1273 and noninferior responses against ancestral SARS-CoV-2 (D614G) at day 29 post boost in participants without detectable prior SARS-CoV-2 infection. Day 29 seroresponses against Omicron BA.4/BA.5 were higher for mRNA-1273.222 than for mRNA-1273 and similar against ancestral SARS-CoV-2 (D614G), both meeting noninferiority criterion. The safety profile of mRNA-1273.222 was similar to that previously reported for mRNA-1273 with no new safety concerns identified. Continued monitoring of neutralization and real-world vaccine effectiveness are needed as additional divergent-virus variants emerge. ClinicalTrials.gov registration: NCT04927065.

Herein, we summarize the interim safety and immunogenicity results of the mRNA-1273.222clinical study and we include neutralization information against variants that emerged after the Omicron BA.4/ BA.5 wave.Rapid evaluation of variant-containing COVID-19 vaccines is important as vaccine updates are likely to be further recommended to enhance protection against COVID-19.). a 379 participants were enrolled and received mRNA-1273; two participants had previously received the primary series but not a first booster dose and another participant had a major protocol deviation, and three were excluded from all analysis sets.Data cutoff dates were 23 September 2022 for mRNA-1273.222at the day 29 interim analysis and 6 July 2022 for the within-study noncontemporaneous mRNA-1273 at the day 91 interim analysis 6 .b, Analysis populations.The full analysis set consisted of all participants who received study vaccine.The safety set consisted of all participants who received study vaccine and was used for all safety analyses except for solicited adverse reactions which were assessed in the solicited safety set.HIV, human immunodeficiency virus.a The per-protocol set for immunogenicity consisted of all participants in the full analysis set who received the planned dose of study vaccination and had antibody data available at prebooster and day 29 and no major protocol deviations.b Seven participants in the mRNA-1273.222and eight participants in the mRNA-1273 arm of the perprotocol immunogenicity set had missing prebooster SARS-CoV-2 information.c Prior SARS-CoV-2 infection based on positive RT-PCR and/or serology test at prebooster baseline.d The per-protocol immunogenicity negative set (PPISnegative) consists of participants in the per-protocol set for immunogenicity (PPIS) who have no serologic or virologic evidence of SARS-CoV-2 infection at prebooster baseline, that is, who are SARS-CoV-2 infection negative, based on both negative RT-PCR tests for SARS-CoV-2 and negative SARS-CoV-2 nucleocapsid antibody test, and is the primary analysis set for immunogenicity.A total of 379 participants received a second booster dose of 50 µg mRNA-1273; two participants had previously received the primary series but not a first booster dose, and another participant had a major protocol deviation.These three participants were excluded from all analysis sets.

Safety
The overall safety profile of the mRNA-1273.222booster is similar to that of the previously described 50 µg mRNA-1273 historical group at 28 days post booster dose 5,6 .Median durations of follow-up days (IQR) were 37.0 (33.0-39.0)for the mRNA-1273.222participants and 127 (125-132) for the historical mRNA-1273 group 6 .Previously reported solicited local and systemic adverse reactions within 7 days after injection for mRNA-1273 are shown in Fig. 2 for comparison with those of mRNA-1273.222.The most frequently reported local adverse reaction after administration of mRNA-1273.222was injection-site pain.The most frequent systemic reactions were fatigue, headache, myalgia and arthralgia (Fig. 2 and Supplementary Table 4).The majority of solicited adverse reactions were mild-to-moderate (grades 1-2) and the most common grade 3 reactions were fatigue and myalgia; no grade 4 reactions occurred.Median duration days (IQR) were 3 (2-4) for local and 3 (1-5) for systemic adverse reactions and persistence beyond 7 days occurred in 1.6% of participants for local reactions and 7.7% for systemic reactions (Supplementary Table 5).Grade 3 events of erythema (1 (0.2%)), headache (1 (0.2%)) and fatigue (4 (0.8%)) persisted beyond 7 days.The frequency of local and systemic adverse reactions was 84.7% and 77.3%, respectively, in participants without prior SARS-CoV-2 infection and 81.2% and 69.6% in those with prior SARS-CoV-2 infection.
The incidence of unsolicited adverse events regardless of the relationship to vaccination ≤28 days after the booster dose of mRNA-1273.222was 23% (Supplementary Table 6) and similar to that reported previously for mRNA-1273 (ref.6); the incidence of unsolicited adverse events considered to be related to study vaccination by the investigator was 8%.Three (0.6%) participants in the mRNA-1273.222group experienced serious adverse events (anginal equivalent and syncope, anemia, fatal event of subarachnoid hemorrhage); none were considered related to study vaccination by the investigators.Medically attended adverse events occurred in 14% of mRNA-1273.222participants; none were considered related to study vaccination by investigators.Two grade 3 events of fatigue, considered related to study vaccination, were reported.No events of myocarditis or pericarditis and no adverse events leading to study discontinuation occurred in this interim analysis.3) and 0, both meeting the prespecified noninferiority criterion.Therefore, all primary and immunogenicity objectives were met (Supplementary Fig. 1).Neutralizing titers against Omicron BA.4/BA.5 and ancestral SARS-CoV-2 (D614G) were also consistently higher with mRNA-1273.222than with mRNA-1273 at day 29 among those ≥65 years and 18-<65 years of age (Extended Data Fig. 2).In participants with evidence of prior SARS-CoV-2 infection, GMTs were higher following the mRNA-1273.222than mRNA-1273 booster against both Omicron BA.4/BA.5 and ancestral SARS-CoV-2 (D614G) with GMRs (95% CI) of 5.11 (4.10−6.36)and 1.84 (1.56−2.18),respectively (Fig. 3 and Supplementary Table 7).
To explore whether time intervals between the first and second booster doses influenced the post booster nAb GMTs, antibody responses against Omicron BA.4/BA.5 and ancestral SARS-CoV-2 (D614G) across quartile time intervals for the mRNA-1273.222and mRNA-1273 groups were analyzed among participants with no previous infection (Extended Data Fig. 3 and Supplementary Table 8).Antibody titers did not appear to increase as the interval between prior doses increased within each treatment group.The GMTs and geometric mean fold-rise (GMFR) for mRNA-1273.222increased at the day 258-289 interval, then decreased over time to ≥312 days against both Omicron BA.4/BA.5 and ancestral SARS-CoV-2 (D614G).The GMTs and GMFR of mRNA-1273 remained generally consistent as the intervals increased.Given the emergence of the Omicron BQ.1.1,XBB.1 and XBB.1.5sublineages with the potential for immune escape, neutralization at day 29 against these variants was assessed in exploratory analyses for mRNA-1273.222as described in the Methods (Fig. 4 and Supplementary Table 11).In a randomly selected subset of mRNA-1273.222participants without prior detectable SARS-CoV-2 infection (n = 40), prebooster GMTs (95% CI) against BA.4/5, BQ.   7.

Discussion
The rapid evolution of antigenically divergent SARS-CoV-2 variants precipitated development of booster immunizations to maintain protection against COVID-19 and regulatory agencies have authorized variant-containing bivalent booster vaccines globally [9][10][11]21 . Givn the pace of viral evolution and need for agility, regulatory agencies authorized Omicron BA.4/BA.5 bivalent boosters based on BA.1 bivalent vaccine clinical trial data 5,6 and BA.4/BA.5 bivalent vaccine animal study information available at the time.The present study provides key clinical safety and immunogenicity data which support authorization of the Omicron BA.4/BA.5 bivalent booster mRNA-1273.222.The rapid manufacturing and deployment of this vaccine, within months, is in response to the speed with which SARS-CoV-2 variants of consequence (BA.4/BA.5)spread and demonstrates that flexible vaccine platforms, such as mRNA, are an important tool as divergent SARS-CoV-2 variants continue to emerge.
The 50 µg bivalent vaccine mRNA-1273.222elicited higher BA.4/ BA.5 neutralizing antibody responses compared to the original 50 µg mRNA-1273 28 days after the booster dose, in participants with and without evidence of SARS-CoV-2 infection on the day of the booster dose.Additionally, mRNA-1273.222exhibited cross-neutralization against divergent variants which are not contained in the vaccine, such as BQ.1.1,XBB.1 and XBB.1.5,although the antibody titers for these variants were lower compared to BA.4/BA.5.These results are consistent with cross-neutralization observed after the BA.1-Omicron-containing mRNA-1273.214bivalent vaccine dose as titers against Omicron BA.4/ BA.5, BQ.1.1 and XBB.1 were lower compared to the matched BA.1 variant in the mRNA-1273.214vaccine 6 .Results from a randomized, active-controlled clinical trial comparing an Omicron BA.1-containing booster vaccine with mRNA-1273, suggest a trend towards improved vaccine efficacy with the variant-containing booster, particularly when the variant is more closely antigenically matched to the variant in the vaccine 16 .The mechanism of immune responses elicited by bivalent boosters is yet to be elucidated.Studies suggest that immunization with Omicron-containing vaccines elicits new germinal centers and de novo B cell populations that likely contribute to enhanced neutralization, and that pre-existing T cell immune responses also contribute to immune responses to SARS-CoV-2 variants 7,22 .
Our interim results also indicate that the incidence of adverse reactions with the Omicron BA. 4  Percentages were based on the number of participants with nonmissing data at baseline and the corresponding timepoint, 95% CI was calculated using the Clopper-Pearson method.g The SRR difference is a calculated common risk difference using inverse-variance stratum weights and the middle point of Miettinen-Nurminen confidence limits of each one of the stratum risk differences.The stratified Miettinen-Nurminen estimate and the CI cannot be calculated when the SRR in both groups is 100%, absolute difference is reported.

Article
https://doi.org/10.1038/s41591-023-02517-yvaccines which appear to be similar to that of the original mRNA-1273 vaccine, and suggest that safety is consistent for mRNA-1273 vaccines regardless of the bivalent variant modification.Longer-term evaluation of the safety of mRNA-1273.222continues in this ongoing study.
Limitations include that the study was not randomized.Given that the Omicron BA.4/BA.5 vaccine update had already been recommended as the standard-of-care 17 , a contemporaneous mRNA-1273 comparator group was not enrolled; instead a historical mRNA-1273 was used to compare the immune responses between mRNA-1273.222and mRNA-1273 (ref.18).Although under such circumstances, use of a historical comparator evaluated using the same immunogenicity assay is advised by the regulatory guidance 19 , differences between the two vaccine groups limit the interpretation of our results despite model-adjusting the neutralizing antibody responses to account for potential confounding factors.The different intervals between prior booster doses and the likelihood that prebooster infections were caused by different circulating variants during the enrollment times between the two groups (Supplementary Fig. 2) could have a distinct impact on the development of immune responses in each group.We used the antinucleocapsid antibody test to assess previous SARS-CoV-2 infection and although a vaccine-induced reduction in seroconversion is possible when using this test 25,26 , the distinction between uninfected and infected persons is becoming less relevant given that the majority of the global population has been infected with SARS-CoV-2, with seroprevalence increases from 16% in February 2021 to 67% by October 2021 (ref.27).Overall seroprevalence rates in high-income countries through March 2022 were 95.9% (92.3-97.8%) in Europe and 99.8% (99.7-99.9%) in the Americas from 4.3% (3.4-5.5%) and 3.6% (2.5-5.2%),respectively in June 2020 (ref.28).mRNA-1273.222antibody responses against BQ.1.1,XBB.1 and XBB.1.5were assessed in subgroups and at only day 29 post booster to enable rapid evaluation, and responses were not assessed for mRNA-1273 as use of this booster has been withdrawn.XBB lineage variants subsequent to XBB.1 and XBB.1.5(for example XBB.1.16)are antigenically similar to XBB.1.5 and neutralization was not assessed separately 6,29,30 .Lastly, this study was not designed to evaluate vaccine effectiveness and while the low number of COVID-19 events post boost precludes an interpretation of efficacy, the incidences are similar to those previously reported for Omicron BA.1 bivalent and original vaccine groups (Supplementary Table 12) 5,6 , and to randomized relative vaccine efficacy trials as well as real-world vaccine effectiveness data 8,16,[31][32][33][34] .
In conclusion, given that SAS-CoV-2 variants are most likely to continue to emerge, our study provides a model for rapid clinical evaluation of a COVID-19 vaccine when a booster update and deployment is needed.Our results indicate that the Omicron BA.4/BA.5 bivalent mRNA-1273.222vaccine increased neutralizing antibody responses against Omicron variants, with no new safety concerns compared to the original vaccine mRNA-1273.Continuous monitoring of cross-neutralization and vaccine effectiveness are needed to ensure updated vaccination strategies against COVID-19.

Online content
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41591-023-02517-y.

Trial registration
The study protocol was approved by the Institutional Review Board on 26 May 2021, and enrollment in part A of the study was initiated on 28 May 2021, before the trial posting date of 14 June 2021 on the clintrials.gov registration website as we were emergently advancing COVID-19 booster vaccine candidates to combat the SARS-CoV-2 pandemic (the objective of this trial).Nonetheless, the trial was registered within 21 days after the first participant was enrolled, compliant per clinicaltrials.govguidance (https://clinicaltrials.gov/ct2/manage-recs/fdaaa).Additionally, in parts F and H of the study presented in this manuscript, the 890 participants were enrolled during 10-23 August 2022 for mRNA-1273.222(Part H) and during 18 February-8 March 2022 (Part F, cohort 2) for mRNA-1273.Moderna received EUA and/or approvals of booster vaccine mRNA-1273 50 µg and bivalent booster candidates mRNA-1273.214by regulatory agencies 17 as well as for mRNA-1273.222presented in this part (H) of the trial 18 .
The trial is being conducted across 23 US sites, in accordance with the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Good Clinical Practice guidelines.The central Institutional Review Board (Advarra, Inc., 6100 Merriweather Drive, Columbia, MD 21044) approved the protocol and consent forms.All participants provided written informed consent.The study was funded by the sponsor, Moderna, Inc. and was involved in the study design as well as the collection, analysis and interpretation of the data.

Participants
Adults with a known history of SARS-CoV-2 infection ≤3 months from screening were excluded.Additional inclusion/exclusion criteria specific to Part H participant eligibility include: Inclusion criteria.Each participant must meet all of the following criteria to be enrolled in this study: (1) Male or female, at least 18 years of age at the time of consent (Screening Visit).
(2) Investigator's assessment that participant understands and is willing and physically able to comply with protocol-mandated follow-up, including all procedures.(3) Participant has provided written informed consent for participation in this study, including all evaluations and procedures as specified in this protocol.( 4) Female participants of nonchildbearing potential may be enrolled in the study.Nonchildbearing potential is defined as surgically sterile (history of bilateral tubal ligation, bilateral oophorectomy, hysterectomy) or postmenopausal (defined as amenorrhea for ≥12 consecutive months before Screening (day 0) without an alternative medical cause).A follicle-stimulating hormone level may be measured at the discretion of the investigator to confirm postmenopausal status.( 5) Female participants of childbearing potential may be enrolled in the study if the participant fulfills all of the following criteria: • Has a negative pregnancy test on the day of vaccination (day 1) • Has practiced adequate contraception or has abstained from all activities that could result in pregnancy for at least 28 days before day 1 • Has agreed to continue adequate contraception through 3 months following vaccination • Is not currently breastfeeding (Adequate female contraception is defined as consistent and correct use of a Food and Drug Administration-approved contraceptive method in accordance with the product label) (6) Participant must have been either previously enrolled in the phase 3 mRNA-1273 COVE trial 23,24 , must have received two doses of mRNA-1273 in that study, with his/her second dose at least 6 months before enrollment in this study, and must be currently enrolled and compliant in that study (that is, has not withdrawn or discontinued early); or participant must have received two doses of mRNA-1273 under the EUA with their second dose at least 6 months before enrollment in this study; or have received a two-dose primary series of mRNA-1273 followed by a 50 µg booster dose of mRNA-1273 in the mRNA-1273 COVE trial or under EUA at least 3 months before enrollment this study; and able to provide proof of vaccination status at the time of screening (day 1).
Exclusion criteria.Participants meeting any of the following criteria at the Screening Visit, unless noted otherwise, will be excluded from the study: (1) Had substantial exposure to someone with SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) in the past 14 days, as defined by the CDC as a close contact of someone who has COVID-19).( 2) Has known history of SARS-CoV-2 infection within 3 months before enrollment.(3) Is acutely ill or febrile (temperature ≥38.0 °C (100.4 °F)) less than 72 hours before or at the Screening Visit or day 1.Participants meeting this criterion may be rescheduled and will retain their initially assigned participant number.(4) Currently has symptomatic acute or unstable chronic disease requiring medical or surgical care, to include substantial change in therapy or hospitalization for worsening disease, at the discretion of the investigator.( 5) Has a medical, psychiatric or occupational condition that may pose additional risk as a result of participation, or that could interfere with safety assessments or interpretation of results according to the investigator's judgment.
https://doi.org/10.1038/s41591-023-02517-y(6) Has a current or previous diagnosis of immunocompromising condition to include human immunodeficiency virus, immune-mediated disease requiring immunosuppressive treatment or other immunosuppressive condition.(7) Has received systemic immunosuppressants or immune-modifying drugs for >14 days in total within 6 months before Screening (for corticosteroids ≥10 mg day −1 of prednisone equivalent) or is anticipating the need for immunosuppressive treatment at any time during participation in the study.(8) Has known or suspected allergy or history of anaphylaxis, urticaria or other important adverse reaction to the vaccine or its excipients.( 9) Has a documented history of myocarditis or pericarditis within 2 months before Screening Visit (day 0). ( 10) Coagulopathy or bleeding disorder considered a contraindication to intramuscular (IM) injection or phlebotomy.(11) Has received or plans to receive any licensed vaccine ≤28 days before the injection (day 1) or a licensed vaccine within 28 days before or after the study injection, with the exception of influenza vaccines, which may be given 14 days before or after receipt of a study vaccine.( 12) Has received systemic immunoglobulins or blood products within 3 months before the Screening Visit (day 0) or plans for receipt during the study.( 13) Has donated ≥450 ml of blood products within 28 days before the Screening Visit or plans to donate blood products during the study.( 14) Plans to participate in an interventional clinical trial of an investigational vaccine or drug while participating in this study.( 15) Is an immediate family member or household member of study personnel, study site staff or sponsor personnel.( 16) Is currently experiencing an SAE in COVE trial at the time of screening for this study.

Safety assessment
The primary safety objective was to evaluate the safety and reactogenicity of 50 µg mRNA-1273.222when administered as a second booster dose.Safety assessments included solicited local and systemic adverse reactions ≤7 days and unsolicited adverse events ≤28 days post booster administration, and serious adverse events, adverse events leading to discontinuation from study vaccine and/or participation, medically attended adverse events and adverse events of special interest from day 1 through the entire study period (~6 months).
Immunogenicity assays.SARS-CoV-2 spike-pseudotyped virus neutralization assay.SARS-CoV-2 nAb in samples were assessed using a validated SARS-CoV-2 spike (S)-pseudotyped virus neutralization assay (PsVNA) in 293/ACE2 cells 14 .The PsVNA quantifies nAb using lentivirus particles that express full-length spike proteins on their surface, and contain a firefly luciferase reporter gene for quantitative measurements of infection in transduced 293T cells expressing high levels of ACE2 (293T/ACE2 cells) by relative luminescence units (RLU).Serial dilution of antibodies was used to produce a dose−response curve.Neutralization was measured as the serum dilution at which RLU was reduced by 50% (ID 50 ) relative to mean RLU in virus control wells (cells + virus but no sample) after subtraction of mean RLU in cell control wells (cells only).Positive controls were included on each assay plate to follow stability over time.

Incidence of SARS-CoV-2 infections
The incidence of symptomatic and asymptomatic SARS-CoV-2 infection was an exploratory objective (Supplementary Table 1).Vaccine effectiveness was not assessed in this trial, but COVID-19 and SARS-CoV-2 infection were actively surveilled through weekly contact and blood draws.SARS-CoV-2 infection is a combination of symptomatic infection (COVID-19) and asymptomatic SARS-CoV-2 infection for participants with negative SARS-CoV-2 status prebooster.Symptomatic infection was evaluated using the primary case definition in the COVE study 24,36 and a secondary case definition based on the Centers for Disease Control and Prevention (CDC) criteria 37 .Asymptomatic SARS-CoV-2 infection was defined as a positive RT-PCR test or a positive serologic test for antinucleocapsid antibody after a negative test at the time of enrollment, in the absence of symptoms.

Statistical analysis
Statistical analysis populations are detailed in Supplementary Table 2 and Fig. 1b.Safety was evaluated in the safety set consisting of all participants who received mRNA-1273.222and solicited adverse reactions in all participants and by prebooster SARS-CoV-2 infection status in the solicited safety set using the same methodology as described for the safety evaluation of the 50 µg mRNA-1273 comparator group 5,6 .The per-protocol immunogenicity set consists of all participants who received the planned booster doses, had prebooster and day 29 antibody data available and no major protocol deviations.Primary immunogenicity objectives were assessed in the per-protocol immunogenicity-SARS-CoV-2-negative set (primary analysis set), those participants without evidence of prior SARS-CoV-2 infection (negative RT-PCR and negative binding antibody against SARS-CoV-2 nucleocapsid at prebooster day 1).Analyses were also performed in participants who had evidence of SARS-CoV-2 infection prebooster (positive binding antibody against SARS-CoV-2 nucleocapsid or positive RT-PCR at booster day 1).
Immunogenicity analysis.The primary immunogenicity objectives were evaluated using a prespecified hierarchical approach which compared 50 µg mRNA-1273.222to 50 µg mRNA-1273 (active control arm in part F, Cohort 2) as second booster doses (Supplementary Fig. 1).For the primary objective on immune response, five hypotheses were specified to be evaluated at day 29 post booster (online protocol and SAP and Supplementary Fig. 1).Interim analysis results for the day 29 hypotheses are presented in this report.Below are the five hypotheses for day 29: (1) 50 µg mRNA-1273.222,as a second booster dose, against Omicron BA.The primary immunogenicity objectives were assessed in the per-protocol immunogenicity-SARS-CoV-2-negative set with a target enrollment of approximately 500 participants for 50 µg mRNA-1273.222.Assuming 40% of participants were excluded from the per-protocol immunogenicity-SARS-CoV-2-negative set (due to a SARS-CoV-2 infection prebooster), with approximately 300 participants in the 50 µg mRNA-1273.222and 260 participants in the 50 µg mRNA-1273 (Part F, Cohort 2-50 µg mRNA-1273) arms in the per-protocol immunogenicity-SARS-CoV-2-negative set, there is approximately 60% power to demonstrate the primary immunogenicity objectives with an alpha of 0.05 (two-sided) at day 29.The assumptions were that the true GMR (mRNA-1273.222second booster versus mRNA-1273 second booster) against Omicron BA.4/5 is 1.5, GMR (mRNA-1273.222second booster versus mRNA-1273 second booster) against ancestral SARS-CoV-2 D614G is 1, the standard deviation of the log-transformed titer is 1.5 and the noninferiority margin for GMR is 1.5.The true SRR against Omicron BA.4/5 after mRNA-1273.222as a second booster dose is 95% (same assumption for 50 µg mRNA-1273), and noninferiority margin for SRR difference against Omicron BA.4/5 is 5%.The true SRR against ancestral SARS-CoV-2 D614G after mRNA-1273.222as a second booster dose is 95% (same assumption for 50 µg mRNA-1273), and the noninferiority margin for SRR difference against ancestral SARS-CoV-2 D614G is 10%.
For the primary immunogenicity objective, an interim analysis was planned at day 29 with a two-sided alpha (0.05) allocated for immunogenicity hypothesis testing.The primary immunogenicity objective was considered met for noninferiority when the lower bound of the 95 CI of the GMR was >0.667 and the SRR difference is >-10%.Superiority was considered met when the lower bound of the 95 CI of GMR is >1.These noninferiority and superiority criteria were chosen based on FDA guidelines 19,38 .Day 29 interim analysis results are presented.The within-study noncontemporaneous mRNA-1273 comparator group (Part F, cohort 2) was enrolled during 18 February-8 March 2022 and data for this group, based on the data cutoff date of 6 July 2022 at the day 91 interim analysis, is used for the immunogenicity comparison.The superiority of the antibody response against Omicron BA.4/BA.5 after a second booster dose of 50 µg mRNA-1273.222compared with 50 µg mRNA-1273 was evaluated only after meeting noninferiority criteria for the four primary objectives 39 : noninferiority of the antibody response against BA.4/BA.5 after the second booster doses of 50 µg mRNA-1273.222versus 50 µg mRNA-1273 based on GMR (1) and SRR difference (2), and noninferiority of the antibody response against ancestral SARS-CoV-2 (D614G) after the second booster doses of 50 µg mRNA-1273.222versus 50 µg mRNA-1273 based on GMR (3) and SRR difference (4).https://doi.org/10.1038/s41591-023-02517-yinfection was the earlier date of positive serology test result based on bAb specific to SARS-CoV-2 nucleocapsid due to infection, or positive RT-PCR, with absence of symptoms.The time to the asymptomatic SARS-CoV-2 infection was calculated as the date of asymptomatic SARS-CoV-2 infection minus the date of injection +1.

Symptomatic SARS-CoV-2 infection (COVID-19).
Symptomatic SARS-CoV-2 infection (COVID-19) was defined as the incidence of the first occurrence of symptomatic SARS-CoV-2 infection measured by RT-PCR of nasal swabs starting 14 days after the booster dose.Surveillance for COVID-19 symptoms is conducted via weekly contact and blood draw, and an illness visit to collect a nasopharyngeal swab was arranged for participants reporting COVID-19 symptoms.
Two definitions of symptomatic SARS-CoV-2 infection (COVID-19) were used including the primary case definition in the COVE trial 23,24 based on a positive post baseline RT-PCR result AND at least TWO systemic symptoms (fever (≥38 °C/≥100.4°F), chills, muscle and/or body aches (not related to exercise), headache, sore throat, new loss of taste/ smell; OR at least ONE of respiratory signs/symptoms (cough, shortness of breath and/or difficulty breathing, OR clinical or radiographical evidence of pneumonia).The second case definition is based on CDC criteria for symptomatic disease defined as a positive post baseline RT-PCR test AND at least ONE systemic or respiratory symptoms (fever (≥38 °C/≥100.4°F), chills, cough, shortness of breath and/or difficulty breathing, fatigue, muscle and/or body aches (not related to exercise), headache, new loss of taste/smell, sore throat, congestion, runny nose, nausea, vomiting or diarrhea) 37 .
The date of a documented COVID-19 case was the later date of a symptom and the date of a positive RT-PCR test, and the two dates were to be within 14 days of each other.The time to the first occurrence of COVID-19 is calculated as the date of documented COVID-19 minus the date of injection +1.Cases are counted starting 14 days after the injection (date of documented COVID-19 minus date of the injection ≥14).
All analyses were conducted using SAS v.9.4 or higher.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Data associated with this study are provided in the paper or supplementary materials.Source data for Figs.

Fig. 1 |
Fig. 1 | Enrollment and analysis populations in the trial.a, Trial profile.Eligible participants were sequentially enrolled as two cohorts to receive single-second booster doses of 50 µg mRNA-1273 (enrolled during 18 February-8 March 2022) or 50 µg mRNA-1273.222(enrolled during 10-23 August 2022).a 379 participants were enrolled and received mRNA-1273; two participants had previously received the primary series but not a first booster dose and another participant had a major protocol deviation, and three were excluded from all analysis sets.Data cutoff dates were 23 September 2022 for mRNA-1273.222at the day 29 interim analysis and 6 July 2022 for the within-study noncontemporaneous mRNA-1273 at the day 91 interim analysis6 .b, Analysis populations.The full analysis set consisted of all participants who received study vaccine.The safety set consisted of all participants who received study vaccine and was used for all safety analyses except for solicited adverse reactions which were assessed in the solicited safety set.HIV, human immunodeficiency virus.a The per-protocol set for immunogenicity consisted of all participants in the full analysis set who

Fig. 3 |
Fig. 3 | nAb titers after 50 µg of mRNA-1273.222and mRNA-1273 administered as second booster doses.a,b, Observed nAb titers against Omicron BA.4/BA.5 (a) and ancestral SARS-CoV-2 (D614G) (b) after 50 µg of mRNA-1273.222and mRNA-1273 administered as second booster doses.Pseudovirus nAb GMTs are provided for all participants regardless of SARS-CoV-2 infection prebooster status, and those with and without previous SARS-CoV-2 infection prebooster.Data are from participants with nonmissing data at the timepoint.Seven participants in mRNA-1273.222and eight participants in the mRNA-1273 group were missing prebooster SARS-CoV-2 status in the per protocol immunogenicity set.Antibody values reported as below the LLOQ (18.5 (1.3 in log 10 scale) for ancestral SARS-CoV-2 (D614G) and 36.7 (1.6 in log 10 scale) for Omicron BA.4/BA.5)were replaced by 0.5 × LLOQ.Values greater than the upper limit of quantification (ULOQ 45,118 (4.7 in log 10 scale) for ancestral SARS-CoV-2 (D614G); 13,705 (4.1 in log 10 scale) for Omicron BA.4/BA.5)were replaced by the ULOQ if actual values were not available.95% CIs were calculated based on the t-distribution of the log-transformed values for GM value, then back-transformed to the original scale for presentation.Data for observed nAb GMTs by prior SARS-CoV-2 infection are provided in Supplementary Table7.

Table 1 | Demographics and study participant characteristics, safety set Characteristics n (%) a mRNA-1273.222 50 µg N = 511 mRNA-1273 50 µg N = 376
Time between second injection of mRNA-1273 in the primary series and the first booster of mRNA-1273 (days) a Percentages based on the number of participants in the safety set.bElecsysassayforbinding antibody to SARS-CoV-2 nucleocapsid.cPreboosterSARS-CoV-2statuswas positive if there was evidence of prior SARS-CoV-2 infection, defined as positive binding antibody against the SARS-CoV-2 nucleocapsid or positive RT-PCR at day 1; negative SARS-CoV-2 status was defined as negative binding antibody against the SARS-CoV-2 nucleocapsid and a negative RT-PCR at day 1.Articlehttps://doi.org/10.1038/s41591-023-02517-yImmunogenicityInparticipants without evidence of prior SARS-CoV-2 infection 28 days after the mRNA-1273.222and mRNA-1273 booster dose, respectively, the observed neutralizing antibodies (nAb), reported as

Table 2 | Primary immunogenicity analysis of Omicron BA.4/BA.5 and ancestral SARS-CoV-2 (D614G) after 50 µg of mRNA- 1273.222 and mRNA-1273 administered as second booster doses in participants with no prior SARS-CoV-2 infection
Antibody values assessed by pseudovirus neutralizing antibody assay reported as below the LLOQ (18.5 for ancestral SARS-CoV-2 (D614G) and 36.7 for Omicron BA.4/BA.5)are replaced by 0.5 × LLOQ.Values greater than ULOQ (45,118 for ancestral SARS-CoV-2 (D614G) and 13,705 for Omicron BA.4/BA.5)are replaced by the ULOQ if actual values are not available.Includes participants with no prior SARS-CoV-2 infection, primary analysis set.N1 = Number of participants with nonmissing data at baseline (prebooster) and the corresponding post baseline timepoint.a Number of participants with nonmissing data at the timepoint (prebooster baseline or post baseline).b GMT, GM FR of nAb at day 29 post baseline timepoint over predose day 1 with corresponding 95% CI based on the t-distribution of log-transformed values or difference in the log-transformed values for GMT value and GMT fold-rise, respectively, then back-transformed to the original scale.c Log-transformed antibody levels are analyzed using an ANCOVA model with the treatment variable as fixed effect, adjusting for age group (<65, ≥65 years) and prebooster titers.The resulting least squares (LS) means, difference of LS means and 95% CI are back-transformed to the original scale.d 95% CI was calculated by stratified Miettinen-Nurminen method adjusted by age group.e Exceeded noninferiority criteria and met superiority criteria including lower bound CI > 1 and testing sequence.f Seroresponse at a participant level defined as a change from <LLOQ to ≥4 × LLOQ, or at least a fourfold rise if baseline (preinjection 1/prebooster is ≥LLOQ; comparison to preinjection 1/prebooster baselines as indicated.

nAb titers against Omicron variants after 50 µg of mRNA-1273.222 administered as a second booster dose by prior SARS-CoV-2 infection status at prebooster
March 2022) or 50 µg bivalent mRNA-1273.222(enrolled between 10 August and 23 August 2022).Data are reported for the mRNA-1273 group (Part F, cohort 2) based on the data cutoff date of 6 July 2022 at the day 91 interim analysis 6 , and for mRNA-1273.222(Part H) based on the 29-day interim analysis data cutoff date of 23 September 2022.

Neutralizing Antibody Titers and Geometric Fold-Rises by Time Intervals.
2 and 3 and Extended Data Figs.2 and 3and the protocol and statistical analysis plan are provided as Supplementary Information.SARS-COV-2 variant sequences were obtained from GISAID Overview of Variants/Mutations, https:// covariants.org/variants(2023).As the trial is ongoing, access to patient-level data and supporting clinical documents by qualified external researchers may be available upon request and subject to review once the trial is complete.A materials transfer and/or data access agreement with the sponsor will be required for accessing shared data.Such requests can be made to Dr. Spyros Chalkias, Moderna Inc., 200 Technology Square, Cambridge, MA 02139, USA.Source data are provided with this paper.Distribution of neutralizing antibody titers (ID50 log 10 ) in the pseudovirus assay against Omicron BA.4/BA.5 (Panel A) and ancestral SARS-CoV-2 (D614G) (Panel B) are shown for serum samples collected before the second booster dose of 50-µg of mRNA-1273.222or50-µg of mRNA-1273 (prebooster), and at 28 days after the second booster dose (day 29) in those with and without prior SARS-CoV-2 infection.The circles are values from individual serum samples.Pseudovirus neutralizing antibody assay lower limits of quantification (LLOQ) are 18.5 (1.3 log 10 ) for ancestral SARS-CoV-2 [D614G] and 36.7 (1.6 log 10 ) for Omicron BA.4/BA.5;upperlimits of quantification (ULOQ) are 45,118 (4.7 log 10 ) for ancestral SARS-CoV-2 [D614G] and 13,705 (4.1 log 10 ) for Omicron BA.4/BA.5.Boxes and horizontal bars denote interquartile (IQR) ranges and median endpoint titers; whisker endpoints are the maximum and minimum values below or above the median ±1.5 times the IQR.Refer to Table2for the observed geometric mean titers and geometric mean fold-rises, and the estimated neutralizing antibody geometric mean titers and geometric mean ratios (ANCOVA model-based) following the second booster doses of 50-µg mRNA-1273 or 50-µg of mRNA-1273.214.Observed neutralizing antibody titers (GM titer log10) in the pseudovirus assay against Omicron BA.4/BA.5 (a) and ancestral SARS-CoV-2 (D614G) (b) are shown for serum samples collected before the second booster dose of 50-µg of mRNA-1273.222or 50-µg of mRNA-1273 (prebooster) (day 0), and at 28 days (day 29) after the second booster dose in all participants and those ≥18 to <65 or ≥65 years of age in the per-protocol immunogenicity set without evidence of prebooster SARS-CoV-2 infection.N=Number of participants with nonmissing data at the timepoint; CI=Confidence interval; GMT=geometric mean titer; LLOQ=lower limit of quantification; ULOQ=upper limit of quantification; PPIS=Per-protocol immunogenicity set.Antibody values assessed by pseudovirus neutralizing antibody assay reported as below the LLOQ (18.5 [1.3 in log10 scale] for ancestral SARS-CoV-2 (D614G) and 36.7 [1.6 in log10 scale] for Omicron BA.4/BA.5)are replaced by 0.5 × LLOQ.Values greater than ULOQ (45,118 [4.7 in log10 scale] for ancestral SARS-CoV-2 (D614G) and 13,705 [4.1 in log10 scale] for Omicron BA.4/BA.5)are replaced by the ULOQ if actual values are not available.Includes participants in the per-protocol immunogenicity set without evidence of prebooster SARS-CoV-2 infection (PPIS-negative).95% CIs were calculated based on the t-distribution of the log-transformed values for GM value, then back-transformed to the original scale for presentation.Neutralizing antibody titers against Omicron BA.4/BA.5 and ancestral SARS-CoV-2 (a) and geometric fold-rises from baseline levels (b) were assessed in participants without previous infection grouped by quartiles of dosing intervals between the first booster dose of mRNA-1273 and second booster doses of mRNA-1273 and mRNA-1273.222post boost within each booster vaccine group at day 29.95% CIs were calculated based on the t-distribution of the log-transformed values for GM value, then back-transformed to the original scale for presentation.Quartiles 1, 2, 3 and 4 represent quartiles indicated s in panel a for mRNA-1273 and mRNA-1273.222.