Reactogenicity, Safety, and Serological and Cellular Immunogenicity of a Booster Dose of SARS-CoV-2 mRNA Prototype, Variant, and Bivalent Vaccines

doi:10.21203/rs.3.rs-1594631/v1

Waning immunity after two SARS-CoV-2 mRNA vaccinations and the emergence of variants precipitated the need for booster doses. We evaluated safety and serological and cellular immunogenicity through 6 months after a third mRNA vaccination in adults who received the mRNA-1273 primary series in the Phase 1 trial approximately 9 to 10 months earlier. The booster vaccine formulations included 100 mcg of mRNA-1273, 50 mcg of mRNA-1273.351 that encodes Beta variant spike protein, and bivalent vaccine of 25 mcg each of mRNA-1273 and mRNA-1273.351. A third dose of mRNA vaccine appeared safe with acceptable reactogenicity. Vaccination induced rapid increases in binding and neutralizing antibody titers to D614G, Beta, Delta and Omicron variants that persisted through 6 months post-boost, particularly after administration of Beta-containing vaccines. Spike-specific CD4 + and CD8 + T cells increased to levels similar to those following the second dose. Boost vaccination induced broad and durable humoral and T cell responses.

ClinicalTrials.gov numbers NCT04283461 (mRNA-1273 Phase 1) and NCT04785144 (mRNA-1273.351 Phase 1)

Vaccine Development

SARS-CoV-2

COVID-19

vaccine

mRNA-1273

mRNA-1273.351

Beta

Omicron

The mRNA-1273 SARS-CoV-2 vaccine, administered as a primary series of two 100 mcg doses 28 days apart, demonstrated 94% efficacy against COVID-19 illness in the pivotal Phase 3 efficacy trial,¹ and was approved by the US Food and Drug Administration (FDA). mRNA-1273 encodes a prefusion stabilized full-length spike (S-2P) glycoprotein of the prototype SARS-CoV-2 Wuhan-Hu-1 isolate² and induces robust neutralizing antibody responses to that strain and to the D614G variant,^3–5 which was the predominant circulating SARS-CoV-2 variant for most of 2020. Breakthrough infections, including severe infections and hospitalizations, began to occur in individuals that had completed their primary series due to waning of immunity and emergence of variants.^6,7

Initial vaccine-induced neutralizing antibody responses after a primary series of mRNA-1273 are lower against Beta (B.1.351), Delta (B.1.617.2) and the more recent Omicron (B.1.1.529, also called BA.1) variants, than against D614G.^8–14 At six or more months after the primary series, neutralizing antibody titers decline further, and may be undetectable, against these variants.^11,13,15 In response to the emergence of variants with immune-evading mutations, Moderna and other manufacturers have developed variant-specific vaccines.

While a third, and more recently a fourth, dose of a COVID-19 mRNA vaccine directed against the prototype strain (Wuhan-Hu-1) is recommended, evaluations of COVID-19 mRNA vaccines directed against variant strains are needed to determine the optimal approach to boosting, since these variant vaccines may or may not meet emerging needs as other more antigenically distant variants emerge and spread. Thus, we evaluated the longitudinal safety and immunogenicity of three booster mRNA vaccine formulations — a 50 mcg monovalent Beta vaccine (mRNA-1273.351), a bivalent vaccine containing 25 mcg of each of the prototype and Beta vaccine formulations, and the prototype 100 mcg (high dose) mRNA-1273 vaccine — given eight to twelve months after the two-dose mRNA-1273 primary series.^3,4,11,15,16 Here we report the safety and durability of the immune responses elicited by those booster formulations.

Participants

We evaluated three mRNA booster vaccines in participants who received their two-dose primary series as part of the mRNA-1273 Phase 1 dose-ranging trial (NCT04283461, DMID 20 − 0003).^3,4,11,15,16 The phase 1 trial participants were recruited into three age-group strata (18–55 years, 56–70 years, and ≥ 71 years), and received a primary series of two mRNA-1273 vaccinations 28 days apart at doses of 25, 50, 100, or 250 mcg between March and June 2020 (Fig. 1).

The monovalent prototype 100 mcg mRNA-1273 booster vaccine was evaluated in a substudy of the phase 1 trial (monovalent prototype group) and included any participant (any primary series dose) who chose to participate. The two variant-containing booster vaccines were evaluated in a new Phase 1 study (NCT04785144, DMID 21 − 0002) available to DMID 20 − 0003 participants in the 50, 100, or 250 mcg dose groups who chose to leave the DMID 20 − 0003 phase 1 study and join the new trial. Participants in DMID 21 − 0002 were randomized 1:1 to receive a third mRNA vaccination of 50 mcg of Beta-variant (monovalent variant) or a combination of 25 mcg of mRNA-1273 with 25 mcg of the Beta variant mRNA-1273.351 (bivalent group) (Fig. 1).

The characteristics of participants are shown in Table 1. The monovalent prototype group included 48 participants, of which 29, 15, 3, and 1 had received a 25, 50, 100, and 250 mcg mRNA-1273 primary series, respectively (Fig. 1). The monovalent variant and bivalent groups also included 48 total participants, of which 8, 28, and 12 had received a 50, 100, or 250 mcg dose primary series, respectively. Thus, many of the participants who received the mRNA-1273 boost received a lower priming dose of mRNA-1273 compared with participants who received mRNA-1273.351 containing vaccine. Participants in the monovalent prototype group tended to be older and had received their third dose at a median of 9.5 months (IQR 8.8, 10.4) after the second vaccination, while the monovalent variant and bivalent groups received their third dose 10.6 months (IQR 10.0, 11.2) after the second vaccination.

Vaccine Reactogenicity and Safety

Among the solicited local adverse events (AEs), pain at the injection site was most common and observed in 87–96% of participants across groups. Most local reactions were not severe. In the monovalent prototype group, solicited systemic AEs were common (fatigue most commonly) (Fig. 2), with 41 (85%) participants reporting at least one solicited systemic AE and 6 (13%) reporting at least one severe solicited systemic AE (chills and headache most commonly). In the monovalent variant group, 22 of the 25 (88%) participants reported at least one solicited systemic AE (fatigue most commonly). Of those with solicited systemic AEs, 2 (8%) reported a severe event (headache and nausea). In the bivalent group, 20 of the 23 (87%) participants reported at least one solicited systemic AE (fatigue and myalgia most commonly). Of these solicited systemic AEs, 3 (13%) reported a severe event (fever [peak 39.1°C], fatigue, and headache).

Unsolicited adverse events reported within 28 days were usually mild to moderate in severity (Supplementary Results and Tables S2 – S6). Severe related unsolicited adverse events in the monovalent variant group included a participant with vomiting and another participant with medically-attended generalized pruritic rash beginning 11 days after vaccination. This decreased in severity but required intermittent antihistamine use.

No serious adverse events related to vaccination occurred and no prespecified halting rules were met. A 58-year-old individual with an underlying history of hypertension, alcohol use, seasonal allergies, anxiety, and brain aneurysm had a serious unrelated adverse event of upper extremity paresthesias beginning 30 days after bivalent vaccination (Supplement).

Vaccine Immunogenicity

SARS-CoV-2 IgG Binding Antibody Geometric Mean Area Under the Curve

Prior to the third dose, IgG binding antibodies expressed as geometric mean (GM) of the area under the curve (AUC) to the ancestral 614D (Wa-1) strain S-2P were observed in all participants [monovalent prototype group 6,841 AUC (95% CI 5491, 8522); monovalent variant group 16,505 (95% CI 12,408, 21,954); bivalent group 15,154 (95% CI 12,409, 18,507)], indicating binding antibody persistence at a median of 9.5 and 10.6 months following the primary series (Fig. 3, Supplementary Table S9). Pre-boost S-2P IgG levels against all tested variants correlated with the primary vaccine series dose, with numerically higher titers persisting in participants that received higher primary series doses; however, non-overlapping confidence intervals were only observed in comparison to those that had received a 25 mcg prime series (Supplementary Table S8).

Following a booster vaccination, a similar and robust anamnestic response was observed in all three groups to 614D S-2P that peaked at Day 15 [monovalent prototype 63,245 AUC (95% CI 61,435, 65,109); monovalent variant 64,583 (95% CI 61,916, 67,364); bivalent 64,854 (95% CI 62,911, 66,858)] and persisted at high levels through D181 (Fig. 3, Supplementary Table S9). Pre-boost IgG binding levels to the Beta variant S-2P antigen were approximately one-third of 614D binding. However, in all groups, the third dose of mRNA vaccine induced similar anamnestic responses against B.1.351 [monovalent prototype 48,242 AUC (95% CI 45,834, 50,778); monovalent variant 52,724 (95% CI 49,461, 56,202); bivalent 50,658 (95% 47,661, 53,843)] that were about 80% of the level of Day 15 responses to 614D (Fig. 3, Supplementary Table S9), and persisted through D181. IgG binding to Omicron (BA.1) was particularly low prior to boosting (< 20% of 614D) but did increase to about 60% of 614D peak binding. Interestingly, the monovalent prototype vaccine induced responses, across 614D and the tested variants, tended to be less durable and more variable than generally observed with the two variant-containing formulations.

SARS-CoV-2 Pseudovirus Neutralization ID₅₀ Titers

The pseudovirus neutralization responses against D614G spike were measured in two laboratories using a similar and highly concordant assay:¹³ a fit-for-purpose assay for monovalent prototype booster samples and a validated assay for the monovalent variant and bivalent samples. Prior to the third dose, neutralizing activity ID₅₀ against pseudovirus expressing D614G was low in most participants [monovalent prototype GMT 39 (95% CI 28, 54); monovalent variant GMT 127 (95% CI 96, 168); bivalent GMT 110 (95% CI 84, 143)] (Fig. 4, Supplemental Table S12). Detectable titers were observed in most, but not all, of those who had received 25 or 50 mcg primary doses and in all 100 or 250 mcg primary doses participants (Supplemental Table S11). At 14 days after the third dose, the D614G ID₅₀ GMTs increased substantially across groups [monovalent prototype 2,842 (95% CI 2,270, 3,560); monovalent variant 2,111 (95% CI 1,615, 2,760); bivalent 2,435 (95% CI 1,912, 3,102)]. Post-boost titers were higher than peak neutralizing titers observed following receipt of the second 100 mcg mRNA-1273 dose [1,399 (95% CI 1,111, 1,762)] and were similar to the 50 mcg mRNA-1273 third dose comparator [2,321 (95% CI 1,890, 2,851)]. Notably, neutralizing titers to D614G were maintained at much higher levels through D181 than after the original primary vaccination series, suggestive of improved durability through 6 months after a third dose.

Pre-boost neutralizing ID₅₀ activity against the Beta variant was low to undetectable (Fig. 4, Supplemental Tables S11, S12). Post-boost Beta responses were robust in all groups, peaking at D15/D29 [monovalent prototype 865, (95% CI 672, 1,113); monovalent variant 1,100 (95% CI 867, 1,395); bivalent 866 (95% CI 659, 1,139)]. These titers were similar to the Phase 2 comparison group [539 (95% CI 428, 680)] and exceeded those observed after the 100 mcg primary series [165 (95% CI 105, 259)] and persisted at high levels through D181, particularly in the arms that received Beta variant vaccine.

Pre-boost pseudovirus neutralizing titers for the Omicron (BA.1) spike variant were low to undetectable in most participants that received a 25 or 50 mcg primary series (Fig. 4, Supplemental Tables S11-12) while those that had received a 100 or 250 mcg primary series had somewhat more persistent neutralizing activity. Omicron titers after boost were typically lower than those observed against other variants [monovalent prototype 565 (95% CI 406, 787); monovalent variant 745 (95% CI 532, 1044); bivalent 567 (95% CI 387, 831)]. Although not statistically significant, higher GMTs with smaller 95% confidence intervals were generally observed in groups that had received Beta-variant containing vaccine at and after Day 91 for Omicron. For example, monovalent variant recipients tended to maintain the highest GMT to Omicron at Day 181 [364 (95% CI 223, 595)] in comparison to monovalent prototype recipients [151 (95% CI 100, 230)].

SARS-CoV-2 Focus Reduction Neutralization Test (FRNT) ID₅₀ Titers

Pre-boost FRNT ID₅₀ levels against D614G were low in all participants [monovalent prototype 28 (95% CI 21, 38); monovalent variant 108 (95% CI 82, 144); bivalent 99 (95% CI 73, 135)] (Supplemental Figure S3, Supplemental Table S14). FRNT ID₅₀ post-boost responses were robust in all groups [monovalent prototype 1,673 (95% CI 1307, 2142); monovalent variant 1,863 (95% CI 1,475, 2,354); bivalent 2,072 (95% CI 1,723, 2,492)] and persisted through Day 181 in all groups.

Pre-boost FRNT titers against Beta were low to undetectable in all groups [monovalent prototype 14 (95% CI 12, 17); monovalent variant 21 (95% CI 15, 29); bivalent 18 (95% CI 13,25)] (Supplemental Figure S3, Supplemental Table S14). Post-boost FRNT responses were robust [monovalent prototype 664 (95% CI 487, 904); monovalent variant 1,142 (95% CI 888, 1,469); bivalent 840 (95% CI 624, 1130)] but were numerically lower than D614G. Prior to boost, antibodies against Omicron were not detected in any participant by FRNT. Peak titers after boost were not different between the groups [monovalent prototype 127 (95% CI 90, 179), monovalent variant 221 (95% CI 170, 287), bivalent 179 (95% CI 134, 238)], but neutralizing antibody against Omicron by FRNT remained higher at Day 181 in those receiving Beta-variant vaccine than in those that received prototype vaccine [monovalent prototype 29 (95% CI 21, 41), monovalent variant 82 (53, 127), bivalent 75 (95% CI 48, 118)] (Supplemental Figure S3, Supplemental Table S14).

SARS-CoV-2 CD4 + and CD8 + T Cell Responses

A validated intracellular cytokine staining assay detected Th1 and Th2 CD4 + and CD8 + T cells following ex vivo stimulation with overlapping 15-mer peptides spanning either prototype (614D) or Beta spike protein sequences. Similar to a previous report,⁴ robust spike-specific IFN-γ and/or IL-2 CD4 + T cell responses were observed at 14 days after the primary series (Fig. 5; Supplemental Table S15). Spike-specific CD4 T cell responses were cross-reactive against the Beta spike with similar magnitudes and response rates compared to the vaccine-matched spike. At the time of boost, spike-specific CD4 T cells declined but remained detectable in nearly all subjects (Fig. 5; Supplementary Table S15).

Spike-specific IFN-γ and/or IL-2 CD4 + T cells recognizing both 614D and Beta increased 2.15–2.79 fold following the boost, returning to similar magnitudes (median 0.43–0.68% of CD4 T cells) and response rates (> 96%) as observed at 14 days after the primary series (Fig. 5, Supplementary Table S15). Spike-specific CD4 T cells were polyfunctional with high expression of IFN-g, IL-2, TNF-a, and CD154 (CD40L) (Supplementary Table S15). Overall, the Th1 CD4 T cell responses were primarily comprised of effector memory and to a lesser extent central memory cells (Supplementary Figure S5). CD4 + T cell Th2 responses, as measured by spike-specific IL-4 expression, were modest (0.01–0.02% of CD4 T cells) and substantially lower than Th1 responses (Fig. 5, Supplementary Figure S4).

Notably, boosting with the monovalent variant (Beta) mRNA vaccine did not improve cross-reactive responses. Even with the Beta-specific peptides, the variant-recognizing spike-specific CD4 T cell responses were not significantly improved in monovalent variant or bivalent participants (Supplemental Figures S9–10). However, all three groups tended to have higher spike-specific CD4 T cell responses to prototype-matched as compared to Beta variant peptides.

Spike-specific IFN-γ and/or IL-2 expressing CD8 + T cells were 0.06–0.09% of the total, were primarily effector memory cells, and were detected in 23–36% of subjects at 2 weeks post-2nd vaccination (Fig. 5, Supplementary Table S16) but then declined. After the boost, spike-specific CD8 + T cells were detected in 40–48% of vaccine recipients with median frequencies of 0.06–0.08%. When spike-specific CD8 + T cells were observed, they cross-reacted against Beta, regardless of group (Fig. 5).

Despite the high efficacy of the mRNA-1273 vaccine, particularly against severe COVID-19, demonstrated in the Phase 3 trial,^1,17 declines in efficacy over time and against emerging variants have been observed.⁷ Emergence of the Beta variant (1.351), against which sera from mRNA-1273 two-dose vaccine recipients had lower cross-neutralization,¹⁵ spurred development of mRNA-1273.351. Concern over waning immunity, particularly for the participants who received the lowest doses of mRNA-1273 (25 or 50 mcg) in the original Phase 1 study, prompted a protocol amendment to offer a boost of 100 mcg mRNA-1273 to Phase 1 study participants (monovalent prototype). Participants who received doses of 50, 100, or 250 mcg in the Phase 1 study also had the option to join a new trial and be randomized to receive either a monovalent variant or a bivalent booster vaccine. The study provided an opportunity to assess humoral and cellular immunological responses after boosting with prototype vaccine and with Beta-containing monovalent and bivalent vaccines.

We previously reported that mRNA-1273 induced binding and neutralizing antibodies against the ancestral 614D strain in participants in the DMID 20 − 0003 Phase 1 trial that persisted through 180 days after the second 100 mcg dose.¹⁵ Extending those observations, we found that binding and neutralizing antibodies (614D/614G) continued to decline but remained readily detectable at 10–11 months (prior to third dose) in all subjects that received the initial 100 mcg primary series (Supplementary Tables S8, S11, S13). In contrast, only 64% of 100 mcg primary series recipients had pseudovirus neutralizing activity against the Beta variant and Omicron variant pseudovirus neutralizing titers were detectable in 48% of the participants who had received a 100 mcg primary series. Neutralizing antibodies to Omicron were not detected prior to the boost by FRNT.

A robust anamnestic response was observed 14 days after boosting, with all 3 vaccines raising serum IgG binding titers and neutralizing activity against 614D/614G to levels observed after a 50 mcg boost of mRNA-1273 (EUA booster formulation) (Figs. 3–4, Supplementary Table S9). Prototype, variant monovalent and bivalent vaccination resulted in strong functional antibody responses that persisted through 6 months by pseudovirus neutralization assay (Fig. 4, Supplementary Figure S3, Supplementary Table S12, S14). Three individuals that had received monovalent prototype vaccine had loss of Omicron pseudovirus neutralizing titers by Day 181, and Omicron titers became undetectable by FRNT by Day 181 in one-third of participants that received monovalent prototype vaccine, but in only about ten percent of participants that received a Beta-variant vaccine. Although this difference in the durability of the response after prototype boost vaccination may have been impacted by initial primary vaccine series (many of the individuals had received 25 or 50 mcg doses in the prototype group), the difference in the durability at 6 months is similar to what has been observed in another study of a bivalent-Beta-prototype vaccine booster that has been prepublished.¹⁸ Additional confirmation of these data are needed, but they do raise the possibility of improving the durability and breadth of the immunological response with a variant mRNA vaccine.

Support for humoral immune responses as a correlate of protection is provided by analysis of data from several vaccine studies,^19,20 including an assessment of the mRNA-1273 Phase 3 COVE study participants that evaluated the correlation between binding and neutralizing antibody levels with risk of symptomatic COVID-19 through 3–4 months post second vaccination.²¹ In the COVE mRNA-1273 study, one analysis estimated vaccine efficacy of 51% (95% CI -51–83%) against COVID-19 in subjects with an undetectable pseudovirus neutralizing antibody ID₅₀ at Day 57. In this study, neutralizing antibodies had waned to an undetectable level by the time of the booster in some individuals, particularly those that had received 25 or 50 mcg doses. In the current study, a third dose of vaccine resulted in GMTs that reached or exceeded 500 for all variants, which exceeds levels that correlate with 90.7% efficacy (95%CI 86.7, 93.6%) to the D614G variant. Notably, GMTs were maintained above 100 through 6 months for all variants tested, with the Beta and bivalent vaccines eliciting somewhat higher and more durable responses to Beta and Omicron variants through 6 months post-boosting. Although it should be noted that antibody titers prior to the third dose of mRNA-1273 were lower than those observed in the groups that received vaccines containing the Beta variant. Data are needed regarding the correlates of protection against the Omicron variants that have now spread globally. While binding and pseudovirus neutralization titers through Day 57 in the Phase 3 trial correlated with protection,²² the correlation was incomplete, suggesting a potential role of T cells.

A two-dose vaccination regimen with the mRNA-1273 vaccine elicits predominantly Th1-biased CD4 + T cell responses irrespective of age and dosage,⁴ which even in the lower dosage groups (25 and 50 mcg) has been shown to persist through at least 6 months after the second dose.¹⁹ The T cell data described here demonstrates the persistence of TH1-biased CD4 + cells in a majority of participants through approximately 10–11 months post vaccination, while Th2 CD4 + T cells are less frequently detected and at a lower magnitude.⁴ TH1-biased CD4 + responses after the third dose were similar across the groups and were similar to those observed after the second dose. Effector, and to a lesser extent, central memory CD4 + T cell responses were also detected in a majority of study participants prior to the administration of the third dose and increased with the third dose (Supplementary Figure S7). Spike-specific CD8 + T cells were less frequently detected at the time of boost but were detected in 40–48% of vaccine recipients after the boost. No clear difference was observed in the CD4 + or CD8 + responses to the Beta-variant peptides with use of the monovalent variant or bivalent vaccines. There was significant cross-reactivity in spike-specific CD4 T cell responses detected among all vaccinated subjects, even after the original prototype vaccine series, suggesting that CD4 T cell responses are sufficiently polyclonal to tolerate the reduction or loss of recognition among the Beta variant epitopes.

All three booster formulations appeared safe with acceptable reactogenicity that was similar to the currently approved 50 mcg boost. The most common solicited adverse reactions after the third dose of mRNA vaccine were injection site pain, fatigue, myalgia and chills, and the frequency of these events was similar across the three booster vaccine strategies (monovalent prototype, monovalent variant, and bivalent groups). Most adverse reactions were mild or moderate, with severe solicited events occurring in 8–13% of participants across the 3 groups. A single participant developed a related severe pruritic generalized rash after monovalent variant vaccine that gradually improved. Additionally, a single participant in the bivalent vaccine group had a transient ischemic attack which was considered unrelated to vaccine at Day 30.

Limitations to this study bear consideration. The most important limitation is that in the Phase 1 study, participants received 25–250 mcg primary series doses. The primary series dose impacted the durability of binding and neutralizing antibodies observed prior to the third dose but did not appear to significantly impact the responses observed post-boost. Additionally, different mRNA doses were used for the boost (100 mcg in monovalent prototype group, 50 mcg total in monovalent variant and bivalent groups). These differences could have impacted antibody responses and durability. Participants here received a third dose of mRNA vaccine at a median of 9.5–10.6 months after their second vaccination, while booster vaccinations are now recommended for adults in the US as soon as 5 months after the primary series. We partially addressed this by providing a comparison of serological responses observed in 50 mcg mRNA-1273 boosted P201 participants.²² The mRNA vaccine utilized here in the monovalent variant and bivalent groups was based on the Spike protein of the Beta variant. As such, the ability to extrapolate to vaccines developed in response to other variants (e.g., Delta, Omicron) is limited. Although preliminary data demonstrate that binding and pseudovirus neutralizing antibody titers obtained from the Phase 3 clinical trial of mRNA-1273 correlate with protection, the threshold predictive of protection against current and emerging variants is unknown. The Omicron analyses performed here were for the B.1.1.529 isolate. Recently the BA.2 variant has become predominant in the US and data are lacking. Finally, data here are from a small study of a third dose of mRNA in a relatively homogeneous racial and ethnic group of participants and quantitative comparisons are limited.

In conclusion, humoral and cellular immune responses elicited by the mRNA-1273 vaccine persist at least through 10–11 months after the primary series against the ancestral strain. A third dose of vaccine serves to increase levels of binding antibodies that recognize the Spike proteins of the ancestral virus and several variants including Beta, Delta, and Omicron. Booster vaccination with the prototype, variant, and bivalent vaccine strategies all resulted in strong functional neutralizing antibody responses to the prototype and tested variant strains. The Beta and bivalent vaccines elicited responses to Beta and Omicron variants that were numerically higher at 6 months post-boosting. No particular difference was observed in the cellular responses to epitopes within the Beta-mutated peptides with use of the monovalent or bivalent Beta variant vaccines. Novel monovalent variant or multivalent mRNA vaccine refinements may lead to improvements in cross-neutralization and durability and should be considered in future booster vaccine development.

Trial Designs and Participants

Additional details of the design, conduct, oversight, analyses, and results of the trials can be found in the protocols, statistical analysis plans, and Supplement. The DMID 20 − 0003 substudy (NCT04283461) and the DMID 21 − 0002 (NCT04785144) trial protocols were reviewed and approved by the Advarra institutional review board. Both protocols permitted interim analyses to inform vaccination strategies and public health. This report includes findings through 181 days after the third mRNA vaccination. The booster vaccination evaluations were conducted at the Kaiser Permanente Washington Health Research Institute in Seattle, the Emory University School of Medicine in Atlanta, and the VRC at National Institutes of Health in Bethesda.

Participants were healthy adults who must have received two doses of mRNA-1273 in the Phase 1 study; other eligibility criteria are included in the protocols (Supplement). All third doses of mRNA vaccine were administered between March and April 2021 and recipients will be followed for safety, reactogenicity, and immunogenicity for twelve months after the third dose of mRNA.

Immune responses observed in these monovalent prototype, monovalent variant, and bivalent groups were compared with two other groups that had received an initial two-dose 100 mcg mRNA-1273 primary series: peak neutralization responses after a 100 mcg primary series in 33 participants in the Phase 1 trial (peak pseudoneutralizing antibody titers were also evaluated in those that received a 25 or 50 mcg primary series); and sera from a Phase 2 randomized placebo-controlled trial sponsored by Moderna, protocol P201(NCT04405076)^5,22 who received a third dose of 50 mcg mRNA-1273 six to eight months after completion of their two-dose 100 mcg mRNA-1273 primary series. These specimens were tested with the same assays in the same previously mentioned laboratories to provide comparisons.

Trial Procedures

For each protocol, follow-up visits were scheduled for 7, 14, 28, 90, 180, and 365 days after the booster vaccination. Participants recorded local and systemic reactions, using a memory aid, through 7 days after the booster vaccination. Adverse events were graded according to a standard toxicity grading scale (Supplementary Table 1).

Assessment of SARS-CoV-2 Binding Antibody and Neutralizing Responses

For this interim analysis, we tested sera obtained on the day of and prior to the third vaccination through 14 days after this dose. Of the 48 participants in the DMID 20 − 0003 booster substudy, all but four had received a 25 mcg or 50 mcg dose of mRNA-1273 for the primary series; for consistency, we restricted the assessment of humoral immune responses to participants in those dose groups. For the DMID 21 − 0002 trial, we tested samples from all participants.

We used a 4-plex Mesoscale Discovery (MSD) platform to detect binding IgG antibodies that has completed validation testing and is undergoing regulatory review.^11,23 IgG binding against 614D and variant spike proteins were evaluated using a fit-for-purpose MSD 10-plex assay.^11,23

Neutralizing antibody responses were assessed by two laboratories using the same SARS-CoV-2 native spike-pseudotyped lentivirus reporter pseudovirus single-round-of-infection neutralization assay (PsVN), and neutralization titers were expressed as the serum inhibitory dilution required to achieve 50% neutralization (ID₅₀).^4,13 A similar focus-reduction neutralization test (FRNT) assay to that previously utilized, which uses live SARS-CoV-2, assessed vaccine-induced neutralizing activity against the D614G, Beta, and Omicron variants.^4,24 The assay used similar methodology to that described for the cell line VeroE6-TMPRSS2.¹³

T Cell Analyses

A validated intracellular cytokine staining was used to examine SARS-CoV-2-specific CD4 + and CD8 + T-cell responses using 27-color flow cytometry.^25,26 Previously cryopreserved specimens were stimulated with four pools of peptides (15 amino acids overlapping by 11 amino acids): conserved S1, conserved S2, beta mutations and original matched, which together covered the spike sequence of the original 614D/Wuhan (Wu-1) and the beta variant. The peptides were pooled according to sequence into the peptides that were conserved between the 614D and beta S1 and S2 subunits, respectively (conserved S1 and conserved S2). The other 2 peptide pools contained just the peptides with mutations in the beta sequence (beta mutations) and a matching set of unmutated peptides (original matched). The interpretation of responses is shown in the Supplement.

Disclosures and Acknowledgements

Moderna, Inc. provided mRNA-1273 and mRNA-1273.351 for the booster vaccinations but did not provide financial support for the clinical trials. Employees of Moderna, Inc., collaborated on protocol development and the Investigational New Drug (IND) application and participated in weekly protocol team calls. The NIAID ultimately made all decisions regarding trial design and implementation. The trial was supported by the Infectious Diseases Clinical Research Consortium through the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, under award number UM1AI148684, and by award numbers UM1AI148373 (Kaiser Washington), UM1AI148576 (Emory University), and HHSN272201500002C (Emmes). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

AUTHOR CONTRIBUTIONS

Concept design: EJA, LAJ, NGR, ATW, MatM, MamM, WB, PS, CBC, BL, HB, JHB, JA, KA, KMN, PCR; Data collection: EJA, LAJ, NGR, ATW, DM, NAD-R, MS, KWC, SOC, MatM, MamM, WB, PS, CBC, SOD, SDS, CMP, JH, JA, KA, AE, CAR, PAR, SK, DSG, RC, AMD, JEL, JRM, SCDR, MJME; Analysis/interpretation of data: EJA, LAJ, NGR, ATW, DM, NAD-R, MS, KWC, SOC, MatM, MamM, WB, PS, CBC, SOD, SDS, BL, HB, RP, CMP, JH, JHB, JA, KA, AE, CAR, PAR, SK, DSG, RC, AMD, JEL, JRM, SCDR, KMN, MJME, PCR; Write/review/intellectual contribution: EJA, LAJ, NGR, ATW, DM, NAD-R, MS, KWC, SOC, MatM, MamM, WB, PS, CBC, SOD, SDS, BL, HB, RP, CMP, JH, JHB, JA, KA, AE, CAR, PAR, SK, DSG, RC, AMD, JEL, JRM, SCDR, KMN, MJME, PCR; All authors approved the final version of the manuscript.

COMPETING INTERESTS

E.J.A has consulted for Pfizer, Sanofi Pasteur, Janssen, GSK, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on data and safety monitoring boards for Kentucky BioProcessing, Inc. and Sanofi Pasteur. He also serves on safety monitoring committees for WCG-ACI Clinical. His institution has also received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines.

S.K’s institution has received funding from CDC to conduct clinical research unrelated to this manuscript, from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines, and from Pfizer to conduct clinical trials of Pfizer COVID-19 vaccine.

C.A.R.’s institution has received funds to conduct clinical research unrelated to this manuscript from BioFire Inc, GSK, MedImmune, Micron, Janssen, Merck, Novavax, PaxVax, Regeneron, Sanofi-Pasteur. She is co-inventor of patented RSV vaccine technology unrelated to this manuscript, which has been licensed to Meissa Vaccines, Inc. Her institution has received funds from NIH, Moderna, Pfizer, and Janssen to conduct clinical trials of COVID-19 vaccines.

L.A.J.’s institution has received funding from CDC to conduct epidemiologic assessments of vaccine safety and from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines.

CBC’s institution has received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines, funding from CDC to conduct COVID-19 vaccine safety assessments, and unrelated funding for C.difficile immunotherapeutics from Merck. CBC receives consultation fees from Altimmune, Janssen, Premier Healthcare, Horizon Pharma and royalties from UpToDate.

BL, HB, and RP are all employees of Moderna.

NGR’s institution has received funding from Lilly, Sanofi Pasteur, Merck, Quidel and Pfizer. She also serves on safety monitoring committees for ICON and EMMES.

PAR’s institution has received funding from NIH to conduct clinical trials on Moderna and Novavax COVID-19 vaccines and from CDC to assess different sampling techniques for COVID-19 diagnostics.

Baden, L. R. et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med 384, 403–416, doi:10.1056/NEJMoa2035389 (2021).
Wrapp, D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science (New York, N.Y 367, 1260–1263, doi:10.1126/science.abb2507 (2020).
Jackson, L. A. et al. An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med 383, 1920–1931, doi:10.1056/NEJMoa2022483 (2020).
Anderson, E. J. et al. Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N Engl J Med 383, 2427–2438, doi:10.1056/NEJMoa2028436 (2020).
Chu, L. et al. A preliminary report of a randomized controlled phase 2 trial of the safety and immunogenicity of mRNA-1273 SARS-CoV-2 vaccine. Vaccine 39, 2791–2799, doi:10.1016/j.vaccine.2021.02.007 (2021).
Fowlkes, A. et al. Effectiveness of COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Frontline Workers Before and During B.1.617.2 (Delta) Variant Predominance - Eight U.S. Locations, December 2020-August 2021. Mmwr 70, 1167–1169, doi:10.15585/mmwr.mm7034e4 (2021).
Baden, L. R. et al. Phase 3 Trial of mRNA-1273 during the Delta-Variant Surge. N Engl J Med, doi:10.1056/NEJMc2115597 (2021).
Shen, X. et al. Neutralization of SARS-CoV-2 Variants B.1.429 and B.1.351. N Engl J Med 384, 2352–2354, doi:10.1056/NEJMc2103740 (2021).
Wu, K. et al. Serum Neutralizing Activity Elicited by mRNA-1273 Vaccine. N Engl J Med 384, 1468–1470, doi:10.1056/NEJMc2102179 (2021).
Maeda, K. et al. Correlates of Neutralizing/SARS-CoV-2-S1-binding Antibody Response with Adverse Effects and Immune Kinetics in BNT162b2-Vaccinated Individuals. medRxiv, doi:10.1101/2021.07.27.21261237 (2021).
Pegu, A. et al. Durability of mRNA-1273 vaccine-induced antibodies against SARS-CoV-2 variants. Science (New York, N.Y 373, 1372–1377, doi:10.1126/science.abj4176 (2021).
Edara, V. V. et al. Infection and Vaccine-Induced Neutralizing-Antibody Responses to the SARS-CoV-2 B.1.617 Variants. N Engl J Med 385, 664–666, doi:10.1056/NEJMc2107799 (2021).
Pajon, R. et al. SARS-CoV-2 Omicron Variant Neutralization after mRNA-1273 Booster Vaccination. N Engl J Med 386, 1088–1091, doi:10.1056/NEJMc2119912 (2022).
Lyke, K. E. et al. SARS-CoV-2 Omicron Neutralization After Heterologous Vaccine Boosting. medRxiv, 2022.2001.2013.22268861, doi:10.1101/2022.01.13.22268861 (2022).
Doria-Rose, N. et al. Antibody Persistence through 6 Months after the Second Dose of mRNA-1273 Vaccine for Covid-19. N Engl J Med 384, 2259–2261, doi:10.1056/NEJMc2103916 (2021).
Widge, A. T. et al. Durability of Responses after SARS-CoV-2 mRNA-1273 Vaccination. N Engl J Med 384, 80–82, doi:10.1056/NEJMc2032195 (2021).
El Sahly, H. M. et al. Efficacy of the mRNA-1273 SARS-CoV-2 Vaccine at Completion of Blinded Phase. N Engl J Med, doi:10.1056/NEJMoa2113017 (2021).
Chalkias, S. et al. Safety, Immunogenicity and Antibody Persistence of a Bivalent Beta-Containing Booster Vaccine. Research Square (2022).
Feng, S. et al. Correlates of protection against symptomatic and asymptomatic SARS-CoV-2 infection. Nat Med 27, 2032–2040, doi:10.1038/s41591-021-01540-1 (2021).
Fong, Y. et al. Immune Correlates Analysis of a Single Ad26.COV2.S Dose in the ENSEMBLE COVID-19 Vaccine Efficacy Clinical Trial. medRxiv, 2022.2004.2006.22272763, doi:10.1101/2022.04.06.22272763 (2022).
Gilbert, P. B. et al. Immune correlates analysis of the mRNA-1273 COVID-19 vaccine efficacy clinical trial. Science (New York, N.Y 375, 43–50, doi:10.1126/science.abm3425 (2022).
Choi, A. et al. Safety and immunogenicity of SARS-CoV-2 variant mRNA vaccine boosters in healthy adults: an interim analysis. Nat Med, doi:10.1038/s41591-021-01527-y (2021).
Atmar, R. L. et al. Heterologous SARS-CoV-2 Booster Vaccinations - Preliminary Report. medRxiv, doi:10.1101/2021.10.10.21264827 (2021).
Vanderheiden, A. et al. Development of a Rapid Focus Reduction Neutralization Test Assay for Measuring SARS-CoV-2 Neutralizing Antibodies. Curr Protoc Immunol 131, e116, doi:10.1002/cpim.116 (2020).
Sadoff, J. et al. Interim Results of a Phase 1-2a Trial of Ad26.COV2.S Covid-19 Vaccine. N Engl J Med 384, 1824–1835, doi:10.1056/NEJMoa2034201 (2021).
Cohen, K. W. et al. Longitudinal analysis shows durable and broad immune memory after SARS-CoV-2 infection with persisting antibody responses and memory B and T cells. Cell Rep Med 2, 100354, doi:10.1016/j.xcrm.2021.100354 (2021).

Table 1. Characteristics of the Participants at Enrollment.*

Vaccine Formulation for the Third Dose	Monovalent Prototype 100 mcg mRNA‑1273 N=48	Monovalent Variant 50 mcg mRNA‑1273.351 N=25	Bivalent 25 mcg mRNA‑1273 + 25 mcg mRNA‑1273.351 N=23
Characteristic
Sex — no. (%)
Male	25 (52)	14 (56)	11 (48)
Female	23 (48)	11 (44)	12 (52)
Age — yr	56.3±16.7	45.0±19.0	43.7±17.0
Age group — no. (%)
18-55 years	18 (38)	17 (68)	16 (70)
56-70 years	16 (33)	4 (16)	4 (17)
71+ years	14 (29)	4 (16)	3 (13)
Race or ethnic group — no. (%)†
American Indian or Alaska Native	0	1 (4)	0
Asian	2 (4)	1 (4)	0
Black	0	1 (4)	1 (4)
White	45 (94)	21 (84)	22 (96)
More than one race	1 (2)	0	0
Unknown	0	1 (4)	0
Hispanic or Latino ethnic group —no. (%)	3 (6)	4 (16)	1 (4)
Body-mass index‡	24.4±3.0	24.9±3.3	25.5±3.1
mRNA-1273 vaccine dose primary two dose series — no. (%)
25 mcg§	29 (60)	0	0
50 mcg	15 (31)	4 (16)	4 (17)
100 mcg	3 (6)	15 (60)	13 (57)
250 mcg	1 (2)	6 (24)	6 (26)
Time interval between second dose of mRNA-1273 and the booster dose — days (months)
Mean (SD)	294±30 (9.7±1)	321±25 (10.5±0.8 )	319±26 (10.5±0.8 )
Range	257, 371 (8.4, 12.2)	269, 359 (8.8, 11.8)	262, 359 (8.6, 11.8)
Median	289 (9.5)	324 (10.6)	324 (10.6)
Q1, Q3	267, 317 (8.8, 10.4)	304, 341 (10.0, 11.2)	303, 342 (10.0, 11.2)

* Plus–minus values are means ±SD.

† Race or ethnic group was reported by the participants.

‡ The body-mass index is the weight in kilograms divided by the square of the height in meters. For the homologous boost cohort this calculation was based on the weight and height measured at the time of screening for the DMID 20-0003 protocol, for the heterologous boost groups it was based on weight and height measured at the first visit for the DMID 21-0002 protocol.

§ Participants who received a 25 mcg mRNA-1273 vaccine dose in their primary series were not eligible to receive a mRNA-1273.351 containing 3^rd dose.

SUPPLEMENTARYDATA26APR2022.docx

Reactogenicity, Safety, and Serological and Cellular Immunogenicity of a Booster Dose of SARS-CoV-2 mRNA Prototype, Variant, and Bivalent Vaccines

Status:

Version 1

Abstract

Figures

Introduction

Results

Participants

Vaccine Reactogenicity and Safety

Vaccine Immunogenicity

SARS-CoV-2 IgG Binding Antibody Geometric Mean Area Under the Curve

SARS-CoV-2 Pseudovirus Neutralization ID₅₀ Titers

SARS-CoV-2 Focus Reduction Neutralization Test (FRNT) ID₅₀ Titers

SARS-CoV-2 CD4 + and CD8 + T Cell Responses

Conclusions

Methods

Trial Designs and Participants

Trial Procedures

Assessment of SARS-CoV-2 Binding Antibody and Neutralizing Responses

T Cell Analyses

Disclosures and Acknowledgements

Declarations

References

Table 1

Supplementary Files

Status:

Version 1

Reactogenicity, Safety, and Serological and Cellular Immunogenicity of a Booster Dose of SARS-CoV-2 mRNA Prototype, Variant, and Bivalent Vaccines

Status:

Version 1

Abstract

Figures

Introduction

Results

Participants

Vaccine Reactogenicity and Safety

Vaccine Immunogenicity

SARS-CoV-2 IgG Binding Antibody Geometric Mean Area Under the Curve

SARS-CoV-2 Pseudovirus Neutralization ID50 Titers

SARS-CoV-2 Focus Reduction Neutralization Test (FRNT) ID50 Titers

SARS-CoV-2 CD4 + and CD8 + T Cell Responses

Conclusions

Methods

Trial Designs and Participants

Trial Procedures

Assessment of SARS-CoV-2 Binding Antibody and Neutralizing Responses

T Cell Analyses

Disclosures and Acknowledgements

Declarations

References

Table 1

Supplementary Files

Status:

Version 1

SARS-CoV-2 Pseudovirus Neutralization ID₅₀ Titers

SARS-CoV-2 Focus Reduction Neutralization Test (FRNT) ID₅₀ Titers