From May 29 to August 13, 2021, we enrolled 458 participants (154, 150, and 154 in each of the three stages) (Figs. S1, S2, and S3). The last visit (trial day 29) occurred on September 13, 2021. One participant (in group 7) did not receive a booster vaccination. The demographic characteristics of the participants were similar across trial groups (Table 1). The interval between primary and booster vaccinations was shortest among participants who were boosted with mRNA-1273, a finding that reflected the temporal progression of enrollment across the sequential trial stages. Two participants (one each in group 4 and group 6) who had serologic evidence of previous SARS-CoV-2 infection (the presence of antibody against nucleocapsid protein) and 1 participant (in group 5) who was found to have Covid-19 2 days before trial day 29 were included in the analyses.
Two serious adverse events that were deemed by the investigators to be unrelated to trial vaccination were reported. One event (acute renal failure caused by rhabdomyolysis associated with a fall) was reported 30 days after the mRNA-1273 booster, and the other (acute cholecystitis) occurred 24 days after the Ad26.COV2.S booster. No prespecified trial-halting rules were met, and no new-onset chronic medical conditions occurred through trial day 29. One related adverse event of special interest (severe vomiting that led to a medically attended visit the day after vaccination) occurred in group 5 (Ad26.COV2.S booster). Participants with unsolicited adverse events of any grade that were deemed by investigators to be related to a trial vaccine were reported in 24 of 154 participants (16%) who received mRNA-1273, in 18 of 150 participants (12%) who received Ad26.COV2.S, and in 22 of 153 participants (14%) who received BNT162b2 (Tables S4, S5, and S6). Most adverse events were mild or moderate. Four participants had severe adverse events that were deemed by the investigators to be related to a trial vaccine: in 1 participant with vomiting who had received mRNA-1273 (group 1) and in 3 participants who had received Ad26.COV2.S (1 with vomiting and 1 with fatigue in group 5 and 1 with an abnormal feeling and insomnia in group 6).
Shown are local (injection-site) and systemic reactions that were reported within 7 days after the administration of the mRNA-1273 (Panel A), Ad26.COV2.S (Panel B), and BNT162b2 (Panel C) boosters, according to the primary immunization regimen. Local and systemic reactions after the booster injection were graded as mild (does not interfere with activity), moderate (interferes with activity), or severe (prevents daily activity).
Solicited injection-site adverse events were common, with local pain or tenderness being reported in 75 to 86% of mRNA-1273 recipients, in 71 to 84% of Ad.26COV2.S recipients, and in 72 to 92% of BNT162b2 recipients (Figure 1 and Tables S7, S8, and S9). Most injection-site reactions were graded as mild, with only 2 (1 in an mRNA-1273 recipient and 1 in an Ad.26COV2.S recipient) that were reported as severe. Malaise, myalgias, and headaches were also commonly reported (Tables S10, S11, and S12). The proportions of all 457 participants in all three stages who reported having a severe systemic solicited event were as follows: malaise or fatigue, 2.0 to 4.5%; myalgia, 0 to 3.3%; headache, 0.7 to 3.3%; nausea, 0 to 2.7%; chills, 0 to 3.3%; arthralgia, 0.6 to 2.0%; and fever, 0.7 to 2.7%. Solicited adverse events were most likely to occur within 3 days after booster vaccination; no clear patterns of frequency were noted for solicited or unsolicited adverse events according to the primary vaccine or age group (Tables S4 through S12).
Binding Antibody Response
Shown are box plots of IgG binding antibody titers against SARS-CoV-2 and pseudovirus neutralizing antibody titers on day 1 (prebooster) and on days 15 and 29, according to whether the participant received the mRNA-1273 (Panel A), Ad26.COV2.S (Panel B), or BNT162b2 (Panel C) booster vaccine. The primary vaccination regimens are listed above the box plots. Binding antibody responses were measured against the wild-type (WA1 S-2P) control variant on a 4-plex electrochemiluminescence immunoassay analyzer (ECLIA), and neutralizing antibody titers were measured against the D614G mutation of the SARS-CoV-2 spike protein. Titers were bridged to international standards and reported as binding antibody units per milliliter and international units for the 50% inhibitory dose (IU50) per milliliter. Data points for individual participants are shown as gray circles. In each box plot, the horizontal line represents the median value, with the top and bottom of the box indicating the 75th percentile and 25th percentile, respectively; the whiskers indicate values that are within 1.5 times the interquartile range. The red dots represent participants who had detectable antibody against the SARS-CoV-2 nucleocapsid protein at enrollment, indicative of previous SARS-CoV-2 infection.
All the participants but one (who had been Ad26.COV2.S primed) had evidence of binding antibody against the SARS-CoV-2 full-length spike glycoprotein trimer (S-2P) in the WA1 strain before booster vaccination (Figure 2). The binding antibody titers against S-2P were lower by a factor of 3 to 15 in participants who had received primary vaccination with single-dose Ad26.COV2.S than in those who had received either of the mRNA vaccines (mRNA-1273 or BNT162b2) (Table 2 and Tables S13 through S30). All the groups had an increase in the binding antibody level after boosting. Among the participants who had received an mRNA booster, an increase in the binding antibody titer by a factor of 2 or more occurred in 98 to 100% of participants who were Ad26.COV2.S primed, in 96 to 100% of those who were mRNA-1273 primed, and in 98 to 100% of those who were BNT162b2 primed. By day 15, the geometric mean binding antibody titer had increased by a factor of 5 to 55; increases were greatest in the participants who had received a BNT162b2 or an mRNA-1273 booster after Ad26.COV2.S primary vaccination (by factors of 34 and 55, respectively). The Ad26.COV2.S booster increased binding antibody titers in all the participants, but the Ad26.COV.2-primed recipients had a level that was lower by a factor of 7 to 10 than those in participants who had received an mRNA vaccine as the priming regimen. Binding antibody levels peaked at day 15 for mRNA-boosted groups and were similar or declining on day 29, whereas binding antibody levels in the Ad26.COV2.S-boosted groups on day 29 were similar to or higher than those measured on day 15.
Before booster administration, binding antibody levels against the delta variant were 34 to 45% lower than levels against WA1 S-2P according to the same 10-plex assay (Tables S31 through S36). After receiving a booster, all the participants had detectable binding antibody against the delta variant at a level that was 15 to 36% lower than that against the WA1 strain. Binding antibody levels in serum samples obtained from participants in the older age group were similar to those in the younger age group. Serologic responses to WA1 and beta S-2P on 4-plex ECLIA (Tables S13 through S24 and S37 through S42) and WA1 and delta S-2P proteins on the 10-plex ECLIA are reported in Tables S25 through S36.
Neutralizing Antibody Response
All serum samples obtained from participants who had received mRNA-1273 as the primary vaccine had prebooster neutralizing activity against D614G S-2P, whereas serum samples obtained from 24 participants (16%) who had received Ad26.COV2.S and from 5 (3%) who had received BNT162b2 had no detectable neutralizing activity against the D614G mutation. Serum neutralization levels (as measured in IU50 per milliliter) before booster vaccination were lower than levels in mRNA-1273–primed recipients by a factor of 10 for Ad26.COV2.S-primed recipients and by a factor of 3 for BNT162b2-primed recipients, regardless of the interval between primary and booster vaccination (Table 2 and Tables S43 through S48).
The kinetics of postbooster neutralizing antibody responses were similar to those observed for binding antibody responses. On day 15, postbooster neutralization titers ranged from 676 to 902 IU50 per milliliter for participants boosted with mRNA-1273, 31 to 382 IU50 per milliliter for those boosted with Ad26.COV2.S, and 344 to 694 IU50 per milliliter for those boosted with BNT162b2. The factor increases in the geometric mean neutralization titers were greatest for Ad26.COV2.S-primed recipients, followed by recipients of primary BNT162b2 and mRNA-1273. In general, postbooster titers were highest in recipients of primary mRNA-1273, followed by primary BNT162b2 and Ad26.COV2.S, regardless of the booster vaccine administered. Recipients of an mRNA booster had a neutralization response that was higher by a factor of 4 than those who were boosted with Ad26.COV.S (Tables S55 through S60).
In general, prebooster serum neutralization levels were lower against the delta and beta variants than those against the D614G mutation and were below the limit of detection in many participants (Tables S67 through S72 and S79 through S84). All but 2 participants who had received Ad26.COV2.S as both the primary and booster vaccine had measurable neutralizing antibody against the delta variant after booster vaccination; similar findings were observed when ID80 neutralization levels were assessed (Tables S73 through S78 and S85 through S90).
Spike-specific T cells are shown in box plots before the administration of a homologous or heterologous booster vaccine on day 1 and after boosting on day 15. The boosters are shown at the top of each column, and the primary vaccines that each participant received are listed directly above each box plot. Circles indicate positive responses, and triangles indicate negative responses. Red symbols denote participants who had detectable antibody against the SARS-CoV-2 nucleocapsid protein at enrollment, indicative of previous SARS-CoV-2 infection. The responses are depicted as the background-subtracted percentage of spike-specific Th1 (interferon-γ, interleukin-2, or both) CD4+ T cells (top row), spike-specific Th2 (interleukin-4, interleukin-5, or interleukin-13) CD4+ T cells (middle row), and Th1 CD8+ T cells (bottom row). (Background subtraction refers to the subtraction of the values of the negative control sample from the peptide-stimulated sample.) The number of participants with a positive response among those tested is indicated as a fraction above each plot. Dashed lines link individual responses before and after the administration of the booster vaccine. The horizontal bar in each box indicates the median of all responses tested.
SARS-CoV-2 spike-specific Th1 (interferon-γ, interleukin-2, or both) CD4+ T cells were detected in 69% of participants at baseline, with higher response rates and amounts among the mRNA-primed participants (Figure 3). At day 15, an increase in the spike-specific Th1 responses occurred after boosting in all groups except those receiving the homologous Ad26.COV2.S regimen. Spike-specific Th2 (interleukin-4, interleukin-5, or interleukin-13) CD4+ T cells were infrequent (and at low levels) or absent in most subgroups. Th1-type CD4+ T-cell responses were predominant before and after homologous or heterologous boosting.
Spike-specific Th1 CD8+ cells were detected in 74 to 90% of the Ad26.COV2.S-primed recipients, as compared with levels of 10 to 30% in mRNA-primed recipients. Booster immunization increased the response rate and amount of spike-specific CD8+ T cells in all groups, except for the Ad26.COV2.S-primed participants who received homologous Ad26.COV2.S boosting, in whom no appreciable change above the already high prebooster response was noted. The highest amounts of spike-specific CD8+ T cells were observed in the Ad26.COV2.S-primed group, regardless of booster, both before boosting and at 15 days.