Older Adults Mount Less Durable Humoral Responses to Two Doses of COVID-19mRNAVaccine but Strong Initial Responses to a Third Dose Francis Mwimanzi,1 Hope R. Lapointe,2 Peter K. Cheung,1,2 Yurou Sang,1 Fatima Yaseen,1 Gisele Umviligihozo,1 Rebecca Kalikawe,1 Sneha Datwani,1 F. Harrison Omondi,1,2 Laura Burns,3 Landon Young,3 Victor Leung,4,5 Olga Agafitei,1 Siobhan Ennis,1 Winnie Dong,2 Simran Basra,1 Li-Yi Lim,1 Kurtis Ng,1 Ralph Pantophlet,1 Chanson J. Brumme,2,4 Julio S. G. Montaner,2,4 Natalie Prystajecky,5,6 Christopher F. Lowe,3,5 Mari L. DeMarco,3,5 Daniel T. Holmes,3,5 Janet Simons,3,5 Masahiro Niikura,1 Marc G. Romney,3,5,a Zabrina L. Brumme,1,2,a and Mark A. Brockman1,2,a 1Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada; 2British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada; 3Division of Medical Microbiology and Virology, St Paul’s Hospital, Vancouver, Canada; 4Department of Medicine, University of British Columbia, Vancouver, Canada; 5Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada; and 6British Columbia Centre for Disease Control Public Health Laboratory, Vancouver, Canada Background. Third coronavirus disease 2019 (COVID-19) vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults. Methods. Wemeasured circulating antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and omicron (BA.1) strains from prevaccine up to 1 month following the third dose, in 151 adults aged 24–98 years who received COVID-19 mRNA vaccines. Results. Following 2 vaccine doses, humoral immunity was weaker, less functional, and less durable in older adults, where a higher number of chronic health conditions was a key correlate of weaker responses and poorer durability. One month after the third dose, antibody concentrations and function exceeded post–second-dose levels, and responses in older adults were comparable inmagnitude to those in younger adults at this time. Humoral responses against omicron were universally weaker than against the ancestral strain after both the second and third doses. Nevertheless, after 3 doses, anti-omicron responses in older adults reached equivalence to those in younger adults. Onemonth after 3 vaccine doses, the number of chronic health conditions, but not age, was the strongest consistent correlate of weaker humoral responses. Conclusions. Results underscore the immune benefits of third COVID-19 vaccine doses, particularly in older adults. Keywords. COVID-19; vaccine; mRNA; SARS-CoV-2; humoral immunity; older adults; binding antibodies; ACE2 displacement; viral neutralization; omicron. Older adults are at increased risk of lethal coronavirus disease 2019 (COVID-19) following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [1–3]. While 2 COVID-19mRNA vaccine doses broadly protect against hospi- talization and death [4–6], weaker vaccine-induced immunity observed in the elderly and other groups [7–12] led to their pri- oritization for third doses [13–16]. Vaccine-induced antibodies also decline over time, which can increase the risk of postvac- cination infections [17–19], particularly with the more trans- missible and immune-evasive omicron variant [20–22]. We and others have shown that older age is associated with weaker antibody responses to COVID-19 mRNA vaccines [10– 12]. We previously characterized longitudinal humoral re- sponses up to 3 months after the second vaccine dose in 151 adults 24 to 98 years of age [12]. Here, we examine binding and neutralizing antibody responses up to 6 months after the second dose, and at 1 month after the third dose. We also eval- uate binding antibodies, ACE2 displacement, and virus neu- tralization against omicron (BA.1). Characterization of the immunological benefits of a third dose is critical to promote continued public uptake, particularly in light of recent omicron-driven infection waves. METHODS Study Design We conducted a prospective longitudinal cohort study in British Columbia, Canada, to examine SARS-CoV-2 specific humoral responses following vaccination with Comirnaty Received 23 February 2022; editorial decision 05 May 2022; accepted 10 May 2022; published online 11 May 2022 Presented in part: The 31st Annual Canadian Conference on HIV/AIDS Research, April 25, 2022, virtual. aM. G. R., Z. L. B., and M. A. B. contributed equally. Correspondence: Mark A. Brockman, PhD, Professor, Faculty of Health Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6 (mark_brockman@ sfu.ca). The Journal of Infectious Diseases® 2022;226:983–94 © The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, pro- vided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com https://doi.org/10.1093/infdis/jiac199 COVID-19 Vaccine in Older Adults • JID 2022:226 (15 September) • 983 The Journal of Infectious Diseases MA J O R A R T I C L E mailto:mark_brockman@sfu.ca mailto:mark_brockman@sfu.ca https://creativecommons.org/licenses/by-nc-nd/4.0/ https://doi.org/10.1093/infdis/jiac199 (BNT162b2 -BioNTech/Pfizer) or Spikevax (mRNA-1273- Moderna). Our cohort (total n= 151) included 81 health care workers (HCW) and 56 older adults (including 18 residents of long-term care or assisted living facilities) who were COVID-19 naive at study entry, and 14 individuals (including 8 HCW and 6 older adults) with anti-SARS-CoV-2 nucleocap- sid (N) antibodies at study entry (COVID-19 convalescent group) [12]. Serum and plasma were collected prior to vaccina- tion; 1 month after the first dose; 1, 3, and 6 months after the second dose; and 1 month following the third dose (see Table 1 for exact collection timings) [12]. Ethics Approval Written informed consent was obtained from all participants or their authorized decision makers. This study was approved by the University of British Columbia/Providence Health Care and Simon Fraser University Research Ethics Boards. Data Sources Sociodemographic, health, and vaccine information was col- lected by self-report and confirmed through medical records where available. Chronic health conditions were defined as hy- pertension, diabetes, asthma, obesity (body mass index ≥30), chronic diseases of lung, liver, kidney, heart, or blood, cancer, and immunosuppression due to chronic conditions or medication, to generate a score ranging from 0 to 11 per partic- ipant [12]. Binding Antibody Assays We measured total binding antibodies against SARS-CoV-2 N and spike (S) receptor binding domain (RBD) in serum using the Roche Elecsys Anti-SARS-CoV-2 and Anti-SARS-CoV-2 S assays, respectively, on a Cobas e601 module analyzer (Roche Diagnostics). Following SARS-CoV-2 infection, both assays should be positive, whereas postvaccination only S should be positive, allowing identification of convalescent indi- viduals. Both tests are electrochemiluminescence sandwich im- munoassays, and report results in arbitrary units (AU)/mL, calibrated against an external standard. For the S assay, the manufacturer indicates that AU values can be considered equivalent toWorld Health Organization-defined international binding antibody units [23]. For the S assay, sera were tested undiluted, with samples above the upper limit of quantification (ULOQ) retested at 1:100 dilution, allowing a 0.4–25 000 U/mL measurement range. We also quantified plasma immunoglob- ulin G (IgG) binding antibodies against RBD using the V-plex SARS-CoV-2 (IgG) Panel 22 ELISA kit (Meso Scale Diagnostics), which features the ancestral (Wuhan) and omi- cron (BA.1) RBD antigens, on a Meso QuickPlex SQ120 instru- ment. Plasma was diluted 1:10 000 as directed, with results reported in AU/mL. Table 1. Participant Characteristics and Sampling Information Variable Category Characteristic Health Care Workers (n=81) Older Adults (n=56) COVID-19 Convalescent at Study Entry (n=14) Sociodemographic/health Age, y, median (IQR) 41 (35–51) 78 (73–83) 48 (36–87) Female sex, n (%) 61 (75) 38 (68) 10 (71) White/Caucasian ethnicity, n (%) 37 (46) 43 (77) 7 (50) Chronic health or immunosuppressive conditions, median (IQR) 0 (0–0) 1 (0–2) 0 (0–1) Vaccine information Comirnaty, first mRNA vaccine, n (%) 80 (99) 48 (86) 13 (93) Comirnaty, second mRNA vaccine, n (%) 79 (98) 46 (82) 13 (93) Time between first and second doses, d, median (IQR) 97 (91–102) 76 (45–85) 112 (87–118) Comirnaty, third mRNA vaccine, n (%)a 32/61 (52) 19/47 (40) 3/6 (50) Time between second and third dose, d, median (IQR) 210 (200–241) 169 (160–231) 189 (170–194) Specimen collection Specimens collected prevaccine, n (%) 80 (99) 49 (88) 13 (93) Specimens collected 1 mo after first dose, n (%) 79 (98) 49 (88) 13 (93) Day of specimen collection 1 mo after first dose, median (IQR) 28 (27–30) 30 (28–32) 31 (28–32) Specimens collected 1 mo after second dose, n (%) 81 (100) 55 (98) 14 (100) Day of specimen collection 1mo after second dose,median (IQR) 29 (29–32) 29 (29–31) 32 (30–36) Specimens collected 3 mo after second dose, n (%) 79 (98) 53 (95) 13 (93) Day of specimen collection 3mo after second dose,median (IQR) 90 (90–91) 90 (89–92) 90 (87–91) Specimens collected 6 mo after second dose, n (%) 78 (96) 40 (71) 10 (71) Day of specimen collection 6mo after second dose,median (IQR) 181 (179–182) 176 (167–182) 180 (179–181) Specimens collected 1 mo after third dose, n (%) 61 (75) 47 (84) 6 (38) Day of specimen collection 1 mo after third dose, median (IQR) 30 (29–31) 32 (29–33) 30 (29–30) COVID-19 postvaccination Anti-N seroconversion during study follow-up, n (%) 6 (7.4) 2 (3.6) … Abbreviations: COVID-19, coronavirus disease 2019; IQR, interquartile range; N, nucleocapsid. aDenominators are the number of specimens collected 1 month after third dose. 984 • JID 2022:226 (15 September) • Mwimanzi et al ACE2 Competition Assay We assessed the ability of plasma antibodies to block the RBD-ACE2 receptor interaction by competition enzyme- linked immunosorbent assay (ELISA; Panel 22 V-plex SARS-CoV-2 [ACE2]; Meso Scale Diagnostics). ACE2 compe- tition assays represent a higher-throughput method to estimate potential virus neutralizing activity [24]. Plasma was diluted 1:20 as directed and results reported as percent ACE2 displacement. Live Virus Neutralization Neutralizing activity in plasma was examined using a live SARS-CoV-2 infectivity assay as previously described [12] with isolate USA-WA1/2020 (BEI Resources) and a local omi- cron isolate (BA.1 strain; GISAID accession No. EPI_ISL_9805779) on VeroE6-TMPRSS2 (JCRB-1819) target cells. Viral stock was diluted to 50 TCID50 (50% tissue culture infectious dose)/200 µL in the presence of serial 2-fold dilutions of plasma (from 1/20 to 1/2560), incubated for 1 hour, and add- ed to target cells in 96-well plates in triplicate. Viral cytopathic effects were recorded 3 days postinfection. Neutralizing activity is reported as the highest reciprocal plasma dilution able to pre- vent cytopathic effects in all 3 wells. Samples exhibiting partial or no neutralization at 1/20 dilution were coded as below the limit of quantification (BLOQ) in this assay. Statistical Analysis Comparisons of binary variables were performed using Fisher exact test. Comparisons of continuous variables were per- formed using the Mann-Whitney U test (for unpaired data) or Wilcoxon test (for paired data). Multiple linear regression was used to investigate the relationship between sociodemo- graphic, health, and vaccine-related variables, and humoral outcomes. Variables included age (per year), sex at birth (fe- male as reference group), ethnicity (non-white as reference), number of chronic health conditions (per additional), mRNA vaccine received (Comirnaty as reference), interval between doses (per day), sampling date following the most recent dose (per day), and convalescent status (COVID-19 naive as refer- ence). Binding antibody and viral neutralization-related depen- dent variables were log-transformed prior to model input. Independent variables were examined for multi collinearity by calculating the bivariate correlation between all pairs of var- iables. Here, coefficients less than −0.75 or greater than 0.75 would have been considered highly collinear; however, no var- iables in any model met this threshold. No interaction terms were considered. Serum antibody half-lives were calculated by fitting exponential curves to antibody concentrations at 1, 3, and 6 months after the second dose. All tests were 2-tailed, with P, .05 considered statistically significant. Analyses were conducted using Microsoft Excel and Prism version 9.2.0 (GraphPad). RESULTS Participant Characteristics HCW, older adults, and COVID-19 convalescent individuals were a median of 41, 79, and 48 years old, respectively, and pre- dominantly female (Table 1). Older adults were predominantly (77%) of white ethnicity (compared to 46% of HCW). Older adults also had a higher number of chronic conditions (a medi- an of 1, interquartile range [IQR] 0–2, range 0–5 in this group vs a median of 1, IQR 0–0, range 0–3 in HCW). All participants received 2 COVID-19 mRNA vaccine doses between December 2020 and July 2021, where the dose interval was up to 112 days as per national guidelines to delay second doses due to initially limited vaccine supply. Overall, more than 90% of first and sec- ond doses were Comirnaty. At the time of writing, 114 partic- ipants had received a third dose between October and December 2021, on average 7 months following their second dose. For Spikevax third doses (53% overall), those aged ≥70 years received a full dose, whereas those ,70 years received a half-dose, as per national guidelines. An additional 6 (7.4%) HCW and 2 (3.6%) older adults de- veloped anti-N antibodies during follow-up (Table 1). Three of these postvaccination infections, all in HCW, occurred be- tween December 2021 and Jan 2022 and were likely omicron BA.1 [25]. In analyses that span the whole study, these partic- ipants are retained in their original “COVID-19–naive at study entry” group, but identified in the figures at their first postinfec- tion visit. In analyses of third-dose responses, they are classified as “prior COVID-19.” After 2-Dose Vaccination, Lower Binding Antibodies Are Associated With Older Age and Chronic Conditions But Older Adults Mount Strong Third-Dose Responses We measured total anti-RBD binding antibody concentrations in serum before and after immunization (Figure 1A). As re- ported previously [12], antibody concentrations in older adults were significantly lower than those in HCW 1 month after the first dose (a median of 2.00 [IQR, 1.75–2.25] log10 U/mL in HCW vs a median of 1.50 [IQR, 1.05–1.99] in older adults), as well as 1 month after the second dose (a median of 4.02 [IQR, 3.88–4.25] in HCW vs a median of 3.74 [IQR, 3.49– 3.91] in older adults) (Mann-Whitney; both P, .0001). Three months after the second dose, antibody concentrations had declined by approximately 0.4 log10 on average, to a medi- an of 3.63 (IQR, 3.44–3.83) in HCWversus a median 3.32 (IQR, 3.04–3.56) in older adults (Mann-Whitney P, .0001 for com- parison between groups). Six months after the second dose, an- tibody concentrations had declined by a further approximately 0.3 log10 on average, to a median of 3.30 (IQR, 3.09–3.47) in HCW versus a median 2.96 (IQR, 2.68–3.20) in older adults (P, .0001). Thus, following 2-dose COVID-19 mRNA vaccination, antibody concentrations remained consistently and significantly lower in older compared to younger adults. COVID-19 Vaccine in Older Adults • JID 2022:226 (15 September) • 985 A B -1 0 1 2 3 4 5 An ti- RB D Ab c on c (lo g 1 0 AU /m L) : E le cs ys a ss ay H C W O ld er A du lts C on v. H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts prevax 1 mo after 1st 1 mo after 2nd 3 mo after 2nd 6 mo after 2nd 1 mo after boost 1 < .0001 < .0001 < .0001 < .0001 .3 n=80 49 13 79 49 13 81 55 14 79 53 13 78 42 10 61 47 6 < .0001 < .0001 < .0001 < .0001 < .0001 < .0001 < .0001 .3 .06 .027 ULOQ LLOQ .3 .6 .3 C on v. C on v. C on v. C on v. C on v. 4 8 16 32 64 128 256 512 1024 2048 4096 ne ut ra liz at io n: re ci pr oc al d ilu tio n H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts H C W O ld er A du lts prevax 1 mo after 1st 1 mo after 2nd 3 mo after 2nd 6 mo after 2nd 1 mo after boost 1 .08 < .0001 < .0001 .002 .6 n=19 12 8 79 49 13 80 52 14 79 53 13 78 42 10 61 47 6 0.006 < .0001 < .0001 < .0001 < .0001 < .0001 ns .009 .002 .005 LLOQ ULOQ .6 .5 .6 C on v. C on v. C on v. C on v. C on v. C on v. Figure 1. Longitudinal antibody binding and neutralization responses to spike RBD following 1, 2, and 3 COVID-19 vaccine doses. A, Binding antibody responses to the SARS-CoV-2 spike RBD in serum, in HCW (blue circles) and older adults (orange circles) who were COVID-19 naive at study entry, as well as COVID-19 convalescent indi- viduals (black circles) at 6 time points: prior to vaccination (pre-vax); 1 month following the first dose; 1, 3, and 6 months following the second dose; and 1 month following the third vaccine dose. Individuals with postvaccination infections are indicated by red dots at their first N-seropositive time point. Participant numbers are provided at the bottom of the plot. A thick horizontal red bar represents the median; thinner horizontal red bars represent the interquartile range. P values were computed using the Mann-Whitney U test (for comparisons between groups) or the Wilcoxon matched pairs test (for comparisons across time points within a group) and are uncorrected for multiple comparisons. B, As in (A) but for virus neutralization activity, defined as the lowest reciprocal plasma dilution at which neutralization was observed in all wells of a triplicate assay. Plasma samples showing neutralization in fewer than 3 wells at a 1/20 dilution were coded as LLOQ. The highest dilution tested was 1/2560, which corresponds to the ULOQ. Note that only a subset of prevaccine plasma samples was assayed for this activity. Abbreviations: conc, concentration; Conv, convalescent; COVID-19, coronavirus disease 2019; HCW, health care worker; LLOQ, lower limit of quantification; N, nucleocapsid; prevax, prevaccination; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ULOQ, upper limit of quantification. 986 • JID 2022:226 (15 September) • Mwimanzi et al By contrast, antibody concentrations in COVID-19 convalescent individuals remained consistently higher than in COVID-19 na- ive individuals after 2 doses. Sixmonths after the second dose, for example, convalescent individuals showed median responses of 3.50 (IQR, 3.40–3.71) log10 U/mL (compared to a median of 3.30 [IQR, 3.09–3.47], P= .027 in HCW; and a median of 2.96 [IQR, 2.68–3.20], P, .0001 in older adults). Multivariable analyses of antibody concentrations after 2 doses, that adjusted for sex, ethnicity, number of chronic health conditions, first-dose vaccine brand, dosing interval, and speci- men collection date, confirmed that older age remained inde- pendently associated with lower antibody concentrations at 1 and 3 months after the second dose (Supplementary Table 1). One month after the second dose, for example, each decade of older age was associated with an approximately 0.06 log10 lower antibody concentration (P= .0067). A higher number of chronic conditions was also independently associated with lower antibody concentrations at both these time points. Six months after the second dose, a higher number of chronic con- ditions remained the strongest independent correlate of lower responses, with each condition associated with a 0.14 log10 low- er antibody concentration (P= .0001). A longer dose interval was associated with higher antibody concentrations at all time points after the second dose (all P, .05), consistent with previous reports [26–28]. COVID-19 convalescent status was also associated withmaintaining 0.26 log10 higher antibody concentrations at 3 and 6 months after the second dose (both P, .05), consistent with superior durability of hybrid (com- bined infection and vaccine-induced) immunity [29–31]. In both HCW and older adults, the third dose boosted anti- body concentrations at least approximately 0.3–0.4 log10 higher than peak values observed after 2 doses (Wilcoxon paired test P, .0001 for both groups). Binding antibodies in HCW rose to a median of 4.31 (IQR, 4.13 to ULOQ) while those in older adults rose to a median of 4.33 (IQR, 4.14 to ULOQ) (P= .33), indicating that older and younger adults mounted comparable initial binding antibody responses to third doses. In multivari- able analyses of third-dose responses, a higher number of chronic conditions was the sole significant correlate of lower antibody concentrations (P= .0078), while having received a Spikevax third dose was associated with higher antibody con- centrations (P= .0091) (Supplementary Table 2). After 2-Dose Vaccination, Weaker Virus Neutralizing Activity Is Associated With Age and Chronic Conditions But Older Adults Mount Strong Third-Dose Responses We next quantified the ability of plasma to prevent target cell infection by the ancestral (USA-WA1/2020) SARS-CoV-2 strain in a live virus neutralization assay (Figure 1B). Neutralizing activity is reported as the highest reciprocal plas- ma dilution capable of preventing viral cytopathic effects in all triplicate assay wells. As previously reported [12], 1 vaccine dose largely failed to induce neutralization in COVID-19 naive individuals, although 2 doses induced this activity in most par- ticipants, albeit at consistently lower levels in older compared to younger adults. One month after the second dose, for example, the median reciprocal dilution to achieve neutralization was 160 (IQR, 80–160) in HCW versus 40 (IQR, 20–80) in older adults (P, .0001). Three months after the second dose, neutralizing ac- tivity had declined by more than 2-fold on average, to a median reciprocal dilution of 40 (IQR, 20–80) in HCW versus a median of 20 (IQR, BLOQ to 40) in older adults (P, .0001). Six months after the second dose, neutralizing activity had declined to BLOQ in 58% of HCW and 83% of older adults (Mann-Whitney P= .0048 for comparison between groups). COVID-19 convalescent individuals, by contrast, maintained significantly higher neutraliz- ing activity compared to naive individuals at all time points fol- lowing 2-dose vaccination. Multivariable analyses confirmed that older age remained significantly associated with weaker neu- tralizing activity at 1 and 3 months after 2-dose vaccination, while COVID-19 convalescent status was associated with superior neu- tralization at all time points following 2-dose vaccination (all P≤ .0002; Supplementary Table 1). A third vaccine dose boosted neutralizing activity in both HCW and older adults to levels that were 2- and 8-fold higher than peak post–second-dose values, respectively (Wilcoxon paired test P≤ .006 for both groups; Figure 1B). Specifically, the median reciprocal dilution in HCW and older adults rose to 320 (IQR, 160–320) and 320 (IQR, 80–320), respectively (P= .6), indicating that older adults mounted comparable ini- tial neutralizing responses to younger adults after the third dose. A multivariable analysis identified prior COVID-19 as the strongest independent predictor of higher neutralization af- ter the third dose (P= .0044; Supplementary Table 2). Chronic Conditions Are Associated With Faster Binding Antibody Decline After 2 Vaccine Doses We next assessed antibody decline after 2-dose vaccination (Figure 2A). Assuming exponential decay and restricting the analysis to participants with a complete longitudinal data series with no values above the ULOQ, we estimated antibody concen- tration half-lives to be a median of 59 (IQR, 52–75) days in HCW versus a median of 52 (IQR, 45–65) days in older adults (P= .016; Figure 2B). This suggests that, in addition to overall weaker responses to 2-dose vaccination compared to younger adults, antibody concentrations in older adults also decline more rapidly. In multivariable analyses, however, a higher num- ber of chronic conditions emerged as the sole independent cor- relate of antibody decline, with each additional condition associated with a 5-day shorter half-life (P= .017; Table 2). COVID-19 convalescent status was associated with a 14-day longer antibody half-life after adjustment for other factors (P= .056), consistent with more durable hybrid immunity [29–31]. COVID-19 Vaccine in Older Adults • JID 2022:226 (15 September) • 987 http://academic.oup.com/jid/article-lookup/doi/10.1093/infdis/jiac199#supplementary-data http://academic.oup.com/jid/article-lookup/doi/10.1093/infdis/jiac199#supplementary-data http://academic.oup.com/jid/article-lookup/doi/10.1093/infdis/jiac199#supplementary-data http://academic.oup.com/jid/article-lookup/doi/10.1093/infdis/jiac199#supplementary-data Omicron-Specific Humoral Responses Given the rise of omicron, we compared peak antibody re- sponses against this strain in plasma at 1 month after the sec- ond and third vaccine doses. Here, all participants with prior COVID-19, regardless of infection timing, were included in the convalescent category. Overall, omicron-specific anti-RBD IgG binding antibodies, measured using the Meso Scale Diagnostics V-Plex assay, were on average 0.4 to 0.5 log10 U/mL lower than those against the wild type (WT; ances- tral Wuhan strain) RBD after 2 and 3 doses (all within-group comparisons P≤ .0002; Figure 3A). Nevertheless, the third dose universally boosted omicron-specific anti-RBD IgG con- centrations to an average of 0.5 log10 higher than levels induced by 2 doses (all within-group comparisons P, .05). Consistent with total anti-RBD binding antibody concentrations quanti- fied using the Roche assay (Figure 1A), anti-RBD IgG concen- trations against WT were significantly higher in HCW compared to older adults after 2 doses (P, .0001) but reached equivalence after 3 doses (P= .4) (Figure 3A). The latter result further confirms that initial third-dose binding antibody re- sponses were comparable in older and younger adults (as high- er dilutions used in the Meso Scale assay allow quantification over a larger dynamic range than the Roche assay). Omicron-specific anti-RBD IgG concentrations followed a similar pattern, with HCW showing marginally higher levels compared to older adults after 2 doses (P= .09), but equivalent levels after 3 doses (P= .49). A multivariable analysis of A B Figure 2. Decay rates of serum binding antibody responses to spike RBD following 2 COVID-19 vaccine doses. A, Temporal declines in serum binding antibody responses to spike RBD following 2 vaccine doses in HCW (blue) and older adults (orange) who were COVID-19 naive at study entry, as well as COVID-19 convalescent participants (black). Only participants with a complete longitudinal data series with no values above the ULOQ are shown. B, Binding antibody half-lives following 2 COVID-19 vaccine doses, calculated by fitting an exponential curve to each participant’s data shown in (A). Participant numbers are indicated at the bottom of the plot. Red bars and whiskers represent the median and interquartile range. P values were computed using the Mann-Whitney U test and are uncorrected for multiple comparisons. Abbreviations: Ab, antibody; conc, concentration; Conv, convalescent; COVID-19, coronavirus disease 2019; CW, health care worker; RBD, receptor-binding domain; ULOQ, upper limit of quantification. Table 2. Multivariable Analysis of the Relationship Between Sociodemographic, Health, and Vaccine-Related Variables and Serum Antibody Half-Life Following 2-Dose COVID-19 mRNA Vaccination Outcome Measure Variable Estimate 95% CI P Value Antibody half-life after 2 vaccine doses Age, per y .058 −.17 to .29 .61 Male sex 5.31 −2.57 to 13.18 .18 White ethnicity 3.11 −4.67 to 10.88 .43 No. chronic conditions, per additional −4.62 −8.39 to −.85 .017 Spikevax as first dose 3.37 −13.26 to 20.00 .69 Dose interval, per d .00014 −.16 to .16 .99 COVID-19 convalescenta 13.78 −.37 to 27.93 .056 Abbreviations: CI, confidence interval; COVID-19, coronavirus disease 2019. aParticipants with positive anti-N serology at study entry. 988 • JID 2022:226 (15 September) • Mwimanzi et al A B WT OM WT OM WT OM WT OM WT OM WT OM 1 2 3 4 5 6 an ti- S- RB D Ig G (l og 10 A U/ m L) : M SD a ss ay 1 mo post 2nd HCW (COVID-19 naive) 1 mo post 3rd 1 mo post 2nd 1 mo post 3rd 1 mo post 2nd 1 mo post 3rd Older Adults (COVID-19 naive) Convalescent (incl. post-vax infection) n=75 55 52 45 22 13 < .0001 < .0001 < .0001 < .0001 .0002 .04< .0001< .0001 < .0001 .09 .40 .49 < .0001 WT OM WT OM WT OM WT OM WT OM WT OM 0 10 20 30 40 50 60 70 80 90 100 AC E2 d is pl ac em en t ( % ) 1 mo post 2nd 1 mo post 3rd 1 mo post 2nd 1 mo post 3rd 1 mo post 2nd 1 mo post 3rd n=75 55 51 45 22 13 < .0001 < .0001 < .0001 < .0001 .0002 .04< .0001< .0001 < .0001 < .0001 .08 .2 < .0001 HCW (COVID-19 naive) Older Adults (COVID-19 naive) Convalescent (incl. post-vax infection) Figure 3. Anti-omicron IgG binding and ACE2 displacement activities 1 month after the second and third COVID-19 vaccine doses. A, Binding IgG responses in plasma to the WT (ancestral Wuhan strain) and omicron S-RBD, measured using the MSD V-Plex assay, in HCW (blue circles) and older adults (orange circles) who remained COVID-19 naive throughout the study, as well as individuals with prior COVID-19 regardless of infection timing (COVID-19 convalescent; black circles) at 1 month after the second and third COVID-19 vaccine doses. Participant numbers are shown at the bottom of the plot. A thick horizontal red bar represents the median; thinner horizontal red bars represent the interquartile range. P values were computed using the Wilcoxon matched pairs test (for all within-group comparisons) or the Mann-Whitney U test (for between-group com- parisons) and are uncorrected for multiple comparisons. B, As in (A) but for ACE2 displacement activity, measured using the V-plex SARS-CoV-2 (ACE2) assay, where results are reported in terms of % ACE2 displacement. Abbreviations: COVID-19, coronavirus disease 2019; HCW, health care worker; SD, Meso Scale Diagnostics; OM, omicron; postvax, postvaccination; S-RBD, spike receptor-binding domain; WT, wild type. COVID-19 Vaccine in Older Adults • JID 2022:226 (15 September) • 989 omicron-specific anti-RBD IgG concentrations after the third dose identified a higher number of chronic conditions as the strongest correlate of poorer responses, with each additional condition associated with a 0.12 log10 lower response (P= .0033; Table 3). A longer interval between the first and sec- ond vaccine doses was marginally associated with a lower third dose response (P= .02). We also assessed the ability of plasma to block the interaction betweenWT and omicron RBD and the cellular ACE2 receptor using a competition assay. This activity was significantly weak- er against omicron compared toWT after both 2 and 3 doses in all groups (all within-group comparisons P≤ .0002; Figure 3B), although the discrepancy wasmost pronounced for older adults after 2 doses (where median anti-WT activity was 90% com- pared to 23% against omicron). The third dose universally boosted ACE2 competition activity against omicron (all within-group comparisons P, .05), with, for example, median activity in older adults rising to 66% (from 23%). Consistent with results for anti-RBD IgG antibodies, plasma ability to block the WT-RBD/ACE2 interaction was significantly higher in HCW compared to older adults after 2 doses (P, .0001), but reached equivalence after 3 doses (in fact, activities in older adults were slightly higher at this time point; P= .08). Ability to block the omicron-RBD/ACE2 interaction followed a similar pattern, with HCW exhibiting significantly higher activity compared to older adults after 2 doses (P, .0001), but equiva- lent levels after 3 doses (P= .2). In multivariable analyses, a higher number of chronic conditions was the strongest corre- late of poorer omicron-specific ACE2 competition activity after 3 vaccine doses, with each additional condition associated with an approximately 6% reduction in this activity (P= .0046; Table 3). Male sex, a longer interval between the first and sec- ond doses, and days elapsed since the third dose also correlated with weaker post–third-dose responses (all P, .05). Finally, we compared plasma neutralization of WT and om- icron strains using a live virus assay in a subset of 20 HCW and 21 older adults who remained COVID-19 negative throughout the study (Figure 4). Neutralizing activity against omicron was significantly weaker than that against WT following 2 and 3 doses in both groups (all P, .0001). The third dose neverthe- less boosted omicron-specific neutralization in both groups, where the increase in older adults was particularly pronounced (from a median of BLOQ after the second dose to a median re- ciprocal dilution of 40 after the third; P, .0001). Consistent with anti-RBD IgG and ACE2 competition results, omicron-specific neutralization was significantly lower in older adults compared to HCW after 2 vaccine doses (P= .0003) but reached equivalence after 3 doses (P= .79). DISCUSSION At 1, 3, and 6 months following 2-dose COVID-19 mRNA vac- cination, antibody binding and neutralizing activity were sig- nificantly weaker in older compared to younger adults. Table 3. Multivariable Analyses of the Relationship Between Sociodemographic, Health, and Vaccine-Related Variables and Omicron-Specific Humoral Immunogenicity Measures Following 3-Dose COVID-19 mRNA Vaccination 1 mo After 3rd Dose Humoral Measure Variable Estimate 95% CI P Value Anti-omicron RBD IgG, log10a Age, per y .0035 −.0027 to .0097 .26 Male sex −.14 −.34 to .054 .15 White ethnicity −.018 −.21 to .17 .85 No. chronic conditions, per additional −.12 −.20 to −.041 .0033 Spikevax as third dose, vs Comirnaty .15 −.039 to .34 .12 Interval between 1st and 2nd dose, per d −.0066 −.012 to −.0011 .020 Interval between 2nd and 3rd dose, per d .00043 −.0034 to .0042 .83 Days since 3rd vaccine dose −.0086 −.038 to .021 .56 Prior COVID-19b .1 −.16 to .37 .43 Anti-omicron ACE2 % displacementa Age, per y .29 −.046 to .63 .090 Male sex −12.38 −23.16 to −1.60 .025 White ethnicity −2.36 −12.74 to 8.01 .65 No. chronic conditions, per additional −6.41 −10.80 to −2.03 .0046 Spikevax as third dose, vs Comirnaty 1.69 −8.58 to 11.97 .74 Interval between 1st and 2nd dose, per d −.41 −.71 to −.11 .0079 Interval between 2nd and 3rd dose, per d −.038 −.25 to .17 .72 Days since 3rd vaccine dose −1.82 −3.43 to −.21 .027 Prior COVID-19b 12.28 −2.11 to 26.67 .094 Abbreviations: CI, confidence interval; COVID-19, coronavirus disease 2019; RBD, receptor-binding domain. aMeasured using the Meso Scale Diagnostics V-plex assay system. bIncludes all participants with positive anti-nucleocapsid serology at any time during the study (ie, both pre- and postvaccine COVID-19 cases). 990 • JID 2022:226 (15 September) • Mwimanzi et al Antibody concentrations were also less durable in older adults, al- though responses declined substantially in all groups over time (eg, by 6months after the second dose, neutralization had declined to BLOQ in almost 60% of HCW and .80% of older adults). In multivariable analyses, a higher number of chronic conditions re- mained consistently and independently associated with weaker and less durable binding antibody responses, while a longer inter- val between first and second doses was consistently associated with higher binding antibody responses after the second dose, as previously reported [26–28]. These findings support public health decisions to provide third doses on or before the 6-month mark, with older adults receiving priority. One month after a third COVID-19 vaccine dose, antibody binding and neutralization reached levels that were signifi- cantly higher than peak levels after the second dose, where the magnitude of boosting in older adults was particularly prominent. Indeed, 1 month after the third dose, antibody binding, ACE2 competition activity, and live virus neutraliza- tion in older adults reached equivalence to younger adults. Consistent with recent evidence [20, 22, 32–38], omicron-specific antibody responses were universally weaker than those specific to the ancestral strain after both 2 and 3 vac- cine doses; nevertheless, anti-omicron responses in older adults also reached equivalence to those observed in younger adults 1 month after the third dose. Notably, the number of chronic conditions persisted as an independent correlate of weaker omicron-specific responses, even after 3 doses. Given ongoing transmission of omicron variants, these results clearly under- score the benefits of a third dose, and support public health de- cisions to provide them to adults of all ages [39]. P = .79 P = .0003 P = .14 P < .0001 P < .0001 P < .0001 P < .0001 P < .0001 Figure 4. Anti-omicron neutralization activities 1 month after the second and third COVID-19 vaccine doses. Neutralization activities, reported as the highest reciprocal plasma dilution at which neutralization was observed in all wells of a triplicate assay, against the WT (ancestral WA1/2020 strain) and omicron virus isolates, in a subset of HCW (blue circles) and older adults (orange circles) who remained COVID-19 naive throughout the study. Participant numbers are shown at the bottom of the plot. A thick horizontal red bar represents the median; thinner horizontal red bars represent the interquartile range. P values were computed using the Wilcoxon matched pairs test (for within-group comparisons) or the Mann-Whitney U test (for between-group comparisons) and are uncorrected for multiple comparisons. Abbreviations: COVID-19, coronavirus disease 2019; HCW, health care worker; LLOQ, lower limit of quantification; WT, wild type. COVID-19 Vaccine in Older Adults • JID 2022:226 (15 September) • 991 Like others [29–31], our findings indicate that individuals who have contracted COVID-19 still benefit from vaccination. Compared to naive participants, convalescent individuals dis- played slower antibody decline, and multivariable analyses demonstrated that binding and neutralization activities were higher in this group at 6 months after the second dose. Our study has several limitations. As the precise immune correlates of protection for SARS-CoV-2 transmission and dis- ease severity remain incompletely characterized [40], particu- larly in light of omicron, we cannot directly interpret our results in terms of individual-level protection. Nevertheless, stronger responses are likely to confer increased protection, and the average .0.3–0.4 log10 increase in binding antibodies and 2- to 8-fold average boost in neutralization conferred by third doses again underscores the benefit of this dose. We did not investigate T-cell responses, which may play critical roles in protection against severe COVID-19, particularly in the con- text of variants [41–48]. Our study was not powered to investi- gate differences between the 2 mRNA vaccines [49, 50], nor in full versus half-doses of Spikevax when administered as third doses. Post–third-dose response durability assessments are also needed. In conclusion, while the observation of strong initial binding and neutralizing antibody responses to third COVID-19 vac- cine doses in older adults, including to omicron, are encourag- ing, it will be important to closely monitor third-dose response durability in this population. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Notes Acknowledgments. We thank the leadership and staff of Providence Health Care, including long-term care and assisted living residences, for their support of this study. We thank the phlebotomists and laboratory staff at St Paul’s Hospital, the BC Centre for Excellence in HIV/AIDS, the Hope to Health Research and Innovation Centre, and Simon Fraser University for assistance. Above all, we thank the participants, without whom this study would not have been possible. Disclaimer. The views expressed in this publication are those of the authors and not necessarily those of AAS, NEPAD Agency, Wellcome Trust, or the UK Government Financial support. This work was supported by the Public Health Agency of Canada (grant number 2020-HQ-000120 COVID-19 Immunology Task Force COVID-19 Hot Spots Award to M. G. R., Z. L. B., and M. A. B.); the Canadian Institutes for Health Research (grant numbers GA2-177713 and FRN-175622 Coronavirus Variants Rapid Response Network to M. A. B.); the Canada Foundation for Innovation (Exceptional Opportunities Fund-COVID-19 awards to M. A. B., M. D., M. N., R. P., and Z. L. B.); and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant number R01AI134229 to R. P.). M. L. D and Z. L. B. hold Scholar Awards from the Michael Smith Foundation for Health Research. F. Y. and L. Y. L. were supported by Simon Fraser University Undergraduate Research Awards. G. U. and F. H. O. hold PhD fellowships from the Sub-Saharan African Network for TB/HIV Research Excellence, a Developing Excellence in Leadership, Training and Science (DELTAS) Africa initiative (grant number DEL-15-006). The DELTAS Africa initiative is an independent funding scheme of the African Academy of Sciences’s Alliance for Accelerating Excellence in Science in Africa and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency with funding from the Wellcome Trust (grant number 107752/Z/15/Z) and the UK Government. Funding to pay the Open Access publication charges for this article was provided by the Public Health Agency of Canada. Potential conflicts of interest. All authors: No reported con- flicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. FismanDN, Bogoch I, Lapointe-Shaw L,McCready J, Tuite AR. Risk factors associated with mortality among residents with coronavirus disease 2019 (COVID-19) in long-term care facilities in Ontario, Canada. JAMA Netw Open 2020; 3:e2015957. 2. 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MMWR Morb Mortal Wkly Rep 2021; 70:1163–6. 994 • JID 2022:226 (15 September) • Mwimanzi et al https://doi.org/10.1101/2022.01.26.22269819 https://doi.org/10.1101/2022.01.26.22269819 Older Adults Mount Less Durable Humoral Responses to Two Doses of COVID-19 mRNA Vaccine but Strong Initial Responses to a Third Dose METHODS Study Design Ethics Approval Data Sources Binding Antibody Assays ACE2 Competition Assay Live Virus Neutralization Statistical Analysis RESULTS Participant Characteristics After 2-Dose Vaccination, Lower Binding Antibodies Are Associated With Older Age and Chronic Conditions But Older Adults Mount Strong Third-Dose Responses After 2-Dose Vaccination, Weaker Virus Neutralizing Activity Is Associated With Age and Chronic Conditions But Older Adults Mount Strong Third-Dose Responses Chronic Conditions Are Associated With Faster Binding Antibody Decline After 2 Vaccine Doses Omicron-Specific Humoral Responses DISCUSSION Supplementary Data Notes References