Comparative analysis of COVID-19 vaccine responses and third booster dose-induced neutralizing antibodies against Delta and Omicron variants

The study addresses how COVID-19 vaccine type, dosing interval (short 3-week vs long ~12-week), and heterologous vs homologous schedules affect the magnitude and durability of humoral immunity, particularly neutralizing antibodies against SARS-CoV-2 variants of concern (Delta and Omicron BA.1). In Finland, vaccine roll-out initially used BNT162b2 with a 3-week interval, later extended to 12 weeks, and due to safety concerns and availability, many received heterologous schedules (ChAdOx1 followed by an mRNA vaccine). Prior work suggested higher neutralizing responses with heterologous ChAdOx1→mRNA regimens and documented waning antibodies and reduced neutralization of Omicron after two doses. This study aims to compare antibody kinetics across vaccine combinations and intervals, and to evaluate the effect of a third mRNA booster on neutralization breadth, including Omicron BA.1, to inform optimal vaccination strategies.

The authors reference studies showing: (1) heterologous ChAdOx1 followed by BNT162b2 or mRNA-1273 can elicit stronger neutralizing responses than homologous regimens; (2) antibody levels wane over time after two vaccine doses; (3) Omicron (BA.1/BA.2) harbors numerous spike substitutions leading to reduced neutralization by sera from two-dose vaccinees or previously infected individuals; (4) comparative effectiveness data indicating mRNA-1273 may induce higher antibody levels and vaccine effectiveness than BNT162b2 in some settings; and (5) longer dosing intervals can enhance immunogenicity and durability compared to short intervals. These prior findings motivate evaluating mixed schedules, interval effects, and booster responses against VOCs.

Design and participants: Prospective cohort of 328 Finnish healthcare workers (HCWs). Short-interval cohort (n = 120) received two BNT162b2 doses 3 weeks apart; all later received an mRNA booster (BNT162b2 n = 47; mRNA-1273 n = 73) at a mean of 8.3 months after dose 2. Long-interval cohort (n = 208; interval ~12 weeks, range ~8–16 weeks) received either 2×BNT162b2 (n = 62), 2×mRNA-1273 (n = 72), ChAdOx1→BNT162b2 (n = 52), or ChAdOx1→mRNA-1273 (n = 22). Sampling schedule: Short-interval group: pre-vaccination; 3 weeks post-dose 1; and 3 weeks, 3 months, 6 months, 8–9 months post-dose 2; plus 3 weeks post-dose 3. Long-interval group: pre-vaccination; 3, 6, and 12 weeks post-dose 1; and 2 weeks, 2 months, and 6 months post-dose 2. Immunoassays: SARS-CoV-2 S1 IgG and N IgG measured using in-house EIA/ELISA with S1 and N antigens coated at 5 µg/ml on 96-well plates; sera diluted 1:100; OD at 450 nm; seropositivity thresholds defined using positive/negative controls with EIA units (cut-off indicated). Neutralization assays: Microneutralization test (MNT) using SARS-CoV-2 isolates representing D614G (B.1), Delta (B.1.617.2), and Omicron BA.1 (B.1.1.529). Two-fold serial dilutions starting at 1:20; virus-serum incubation 1 h at 37°C; infection of VeroE6-derived cells for 6 days; fixation, crystal violet staining; ID50 determined as reciprocal dilution inhibiting 50% cytopathic effect. Neutralization positivity defined as complete inhibition at ≥1:20. Virus strains and culture: Clinical isolates F1N25-20 (D614G), F1N37-21 (Delta), F1N51-21 (Omicron BA.1) propagated in VeroE6-derived cells; titers by TCID. Statistics: GraphPad Prism; paired analyses by Wilcoxon signed-rank; group comparisons by Kruskal–Wallis with Dunn’s multiple comparisons; two-sided tests; significance p < 0.05. Structural illustrations generated with UCSF Chimera. Ethics: Approvals from regional ethics boards; written informed consent obtained.

- Two-dose long-interval regimens (2×BNT162b2, 2×mRNA-1273, ChAdOx1→BNT162b2, ChAdOx1→mRNA-1273) induced high S1 IgG and neutralizing antibodies (NAbs) to D614G and Delta. Three weeks after dose 2, geometric mean S1 IgG EIA units: BNT162b2 128, mRNA-1273 158, ChAdOx1→BNT162b2 89, ChAdOx1→mRNA-1273 153. - Antibodies waned over time across all groups; by 2 months post–dose 2, 99% had detectable S1 IgG; at 6 months in selected groups, 94% still had detectable S1 IgG, but levels declined. - Short-interval (3-week) 2×BNT162b2 induced 2–3-fold lower neutralizing titers at 3 weeks post–dose 2 compared with long-interval schedules; differences diminished by 3 months, though 2×mRNA-1273 maintained higher GMTs than short-interval 2×BNT162b2. - In a subset (n = 59) with short-interval 2×BNT162b2: at 3 weeks post–dose 2, 100% neutralized D614G; 98% neutralized Delta; only 24% neutralized Omicron BA.1. By 3 and 6 months, mean NAb titers to D614G and Delta decreased ~2-fold; Omicron BA.1 titers were mostly below detection. - Pre-booster, NAb positivity rates were 90% (D614G), 56% (Delta), and 5% (Omicron BA.1). Three weeks after a third mRNA dose (BNT162b2 or mRNA-1273), 100% neutralized all three variants. - Titer ratios: At 3 weeks post–dose 2, mean NAb titers against D614G were ~2-fold higher than Delta and ~23-fold higher than Omicron BA.1. After the third dose, the gaps narrowed to ~2-fold (vs Delta) and ~6-fold (vs Omicron BA.1), indicating broadened neutralization. - Among long-interval regimens, mRNA-1273 as second dose yielded the highest S1 IgG levels at 3 weeks and 3 months post–dose 2; for Delta neutralization, 2×mRNA-1273 had the highest GMT (reported as 339 at 3 weeks; partial data provided), and short-interval 2×BNT162b2 showed 2–3× lower GMTs than long-interval regimens at 3 weeks. - Prior SARS-CoV-2 infection boosted post–dose 1 responses; age had minimal effect after two doses; booster responses were slightly lower in 55–65 vs 35–54 years, and the youngest (20–34) showed lower S1 IgG post-booster than 35–54 groups in this cohort. - Breakthrough infections after two doses were rare in this cohort (very low rate reported; e.g., 1 PCR-confirmed case in short-interval group).

The findings demonstrate that dosing interval and vaccine type materially affect early neutralizing responses, with longer intervals and mRNA-1273 generally yielding higher titers after two doses. Heterologous ChAdOx1→mRNA schedules performed comparably well to homologous mRNA regimens. However, humoral responses waned across all groups, and two doses provided limited neutralization of Omicron BA.1, aligning with reports of immune evasion. A third mRNA booster restored and broadened neutralization, achieving universal seropositivity against D614G, Delta, and Omicron BA.1 and narrowing titer gaps, supporting booster policies to counter VOCs. The data are consistent with real-world effectiveness studies showing stronger performance of mRNA-1273 and with immunological observations that boosting increases antibody breadth and magnitude. The results suggest that flexible use of licensed vaccines, including heterologous schedules and extended intervals, can achieve robust immunity, and that boosters are important for sustaining protection against evolving variants. Cell-mediated immunity, not assessed here, likely contributes to protection against severe disease across VOCs.

This study shows that: (1) long-interval two-dose regimens and heterologous ChAdOx1→mRNA schedules induce robust antibody responses; (2) short-interval 2×BNT162b2 yields lower early neutralization titers; (3) antibody levels wane over months regardless of regimen; and (4) a third mRNA booster markedly increases antibody levels and restores neutralization breadth to include Omicron BA.1. These findings support booster vaccination to maintain cross-variant protection and indicate that mRNA-1273 and longer dosing intervals may confer higher titers. Future work should assess durability post-booster, cellular immunity across VOCs, responses to newer Omicron sublineages, and optimization of heterologous booster strategies.

- Observational cohort of healthcare workers in Finland may limit generalizability to broader populations (age, comorbidities). - Sample sizes for some vaccine combinations (e.g., ChAdOx1→mRNA-1273) and for neutralization subsets were relatively small, reducing precision. - Neutralization against Omicron was assessed for BA.1 only; other sublineages (e.g., BA.2 and beyond) were not directly tested. - Cellular immunity (T-cell responses) and other immune correlates were not measured. - Follow-up for some groups extended to 6–9 months post–dose 2, but post-booster durability beyond 3 weeks was not assessed. - Potential prior undiagnosed infections (anti-N positive without PCR confirmation) could confound antibody kinetics in a minority of participants.