A randomized controlled trial of heterologous ChAdOx1 nCoV-19 and recombinant subunit vaccine MVC-COV1901 against COVID-19

The study addresses whether a heterologous prime-boost regimen using ChAdOx1 followed by a protein subunit vaccine (MVC-COV1901) can improve immunogenicity and maintain safety compared with homologous ChAdOx1 boosting. ChAdOx1 was widely deployed globally with proven efficacy against symptomatic and severe COVID-19 but raised rare safety concerns (TTS/VITT), motivating consideration of non-adenoviral boosters. Prior trials and observational studies suggested benefits of mix-and-match strategies, but head-to-head randomized data with a protein subunit booster and the effect of dosing interval were limited. This trial evaluates immunogenicity (neutralizing antibodies, binding antibodies, B- and T-cell responses) and reactogenicity for heterologous versus homologous boosting at short (4–6 weeks) and longer (8–10 weeks) prime-boost intervals.

Background evidence indicates two-dose ChAdOx1 shows ~70% efficacy in trials and strong real-world protection against severe outcomes. Rare TTS events after ChAdOx1 prompted exploration of heterologous boosting. Multiple studies have reported superior humoral and cellular responses with ChAdOx1 followed by mRNA vaccines versus homologous ChAdOx1 (e.g., Com-COV and other cohorts), and broader antibody responses after heterologous regimens. Protein-based vaccines like MVC-COV1901 have demonstrated safety and immunogenicity in phase 1/2 trials. Prior pooled analyses for ChAdOx1 suggested longer intervals (12–16 weeks) improved efficacy and immunogenicity, though effects on interval can vary and may be confounded by background infection rates; robust randomized data on interval effects with heterologous protein boosters remained needed.

Design: Investigator-initiated, single-center, single-blind, 1:1 randomized, non-inferiority clinical trial in healthy adults (20–70 years) who had received a first dose of ChAdOx1. Participants were randomized to receive either a homologous ChAdOx1 booster or a heterologous MVC-COV1901 booster. Stratified randomization by prime-boost interval (4–6 weeks vs 8–10 weeks) ensured equal-sized strata. Participants were blinded to vaccine received; administering clinical staff were unblinded; laboratory staff were blinded. Follow-up scheduled to day 168 post-boost; this interim analysis reports through day 28. Primary objective: Non-inferiority of heterologous vs homologous booster by neutralizing antibody GMT at day 28 post-boost, per Taiwan FDA immuno-bridging criteria (lower bound of 95% CI for GMT ratio ≥ 0.67–0.76 as per local standards). Participants: 100 adults (mostly healthy Han Taiwanese). Exclusions included severe disorders; all had no serious AEs after first ChAdOx1. Women of childbearing potential required contraception and negative pregnancy test. Outcomes and assays: Humoral responses measured by ELISA-based surrogate neutralizing antibody assay (MeiBio/Medipro kit; negative cutoff <34.47 IU/mL; sensitivity 92.2%, specificity 93%) prior to and after boosting; live-virus neutralization (PRNT-like) at day 28 against Wuhan and Delta variants. Binding antibodies (S1/RBD) quantified by ELISA. Cellular immunity assessed via ex vivo ELISpot: antibody-secreting B cell ELISpot (IgG, IgA, IgM) to spike; IFN-γ ELISpot for T-cell responses to spike S1 and S2 peptide pools. PBMCs prepared by density gradient centrifugation. Detailed ELISpot protocols included antigen/antibody coating, incubation, detection with alkaline phosphatase-conjugated antibodies, and automated spot counting. Safety: Solicited local/systemic AEs captured through day 7 post-boost; unsolicited AEs through day 28; SAEs and AESIs through day 168. Sample size: Assuming detectable group differences, 80 participants (40/group) provided 80% power at two-sided 5% significance; accounting for missingness and stratification, 100 participants (50 per group; equally split by interval strata) were enrolled. Statistical analysis: Continuous variables summarized with means/medians/SDs; categorical as counts/percentages. Group comparisons used independent t-tests or Mann–Whitney where applicable; one-way ANOVA for within-group/time and multi-group comparisons with post hoc tests. GMTs with 95% CIs reported; geometric means for fold-changes used a substitution of 1 for zero/negative change values. Analyses done with SAS 9.4 and GraphPad Prism. Trial registration: ClinicalTrials.gov NCT05054621 (also referenced identifiers include NCT05036241 in data availability).

- Participants: 100 one-dose ChAdOx1 recipients (22–62 years; well-balanced demographics) randomized 1:1 to ChAdOx1 vs MVC-COV1901 boosters; no withdrawals through day 28. - Safety: Common solicited AEs within 1 week post-boost were injection-site pain (63.0%), fatigue (43.0%), headache (28.0%), myalgia (27.0%) with no significant incidence differences between groups. High-grade fatigue was more frequent with homologous ChAdOx1 vs heterologous MVC-COV1901 (18.0% vs 6.0%; P = 0.0160). No serious AEs in either group through day 28. - Neutralizing antibodies (surrogate ELISA): Baseline GMTs were low and similar (32.2 vs 30.2 IU/mL; P = 0.790). After boosting, MVC-COV1901 recipients had higher GMTs: day 1–3, 202.1 IU/mL; day 28, 235.5 IU/mL. These were 2.6-fold (day 1–3) and substantially higher (reported as up to 21-fold) than homologous ChAdOx1 at the corresponding time points (both P < 0.001). In the article summary, day-28 wild-type GMTs were 236 IU/mL (MVC-COV1901) vs 115 IU/mL (ChAdOx1), GMT ratio 2.1 (95% CI, 1.4–2.9). - Live virus neutralization at day 28: Heterologous MVC-COV1901 showed superiority vs homologous ChAdOx1 against Wuhan strain (between-group P < 0.0010) and Delta variant (P < 0.0001). Reported GMT ratio against Delta was 2.6 (95% CI, 1.8–3.5). - Binding antibodies: S1-binding antibodies increased post-boost with higher responses in heterologous recipients (details summarized alongside Fig. 3; anti-S1 responses rose from baseline to day 10 ± 3 and day 28 ± 3). - Dosing interval effect: In the heterologous group, short interval (4–6 weeks) yielded higher nAb GMTs than long interval (8–10 weeks): at an early post-boost timepoint (reported as day 0), 258.4 vs 158.2 IU/mL (P = 0.250); at day 28, 325.1 vs 170.5 IU/mL (P = 0.050). A similar trend favoring short interval appeared in homologous ChAdOx1 at day 10 ± 3. Interval effects on increased immunogenicity persisted at day 28 for the heterologous schedule but not for homologous. - B-cell responses: Spike-specific antibody-secreting B cells (ELISpot) increased significantly by day 10 ± 3 in both groups (one-way ANOVA, P < 0.0001). IgG predominated; IgA increased; IgM was minimal. Heterologous MVC-COV1901 induced higher frequencies than homologous ChAdOx1 for IgG (P = 0.0007) and IgA (P = 0.0010), not IgM. Spike-specific IgG-secreting cell frequency correlated with surrogate neutralizing titers on day 10 ± 3 (P = 0.0002) and day 28 ± 3 (P = 0.0024). The strongest day 10 ± 3 IgG ASC response occurred in heterologous recipients with the shortest interval (P = 0.0004). - T-cell responses: Heterologous recipients showed significant boosts in IFN-γ ELISpot responses at day 10 ± 3 (P < 0.0001) and day 28 ± 3 (P = 0.046), remaining nearly 15-fold above baseline at day 28. Heterologous responses exceeded homologous at day 10 ± 3 (P = 0.0039) and day 28 ± 3 (P = 0.0051). S1 responses tended to be higher than S2 in both groups. Interval analyses showed higher T-cell responses with shorter intervals in some comparisons and, at day 28 ± 3, a higher fold-change in heterologous long-interval vs homologous short-interval subgroups (P = 0.0234). - Assay correlation: Strong correlation observed between surrogate neutralization ELISA and binding antibody assays; similar fold-increase patterns across methods.

The randomized, participant-blinded trial demonstrates that heterologous boosting with MVC-COV1901 after ChAdOx1 significantly enhances both humoral and cellular immunity compared with homologous ChAdOx1 boosting. Neutralizing antibody titers (against wild-type and Delta) and spike-specific B- and T-cell responses were all higher with the heterologous schedule, with favorable reactogenicity (lower high-grade fatigue and no SAEs). Shorter prime-boost intervals (4–6 weeks) tended to yield higher immunogenicity, particularly in the heterologous group, although interval effects varied by timepoint and endpoint. These findings align with the broader literature showing immunologic advantages of heterologous regimens after adenoviral-vector priming and suggest protein subunit boosters can provide robust, potentially broader protection against variants. The observed strong T-cell and B-cell responses may contribute to enhanced cross-variant immunity. Collectively, the results support mix-and-match strategies using protein subunit vaccines to augment immunity in ChAdOx1-primed individuals while maintaining an acceptable safety profile.

Heterologous prime-boost vaccination with ChAdOx1 followed by MVC-COV1901 was safe and elicited superior humoral and cellular immune responses compared with a homologous ChAdOx1 booster. Benefits were notable at shorter prime-boost intervals. These data support use of protein-based subunit boosters for individuals primed with adenoviral-vector vaccines to enhance protection against ancestral SARS-CoV-2 and variants such as Delta. Ongoing follow-up to days 56 and 168 will clarify durability and kinetics of responses. Future research should assess breadth against emerging variants, optimize prime-boost intervals, evaluate real-world effectiveness, and explore the functional antibody repertoire induced by heterologous schedules.

- Modest sample size (n=100) limits precision and subgroup analyses. - Single-center study population (primarily healthy Han Taiwanese) may limit generalizability. - Interim analysis through day 28 post-boost; durability beyond this timepoint pending. - Single-blind design with unblinded vaccinators; potential for minor bias, though lab staff were blinded. - Reliance on surrogate ELISA neutralization assays for primary immunogenicity, with limited live-virus neutralization timepoints. - Interval effects were exploratory and may be influenced by context; comparisons with other trials are complicated by differing epidemiology and potential background infections.