Heterologous ChAdOx1/BNT162b2 vaccination induces stronger immune response than homologous ChAdOx1 vaccination: The pragmatic, multi-center, three-arm, partially randomized HEVACC trial

Abstract

Background: Several COVID-19 vaccines have been approved. The mRNA vaccine from Pfizer/BioNTech (Comirnaty, BNT162b2; BNT) and the vector vaccine from AstraZeneca (Vaxzevria, ChAdOx1; AZ) have been widely used. mRNA vaccines induce high antibody and T cell responses, also to SARS-CoV-2 variants, but are costlier and less stable than the slightly less effective vector vaccines. For vector vaccines, heterologous vaccination schedules have generally proven more effective than homologous schedules.

Methods: In the HEVACC three-arm, single-blinded, adaptive design study (ClinicalTrials.gov Identifier: NCT04907331), participants between 18 and 65 years with no prior history of SARS-CoV-2 infection and a first dose of AZ or BNT were included. The AZ/AZ and the AZ/BNT arms were randomized (in a 1:1 ratio stratified by sex and trial site) and single-blinded, the third arm (BNT/BNT) was observational. We compared the reactogenicity between the study arms and hypothesized that immunogenicity was higher for the heterologous AZ/BNT compared to the homologous AZ/AZ regimen using neutralizing antibody titers as primary endpoint.

Findings: This interim analysis was conducted after 234 participants had been randomized and 254 immunized (N=109 AZ/AZ, N=115 AZ/BNZ, N=30 BNT/BNT). Heterologous AZ/BNT vaccination was well tolerated without study-related severe adverse events. Neutralizing antibody titers on day 30 were statistically significant higher in the AZ/BNT and the BNT/BNT groups than in the AZ/AZ group, for B.1.617.2 (Delta) AZ/AZ median reciprocal titer 75.9 (99.9% CI 58.0 - 132.5), AZ/BNT 571.5 (99.9% CI 396.6 - 733.1), and BNT/BNT 404.5 (99.9% CI 68.3 - 1024). Similarly, the frequency and multifunctionality of spike-specific T cell responses was comparable between the AZ/BNT and the BNT/BNT groups, but lower in the AZ/AZ vaccinees.

Interpretation: This study clearly shows the immunogenicity and safety of heterologous AZ/BNT vaccination and encourages further studies on heterologous vaccination schedules.

Funding: This work was supported by the Medical University of Innsbruck, and partially funded by NIAID contracts No. 75N9301900065, 75N93021C00016, and 75N93019C00051.

Keywords: BNT162b2; ChAdOx1; Heterologous COVID-19 vaccination; Neutralizing antibodies; SARS-CoV-2; T cells.

Figure 1

Patient recruitment. After screening N=18 participants were excluded due to previous infection, not given consent etc. Participants (N=234) who received an AZ prime were randomized to the AZ/AZ or the AZ/BNT arm. Due to organizational reasons (ordering of vaccine doses etc.) randomization was performed 2-4 days prior to vaccination. N=7 for AZ/AZ and N=3 for AZ/BNT dropped out after randomization but prior to vaccination as they meanwhile received their second dose of vaccine outside the study. These participants did not know at the time point of drop-up which vaccine they would have received in the study. Each of the remaining participants was vaccinated according to their randomization. N=1 participant for the AZ/AZ and AZ/BNT arm each dropped out immediately after boost vaccination reducing the subgroup included in follow-up analysis for safety and immunogenicity to N=108 for AZ/AZ and N=114 for AZ/BNT (modified intention to treat population). Some participants have not yet reached day 30 post boost (N=5 for AZ/AZ, N=3 for AZ/BNT, N= 10 for BNT/BNT). Some participants were unavailable on day 10 (N=4 for AZ/AZ, N=0 for AZ/BNT, N= 2 for BNT/BNT) or day 30 (N=1 for AZ/AZ, N=1 for AZ/BNT, N=2 for BNT/BNT). These participants were not excluded from the study but re-invited for the next study visit. However, this reduced the number of participants analyzed per time point compared to the total number of participants within a group. Numbers of participants per group which are analyzed for day 10 and day 30 are shown in the figure.

Figure 2

Antibody responses are higher after heterologous vaccination compared to homologous AZ vaccination. A. Titers of anti-S IgG at screening (3-7 days prior to boost), day 10 and day 30 post boost vaccination. Dotted line indicates detection limit (7.1 BAU/ml). B. Titers of anti-S IgA at screening and day 30 post boost vaccination. Values are expressed as optical density (OD). Dotted lines indicate cut-off values of the assay (< 0.8 negative; 0.8 – 1.1 borderline positive; > 1.1 positive). C. 50% neutralization titers as determined in a VSV pseudovirus neutralization assay using ancestral (wild type) spike. D. 50% neutralization titers against B.1.1.7, B1.351 and B.1.617.2 variants were quantified in a focus forming assay using replication competent SARS-CoV-2 isolates. Median and individual values are shown. For C and D, titers ≤ 1:16 were considered negative (indicated by the dotted line). Values <1:16 were set to 1:16 and values >1:1024 to 1:1024. Statistical differences were determined using Kruskal-Wallis test followed by uncorrected Dunn's comparison between AZ/AZ and AZ/BNT groups. A p value <0.001 was used as significance level according to stopping rule of the interim analysis as applied for the comparison of the primary endpoint neutralizing antibodies between AZ/AZ and AZ/BNT arms. All other comparisons were exploratory. 95 % confidence intervals (95 % CI) for binding antibodies and 99.9 % CI for neutralizing antibodies are shown in Supplementary Tables S3 and S4. AZ/AZ screening n=109, d10 n=104, d30 n=102; AZ/BNT screening n=115, d10 n=114, d30 n=110; BNT/BNT screening n=30, d10 n=28, d30 n=18.

Figure 3

Spike specific T cell responses are higher after heterologous vaccination. A. Quantiferon IFNγ release assay measuring IFNγ (IU/ml) in blood samples from AZ/AZ (blue circles), AZ/BNT (purple circles), BNT/BNT (red cicrles) vaccine groups at 10 days after boost vaccination. Whole blood samples were either non-stimulated (Nil) or stimulated with specific CD4 (Ag1) and CD4+CD8 (Ag2) SARS-CoV-2 peptide pools from spike antigen (S1 S2 RDB). As positive control mitogen-stimulated samples were also analyzed. B. spike-specific CD4⁺ T cells and CD8⁺ T cells were quantified by AIM (surface OX40⁺CD137⁺ and CD69⁺CD137⁺, respectively). Comparison of spike-specific AIM⁺ CD4⁺ T cell and CD8⁺ T frequencies between AZ/AZ (blue circles), AZ/BNT (purple circles), and BNT/BNT (red circles) vaccine regimens at day 10 post boost are shown. The bars indicate the geometric mean and geometric SD in the analysis of the spike-specific CD4⁺ and CD8⁺ T cell frequencies. C. spike-specific CD4⁺ T cells expressing intracellular CD40L (iCD40L) and producing IFNγ, TNFα, IL-2 or granzyme B (GzB) and spike-specific CD8⁺ T cells producing IFNγ, TNFα, IL-2 or GzB by intracellular cytokine staining (ICS) (D). The dotted green line indicates limit of quantification (LOQ). Proportions of multifunctional activity profiles of the spike-specific CD4⁺ (E) and CD8⁺ T cells (F) evaluated on days 10 post boost. The dark blue, navy blue, turquoise and white colors in the pie charts depict the production of one, two, three, and four functions, respectively. spike specific AIM⁺ CD4 and CD8 T cells against the ancestral spike sequence were compared to the spike sequence derived from the B.1.617.2 (G) as well as the P.1., the B.1.427/B.1.429, the B.1.351 and the B.1.1.7 (H) variants at day 10 post boost. Background-subtracted and log data analyzed in all cases. N as indicated in figure. 95 % confidence intervals are shown in Supplementary Tables S6-S8.