key: cord-0696042-wpuu53t2 authors: Lee, H. K.; Go, J.; Sung, H.; Kim, S. W.; Walter, M.; Knabl, L.; Furth, P. A.; Hennighausen, L.; Huh, J. W. title: Genetic immune response and antibody repertoire of heterologous ChAdOx1-BNT162b2 vaccination in a Korean cohort date: 2022-02-08 journal: nan DOI: 10.1101/2022.02.07.22270617 sha: 876d87512c46a9cb56056573b38fd2c53fd545c3 doc_id: 696042 cord_uid: wpuu53t2 Heterologous ChAdOx1-BNT162b2 vaccination induces a stronger immune response than two doses of BNT162b2 or ChAdOx1. Yet, the molecular transcriptome, the germline allelic variants of immunoglobulin loci and anti-Omicron antibody levels induced by the heterologous vaccination have not been formally investigated. Moreover, there is a paucity of COVID vaccine studies including diverse genetic populations. Here, we show a robust molecular immune transcriptome and antibody repertoire in 51 office workers from the Republic of Korea after a heterologous ChAdOx1-BNT162b2 vaccination or a homologous ChAdOx1-ChAdOx1 vaccination. Anti-spike-specific IgG antibody levels in the heterologous group increased from 14,000 U/ml to 142,000 AU/ml within eight days after the BNT162b2 vaccination. In contrast, antibody levels in the homologous group increased two-fold after the second ChAdOx1 dose. Antibody titers against the Omicron spike protein as compared to the ancestral strain were reduced to a lesser extent in the heterologous group. RNA-seq conducted on immune cells demonstrated a stronger activation of interferon-induced genetic programs in the heterologous cohort. An increase of specific IGHV clonal transcripts encoding neutralizing antibodies was preferentially detected in the heterologous cohort. Enrichment of B cell and CD4+ T cell responses were observed following both heterologous and homologous vaccination using scRNA-seq, but clonally expanded memory B cells were relatively stronger in the ChAdOx1-BNT162b2 cohort. In summary, a heterologous vaccination with ChAdOx1 followed by BNT162b2 provides an innate and adaptive immune response exceeding that seen in homologous ChAdOx1 vaccinations but equivalent to that seen in homologous BNT162b2 vaccination. The ChAdOx1 nCoV-19 vector (AZD1222) and BNT162b2 mRNA vaccines (hereafter This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022 . Here, we use serology and transcriptome analyses to investigate the immune response of office and laboratory workers from a major health care center in the Republic of Korea. Study participants received one ChAd dose followed either by a second ChAd dose or the BNT vaccine. The sequencing depth in this study, and our previous study on individuals receiving two doses of BNT facilitated the identification of an expanded immune response in individuals receiving the heterologous vaccination. A current discussion centers around the immune response elicited by heterologous ChAdOx1-BNT162b2 (referred to ChAd and BNT throughout the manuscript) vaccinations as compared to homologous two ChAd or two BNT doses. Here we compared the molecular immune response elicited by a heterologous vaccine regimen, ChAd followed by BNT, and a homologous two-dose ChAd regimen. Employees (n=51 This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We measured anti-spike IgG antibody levels, circulating cytokines levels and immune transcriptomes (RNA-seq) on peripheral immune cells prior to the vaccination and on days 2-4 (mean 2.8, median 3.0 days) and 7-10 (mean 7.9, median 7.0 days) post vaccination (referred to day 3 and day 8 throughout the manuscript). For selected samples we also conducted scRNA-seq. First, we measured circulating antibody responses in plasma samples from 45 individuals receiving the heterologous ChAd-BNT vaccine regimen using enzyme-linked immunosorbent assay (ELISA) (Figure 2 ). On average, anti-spike (WH04) IgG levels were approximately 70 AU/ml within three days following the initial ChAd dose and 14,000 AU/ml within three days following the BNT vaccine dose (Figure 2A ; Supplementary Table 2 ). Another 10-fold increase was observed at day 8 following the BNT vaccination. In contrast, no further increase in anti-spike IgG levels was observed in the homologous group between day 3 and 8 following the second ChAd dose (Figure 2B; Supplementary Table 2 ). Similar patterns were observed for the spike proteins from the Alpha, Beta, Gamma, Iota, Kappa, Zeta and Delta variants. Antibody concentrations against Omicron were reduced by approximately 70% in the ChAd-BNT group and 75% in the ChAd-ChAd group compared to the level from the ancestral strain ( This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint activity against different variants, including Delta (B.1.617.2) and used the degree of serum antibodies blocking the binding of ACE2 to SARS-CoV-2 variants' spike as a proxy. While neutralizing activity for the ancestral strain and other variants increased between days 3 and 8 after the BNT162b vaccination in the heterologous cohort, no further increase was detected in the homologous cohort after the second ChAd dose ( Figure 2D - Table 2 ). We also found increased neutralizing activity against Delta variant in the BNT-BNT cohort 15 . Upon delivery of the second vaccine dose, CXCL10, IL-10 and IL-1Ra levels were statistically elevated by BNT but not by ChAd ( Figure 3C -F). Only IL-1Ra was induced in both groups ( Figure 3E -F). CXCL10 (IP-10) expression is rapidly activated following vaccination and viral infections and it has been described as a biomarker associated with COVID-19 severity [16] [17] [18] . Its regulation by IFN-g is mediated by the JAK-STAT signaling pathway 19 . To further understand the molecular underpinnings of the stark differences in antibody responses observed in the two cohorts after the second dose, BNT versus ChAd, we This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 14) . The preferential increase in anti-spike antibodies and neutralizing antibodies in the heterologous cohort upon receiving the second (BNT) vaccine, led us to dig deeper and interrogate the expression profiles of specific germline variable gene classes. Our This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Our study adds to a molecular understanding of findings that a heterologous ChAdOx1- This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint 9 pathway than the second ChAd dose or the second BNT dose in the homologous group. Our analysis permitted the preferential activation of specific variable germline classes as well as CDR3 classes in the ChAd-BNT and ChAd-ChAd groups. An increase of specific IGHV clonal transcripts encoding neutralizing antibodies was preferentially detected in the heterologous cohort and the homologous BNT-BNT cohort. The benefits of adding BNT162b2 as the second dose in heterologous vaccination regimens with ChAdOx1 as the primary dose is well established 5, 33 . In extension, recent studies demonstrated that a three-dose heterologous regimen with two doses of CoronoVac immunization followed by BNT162b2 was associated with a 1.4-fold increase of neutralizing antibodies against Omicron as compared to a homologous two-dose BNT162b2 regimen 34,35 . However, neutralizing activity against Omicron was reduced by 7.1-fold and 3.6-fold compared to the ancestral and Delta variants, respectively. Here we show that a two-dose heterologous vaccine regimen with initial ChAd vaccination followed by BNT resulted in Omicron targeted antibody titers less than 3.2-fold reduced than that found against the ancestral variant as compared to 5.9-fold reduction with ChAd followed by ChAd, and a 4.7-fold reduction in a historical cohort receiving BNT-BNT. Further evolution of COVID-19 variants is an open question, but it is likely that additional variants will emerge, with manufacture of targeted vaccines following their identification. Therefore, it is reasonable to examine vaccine regimens for their ability to induce a range of antibody response that may provide coverage for emerging variants. Here the ChAd-BNT regimen appeared to be successful for that purpose. To date, up to 2.5 billion doses of ChAd have been delivered with an additional 500 million doses of alternative adenovirus based vaccines from Johnson and Johnson and Sputnik from Russia 36 . Data presented here supports that from an immunological perspective of a heterologous adenovirus-based mRNA-based regimen is effective and may be a reasonable cost-effective approach for specific populations where public health officials deem that adenovirus-based vaccine side effects are low risk. A heterologous strategy might also foster enhanced immune responses in immunocompromised individuals. for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. There are several limitations to this study. First, our study population was limited to a specific geographic area (South Korea) and a specific genetic population. Second, most study subjects were healthy females of normal weight. Third, the homologous ChAd-ChAd cohort was smaller than the heterologous ChAd-BNT cohort. Fourth, our study did not investigate neutralizing antibodies using live or pseudo-SARS-CoV-2 virus and variants. Table 1 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Whole blood was collected, and total RNA was extracted from the buffy coat and purified using the RNeasy Mini Kit (Qiagen, #74104) according to the manufacturer's instructions. The concentration and quality of RNA were assessed by an Agilent Bioanalyzer 2100 (Agilent Technologies, CA). The raw data were subjected to QC analyses using the FastQC tool (version 0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). mRNA-seq read quality control was done using Trimmomatic 37 (version 0.36) and STAR RNA-seq 38 (version STAR 2.5.4a) using 150 bp paired-end mode was used to align the reads (hg19). HTSeq 39 (version 0.9.1) was to retrieve the raw counts and subsequently, Bioconductor package DESeq2 40 in R (https://www.R-project.org/) was used to normalize the counts across samples and perform differential expression gene analysis. Additionally, the RUVSeq 41 package was applied to remove confounding factors. The data were pre-filtered keeping for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint only genes with at least ten reads in total. The visualization was done using dplyr (https://CRAN.R-project.org/package=dplyr) and ggplot2 42 . The genes with log2 fold change >1 or <-1 and adjusted p-value (pAdj) <0.05 corrected for multiple testing using the Benjamini-Hochberg method were considered significant and then conducted gene enrichment analysis (GSEA, https://www.gsea-msigdb.org/gsea/msigdb). For T-or B-cell receptor repertoire sequencing analysis, trimmed fastq files from bulk RNA-seq were aligned against human V, D and J gene sequences using the default settings with MiXCR 43,44 . CDR3 sequence and the rearranged BCR/TCR genes were identified. The isolated PBMCs were frozen in freezing media (Thermofisher) and stored at - This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint cytokine levels between two groups, data were presented as standard deviation in each group and were evaluated with a two-way ANOVA followed by Tukey's multiple comparisons test using GraphPad PRISM (version 9.0). A value of *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 was considered statistically significant. This study was approved by the Institutional Review Board (IRB) of Asan medical center in Korea (IRB number 2021-0898). Participant information was coded and anonymized. The RNA-seq and scRNA-seq data from this study will be uploaded in GEO before publishing the manuscript. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 14) . Floating bars (min to max); line at mean. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint Figure 1 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint Figure 2 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint Figure 3 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint Figure 4 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint Figure 5 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270617 doi: medRxiv preprint Figure 6 for use under a CC0 license. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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The copyright holder for this preprint this version posted Systems vaccinology of the BNT162b2 mRNA vaccine in humans Correlation between Reactogenicity and Immunogenicity after the ChAdOx1 nCoV-19 and BNT162b2 mRNA Vaccination Safety and effectiveness of BNT162b2 mRNA Covid-19 vaccine in adolescents Serum Antibody Response Comparison and Adverse Reaction Analysis in Healthcare Workers Vaccinated with the BNT162b2 or ChAdOx1 COVID-19 Vaccine Immune responses to the ChAdOx1 nCoV-19 and BNT162b2 vaccines and to natural COVID-19 infections over a three-month period Robust immune response to the BNT162b mRNA vaccine in an elderly population vaccinated 15 months after recovery from COVID-19. medRxiv A dynamic COVID-19 immune signature includes associations with poor prognosis Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events An interferon-gamma-related cytokine storm in SARS patients JAK inhibitors dampen activation of interferon-stimulated transcription of ACE2 isoforms in human airway epithelial cells Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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The copyright holder for this preprint this version posted authors read and approved the manuscript. The authors declare no competing interests.