Mass Cytometry Analysis of High-Dimensional Single-Cell Immune Profiles in ZF2001-Vaccinated Patients Infected with SARS-CoV-2

Abstract

Introduction: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a public health emergency of international concern (PHEIC) by the WHO. ZF2001, a protein subunit vaccine targeting the RBD, was utilized to evaluate its impact on the immune system of COVID-19 patients. This study aimed to investigate peripheral cell profiles one year after three doses of ZF2001 vaccine using single cell mass spectrometry flow cytometry (CyTOF), a technique that allows detailed characterization of the immune response against SARS-COV-2 infection and further evaluation of ZF2001 mechanisms as a prophylactic against chronic disease and reducing mortality.

Methods: This study profiled peripheral blood mononuclear cells (PBMCs) from 16 vaccinated COVID-19 patients (Omicron 5.2) and 8 hDs using CyTOF with a 41-antibody panel. PBMCs isolated via Lymphoprep density gradient underwent metal-tagged antibody staining. Data analysis included FlowJo gating, Seurat/Harmony batch correction, PhenoGraph clustering (k=45), and t-SNE visualization. Statistical assessments employed Wilcoxon tests and Spearman correlation.

Results: Our findings revealed significant differences between infected and healthy individuals one year after three doses of ZF2001. Specifically, infected individuals exhibited: significant elevation of cytotoxic T cells expressing CD8 with a proliferation marker antigen-Kiel 67 (Ki67) and an adhesion molecule (CD138), expansion of B cells and reduction of monocytes expressing CD16, as well as depletion of CD4+ T cells and differentiation of T cells 1 year after the vaccine. These changes suggested that the vaccine was effectively modulating the immune response.

Discussion: Our results provided a detailed single-cell profile of the immune response to SARS-CoV-2 infection in vaccinated patients, highlighting significant changes in immune cell kinetics indicative of an active innate and adaptive immune cell response.

Keywords: COVID-19; CyTOF; ZF2001; immune response; vaccine.

Figure 1

Timeline for vaccination, COVID-19 infection and sample acquisition followed by experimental approach. (A) Vaccination and sample collection schedule. The vaccination regimen with ZF2001 was administered in three doses. The first dose was administered over the course of one week, beginning on April 30, 2021. The second dose commenced on June 8, 2021, and was completed within two months. The third dose was administered between July and September 2021. A COVID-19 infection was recorded on October 17, 2022, followed by the collection of blood samples on October 24, 2022. In this study, 24 individuals were vaccinated, comprising 8 healthy controls and 16 COVID-19-infected participants. Infection of COVID19 had happened on Oct. 17, 2022 and blood sample were collected on Oct 24, 2022. In our study, 24 people were vaccinated. 8 of theme were health control (HD) and 16 were infected by COVID19. (B) Following isolation, peripheral blood mononuclear cells (PBMCs) were labeled with metal-tagged antibodies and analyzed using cytometry by time of flight (CyTOF) to assess immune responses.

Figure 2

Peripheral blood mononuclear cell cluster sorting according to marker expression levels. (A) Heatmap of normalized immune cell marker expressing in 32 immune cell clusters. (B) T-stochastic neighbor embedding (T-SNE) map was colored by defined cell types in control, mild and moderated groups.

Figure 3

Comparative Analysis of T-cell Subsets in Healthy Donors and COVID-19 Patients. (A) t-Distributed Stochastic Neighbor Embedding (t-SNE) plot displaying the distribution of T-cell subsets, colored by cell type. (B) t-SNE projections illustrating major T-cell subsets in healthy donors and COVID-19 patients, colored by group. (C) Box plots depicting the frequency of CD4⁺ T cells, which were significantly higher in healthy donors (p<0.05), while CD8⁺ T cells showed no significant differences between the groups. (D) Box plots indicating reduced frequencies of Th1 cells, Th2 cells and CD4⁺ T naive (TN) cells in COVID-19 patients compared to healthy donors. (E) Box plots showing an increased frequency of Ki67⁺CD8⁺ effector T cells (Te) in COVID-19 patients, whereas CD8⁺ effector memory T cells (Tem) were lower. (F) Bubble plots illustrating proteins with significant differential expression in CD4⁺ T cell subtypes between groups. (G) Bubble plots illustrating proteins with significant differential expression in CD8⁺ T cell subtypes between groups. Dot size represents the negative log10 p-value, with green dots indicating higher expression in healthy donors and red dots indicating lower expression. Only proteins with an absolute log2 fold change over 1 and p<0.05 were selected for display following a unpaired Wilcoxon test. Significance levels were denoted as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Figure 4

B-cell Subsets comparison in Healthy Donors and COVID-19 Patients. (A) t-Distributed Stochastic Neighbor Embedding (t-SNE) plot displaying the distribution of B-cell subsets, colored by cell type. (B) t-SNE projections illustrating major T-cell subsets in healthy donors and COVID-19 patients, colored by group. (C) Box plots depicting the frequency of B cells and their subtypes, which were significantly higher in covid-19 patients (p<0.01). (D) Bubble plots illustrating proteins with significant differential expression in B cell subtypes between groups. Dot size represents the negative log10 p-value, with green dots indicating higher expression in healthy donors and red dots indicating lower expression. Only proteins with an absolute log2 fold change over 1 and p<0.05 were selected for display following a unpaired Wilcoxon test. Significance levels were denoted as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Figure 5

Monocyte cell Subsets comparison in Healthy Donors and COVID-19 Patients. (A) t-Distributed Stochastic Neighbor Embedding (t-SNE) plot displaying the distribution of Monocyte cell subsets, colored by cell type. (B) t-SNE projections illustrating major Monocyte cell subsets in healthy donors and COVID-19 patients, colored by group. (C) Box plots depicting the frequency of Monocyte cells and their subtypes, which were significantly higher in covid-19 patients (p<0.01). (D) Dot plots illustrating proteins with significant differential expression in B cell subtypes between groups. Dot size represents the negative log10 p-value, with green dots indicating higher expression in healthy donors and red dots indicating lower expression. Only proteins with an absolute log2 fold change over 1 and p<0.05 were selected for display following an unpaired Wilcoxon test. Significance levels were denoted as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.