Heterologous Booster Immunization Based on Inactivated SARS-CoV-2 Vaccine Enhances Humoral Immunity and Promotes BCR Repertoire Development

Abstract

Recent studies have indicated that sequentially administering SARS-CoV-2 vaccines can result in increased antibody and cellular immune responses. In this study, we compared homologous and heterologous immunization strategies following two doses of inactivated vaccines in a mouse model. Our research demonstrates that heterologous sequential immunization resulted in more immune responses displayed in the lymph node germinal center, which induced a greater number of antibody-secreting cells (ASCs), resulting in enhanced humoral and cellular immune responses and increased cross-protection against five variant strains. In further single B-cell analysis, the above findings were supported by the presence of unique B-cell receptor (BCR) repertoires and diversity in CDR3 sequence profiles elicited by a heterologous booster immunization strategy.

Keywords: B-cell receptor; SARS-CoV-2; humoral immunity; sequential immunization; vaccine.

Figure 1

Booster immunization significantly increases antigen-specific antibodies in mice. (a) Immunization procedures for mice; Group A: 2×INA (I-I), Group B: 3×INA (I-I-I), Group C: 2×INA+protein (I-I-P), Group D: 2×INA+mRNA (I-I-M). (b) SARS-CoV-2 S1-specific total IgG levels were assessed 14 days post immunization in four groups of mice. (c–h) The neutralizing antibody titers of pseudovirus were analyzed for six different variants of the virus, including WT, Alpha, Beta, Gamma, Delta, and Omicron BA.1. (c) WT, (d) Alpha, (e) Beta, (f) Gamma, (g) Delta, (h) Omicron BA.1. (i) SARS-CoV-2 S1-specific total IgG titers 90 days after immunization. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Heterologous booster immunization elicits stronger antibody-secreting cells and induces IgG antibody class switching while enhancing the T-cell response (Group A: I-I; Group B: I-I-I; Group C: I-I-P; Group D: I-I-M). (a,b) Splenic lymphocytes from four groups of mice were analyzed for their ability to produce ASCs in response to the S1 antigen. (c) Ratios of IgG1 to IgG2a in the four groups of mice. (d,e) GC center reaction. (d) Proportions of GC B-cells in lymph nodes. (e) Proportions of TFH cells in lymph nodes. (f–j) T-cell response of four groups of mice. (f,g) S1-specific IFN-γ-secreting cells in splenic lymphocytes. (h,j) S1-specific IL-4-secreting cells in splenic lymphocytes. (j) Proportion of CD8+ splenic lymphocytes.). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Concentration of ten cytokines in the serum of mice sacrificed at the completion of the immunization schedule (Group A: I-I; Group B: I-I-I; Group C: I-I-P; Group D: I-I-M). (a) IL-1β, (b) IL-2, (c) IL-4, (d) IL-5, (e) IL-6, (f) IL-10, (g) IL-12, (h) CXCL-1, (i) IFN-γ, (j) TNF-α. * p < 0.05, ** p < 0.01.

Figure 4

Flow cytometric sorting of antigen-specific B-cells and analysis of lineage distribution (Group A: I-I; Group B: I-I-I; Group C: I-I-P; Group D: I-I-M). (a) Flow cytometry sorting of antigen-specific B-cells. (b,c) Somatic hypermutation in IgH- and IgK-encoding genes. (d–f) Distribution of germline genes encoding IgH. (e–g) Distribution of germline genes in IgK. (h,i) Distribution of germline high-frequency gene subclasses encoding IgH and IgK.

Figure 5

Heterologous boosting increases the diversity of CDR3 (Group A: I-I; Group B: I-I-I; Group C: I-I-P; Group D: I-I-M). (a,b) Number of common and unique CDR3 sequences in the four groups. (a) H-CDR3. (b) K-CDR3. Green ovals represent group A, blue ovals represent group B, red ovals represent group C, and yellow ovals represent group D. The overlap between them indicates the number of shared sequences. (c,d) Length distribution of CDR3. (c) H-CDR3. (d) K-CDR3.