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. 2022 Apr:569:56-63.
doi: 10.1016/j.virol.2022.03.001. Epub 2022 Mar 6.

Kappa-RBD produced by glycoengineered Pichia pastoris elicited high neutralizing antibody titers against pseudoviruses of SARS-CoV-2 variants

Taotao Mi  1 , Tiantian Wang  2 , Huifang Xu  2 , Peng Sun  2 , Xuchen Hou  2 , Xinwei Zhang  3 , Qian Ke  4 , Jiawen Liu  2 , Shengwei Hu  5 , Jun Wu  6 , Bo Liu  7
Affiliations

Kappa-RBD produced by glycoengineered Pichia pastoris elicited high neutralizing antibody titers against pseudoviruses of SARS-CoV-2 variants

Taotao Mi et al. Virology. 2022 Apr.

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kappa (B.1.617.1) variant represented the main variant of concern (VOC) for the epidemic in India in May 2021. We have previously established a technology platform for rapidly preparing SARS-CoV-2 receptor-binding domain (RBD) candidate vaccines based on glycoengineered Pichia pastoris. Our previous study revealed that the wild-type RBD (WT-RBD) formulated with aluminum hydroxide and CpG 2006 adjuvant effectively induces neutralizing antibodies in BALB/c mice. In the present study, a glycoengineered P. pastoris expression system was used to prepare recombinant kappa-RBD candidate vaccine. Kappa-RBD formulated with CpG and alum induced BALB/c mice to produce a potent antigen-specific antibody response and neutralizing antibody titers against pseudoviruses of SARS-CoV-2 kappa, delta, lambda, beta, and omicron variants and WT. Therefore, the recombinant kappa-RBD vaccine has sufficient potency to be a promising COVID-19 vaccine candidate.

Keywords: Receptor-binding domain (RBD); SARS-CoV-2 kappa; Vaccine; Yeast.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
SARS-CoV-2 receptor-binding domain (RBD) glycoprotein constructs. (a) Schematic of the S protein of SARS-CoV-2 kappa showing the position of the N-terminal domain (NTD), RBD, subdomains 1 and 2 (SD1 and SD2), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), and transmembrane region (TM). (b) Mutations in the RBD of different SARS-CoV-2 variants. (c) SDS-PAGE analysis of various glycoengineered yeast/RBD clones. Each colony sample in Lanes 1-2-3-4-5-6-7-8. M: size markers; RBD: SARS-CoV-2 RBD. (d) SDS-PAGE analysis of the RBD purification process. (e) SDS-PAGE and western blotting results of RBD digested with PNGase F. (f) BLI profiles measuring the interaction between recombinant RBD and ACE2. A11, A12: 50 nM RBD; C11, C12: 100 nM RBD; D11, D12: 200 nM RBD; F11, F12: 400 nM RBD; G11, G12: baseline.
Fig. 2
Fig. 2
Humoral immune responses to RBD vaccination by BALB/c mice. (a) Immunological strategy for BALB/c mice. (b) Changes in the bodyweight of mice from different test and control groups. (c) Antibody titers after initial immunization with vaccines containing different adjuvant components and different antigen doses. (d) Antibody titers after the second immunization with vaccines containing different adjuvant components and different antigen doses. Specific (e) IgG1, (f) IgG2a, (g) IgG2b, and (h) IgG3 antibody titers after the second immunization with vaccines containing different adjuvant components and different antigen doses. The P-values were determined by an independent t-test (*P < 0.05, **P < 0.01, ***P < 0.005). Normal saline: 0.9% (w/v) NaCl.
Fig. 3
Fig. 3
Cellular immune responses by BALB/c mice to vaccination with 10 μg RBD. (a) Splenic IFN-γ, IL-2, and IL-4 ELIspot responses to the SARS-CoV-2 RBD antigen. (b) Splenic IFN-γ, IL-2, and IL-4 ELIspot responses to CpG. SFCs: Spot-forming cells.
Fig. 4
Fig. 4
Neutralizing activity against the pseudoviruses (SARS-CoV-2 kappa, lambda, delta, and beta) following vaccination with RBD. The P-values were determined by an independent t-test (*P < 0.05, **P < 0.01, ***P < 0.005). IC50: half maximal inhibitory concentration.
Fig. 5
Fig. 5
Neutralization by immune serum against pseudoviruses of omicron and wild-type (WT) SARS-CoV-2. The P-values were determined by an independent t-test (*P < 0.05, **P < 0.01, ***P < 0.005). IC50: half maximal inhibitory concentration.
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