Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1

Abstract

Introduction: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies.

Methods: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model.

Results: One dose of 10-μg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta.

Discussion: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system.

Keywords: C1; SARS-CoV-2; Thermothelomyces heterothallica; filamentous fungus; hamster; receptor-binding domain; vaccine.

Figure 1

Production and analysis of antigens used for immunization. (A) RBD-Spytag in C1 fermentation process and C-tag affinity–purified RBD-Spytag analyzed in SDS-PAGE with Coomassie Blue dye staining. Lane 1, MW protein marker; lane 2, 98 h of fermentation supernatant; lane 3, 116 h of fermentation supernatant, which is the starting material for the affinity purification; lanes 4–6, final purified and dialyzed RBD-Spytag protein of 6.5, 3.2, and 1.3 µg, respectively, loaded on SDS-PAGE. The RBD-Spytag, corresponding to the correct size of 24-kDa protein, is marked with an arrow. (B) SDS-PAGE analysis was performed on the SARS-CoV-2 spike RBD antigens or RBD/nanoparticle mixtures used for immunization (left panel). For comparison, nanoparticles were also analyzed separately (right panel). Asterisks indicate contaminating yeast proteins. The abbreviations ST, SC, and NP stand for SpyTag, SpyCatcher, and nanoparticles, respectively. (C) Antigen combinations tested in the Syrian hamster model. (D) Schematic representation of the study design (Created with BioRender.com).

Figure 2

SARS-CoV-2 RBD-nano vaccine induces high neutralizing antibodies titers. Antibodies induced by the different vaccine formulations and neutralizing antibodies titers were quantified at (A, B) 28 days, (C, D) 42 days after receiving the first immunization dose, and (E, F) at the day of necropsy 46 days after immunization. IgG antibodies (A, C, E) were detected by RBD-ELISA, and dotted lines indicate the assay cutoff value $(\overline{x}+2SD)$ based on the control group. Neutralizing antibodies titers (B, D, F) are expressed as the reciprocal of the dilution that gave a 50% reduction of stained cells. P-values were calculated by a two-way ANOVA test; mean ± SD are presented. ****p< 0.0001; ***p< 0.001; **p< 0.01; *p< 0.05.

Figure 3

Neutralizing antibodies induced by SARS-CoV-2 RBD-nano vaccine neutralize SARS-CoV-2 VOCs. Virus neutralization assay against D614G, Alpha, Beta, Gamma, Delta, Omicron BA.1, and Omicron BA.5 variants of concern at (A) day of 42 and (B) day 46 were done using vesicular stomatitis virus pseudotyped with the respective spike protein. Titers are expressed as the reciprocal dilution that reduced entry to 50%. P-values were calculated using a one-way ANOVA analysis. ****p< 0.0001; ***p< 0.001; **p< 0.01; *p< 0.05.

Figure 4

Viral load in the lung is reduced after vaccination with RBD-nano plus adjuvant. Viral titers were calculated in (A) lung and (B) nasal turbinate using TCID₅₀. P-values were calculated using a Brown–Forsythe and Welch ANOVA test. ****p< 0.0001; ***p< 0.001; **p< 0.01; *p< 0.05.

Figure 5

Histopathological lesions and viral antigen in the lungs of SARS-CoV-2–infected hamsters. (A, B) Representative images of a PBS-treated infected control hamster showing multifocal areas of inflammation in alveoli [black arrowheads and inset in (A)] associated with abundant viral antigen in pneumocytes [brown signal, black arrowheads, and inset in (B)]. Inflammatory infiltrates are also present in the airways [white arrowheads in (A)], but only occasional bronchial epithelial cells are positive for viral antigen [white arrowheads in (B)]. (C, D) Representative image of a hamster vaccinated with an RBD vaccine (RBD-nano + Alum) showing inflammatory lesions and viral antigen exclusively in the main airways (white arrowheads, inserts). (A, C) Hematoxylin and eosin stain. (B, D) Immunohistochemistry for SARS-CoV-2 nucleocapsid protein. Insets show 400× magnification of areas delineated by rectangles in the overview images. (E) Semiquantitative score of histological lesions induced by SARS-CoV-2 infection. (F) Semiquantitative score of SARS-CoV-2 antigen present in alveoli and airways. P-values for were calculated using a one-way ANOVA analysis. ****p< 0.0001; ***p< 0.001; **p< 0.01; *p< 0.05.