Oral Immunization with Escherichia coli Nissle 1917 Expressing SARS-CoV-2 Spike Protein Induces Mucosal and Systemic Antibody Responses in Mice

Abstract

As of October 2022, the COVID-19 pandemic continues to pose a major public health conundrum, with increased rates of symptomatic infections in vaccinated individuals. An ideal vaccine candidate for the prevention of outbreaks should be rapidly scalable, easy to administer, and able to elicit a potent mucosal immunity. Towards this aim, we proposed an engineered Escherichia coli (E. coli) Nissle 1917 (EcN) strain with SARS-CoV-2 spike protein (SP)-coding plasmid, which was able to expose SP on its cellular surface by a hybridization with the adhesin involved in diffuse adherence 1 (AIDA1). In this study, we presented the effectiveness of a 16-week intragastrically administered, engineered EcN in producing specific systemic and mucosal immunoglobulins against SARS-CoV-2 SP in mice. We observed a time-dependent increase in anti-SARS-CoV-2 SP IgG antibodies in the sera at week 4, with a titre that more than doubled by week 12 and a stable circulating titre by week 16 (+309% and +325% vs. control; both p < 0.001). A parallel rise in mucosal IgA antibody titre in stools, measured via intestinal and bronchoalveolar lavage fluids of the treated mice, reached a plateau by week 12 and until the end of the immunization protocol (+300, +47, and +150%, at week 16; all p < 0.001 vs. controls). If confirmed in animal models of infection, our data indicated that the engineered EcN may be a potential candidate as an oral vaccine against COVID-19. It is safe, inexpensive, and, most importantly, able to stimulate the production of both systemic and mucosal anti-SARS-CoV-2 spike-protein antibodies.

Keywords: COVID-19; IgA; engineered probiotics; oral vaccine.

Scheme 1

Immunization protocol and sample collection during the 16 weeks.

Figure 1

EcN-pAIDA1-SP expresses SARS-CoV-1 spike protein on the cell surface as shown by immunofluorescence and brightfield (100× magnification) (A) and Western blot analysis for SARS-CoV-2 spike protein S1 quantification (B,C), (see Supplementary Figure for uncropped blot). Results are expressed as mean ± SD of n = 4 experiments performed in triplicate.

Figure 2

Expression of SARS-CoV-1 spike protein in fecal samples of the EcN-pAIDA1-SP, EcN-pAIDA1, and control mice. Western blot analysis for SARS-CoV-2 spike protein S1 quantification (A,B). Results are expressed in mean ± SD of n = 4 experiments performed in triplicate. (*** p < 0.001 vs. control group). (See Supplementary Figure for uncropped blots).

Figure 3

E. coli Nissle pAIDA1-Spike triggered IgG(s) production after oral administration. IgG titre in sera samples of mice treated with control (black), EcN-pAIDA1 2 × 10⁹ CFU (red), and EcN-pAIDA1-SP 2 × 10⁹ CFU (blue) at weeks 0, 2, 4, 6, 8, 10, 12, 14, and 16. Results are expressed in mean ± SD of n = 4 experiments performed in triplicate. (** p < 0.01 vs. control group, *** p < 0.001 vs. control group).

Figure 4

E. coli Nissle pAIDA1-Spike triggered IgA(s) production after oral administration. IgA titre in samples of mice treated with control (black), EcN-pAIDA1 2 × 10⁹ CFU (red), and EcN-pAIDA1-SP 2 × 10⁹ CFU (blue) at weeks 0, 4, 8, 12, and 16. (A) IgA titre in fecal samples of mice treated with gavage of PBS (black), EcN-pAIDA1 d 2 × 10⁹ CFU (red), and EcN-pAIDA1-SP 2 × 10⁹ CFU (blue). (B) IgA titre in GLF samples of mice treated with gavage of PBS (black), EcN-pAIDA1 d 2 × 10⁹ CFU (red), and EcN-pAIDA1-SP 2 × 10⁹ CFU (blue). (C) IgA titre in BALF samples of mice treated with gavage of PBS (black), EcN-pAIDA1 d 2 × 10⁹ CFU (red), and EcN-pAIDA1-SP 2 × 10⁹ CFU (blue). Results are expressed in mean ± SD of n = 8 experiments performed in triplicate. (* p < 0.05 vs. control group, ** p < 0.01 vs. control group, *** p < 0.001 vs. control group).

Figure 5

Immunofluorescence staining and their respective quantification corresponding to (A,B) CD103 and (C–F) CD138 at the colon and spleen sites, showing the effects of the EcN-pAIDA1-SP on mucosal dendritic cells in the gut (A) and on plasma cell population in the colonic mucosa and at the spleen site. All the samples of EcN-pAISA1-SP immunized group, EcN-pAIDA1 group, and control group were collected at week 16. Nuclei were stained using Hoechst staining. Results are expressed as mean ± SD of n = 5 experiments performed in triplicate. *** p < 0.001 vs. control. Scale bar = 20 μm (A,C); 100 μm (D).

Figure 6

Specific anti-SARS-CoV-2 spike IgG antibody avidity detected in sera samples of EcN-pAISA1-SP immunized group at weeks 4, 8, 12, and 16 (A). Specific anti-SARS-CoV-2 spike IgA antibody avidity detected in BALFs samples of EcN-pAIDA1-SP immunized group at weeks 4, 8, 12, and 16 (B). Specific anti-SARS-CoV-2 spike IgA antibody avidity detected in GLFs samples of EcN-pAISA1-SP immunized group at weeks 4, 8, 12, and 16 (C). Results were calculated as ODurea/ODreference and express as a percentage of n = 4 experiments performed in triplicate.

Figure 7

Representative images of hematoxylin-and-eosin (H&E) stained colon sections (A) and relative histological damage score showing the effect of the EcN-pAIDA1-SP (B); magnification 4×; samples collected at t = 16 weeks. Serum levels of LPS after oral administration of control, EcN-pAIDA1, and EcN-pAIDA1-SP; samples collected at t = 16 weeks (C). Results are expressed as mean ± SD of n = 4 experiments performed in triplicate.