SARS-CoV-2 Spike Protein-Expressing Enterococcus for Oral Vaccination: Immunogenicity and Protection

Abstract

The declaration of the conclusion of the COVID-19 pandemic notwithstanding, coronavirus remains prevalent in circulation, and the potential emergence of novel variants of concern introduces the possibility of new outbreaks. Moreover, it is not clear how quickly and to what extent the effectiveness of vaccination will decline as the virus continues to mutate. One possible solution to combat the rapidly mutating coronavirus is the creation of safe vaccine platforms that can be rapidly adapted to deliver new, specific antigens in response to viral mutations. Recombinant probiotic microorganisms that can produce viral antigens by inserting specific viral DNA fragments into their genome show promise as a platform and vector for mucosal vaccine antigen delivery. The authors of this study have developed a convenient and universal technique for inserting the DNA sequences of pathogenic bacteria and viruses into the gene that encodes the pili protein of the probiotic strain E. faecium L3. The paper presents data on the immunogenic properties of two E. faecium L3 vaccine strains, which produce two different fragments of the coronavirus S1 protein, and provides an assessment of the protective efficacy of these oral vaccines against coronavirus infection in Syrian hamsters.

Keywords: SARS-CoV-2 spike protein; mucosal vaccines; probiotic strain E. faecium L3; recombinant probiotic-based vaccines; vaccine efficacy.

Figure 1

Experimental setup.

Figure 2

The structure of the coronavirus inserts in a live probiotic vaccine.

Figure 3

Analysis of specific IgG in sera by ELISA. The OD450 nm values of sera diluted 1:100 are shown. The results are presented as the mean ± SEM (n = 5/group). *—A statistically significant difference between the two groups was identified using a Mann–Whitney U test.

Figure 4

Analysis of specific IgG in pooled blood sera in ELISA with SA and SB proteins as antigens (n = 5/group). After administering the Vac1 (A,C) and Vac2 (B,D) vaccines, blood sera from individual animals were combined and analyzed using an ELISA assay to assess the presence of IgG antibodies against recombinant SA and SB proteins, which are similar to the coronavirus components in Vac1 and Vac2, respectively. (A,B) Comparison of sera in ELISA based on OD450 values. The optical density at 450 nm (OD₄₅₀) was determined for serum diluted 1:20. (C,D) Comparison of sera in ELISA based on the end-point ELISA titers. To determine the titer, the serum dilution was considered at which the OD450 was five times higher than that of the serum from an untreated hamster (OD = 0.240).

Figure 5

Analysis of SARS-CoV-2 virus load in the lungs on days 3 and 6 after oral vaccination. Syrian hamsters were orally administered Vac1, Vac2, and E. faecium L3 according to the schedule in Materials and Methods. Hamsters were orally challenged with SARS-CoV-2 28 days after the second vaccination course. After 3 (n = 3/group) and 6 (n = 3/group) days from the onset of infection, the lungs were obtained. The viral load (A), decrease in viral load (B), and inhibition coefficient (C) were determined. •—A statistically significant difference between the two groups was identified using a Mann–Whitney U test.

Figure 6

Histological and macroscopic analysis of lung tissue. The Syrian hamster lungs were examined on day 6 using Hematoxylin and eosin stain: untreated and uninfected control animals (a); animals that were orally vaccinated with E. faecium L3 (b); live recombinant probiotic vaccines Vac1 (c) and Vac2 (d); untreated and infected hamsters (e) (n = 3/group). The pulmonary pathology in Syrian hamsters after SARS-CoV-2 virus infection on day 3 and 6 post-SARS-CoV-2 virus infection (n = 3/group/point) was quantified using a scoring system (f). Hematoxylin and eosin staining x10. Scale bar corresponds to 200 μm.