Evaluation of Immune Response to Mucosal Immunization with an Oral Probiotic-Based Vaccine in Mice: Potential for Prime-Boost Immunization against SARS-CoV-2

Abstract

Following the conclusion of the COVID-19 pandemic, the persistent genetic variability in the virus and its ongoing circulation within the global population necessitate the enhancement of existing preventive vaccines and the development of novel ones. A while back, we engineered an orally administered probiotic-based vaccine, L3-SARS, by integrating a gene fragment that encodes the spike protein S of the SARS-CoV-2 virus into the genome of the probiotic strain E. faecium L3, inducing the expression of viral antigen on the surface of bacteria. Previous studies demonstrated the efficacy of this vaccine candidate in providing protection against the virus in Syrian hamsters. In this present study, utilizing laboratory mice, we assess the immune response subsequent to immunization via the gastrointestinal mucosa and discuss its potential as an initial phase in a two-stage vaccination strategy. Our findings indicate that the oral administration of L3-SARS elicits an adaptive immune response in mice. Pre-immunization with L3-SARS enhances and prolongs the humoral immune response following a single subcutaneous immunization with a recombinant S-protein analogous to the S-insert of the coronavirus in Enterococcus faecium L3.

Keywords: Enterococcus faecium L3; S protein; SARS-CoV-2; immune response; mucosal vaccines; prime-boost immunization; probiotic-based vaccines.

Figure 1

Assessing L3-SARS persistence in vaccinated mice’s gastrointestinal tract. Fecal samples (100 μg) were homogenized in 1 mL PBS, followed by 2 min of settling to remove larger particles. Afterward, 10 μL of supernatant was aseptically plated on agar and incubated at 37 °C for 24 h to enumerate colonies.

Figure 2

S-specific serum and local immune response after oral immunization with enterococcal strains. Serum and nasal lavages were collected from mice (n = 10) on the indicated days in the figure. Nasal lavages were collected on day 40 after the start of immunization. (A) Sera were treated with Enterococcus faecium L3 enterococci, and the level of IgG antibodies against recombinant protein S was assessed using ELISA. (B) The level of serum IgA antibodies against recombinant protein S was assessed using ELISA. (C) In nasal lavages, the level of IgA antibodies was assessed using ELISA. Reciprocal antibody titers were expressed as log10 and presented as mean ± SEM on the ordinate axis, (*)—p ≤ 0.05.

Figure 3

Serum titration curves comparing before and after treatment with Enterococcus faecium L3. (A) Titration curves of native control (L3) and immune (L3-SARS) sera. (B) Titration curves of control (L3) and immune (L3-SARS) sera after treatment with Enterococcus faecium L3. To remove nonspecific IgG binding, control and immune sera were incubated with Enterococcus faecium L3 as described in the Materials and Methods section. Graph lines show mean OD450 values for each serum dilution and are presented as mean ± SEM on the ordinate axis, (*)—p ≤ 0.05.

Figure 4

Induction of IFN-γ in spleen lymphocytes after S antigen stimulation in vitro. Mouse splenocytes were stimulated with purified recombinant S protein at a final concentration of 1 μg/mL. After 3 and 6 days, cell culture supernatants were collected to examine the amounts of interferon-gamma (IFN-γ) using commercially available cytokine quantitative ELISA kits. IFN γ concentration is presented as mean ± SEM after subtracting the values of non-stimulated splenocytes obtained from the same groups of mice, (*)—p ≤ 0.05.

Figure 5

S-specific serum immune response to heterologous prime-boost with initial oral L3-SARS and subsequent subcutaneous protein S immunization. At 12 day (A,B) and 150 day (C) intervals, prime-boost vaccination. Levels of IgG (A,C) and IgA (B) antibodies against recombinant protein S were assessed using ELISA. Reciprocal antibody titers were expressed as log10 and presented as mean ± SEM, (*)—p ≤ 0.05.

Figure 6

The schedule of the vaccination.

Figure 7

The schedule of the prime-boost effect of enteral vaccination.