Boosting Vaccine-Elicited Respiratory Mucosal and Systemic COVID-19 Immunity in Mice With the Oral Lactobacillus plantarum

Abstract

Boosting and prolonging SARS-CoV-2 vaccine-elicited immunity is paramount for containing the COVID-19 pandemic, which wanes substantially within months after vaccination. Here we demonstrate that the unique strain of probiotic Lactobacillus plantarum GUANKE (LPG) could promote SARS-CoV-2-specific immune responses in both effective and memory phases through enhancing interferon signaling and suppressing apoptotic and inflammatory pathways. Interestingly, oral LPG administration promoted SARS-CoV-2 neutralization antibodies even 6 months after immunization. Furthermore, when LPG was given immediately after SARS-CoV-2 vaccine inoculation, specific neutralization antibodies could be boosted >8-fold in bronchoalveolar lavage (BAL) and >2-fold in sera, T-cell responses were persistent and stable for a prolonged period both in BAL and the spleen. Transcriptional analyses showed that oral application of LPG mobilized immune responses in the mucosal and systemic compartments; in particular, gut-spleen and gut-lung immune axes were observed. These results suggest that LPG could be applied in combination with SARS-CoV-2 vaccines to boost and prolong both the effective and memory immune responses in mucosal and systemic compartments, thereby improving the efficacy of SARS-CoV-2 vaccination.

Keywords: COVID-19; Lactobacillus plantarum; accquired immunity; adjuvant; gut-lung axis; memory immunity; probiotics; vaccine.

Figure 1

Oral LPG administration 6 months after immunization improved humoral immune responses in mice. Experimental schedule (A): ICR mice were vaccinated with DNA vaccine at weeks 0 and 4, their binding (B) and neutralization (C) antibody responses were monitored by ELISA and pseudovirus inhibition assays respectively, from 5 weeks to 24, these mice were then orally administrated with PBS or LPG and observed for 42 days. The antibody titers immediately before antibiotic treatment were set as baseline. Sera RBD binding (D) and neutralization (F) antibodies on day 7, 14, 28, and 42 post administration of LPG or PBS were assessed by ELISA and pseudovirus inhibition assay, respectively. “Index” on the Y-axis represents the ratios of titers of binding antibodies (E) and neutralization (G) at indicated time points on the X-axis to their counterparts at baseline and reflects the adjusted relative values after the removal of baseline differences. D: Day; W: Week. Titer data are presented as geometric mean titer ± geometric standard deviation. Mann-Whitney tests were performed to analyze differences between experimental groups **P < 0.01.

Figure 2

Oral LPG administration immediately after immunization improved humoral and cellular immune responses in mice. Experimental schedule (A). ICR mice were primed and boosted with vaccines followed by oral administration of LPG or PBS as described in the Methods. Serum antibody responses were assessed at baseline (immediately before antibiotic treatment) and day 7 post intragastric administration (D7) for RBD-specific binding antibodies by ELISA (B) and pseudovirus neutralization assays (C). BAL antibody responses were assessed on day 7 post intragastric administration for RBD-specific binding antibodies by ELISA (D) and pseudovirus neutralization assays (E). Assessments of RBD-specific T cell responses. T-cell responses in splenocytes (F) and BAL cells (G) were determined on day 3, 7, and 14 post intragastric administration. BAL: Bronchoalveolar lavage; D: Day; W: Week. Titer data are presented as geometric mean titer ± geometric standard deviation; ELISpot counts were expressed as mean ± s.e.m. Mann-Whitney tests were performed to analyze differences between experimental groups. **P < 0.01, ***P < 0.001, and ns, not significant.

Figure 3

Oral LPG administration down-regulates inflammation and apoptosis but promotes IFN signaling in mouse lymphoid tissues. Experimental schedule (A). ICR mice (n = 20/group) were treated as described in the Methods, and different tissues were collected for RNA sequencing. GSEA revealed inhibition of TNF-α signaling (top) and apoptosis (middle) gene sets in lymphoid tissues in the LPG group compared with the PBS group, and enhancement of IFN response (bottom) gene sets in lymphoid and small intestine tissues in the LPG group compared with the PBS group (B). IL-6 and TNF-α in BAL were quantified from mice sacrificed on day 3, 7, and 14 (C). Heatmap shows log2 RPKM values of a selection of genes associated with B cells (D) and T cells (E) from the spleen and small intestine. BAL, bronchoalveolar lavage; ES, enrichment score; FDR-q, false discovery rate q value; NES, normalized enrichment score.