Trained immunity induction by the inactivated mucosal vaccine MV130 protects against experimental viral respiratory infections

Abstract

MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity.

Keywords: Candida albicans; human monocytes; influenza; macrophages; mucosal; polybacterial immunomodulator; respiratory infection; trained immunity; vaccine; viral infection.

Graphical abstract

Figure 1

MV130 protects against viral respiratory infections (A) Graphical outline of the in vivo models of MV130 (10⁹ bacteria/mL) treatment followed by i.n. viral infection. (B) Weight loss after VACV i.n. infection (5 × 10⁴ PFU/mouse). Mean ± SEM are presented. (C) Lung viral load on day 3 post VACV infection. Individual data and the mean ± SEM are presented. (D and E) Weight loss (D) and survival (E) of mice infected i.n. with influenza A virus (2 × 10³ PFU/mouse = 2 × LD₅₀). ‡ indicates that the excipient group had to be excluded from the graph from day 6 because mice started to die (D). In (B) and (D), weights were recorded daily and compared using a two-way ANOVA test. In (C), lung viral titers were compared using an unpaired Student's t test. In (E), survival curves were compared with a log rank (Mantel-Cox) test. Results from a pool of two independent experiments with n = 16 (B) and n = 15 (D and E) mice per group. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 2

MV130 modulates lung immune landscape and promotes long-term protection against viral infections (A) Graphical outline for data in (B) and (C). (B and C) Absolute numbers of the indicated lung immune cell populations, myeloid (B) and lymphoid (C), on day 1 and day 7 after the last MV130 (10⁹ bacteria/mL) administration, were analyzed by flow cytometry. Individual data (n = 5) and the mean ± SEM are shown. ^∗∗p < 0.01; ^∗∗∗p < 0.001 (unpaired Student's t test comparing excipient and MV130 in the same time point). (D) Graphical outline for data in (E), (F), and (G). (E and F) Absolute numbers of the indicated lung immune cell populations, myeloid (E) and lymphoid (F), 3 months after the last MV130 (10⁹ bacteria/mL) administration, were analyzed by flow cytometry. Individual data (n = 10) and the mean ± SEM are shown. ^∗∗∗p < 0.001 (unpaired Student's t test comparing excipient and MV130). (G) Weight loss of mice infected i.n. with influenza A virus (10³ PFU/mouse). Results from a pool of two independent experiments (mean ± SEM) are shown (n = 16 in excipient group and 16 in MV130 group). ‡ indicates that the excipient group had to be excluded from the graph from day 10 because mice started to die. Weights were compared using a two-way ANOVA test comparing MV130 and excipient groups. ^∗∗p < 0.01.

Figure 3

MV130 shows key features of trained immunity (A) Graphical outline: wild-type C57BL/6 and Rag1^−/− mice were treated with MV130 (10⁹ bacteria/mL) or excipient at day −7 and day −4, followed by intravenous infection with 3 × 10⁵ (B) or 1.5 × 10⁵ (C) C. albicans at day 0. (B and C) Survival of wild-type C57BL/6 (B) and Rag1^−/− (C) mice in two pooled independent experiments with n = 19 per group (B) and with n = 20 for excipient-treated and n = 19 for MV130-treated (C) mice. Survival curves were compared with log rank (Mantel-Cox) test. ^∗∗p < 0.01. (D) Graphical outline of the effect of in vivo inhibition with metformin (or not) in mice pretreated with MV130 (10⁹ bacteria/mL) or excipient and subsequently challenged with influenza A virus (2 × 10³ PFU/mouse = 2 × LD₅₀). (E and F) Weight loss (E) and survival (F) of mice following the treatments indicated in (D). Results from a pool of two independent experiments are shown, with n = 13 for excipient, n = 11 for MV130, n = 12 for excipient + metformin, and n = 13 for MV130 + metformin treated mice. Weights were compared using a two-way ANOVA test comparing MV130 and metformin + MV130 groups. Survival curves were compared with log rank (Mantel-Cox) test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (G) Graphical outline for data in (H) and (I). (H and I) Weight loss (H) and survival (I) of mice following 2 weeks of treatment with MV130/excipient as shown in (G). Results from a pool of two independent experiments are shown (n = 20 in excipient group and 18 in MV130 group). (E and H) data are shown as mean ± SEM. Weights were compared using a two-way ANOVA test comparing MV130 and excipient groups. Survival curves were compared with log rank (Mantel-Cox) test. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

MV130 trains bone marrow myeloid progenitors (A) Graphical outline of the in vivo model of MV130 treatment followed by myeloid progenitor (MPP) sorting and subsequent ATAC-seq analysis. (B and C) ATAC-seq analysis in MPPs sorted from bone marrow of mice 1 week after the last challenge with MV130 or its excipient. Bar plot showing enrichment results for all gene sets in the Hallmark collection. Asterisks denote significant enrichment (FDR q value <0.25). Positive enrichment score (in red) suggests association between gene sets and regions with increased chromatin accessibility in MV130-treated samples. Negative enrichment score is in blue (B). GSEA for TNFα signaling, inflammatory response, and DNA repair with their equivalent volcano plots showing biological pathways and genes differentially regulated in excipient-treated and MV130-treated mice (C). (D) Graphical outline of the in vivo model of MV130 treatment followed by BMDM differentiation and challenge. (E) TNFα production by ELISA in the supernatants of BMDM in response to LPS treatment. Individual data (n = 9) and the mean ± SEM from two independent experiments. Unpaired Student's t test between MV130 and excipient conditions. ^∗p < 0.05; ^∗∗p < 0.01.

Figure 5

MV130 trains human monocytes in vitro (A) Schematic representation of the in vitro human monocyte training with MV130. (B and C) Measurement of both TNFα (B) and IL-6 (C) levels in the supernatants of human monocytes treated as indicated in (A) by ELISA. Each dot represents an independent experiment using monocytes from different donors (n = 6) in independent experiments; mean ± SEM are presented. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 using paired Student's t test. (D and E) ATAC-seq analysis in human monocytes 5 days after challenge with MV130 or its excipient. Bar plot showing enrichment results for all gene sets in the Hallmark collection. Asterisks denote significant enrichment (FDR q value <0.25). Positive enrichment score (in red) suggests association between gene sets and regions with increased chromatin accessibility in MV130-treated samples. Negative enrichment score is in blue (D). GSEA for inflammatory response and TNFα signaling with their equivalent volcano plots showing biological pathways and genes differentially regulated in excipient- and MV130-treated cells (E). (F) Oxygen consumption rate (OCR) of human monocytes incubated with excipient or MV130 was analyzed by Seahorse extracellular flux assay at day 5 post challenge. Basal respiration rate (BRR) was defined as OCR before inhibitor addition. Maximal respiration rate (MRR) was defined as the OCR after addition of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Spare respiration capacity (SRC) was defined as the difference between MRR and BRR. Extracellular acidification rate (ECAR) was measured in the absence of inhibitor. Error bars represent SEM. ^∗p < 0.05. (G) Analysis of lactate production in the supernatant of human monocytes 24 h (left, n = 15) and 7 days (right, n = 9) after training with MV130 or excipient. Each dot represents an independent experiment using monocytes from different donors; dots from the same experiment are paired. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 using paired Student's t test.