A Combination of Polybacterial MV140 and Candida albicans V132 as a Potential Novel Trained Immunity-Based Vaccine for Genitourinary Tract Infections

Abstract

Recurrent urinary tract infections (RUTIs) and recurrent vulvovaginal candidiasis (RVVCs) represent major healthcare problems with high socio-economic impact worldwide. Antibiotic and antifungal prophylaxis remain the gold standard treatments for RUTIs and RVVCs, contributing to the massive rise of antimicrobial resistance, microbiota alterations and co-infections. Therefore, the development of novel vaccine strategies for these infections are sorely needed. The sublingual heat-inactivated polyvalent bacterial vaccine MV140 shows clinical efficacy for the prevention of RUTIs and promotes Th1/Th17 and IL-10 immune responses. V132 is a sublingual preparation of heat-inactivated Candida albicans developed against RVVCs. A vaccine formulation combining both MV140 and V132 might well represent a suitable approach for concomitant genitourinary tract infections (GUTIs), but detailed mechanistic preclinical studies are still needed. Herein, we showed that the combination of MV140 and V132 imprints human dendritic cells (DCs) with the capacity to polarize potent IFN-γ- and IL-17A-producing T cells and FOXP3+ regulatory T (Treg) cells. MV140/V132 activates mitogen-activated protein kinases (MAPK)-, nuclear factor-κB (NF-κB)- and mammalian target of rapamycin (mTOR)-mediated signaling pathways in human DCs. MV140/V132 also promotes metabolic and epigenetic reprogramming in human DCs, which are key molecular mechanisms involved in the induction of innate trained immunity. Splenocytes from mice sublingually immunized with MV140/V132 display enhanced proliferative responses of CD4+ T cells not only upon in vitro stimulation with the related antigens contained in the vaccine formulation but also upon stimulation with phytohaemagglutinin. Additionally, in vivo sublingual immunization with MV140/V132 induces the generation of IgG and IgA antibodies against all the components contained in the vaccine formulation. We uncover immunological mechanisms underlying the potential mode of action of a combination of MV140 and V132 as a novel promising trained immunity-based vaccine (TIbV) for GUTIs.

Keywords: candida albicans V132; dendritic cells; polybacterial preparation MV140; recurrent urinary tract infections (RUTIs); recurrent vulvovaginal candidiasis (RVVCs); trained immunity-based vaccines (TIbVs).

Conflict of interest statement

OP has received fee for lectures or participation in Advisory Boards from Allergy Therapeutics, Amgen, AstraZeneca, Diater, GSK, Inmunotek SL, Novartis, Sanofi Genzyme, Stallergenes and Regeneron. OP has received research grants from Inmunotek SL and Novartis SL. JS is the founder and CEO of Inmunotek SL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SI declared a shared affiliation, with no collaboration, with several of the authors, LC, CO, CV and OP, to the handling editor at the time of review.

Copyright © 2021 Martin-Cruz, Sevilla-Ortega, Benito-Villalvilla, Diez‐Rivero, Sanchez-Ramón, Subiza and Palomares.

Figures

Figure 1

MV140/V132 induces hmoDCs maturation and production of pro-inflammatory cytokines and IL-10. (A) Cytokine production after stimulation of hmoDCs with control excipients (containing all excipients except the bacteria and yeast), V132, MV140, and MV140/V132 for 18 h quantified in cell-free supernatants by ELISA. (B) Cytokine levels of V132-treated hmoDCs plus cytokine levels of MV140-treated hmoDCs compared with cytokine levels of MV140/V132-stimulated hmoDCs. (C) Percentage of CD83- and HLA-DR-positive cells after stimulation with control excipients, V132, MV140 and MV140/V132 for 18 h. (D) Flow cytometry representative dot plots for the expression of surface markers CD83 and HLA-DR after stimulation of hmoDCs with the indicated stimulus. (E) Mean Fluorescence Intensity (MFI) values for CD83 and HLA-DR. Results are mean ± s.e.m. of 6-8 (A), 6 (B), and 7-8 (C, E) independent experiments. Paired Student t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2

MV140/V132-activated hmoDCs induce T cell proliferation and Th1, Th17, and IL-10 producing T cells. (A) Representative dot plots of proliferating CFSE-labeled allogeneic naive CD4+ T cells after 3 days of co-culture with control excipients-, V132-, MV140-, and MV140/V132-stimulated hmoDCs and the graph with the frequency of proliferating cells (right side). (B) Cytokines produced by allogeneic naive CD4+ T cells primed by hmoDCs in the presence of the indicated stimulus after 3 days quantified in cell-free supernatants by ELISA. (C) Levels of IL-17A and IL-10 for V132- plus MV140-stimulated co-cultures compared to MV140/V132-stimulated co-cultures. Results are mean ± s.e.m. of 5 (A), and 8 to 12 (B, C) independent experiments. Paired Student t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

MV140/V132-activated hmoDCs generate IFN-γ-, IL-17A-, and IL-10-producing T cell production as well as FOXP3+ Treg cells. (A) Percentage of CD3+CD4+ T cells producing IFN-γ, IL-10 and IL-17A generated after 3 days of co-culture with control excipients-, V132-, MV140-, and MV140/V132-stimulated hmoDCs as determined by intracellular staining and flow cytometry analysis. Representative dot plots are shown for the intracellular staining after flow cytometry analysis. (B) Percentage of induced CD4+CD25highCD127-FOXP3+ Treg cells gated on CD4+ T cells after 3 days of co-culture with allogeneic control excipients-, V132-, MV140-, and MV140/V132-activated hmoDCs. Results are mean ± s.e.m. of 7-8 (A), and 5 (B) independent experiments. Paired Student t test, *P < 0.05, and **P < 0.01.

Figure 4

Activation of MAPKs, NF-κB, and mTOR signaling pathways in hmoDCs stimulated with control excipients, V132, MV140, or MV140/V132. (A, B) Western blot analysis of protein extracts from hmoDCs stimulated for 30 min in the indicated conditions. Quantification of the reactive phosphorylated bands by scanning densitometry. β-actin was used as a loading control. One representative example is shown. Results are mean ± s.e.m. of four to seven independent experiments. Paired Student t test, *P < 0.05, and **P < 0.01.

Figure 5

MV140/V132 induces metabolic and epigenetic reprogramming in hmoDCs. (A) Glucose consumption by hmoDCs treated with control excipients, V132, MV140 and MV140/V142 after 18 h and calculated metabolic rate. (B) Increase in lactate production in cell-free supernatants from MV140/V132-stimulated hmoDCs relative to MV140-activated hmoDCs. (C) Relative changes in fluorescence intensity of stimulated hmoDCs stained with MitoTracker Red. (D) Mitochondrial mass determination as the ratio of mitochondrial (mt) and nuclear (n) DNA as expressed by mt COXII/n GAPDH. (E) Messenger RNA expression levels of HIF-1α gene in hmoDCs stimulated for 18 h. Arbitrary units (A.u.) are 2−ΔCT values multiplied by 104, with ΔCT defined as the difference between the cycle threshold value for the gene of interest and EF1α. (F) Cytokine production by V132-, MV140-, and MV140/V132-activated hmoDCs in the presence of 5′-methylthioadenosine (MTA) or pargyline as inhibitors of histone methyltransferases and demethylases, respectively. Results are mean ± s.e.m. of 6 (A), 4 (B), 7 (C), 4 (D), 5 (E), and 4-7 (F) independent experiments. Paired Student t test, *P < 0.05, **P < 0.01, and ***P < 0.01.

Figure 6

Sublingual immunization of BALB/c mice with MV140/V132 enhances splenic T cell responses and induces specific antibodies against all the components included in the formulation. (A) Scheme of the sublingual immunization protocol and analysis of induced systemic responses. (B) Proliferation of CFSE-labeled CD4+ T cells from splenocytes isolated from mice immunized sublingually with MV140/V132 or control excipients after in vitro stimulation with V132, MV140, V140/V132, phytohaemagglutinin (PHA), or control (containing all excipients except the bacteria and yeast). (C) Serum IgG and IgA antibodies specific for (C)albicans, K pneumoniae, E coli, E faecalis and P. vulgaris from mice immunized with MV140/V132 or with control excipients. (D) Fold change of specific IgG and IgA antibodies generated in mice immunized with MV140/V132 relative to control mice. (E) Fold change of specific IgG and IgA antibodies for K pneumoniae, E coli, E faecalis and P. vulgaris generated in mice immunized with MV140/V132 or MV140 alone relative to control mice. (F) Fold change of specific IgG and IgA antibodies for C albicans generated in mice immunized with MV140/V132 or V132 alone relative to control mice. Results are mean ± s.e.m. of 7-8 (B) and 5-6 (C–F) individual mice per condition of one single experiment. Unpaired t test, *P < 0.05, **P < 0.01, and ***P < 0.001.