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To discuss recently discovered mechanisms of action of some bacterial vaccines that may account for their clinical benefit in the prevention of recurrent wheezing and asthma exacerbations in infants and early childhood.Trained immunity has been shown to confer innate immune cells with a quite long-term nonspecific protection against a broad spectrum of pathogens. Inducers of trained immunity include some bacterial vaccines. Trained immunity-based vaccines (TIbV) of bacterial origin have the capability to induce nonspecific responses to a variety of pathogens, including respiratory viruses, in addition to their nominal bacterial antigens. Clinical data, from epidemiological surveys to well designed randomized clinical trials, indicate that TIbV formulated with bacteria prevent respiratory tract infections of viral cause, such as those associated with recurrent wheezing or asthma exacerbation, in children. Administration of these vaccines by the mucosal route may be important for their outcome in respiratory infections.Mucosal bacterial immunotherapy, including certain TIbV, confer protection against a broad spectrum of pathogens, such as viruses, through a mechanism mediated by trained immunity. Clinical studies on the use of these preparations against recurrent wheezing reflect these mechanistic effects. These findings open a new avenue for the development of new strategies for this condition.

The authors thank the patients and their families for participating in this study. They are grateful to the members of the David Sancho laboratory, especially to Carlos del Fresno, for helpful critical discussion. Rationale: Recurrent wheezing in children represents a severe public health concern. Wheezing attacks (WA), mainly associated with viral infections, lack effective preventive therapies. Objectives: To evaluate the efficacy and safety of mucosal sublingual immunotherapy based on whole inactivated bacteria (MV130) in preventing WA in children. Methods: A Phase 3 randomized, double-blind, placebo-controlled, parallel-group trial including a cohort of 120 children

Summary 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., Graphical abstract, Highlights • MV130 reduces morbi-mortality in mouse models of viral respiratory infection • MV130 modulates mouse immunity complying with key features of trained immunity • MV130 promotes reprogramming of mouse bone marrow progenitors • MV130 induces trained immunity in human monocytes, Brandi et al. investigate the mechanism of action of an inactivated polybacterial mucosal immunomodulator, MV130, which confers protection in patients suffering from recurrent respiratory infections. They show that MV130 induces trained immunity both in mouse bone marrow progenitors, resulting in protection from viral infection, and in human monocytes in vitro.

Infectious complications are a major cause of morbidity and mortality in B-cell hematological malignancies (HM). Prophylaxis for recurrent infections in HM patients with antibody deficiency consists of first-line antibiotics and when unsuccessful, gammaglobulin replacement therapy (IgRT). Recent knowledge of trained immunity-based vaccines (TIbV), such as the sublingual polybacterial formulation MV130, has shown a promising strategy in the management of patients with recurrent infections. We sought to determine the clinical benefit of MV130 in a cohort of HM patients with recurrent respiratory tract infections (RRTIs) who underwent immunization with MV130 for 3 months. Clinical information included the frequency of infections, antibiotic use, number of visits to the GP and hospitalizations previous and after MV130 immunotherapy. Improvement on infection rate was classified as: clear (>60% reduction of infection), partial (26%–60%) and low (≤25%) improvement. Fifteen HM patients (aged 42 to 80 years; nine females) were included in the study. All patients reduced their infection rate. Analysis of paired data revealed that the median (range, min - max) of respiratory infectious rate significantly decreased from 4.0 (8.0–3.0) to 2.0 (4.0–0.0) (pp = 0.002) during 12 months after initiation of treatment with MV130. The number of infectious-related GP or emergency room visits declined from 4.0 (8.0–2.0) to 2.0 (3.0–0.0) (pp = 0.032). Regarding safety, no adverse events were observed. On the other hand, immunological assessment of serum IgA and IgG levels demonstrated an increase in specific antibodies to MV130-contained bacteria following MV130 immunotherapy. In conclusion, MV130 may add clinical benefit reducing the rate of infections and enhancing humoral immune responses in these vulnerable patients.

Recurrent respiratory tract infections (RRTIs) are the first leading cause of community-and nosocomial-acquired infections. Antibiotics remain the mainstay of treatment, enhancing the potential to develop antibiotic resistances. Therefore, the development of new alternative approaches to prevent and treat RRTIs is highly demanded. Daily sublingual administration of the whole heat-inactivated polybacterial preparation (PBP) MV130 significantly reduced the rate of respiratory infections in RRTIs patients, however, the immunological mechanisms of action remain unknown. Herein, we study the capacity of MV130 to immunomodulate the function of human dendritic cells (DCs) as a potential mechanism that contribute to the clinical benefits. We demonstrate that DCs from RRTIs patients and healthy controls display similar ex vivo immunological responses to MV130. By combining systems biology and functional immunological approaches we show that MV130 promotes the generation of Th1/Th17 responses via receptor-interacting serine/threonine-protein kinase-2 (RIPK2)-and myeloid-differentiation primary-response gene88 (MyD88)-mediated signalling pathways under the control of IL-10. In vivo BALB/c mice sublingually immunized with MV130 display potent systemic Th1/Th17 and IL-10 responses against related and unrelated antigens. We elucidate immunological mechanisms underlying the potential way of action of MV130, which might help to design alternative treatments in other clinical conditions with high risk of recurrent infections. This work was supported by grant IPT-2012-0639-090000 from INNPACTO and MINECO, Spain to Inmunotek S.L. The authors' laboratories are supported by grants SAF2014-52706-R to O.P. from MINECO, Spain, and SAF2016-79040R to D.S. from MINECO and European Fund for Regional Development, Spain. O.P. is a Ramon y Cajal Scholar funded by MINECO and the European Social Fund. L.C. is a recipient of a European Respiratory Society Fellowship (RESPIRE2-2013-3708). We thank to Juan Lopez-Relano and Sarai Martinez-Cano for excellent technical assistance with mice experiments. Sí

Background: Recurrent wheezing affects one third of children being a global health problem with considerable expenditure and impact on quality of life. Virtually all wheezing attacks (WA) in young children are of viral etiology. Their prevention is a major concern as effective therapies are still lacking. Aims: To evaluate the efficacy and safety of a mucosal whole-cell inactivated polybacterial preparation (MV130) in the prevention of WA, and to investigate the underlying mechanism of action. Methods: A randomized, double-blind, placebo-controlled, parallel-group study, including 120 children Results: A reduction of 40% in the number of WA in the active vs the placebo group (p Administration of MV130 conferred protection to intranasal viral infection in mice. This non-specific resistance was mediated by trained immunity. Conclusion: We provide evidence for the first time that a mucosal (sublingual) polybacterial formulation induces trained immunity, confers broad protection beyond the pathogens included in the preparation and shows clinical efficacy against a virus-related condition such as WA, with an excelent safety profile.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4+ effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease., The authors present an extensive profile of host transcriptional respones to a diverse group of pathogens and allergens. In doing so, they identify TH1, type I IFN, TH17, and TH2 responses, that underlie each immune response in both the blood and lung, which represents a global profile of host-pathogen immune responses.

Summary The rapid transit from hypoxia to normoxia in the lung that follows the first breath in newborn mice coincides with alveolar macrophage (AM) differentiation. However, whether sensing of oxygen affects AM maturation and function has not been previously explored. We have generated mice whose AMs show a deficient ability to sense oxygen after birth by deleting Vhl, a negative regulator of HIF transcription factors, in the CD11c compartment (CD11cΔVhl mice). VHL-deficient AMs show an immature-like phenotype and an impaired self-renewal capacity in vivo that persists upon culture ex vivo. VHL-deficient phenotype is intrinsic in AMs derived from monocyte precursors in mixed bone marrow chimeras. Moreover, unlike control Vhlfl/fl, AMs from CD11cΔVhl mice do not reverse pulmonary alveolar proteinosis when transplanted into Csf2rb−/− mice, demonstrating that VHL contributes to AM-mediated surfactant clearance. Thus, our results suggest that optimal AM terminal differentiation, self-renewal, and homeostatic function requires their intact oxygen-sensing capacity., Graphical Abstract, Highlights • Hypoxia and glycolysis signatures are downregulated during alveolar macrophage maturation • VHL-deficient alveolar macrophages induce HIF-target genes and are more glycolytic • VHL-deficient alveolar macrophages show an intrinsic immature-like phenotype • VHL promotes alveolar macrophage self-renewal and surfactant clearance capacity, Izquierdo et al. show that the absence of VHL in lung alveolar macrophages prevents their terminal maturation. VHL-deficient alveolar macrophages induce HIF-target gene expression and are partially desensitized to oxygen. Moreover, they have a decreased self-renewal and surfactant clearance capacity, suggesting that oxygen sensing contributes to alveolar macrophage functional maturation.

Melioidosis, a severe human disease caused by the bacterium Burkholderia pseudomallei, has a wide spectrum of clinical manifestations ranging from acute septicemia to chronic localized illness or latent infection. Murine models have been widely used to study the pathogenesis of infection and to evaluate novel therapies or vaccines, but how faithfully they recapitulate the biology of human melioidosis at a molecular level is not known. In this study, mice were intranasally infected with either high or low doses of B. pseudomallei to generate either acute, chronic, or latent infection and host blood and tissue transcriptional profiles were generated. Acute infection was accompanied by a homogeneous signature associated with induction of multiple innate immune response pathways, such as IL-10, TREM1, and IFN signaling, largely found in both blood and tissue. The transcriptional profile in blood reflected the heterogeneity of chronic infection and quantitatively reflected the severity of disease. Genes associated with fibrosis and tissue remodeling, including matrix metalloproteases and collagen, were upregulated in chronically infected mice with severe disease. Transcriptional signatures of both acute and chronic melioidosis revealed upregulation of iNOS in tissue, consistent with the expression of IFN-γ, but also Arginase-1, a functional antagonist of the iNOS pathway, and was confirmed by immunohistochemistry. Comparison of these mouse blood datasets by pathway and modular analysis with the blood transcriptional signature of patients with melioidosis showed that many genes were similarly perturbed, including Arginase-1, IL-10, TREM1, and IFN signaling, revealing the common immune response occurring in both mice and humans.

DCs are necessary and sufficient for induction of allergic airway inflammation. CD11b+ DCs direct the underlying Th2 immunity, but debate surrounds the function of CD103+ DCs in lung immunity and asthma after an allergic challenge. We challenged Batf3-/- mice, which lacked lung CD103+ DCs, with the relevant allergen house dust mite (HDM) as a model to ascertain their role in asthma. We show that acute and chronic HDM exposure leads to defective Th1 immunity in Batf3-deficient mice. In addition, chronic HDM challenge in Batf3-/- mice results in increased Th2 and Th17 immune responses and exacerbated airway inflammation. Mechanistically, Batf3 absence does not affect induction of Treg or IL-10 production by lung CD4+ T cells following acute HDM challenge. Batf3-dependent CD103+ migratory DCs are the main source of IL-12p40 in the mediastinal lymph node DC compartment in the steady state. Moreover, CD103+ DCs selectively increase their IL-12p40 production upon HDM administration. In vivo IL-12 treatment reverts exacerbated allergic airway inflammation upon chronic HDM challenge in Batf3-/- mice, restraining Th2 and Th17 responses without triggering Th1 immunity. These results suggest a protective role for lung CD103+ DCs to HDM allergic airway inflammation through the production of IL-12. We are grateful to members of the DS lab for discussions and critical reading of the manuscript. We thank the staff at the CNIC facilities for technical support, Jesus Sanchez-Ruiz for the graphical illustration, Ruth Conde-Garrosa for technical assistance, Antonio Molina-Iracheta for histological quantification and analysis, and Simon Bartlett for editorial assistance. LC is the recipient of a European Respiratory Society Fellowship (RESPIRE2-2013-3708). Work in the DS laboratory is funded by the CNIC and grants from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and European Fund for Regional Development (FEDER) (SAF-2013-42920-R and SAF-2016-79040-R); the European Commission (635122-PROCROP H2020); and the Fondation ACTERIA. The CNIC is supported by the MEIC and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí

Background: Asthma is a chronic inflammatory disease associated to eosinophilia, with variable and often reversible airway obstruction accompanied by airway hyperreactivity. Following inhalation of allergens, conventional lung dendritic cells (DCs), which consist of CD11b + and CD103 + DCs in mice, migrate to the local lymph nodes (LN). It has been suggested that upon exposure to allergens, CD11b + DCs interact with naive T cells in the lung-draining LN, providing the first instruction for T helper cell 2 (Th2) differentiation characteristic in asthma; however, the role of CD103 + DCs in airway inflammation remains controversial. Aim: Investigate the role of lung CD103 + DCs in HDM-induced asthma. Methods: WT and Batf3 -/- mice, used as a model of CD103 + DC impairment, were exposed to HDM, and the development features of asthma such as increase in BAL infiltrates, Th2 and Th17 cytokine production, mucus secretion and IgE levels in sera analysed. Results: Batf3-deficient mice show a significant increase in total BAL infiltrates. In addition, mice deficient for CD103 + DCs develop more perivascular and peribronchial infiltration than WT mice, which correlates with an increase in Th2 and Th17 cytokine production. To further investigate the mechanism, the production of IL-12 by lung migratory DCs was evaluated in the lung-draining LN following exposure to HDM. Only CD103 + DCs were able to induce the production of IL-12, which in turn enhances Th1 differentiation, attenuating Th2 and Th17 response and main features of asthma. Conclusion: Batf3 -dependent DCs are essential providers of IL-12 that controls induction of Th1 immunity in response to HDM, thus dampening allergic airway inflammation.

Introduction: Asthma is a chronic inflammatory disease associated to eosinophilia, airway obstruction and airway hyperreactivity. Following inhalation of allergens, conventional lung dendritic cells (DCs), which consist of CD11b + DCs and CD103 + DCs in mice, migrate to the local lymph nodes (LN). Upon exposure to allergens, CD11b + DCs provide the first instruction for T helper cell 2 (Th2) differentiation characteristic in asthma; however, the role of CD103 + DCs in airway inflammation remains controversial. Objectives: Investigate the role of lung CD103 + DCs in HDM-induced asthma. Material and Methods: WT and Batf3 -/- mice, used as a model of CD103 + DC impairment, were chronically exposed to house dust mite (HDM), and the development of characteristic features of asthma was analysed. Results: Batf3-deficient mice show a significant increase in total BAL infiltrates, including mainly an influx of eosinophils and neutrophils. In addition, mice deficient for CD103 + DCs develop more perivascular and peribronchial infiltration than WT mice, which correlates with an increase in Th2 and Th17 cytokine production by lung-draining LN and splenocytes. To further investigate the mechanism, the production of IL-12 by both subsets of lung migratory DCs (CD103 + and CD11b + ) was evaluated in the lung-draining LN following exposure to HDM. Only CD103 + DCs were able to increase the production of IL-12, which in turn enhances Th1 differentiation, attenuating Th2 and Th17 response and main features of asthma. Conclusion: Batf3 -dependent DCs are essential providers of IL-12 that controls induction of Th1 immunity in response to HDM, thus dampening allergic airway inflammation.

There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice.

Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo . The B. pseudomallei Δ relA Δ spoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the Δ relA Δ spoT mutant resulted in partial protection against infection with wild-type B. pseudomallei . In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei , and the Δ relA Δ spoT mutant may be a promising live-attenuated vaccine candidate.

Burkholderia pseudomallei is a Gram-negative bacterium which is the causative agent of melioidosis, a disease which carries a high mortality and morbidity rate in endemic areas of South East Asia and Northern Australia. At present there is no available human vaccine that protects against B. pseudomallei, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. This review considers the multiple elements of melioidosis vaccine research including: (i) the immune responses required for protective immunity, (ii) animal models available for preclinical testing of potential candidates, (iii) the different experimental vaccine strategies which are being pursued, and (iv) the obstacles and opportunities for eventual registration of a licensed vaccine in humans.

Burkholderia pseudomallei is the etiological agent of human melioidosis, a disease with a broad spectrum of clinical manifestations ranging from fatal septicemia to chronic localized infection or asymptomatic latent infection. Most clinical and immunological studies to date have focused on the acute disease process; however, little is known about pathology and immune response in chronic melioidosis. Here, we have developed a murine model of chronic disease by challenging C57BL/6 mice intranasally with a low dose of B. pseudomallei and monitoring them up to 100 days postinfection. Bacterial burdens were heterogeneous in different animals at all time points, consistent with the spectrum of clinical severity observed in humans. Proinflammatory cytokines such as gamma interferon (IFN-γ), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were induced during chronic infection, and histopathological analysis showed features in common with human melioidosis. Interestingly, many of these features were similar to those induced by Mycobacterium tuberculosis in humans, such as development of a collagen cord that encapsulates the lesions, the presence of multinucleated giant cells, and granulomas with a caseous necrotic center, which may explain why chronic melioidosis is often misdiagnosed as tuberculosis. Our model now provides a relevant and practical tool to define the immunological features of chronic melioidosis and aid in the development of more effective treatment of this disease in humans.

The evaluation of new therapies to treat allergic asthma makes frequent use of histological studies. Some of them are based on microscope observation of stained paraffin lung sections to quantify cellular infiltrate, an effect directly related to allergic processes. Currently, there is no software tool available for doing this quantification automatically. This paper presents a methodology and a software tool for the quantification of cellular infiltrate in lung tissue images in an allergic asthma mouse model. The image is divided into regions of equal size, which are then classified by means of a segmentation algo rithm based on texture analysis. The classification uses three discriminant functions, built from parameters derived from the histogram and the co occurrence matrix. These functions were calculated by means of a stepwise discriminant analysis on 79 samples from a training set. Results provided a correct classification of 96.8% on an independ ent test set of 251 samples labeled manually. Regression analysis showed a good agree ment between automatic and manual methods. A reliable and easy to implement method has been developed to provide an automatic method for quantifying micros copy images of lung histological studies. Results showed similar accuracy to that pro vided by an expert, while allowing analyzing a much larger number of fields in a repea table way Contract grant sponsor: CD TEAM project, CENIT program, Ministerio de Industria Publicado

Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes — Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes — quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an “accidental pathogen”, where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts., Author Summary Bacterial transcriptomes are dynamic, context-specific and condition-dependent. Infection by the soil bacterium, Burkholderia pseudomallei, causes melioidosis, an often fatal infectious disease of humans and animals. Possessing a large multi-chromosomal genome, B. pseudomallei is able to persist and survive in a multitude of environments. To obtain a comprehensive overview of B. pseudomallei expressed transcripts, we initiated whole-genome transcriptome profiling covering a broad spectrum of conditions and exposures — a so-called “condition compendium”. Using the compendium, we confirmed many previously-annotated genes and operons, and also identified hundreds of novel transcripts including anti-sense transcripts and non-coding RNAs. By systematically examining genes exhibiting highly similar expression patterns, we ascribed putative functions to previously uncharacterized genes, and identified novel regulatory elements controlling these expression patterns. We also used the compendium to elucidate candidate virulence pathways associated with quorum-sensing and infection in mice. Our study showcases the power of a B. pseudomallei condition compendium as a valuable resource for understanding microbial physiology and the pathogenesis of melioidosis.