Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Enhancing colistin efficacy against Salmonella infections with a quinazoline-based dual therapeutic strategy.

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.

Quillaja brasiliensis nanoparticle adjuvant formulation improves the efficacy of an inactivated trivalent influenza vaccine in mice.

The threat of viral influenza infections has sparked research efforts to develop vaccines that can induce broadly protective immunity with safe adjuvants that trigger robust immune responses. Here, we demonstrate that subcutaneous or intranasal delivery of a seasonal trivalent influenza vaccine (TIV) adjuvanted with the Quillaja brasiliensis saponin-based nanoparticle (IMXQB) increases the potency of TIV. The adjuvanted vaccine (TIV-IMXQB) elicited high levels of IgG2a and IgG1 antibodies with virus-neutralizing capacity and improved serum hemagglutination inhibition titers. The cellular immune response induced by TIV-IMXQB suggests the presence of a mixed Th1/Th2 cytokine profile, antibody-secreting cells (ASCs) skewed toward an IgG2a phenotype, a positive delayed-type hypersensitivity (DTH) response, and effector CD4+ and CD8+ T cells. After challenge, viral titers in the lungs were significantly lower in animals receiving TIV-IMXQB than in those inoculated with TIV alone. Most notably, mice vaccinated intranasally with TIV-IMXQB and challenged with a lethal dose of influenza virus were fully protected against weight loss and lung virus replication, with no mortality, whereas, among animals vaccinated with TIV alone, the mortality rate was 75%. These findings demonstrate that TIV-IMXQB improved the immune responses to TIV, and, unlike the commercial vaccine, conferred full protection against influenza challenge.

Salmonella-induced immune response reduces recurrence and tumor dissemination in preclinical melanoma model.

Localized melanoma is easy to remove by surgery, resulting in a high five-year relative survival rate. However, when disseminated the disease management is challenging. The use of immunotherapies, such as anti-checkpoint monoclonal antibodies, has improved treatment options but still only a small percentage of patients responds to these expensive treatments. In this work, we apply a bacteria-based immunotherapy using LVR01, an attenuated Salmonella enterica serovar Typhimurium, as neoadjuvant therapy one week before surgery in a preclinical disseminated murine melanoma model. LVR01 administration resulted in tumor growth retardation prior to tumor resection, due to a rapid upregulation of inflammatory genes in the tumor microenvironment. As a consequence, cell infiltration increased, particularly neutrophils, macrophages and NK cells, being the latter involved in Salmonella anti-tumor activity. Besides, tumor-draining lymph node infiltration is characterized by reinvigorated CD4+ and CD8+ lymphocytes. Induced immune response could account for the prevention or delay of tumor recurrence and appearance of metastasis, resulting in a prolonged overall survival after surgery. Furthermore, upon rechallenge mice show partial protection, suggesting the existence of specific memory against melanoma. We propose that neoadjuvant LVR01 treatment could represent an interesting inexpensive alternative that may ease tumor resection, while preventing tumor recurrence in patients with melanoma.

Preclinical Evaluation of LVR01 Attenuated Salmonella as Neoadjuvant Intralesional Therapy in Combination with Chemotherapy for Melanoma Treatment.

Treatment of malignant melanoma has improved in the last few years owing to early detection and new therapeutic options. Still, management of advanced disease remains a challenge because it requires systemic treatment. In such cases, dacarbazine-based chemotherapy has been widely used, despite low efficacy. Neoadjuvant therapies emerge as alternative options that could help chemotherapy to achieve increased benefit. In this work, we evaluate LVR01, an attenuated Salmonella enterica serovar typhimurium, as neoadjuvant intralesional therapy in combination with dacarbazine in a preclinical melanoma model. B16F1 melanoma‒bearing mice received intraperitoneal administration of dacarbazine for 3 consecutive days. LVR01 treatment, consisting of one single intratumoral injection, was applied 1 day before chemotherapy began. This therapeutic approach retarded tumor growth and prolonged overall survival, revealing a strong synergistic antitumor effect. Dacarbazine induced a drastic reduction of secondary lymphoid organ cellularity, which was partially restored by Salmonella, particularly potentiating activated cytotoxic cell compartments. Systemic immune reactivation could be a consequence of the intense inflammatory tumor microenvironment induced by LVR01. We propose that the use of LVR01 as neoadjuvant intralesional therapy could be considered as an interesting strategy with close clinical application to boost chemotherapy effect in patients with melanoma.

Bacterial Lysates as Immunotherapies for Respiratory Infections: Methods of Preparation.

Bacterial lysates, prepared from the microorganisms most frequently involved in human Respiratory Tract Infections (RTIs) have been in the market for several decades, and at present, several different brands are available in many countries worldwide. They all claimed to exert local and systemic immunomodulatory effects but different clinical trials show disparate results between them. The lack of consistency of predicted therapeutic effects has undermined their clinical use and hampered licensing in several countries. One explanation for such lack of consistency in the results is that their methods of preparation are also very different. Here, we review the available literature describing methods of preparation of bacterial lysates, including patent disclosure documents. We found a great variety of methodologies of preparation and a lack of standardized procedures among them. The main conclusion of our study is that there is a clear need for standardized protocols of production to obtain comparable results in clinical trials worldwide.

Correction to: Genome analysis of Salmonella enterica subsp. diarizonae isolates from invasive human infections reveals enrichment of virulence-related functions in lineage ST1256.

An amendment to this paper has been published and can be accessed via the original article.

PhoQ is an unsaturated fatty acid receptor that fine-tunes Salmonella pathogenic traits.

The Salmonella enterica PhoP/PhoQ two-component signaling system coordinates the spatiotemporal expression of key virulence factors that confer pathogenic traits. Through biochemical and structural analyses, we found that the sensor histidine kinase PhoQ acted as a receptor for long-chain unsaturated fatty acids (LCUFAs), which induced a conformational change in the periplasmic domain of the PhoQ protein. This resulted in the repression of PhoQ autokinase activity, leading to inhibition of the expression of PhoP/PhoQ-dependent genes. Recognition of the LCUFA linoleic acid (LA) by PhoQ was not stereospecific because positional and geometrical isomers of LA equally inhibited PhoQ autophosphorylation, which was conserved in multiple S. enterica serovars. Because orally acquired Salmonella encounters conjugated LA (CLA), a product of the metabolic conversion of LA by microbiota, in the human intestine, we tested how short-term oral administration of CLA affected gut colonization and systemic dissemination in a mouse model of Salmonella-induced colitis. Compared to untreated mice, CLA-treated mice showed increased gut colonization by wild-type Salmonella, as well as increased dissemination to the spleen. In contrast, the inability of the phoP strain to disseminate systemically remained unchanged by CLA treatment. Together, our results reveal that, by inhibiting PhoQ, environmental LCUFAs fine-tune the fate of Salmonella during infection. These findings may aid in the design of new anti-Salmonella therapies.

Quinazoline-Based Antivirulence Compounds Selectively Target Salmonella PhoP/PhoQ Signal Transduction System.

The rapid emergence of multidrug resistance among bacterial pathogens has become a significant challenge to human health in our century. Therefore, development of next-generation antibacterial compounds is an urgent need. Two-component signal transduction systems (TCS) are stimulus-response coupling devices that allow bacteria to sense and elaborate adaptive responses to changing environmental conditions, including the challenges that pathogenic bacteria face inside the host. The differential presence of TCS, present in bacteria but absent in the animal kingdom, makes them attractive targets in the search for new antibacterial compounds. In Salmonella enterica, the PhoP/PhoQ two-component system controls the expression of crucial phenotypes that define the ability of the pathogen to establish infection in the host. We now report the screening of 686 compounds from a GlaxoSmithKline published kinase inhibitor set in a high-throughput whole-cell assay that targets Salmonella enterica serovar Typhimurium PhoP/PhoQ. We identified a series of quinazoline compounds that showed selective and potent downregulation of PhoP/PhoQ-activated genes and define structural attributes required for their efficacy. We demonstrate that their bioactivity is due to repression of the PhoQ sensor autokinase activity mediated by interaction with its catalytic domain, acting as competitive inhibitors of ATP binding. While noncytotoxic, the hit molecules exhibit antivirulence effect by blockage of S Typhimurium intramacrophage replication. Together, these features make these quinazoline compounds stand out as exciting leads to develop a therapeutic intervention to fight salmonellosis.

Genome analysis of Salmonella enterica subsp. diarizonae isolates from invasive human infections reveals enrichment of virulence-related functions in lineage ST1256.

BACKGROUND: Salmonella enterica subsp. diarizonae (IIIb) is frequently isolated from the environment, cold-blooded reptiles, sheep and humans; however only a few studies describe the isolation of this subspecies from invasive human infections. The factors contributing to this unusual behavior are currently unknown. RESULTS: We report here the genome features of two diarizonae strains, SBO13 and SBO27, isolated from endocervical tissue collected post-abortion and from cerebrospinal fluid of a newborn child, respectively, in the city of Santa Cruz, Bolivia. Although isolated six years apart, SBO27 in 2008 and SBO13 in 2014, both strains belong to the same sequence type 1256 (ST1256) and show a high degree of genome conservation sharing more than 99% of their genes, including the conservation of a ~ 10 kb plasmid. A prominent feature of the two genomes is the presence of 24 genomic islands (GIs), in addition to 10 complete Salmonella pathogenicity islands (SPI) and fragments of SPI-7, a pathogenicity island first reported in the human-adapted serovar Typhi. Some of the GIs identified in SBO13 and SBO27 harbor genes putatively encoding auto-transporters involved in adhesion, lipopolysaccharide modifying enzymes, putative toxins, pili-related proteins, efflux pumps, and several putative membrane cation transport related-genes, among others. These two Bolivian isolates also share genes encoding the type-III secretion system effector proteins SseK2, SseK3 and SlrP with other diarizonae sequence types (ST) mainly-associated with infections in humans. The sseK2, sseK3 and slrP genes were either absent or showing frameshift mutations in a significant proportion of genomes from environmental diarizonae isolates. CONCLUSIONS: The comparative genomic study of two diarizonae strains isolated in Bolivia from human patients uncovered the presence of many genes putatively related to virulence. The statistically-significant acquisition of a unique combination of these functions by diarizonae strains isolated from humans may have impacted the ability of these isolates to successfully infect the human host.

Temporal evolution of anti-Clostridium antibody responses in sheep after vaccination with polyvalent clostridial vaccines.

Polyvalent clostridial vaccines, composed of a complex mixture of toxoids from up to 9 different species, are highly effective in controlling clostridial diseases in cattle and sheep. Commercially available vaccines usually state that in normal field conditions two doses administered 4 to 6 weeks apart elicit protective antibody levels that will last for one year. However, studies on the development and duration of the antibody response against the different Clostridium species in target animals are scarce and only partial. Evaluating the temporal evolution of the antibody responses upon vaccination in target species is relevant to understand the bases of protective immunity induced by these vaccines and to develop new optimized vaccines. Here, we assessed the antibody response in sheep against each Clostridium component of two different 9-valent Clostridial vaccines over the period of one year. One vaccine was a commercially available vaccine and the other was an experimental vaccine prepared by us with the same antigens that we used to set up a specific ELISA for each Clostridium species. Both vaccines showed similar results, irrespectively of the origin of the antigens used for the ELISAs, with antibody titers that peaked at day 36 after vaccination and large inter individual variations in the magnitude of the response. Antibody titers were maintained up to 90 days and then markedly decreased, becoming even undetectable in some animals 6 months after vaccination. Given that the current scheme of yearly revaccination has largely shown to be effective at controlling the burden of disease, our results strongly suggest that circulating antibody levels cannot completely explain the protective immunity elicited by these vaccines, and prompt for further studies into the correlates of protection of clostridial vaccines.

Salmonella Immunotherapy Improves the Outcome of CHOP Chemotherapy in Non-Hodgkin Lymphoma-Bearing Mice.

We have previously shown that Salmonella immunotherapy is effective to treat B-cell non-Hodgkin lymphoma (B-NHL) in mice. However, this model involves animals with high tumor burden, whereas in the clinics B-NHL patients are usually treated with chemotherapy (CHOP: cyclophosphamide, doxorubicin, vincristine, and prednisone) as first-line therapy prior to immunotherapy. Recently, we have described a NHL-B preclinical model using CHOP chemotherapy to achieve MRD in immunocompetent animals that closely resemble patients' conditions. In this work, we assessed the efficacy of Salmonella immunotherapy in B-NHL-bearing mice undergoing chemotherapy. Salmonella administration significantly delayed tumor growth and prolonged survival of chemotherapy-treated NHL-bearing animals. Mice receiving the CHOP-Salmonella combined therapy showed increased numbers of tumor-infiltrating leukocytes and a different profile of cytokines and chemokines expressed in the tumor microenvironment. Further, Salmonella immunotherapy in CHOP-treated animals also enhanced NK cells cytotoxic activity as well as induced systemic lymphoma-specific humoral and cellular responses. Chemotherapy treatment profoundly impacted on the general health status of recipient animals, but those receiving Salmonella showed significantly better overall body condition. Altogether, the results clearly demonstrated that Salmonella immunotherapy could be safely used in individuals under CHOP treatment, resulting in a better prognosis. These results give strong support to consider Salmonella as a neoadjuvant therapy in a clinical setting.

A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties.

Salmonella enterica serovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to prevent Salmonella dissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) of S Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory properties in vitro and in vivo The aim of this work was to elucidate the underlying cause of the absence of flagella in S Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in the fliE gene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only when fliA was artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences in fliE adjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected in S Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods.

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Infantis Strain SPE101, Isolated from a Chronic Human Infection.

We report a 4.99-Mb draft genome sequence of Salmonella enterica subsp. enterica serovar Infantis strain SPE101, isolated from feces of a 5-month-old breast-fed female showing diarrhea associated with severe dehydration and malnutrition. The infection prolonged for 6 months despite antibiotic treatment.

Flagellin-Mediated Protection against Intestinal Yersinia pseudotuberculosis Infection Does Not Require Interleukin-22.

Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved.

A novel non-Hodgkin lymphoma murine model closer to the standard clinical scenario.

BACKGROUND: Non-Hodgkin lymphomas (NHL) are the most frequent hemato-oncological malignancies. Despite recent major advances in treatment, a substantial proportion of patients relapses highlighting the need for new therapeutic modalities. Promissory results obtained in pre-clinical studies are usually not translated when moving into clinical trials. Pre-clinical studies are mainly conducted in animals with high tumor burden; instead patients undergo chemotherapy as first line of treatment and most likely are under remission when immunotherapies are applied. Thus, an animal model that more closely resembles patients' conditions would be a valuable tool. METHODS: BALB/c mice were injected subcutaneously with A20 lymphoma cells and after tumor development different doses of chemotherapy were assessed to find optimal conditions for minimal residual disease (MRD) establishment. Tumor growth and survival, as well as drugs side effects, were all evaluated. Complete lymphoma remission was monitored in vivo using positron emission tomography (PET), and the results were correlated with histology. Immunological status was assessed by splenocytes proliferation assays in NHL-complete remission mice and by analyzing tumor cell infiltrates and chemokines/cytokines gene expression in the tumor microenvironment of animals with residual lymphoma. RESULTS: Two cycles of CHOP chemotherapy at days 25 and 35 post-tumor implantation induced complete remission for around 20 days. PET showed to be a suitable follow-up technique for MRD condition with 85.7 and 75% of sensibility and specificity respectively. Proliferative responses upon mitogen stimulation were similar in animals that received chemotherapy and wild type mice. Tumors from animals with residual lymphoma showed higher numbers of CD4+ and CD8+ and similar numbers of NK, neutrophils and Tregs infiltrating cells as compared with non-treated animals. Gene expression of several cytokines as well as an array of chemokines associated with migration of activated T cells to tumor sites was upregulated in the tumor microenvironment of animals that received chemotherapy treatment. CONCLUSIONS: We established a NHL-B pre-clinical model using standard chemotherapy to achieve MRD in immunocompetent animals. The MRD condition is maintained for approximately 20 days providing a therapeutic window of time where new immunotherapies can be tested in conditions closer to the clinics.

Sublingual flagellin protects against acute pneumococcal pneumonia in a TLR5-dependent and NLRC4-independent fashion.

AIM: To evaluate efficacy of sublingual flagellin to treat acute pneumonia. MATERIALS & METHODS: Mice were treated sublingually with flagellin and challenged intranasally with a lethal dose of pneumococcus. Flagellins lacking TLR5 or NLRC4 activation domains were used to assess their contribution to protection. RESULTS: Sublingual flagellin protected mice in a TLR5-dependent, NLRC4-independent fashion. Neutrophils were required for protection. Flagellin-stimulated lung epithelial cells recapitulated the lung's transcriptional profile suggesting they could be targeted by flagellin in vivo. CONCLUSION: Ligation of TLR5, a pathogen recognition receptor not naturally engaged by pneumococcus, protects mice from invasive pneumonia when administered via sublingual route. This can be a highly cost-effective alternative therapy against pneumonia.

Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.