Influenza Virus Inactivated by Heavy Ion Beam Irradiation Stimulates Antigen-Specific Immune Responses.

The COVID-19 pandemic has made clear the need for effective and rapid vaccine development methods. Conventional inactivated virus vaccines, together with new technologies like vector and mRNA vaccines, were the first to be rolled out. However, the traditional methods used for virus inactivation can affect surface-exposed antigen, thereby reducing vaccine efficacy. Gamma rays have been used in the past to inactivate viruses. We recently proposed that high-energy heavy ions may be more suitable as an inactivation method because they increase the damage ratio between the viral nucleic acid and surface proteins. Here, we demonstrate that irradiation of the influenza virus using heavy ion beams constitutes a suitable method to develop effective vaccines, since immunization of mice by the intranasal route with the inactivated virus resulted in the stimulation of strong antigen-specific humoral and cellular immune responses.

Elucidation of the Active Form and Reaction Mechanism in Human Asparaginase Type III Using Multiscale Simulations.

l-asparaginases catalyze the asparagine hydrolysis to aspartate. These enzymes play an important role in the treatment of acute lymphoblastic leukemia because these cells are unable to produce their own asparagine. Due to the immunogenic response and various side effects of enzymes of bacterial origin, many attempts have been made to replace these enzymes with mammalian enzymes such as human asparaginase type III (hASNaseIII). This study investigates the reaction mechanism of hASNaseIII through molecular dynamics simulations, quantum mechanics/molecular mechanics methods, and free energy calculations. Our simulations reveal that the dimeric form of the enzyme plays a vital role in stabilizing the substrate in the active site, despite the active site residues coming from a single protomer. Protomer-protomer interactions are essential to keep the enzyme in an active conformation. Our study of the reaction mechanism indicates that the self-cleavage process that generates an N-terminal residue (Thr168) is required to activate the enzyme. This residue acts as the nucleophile, attacking the electrophilic carbon of the substrate after a proton transfer from its hydroxyl group to the N-terminal amino group. The reaction mechanism proceeds with the formation of an acyl-enzyme complex and its hydrolysis, which turns out to be the rate-determining step. Our proposal of the enzymatic mechanism sheds light on the role of different active site residues and rationalizes the studies on mutations. The insights provided here about hASNaseIII activity could contribute to the comprehension of the disparities among different ASNases and might even guide the design of new variants with improved properties for acute lymphoblastic leukemia treatment.

IFN-α/ß Signaling Is Required for CDG-Mediated CTL Generation and B Lymphocyte Activation.

Among cyclic di-nucleotides (CDN), both cyclic di-AMP (CDA) and di-GMP (CDG) are promising adjuvants and immune modulators. These molecules are not only able to induce profuse antibody production but also predominant T helper 1 and cytotoxic CD8 T lymphocytes (CTL) responses, which enable their use for vaccination against intracellular pathogens as well as in cancer immunotherapy. However, for their successful translation into the clinic, a comprehensive understanding of CDN mode of action is still essential. Consistent with evidence in the literature, we show here that IFN-α/ß (Type I IFN) is crucial for CDG-mediated B cell activation. We recently determined the key role of type I IFN signaling for CDA-mediated enhancement of immunogenicity. Based on the biological activities of type I IFN, in this study, we hypothesized that it might also be required for CTL induction by CDG. We disclose here the mode of action of type I IFN signaling in CDG-mediated cross-presentation and subsequent CTL generation.

Safety and efficacy of prophylactic and therapeutic vaccine based on live-attenuated Listeria monocytogenes in hepatobiliary cancers.

Primary liver cancer (PLC) comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represents the third deadliest cancer worldwide with still insufficient treatment options. We have previously found that CD4 T helper 1 (Th1) response is indispensable for the protection against PLC. In the present research, we aimed to test the potent inducers of Th1 responses, live-attenuated Listeria monocytogenes ∆actA/∆inlB strain as preventive/therapeutic vaccine candidate in liver fibrosis, HCC, and CCA. Studies were performed using autochthonous models of HCC and CCA, highly reflecting human disease. L. monocytogenes ∆actA/∆inlB demonstrated strong safety/efficacy in premalignant and malignant liver diseases. The protective mechanism relied on the induction of strong tumor-specific immune responses that keep the development of hepatobiliary cancers under control. Combination therapy, comprising Listeria vaccination and a checkpoint inhibitor blockade significantly extended the survival of HCC-bearing mice even at the advanced stages of the disease. This is the first report on the safety and efficacy of Listeria-based vaccine in liver fibrosis, as well as the first proof of principle study on Listeria-based vaccines in CCA. Our study paves the way for the use of live-attenuated Listeria as safe and efficient vaccine and a potent inducer of protective immune responses in liver fibrosis and hepatobiliary malignancies.

La evaluación preoperatoria en intervenciones quirúrgicas no cardiacas en el Hospital de Especialidades Carlos Andrade Marín / Preoperative evaluation of non-cardiac surgical interventions at Carlos Andrade Marín Specialties Hospital

INTRODUCCIÓN. La evaluación preoperatoria determina el estado de salud del paciente que será sometido a una intervención quirúrgica, minimiza su riesgo y optimiza los recursos humanos y materiales del escenario perioperatorio. OBJETIVO. Determinar el proceso de evaluación preoperatorio y su capacidad para prevenir eventos clínicos adversos durante el postoperatorio inmediato de pacientes adultos sometidos a un procedimiento quirúrgico no cardiaco. MATERIALES Y MÉTODOS. Estudio retrospectivo de corte transversal. Población de 6 250 adultos y muestra de 912 Historias Clínicas electrónicas de pacientes atendidos en el Hospital de Especialidades Carlos Andrade Marín, en el año 2017. Criterios de inclusión: pacientes mayores de 18 años, que se sometieron a una evaluación preoperatoria de manera ambulatoria y fueron operados de intervenciones no cardiacas hasta el mes de enero del 2019, o suspensión de cirugía por contraindicaciones determinadas en la cita médica. Criterios de exclusión: pacientes menores a 18 años, no intervenidos quirúrgicamente, hospitalizados por cualquier motivo entre la revisión y la cirugía, embarazadas e individuos con consulta preoperatoria fuera del hospital. Los programas informáticos utilizados para el registro de datos y su análisis fueron Microsoft Excel e International Business Machines Statistical Package for the Social Sciences. RESULTADOS. El 82,68% (754; 912) presentó un antecedente clínico y el 82,13% (749; 912) uno de tipo quirúrgico. Se reportó un 2,00% (18; 912) de complicaciones postoperatorias, y un caso de muerte pasadas las 72 horas postquirúrgicas. No se encontró correlación estadísticamente significativa p>0,05 entre las conclusiones clínicas del control preoperatorio y la ocurrencia de complicaciones en el postoperatorio. CONCLUSIÓN. Se evidenció que el proceso de evaluación preoperatoria fue realizado de manera sistemática a pacientes con características sociodemográficas y clínicas heterogéneas y no existió correlación estadística entre sus resultados y la presencia de complicaciones perioperatorias.

INTRODUCTION. The preoperative evaluation determines the health status of a patient that will undergo a surgical intervention, minimizes its risk, and optimizes the human and material resources of the perioperative scenario. OBJECTIVE. To determine the preoperative evaluation process and its ability to prevent adverse clinical events during the immediate postoperative period in adult patients undergoing a noncardiac surgical procedure. MATERIALS AND METHODS. This is a retrospective cross-sectional study. The sample of 912 medical records was calculated upon a population of 6 250 adults treated at the Carlos Andrade Marín Specialties Hospital in 2017. Inclusion criteria: patients over 18 years of age, who underwent a preoperative evaluation on an outpatient basis, and were operated on for non-cardiac interventions until January 2019, or suspension of surgery due to contraindications determined in the medical appointment. Exclusion criteria: patients under 18 years of age, not undergoing surgery, hospitalized for any reason between revision and surgery, pregnant women, and individuals with preoperative consultation outside the hospital. The computer programs used for data collection and analysis were Microsoft Excel and the International Business Machines Statistical Package for the Social Sciences. RESULTS. 82,68% (754; 912) had a previous clinical condition and 82,13% (749; 912) had a previous surgical intervention. 2,00% (18; 912) of postoperative complications were reported, and one case of death after 72 postoperative hours. No statistically significant correlation p>0,05 was found between the clinical conclusions of the preoperative control and the occurrence of postoperative complications. CONCLUSION. It was evidenced that the preoperative evaluation process was carried out systematically to patients with heterogeneous sociodemographic and clinical characteristics and there was no statistical correlation between its results and the presence of perioperative complications.

Prophylactic Multi-Subunit Vaccine against Chlamydia trachomatis: In Vivo Evaluation in Mice.

Chlamydia trachomatis is the most frequent sexually-transmitted disease-causing bacterium. Urogenital serovars of this intracellular pathogen lead to urethritis and cervicitis. Ascending infections result in pelvic inflammatory disease, salpingitis, and oophoritis. One of 200 urogenital infections leads to tubal infertility. Serovars A-C cause trachoma with visual impairment. There is an urgent need for a vaccine. We characterized a new five-component subunit vaccine in a mouse vaccination-lung challenge infection model. Four recombinant Pmp family-members and Ctad1 from C. trachomatis serovar E, all of which participate in adhesion and binding of chlamydial elementary bodies to host cells, were combined with the mucosal adjuvant cyclic-di-adenosine monophosphate. Intranasal application led to a high degree of cross-serovar protection against urogenital and ocular strains of C. trachomatis, which lasted at least five months. Critical evaluated parameters were body weight, clinical score, chlamydial load, a granulocyte marker and the cytokines IFN-γ/TNF-α in lung homogenate. Vaccine antigen-specific antibodies and a mixed Th1/Th2/Th17 T cell response with multi-functional CD4+ and CD8+ T cells correlate with protection. However, serum-transfer did not protect the recipients suggesting that circulating antibodies play only a minor role. In the long run, our new vaccine might help to prevent the feared consequences of human C. trachomatis infections.

B cell depletion impairs vaccination-induced CD8+ T cell responses in a type I interferon-dependent manner.

OBJECTIVES: The monoclonal anti-CD20 antibody rituximab is frequently applied in the treatment of lymphoma as well as autoimmune diseases and confers efficient depletion of recirculating B cells. Correspondingly, B cell-depleted patients barely mount de novo antibody responses during infections or vaccinations. Therefore, efficient immune responses of B cell-depleted patients largely depend on protective T cell responses. METHODS: CD8+ T cell expansion was studied in rituximab-treated rheumatoid arthritis (RA) patients and B cell-deficient mice on vaccination/infection with different vaccines/pathogens. RESULTS: Rituximab-treated RA patients vaccinated with Influvac showed reduced expansion of influenza-specific CD8+ T cells when compared with healthy controls. Moreover, B cell-deficient JHT mice infected with mouse-adapted Influenza or modified vaccinia virus Ankara showed less vigorous expansion of virus-specific CD8+ T cells than wild type mice. Of note, JHT mice do not have an intrinsic impairment of CD8+ T cell expansion, since infection with vaccinia virus induced similar T cell expansion in JHT and wild type mice. Direct type I interferon receptor signalling of B cells was necessary to induce several chemokines in B cells and to support T cell help by enhancing the expression of MHC-I. CONCLUSIONS: Depending on the stimulus, B cells can modulate CD8+ T cell responses. Thus, B cell depletion causes a deficiency of de novo antibody responses and affects the efficacy of cellular response including cytotoxic T cells. The choice of the appropriate vaccine to vaccinate B cell-depleted patients has to be re-evaluated in order to efficiently induce protective CD8+ T cell responses.

Interdependencies between Toll-like receptors in Leishmania infection.

Multiple pathogen-associated molecular patterns (PAMPs) on a pathogen's surface imply their simultaneous recognition by the host cell membrane-located multiple PAMP-specific Toll-like receptors (TLRs). The TLRs on endosomes recognize internalized pathogen-derived nucleic acids and trigger anti-pathogen immune responses aimed at eliminating the intracellular pathogen. Whether the TLRs influence each other's expression and effector responses-termed TLR interdependency-remains unknown. Herein, we first probed the existence of TLR interdependencies and next determined how targeting TLR interdependencies might determine the outcome of Leishmania infection. We observed that TLRs selectively altered expression of their own and of other TLRs revealing novel TLR interdependencies. Leishmania major-an intra-macrophage parasite inflicting the disease cutaneous leishmaniasis in 88 countries-altered this TLR interdependency unfolding a unique immune evasion mechanism. We targeted this TLR interdependency by selective silencing of rationally chosen TLRs and by stimulation with selective TLR ligands working out a novel phase-specific treatment regimen. Targeting the TLR interdependency elicited a host-protective anti-leishmanial immune response and reduced parasite burden. To test whether this observation could be used as a scientific rationale for treating a potentially fatal L. donovani infection, which causes visceral leishmaniasis, we targeted the inter-TLR dependency adopting the same treatment regimen. We observed reduced splenic Leishman-Donovan units accompanied by host-protective immune response in susceptible BALB/c mice. The TLR interdependency optimizes TLR-induced immune response by a novel immunoregulatory framework and scientifically rationalizes targeting TLRs in tandem and in sequence for redirecting immune responses against an intracellular pathogen.

Development and Assessment of a Brazilian Pilot Massive Open Online Course in Planetary Health Education: An Innovative Model for Primary Care Professionals and Community Training.

Introduction: Planetary health (PH) has emerged as a leading field for raising awareness, debating, and finding solutions for the health impacts of human-caused disruptions to Earth's natural systems. PH education addresses essential questions of how humanity inhabits Earth, and how humans affect, and are affected by, natural systems. A pilot massive open online course (MOOC) in PH was created in Brazil in 2020. This MOOC capitalized on the global online pivot, to make the course accessible to a broader audience. This study describes the process of course creation and development and assesses the impact evaluation data and student outcomes of the PH MOOC. Methods: The PH MOOC pilot was launched in Brazilian Portuguese, using the TelessaúdeRS-UFRGS platform on 4/27/2020 and concluded on 7/19/2020 with a total load of 80 h. It was composed of 8 content modules, pre and post-test, 10 topics in a forum discussion, and an optional action plan. This study analyzes the course database, profile of participants, answers to questionnaires, forum interaction, and action plans submitted. Results: Two thousand seven hundred seventy-seven participants enrolled in the course, of which 1,237 (44.54%) gave informed consent for this study. Of the 1,237 participants who agreed to participate in the research, 614 (49.8%) completed the course, and 569 (92.67%) were accredited by TelessaúdeRS-UFRGS. The majority of the participants were concerned with climate change, trained in the health area, and worked in primary health care in places that lacked ongoing sustainability programs. Two hundred forty-one action plans were submitted, major topics identified were food and nutrition, infectious diseases, and garbage and recycling. Discussion: The use of the PH lens and open perspective of the course centered the need to communicate planetary health topics to individuals. The local plans reflected the motto of "think global and act local." Brazil presents a context of an unprecedented social, political, and environmental crisis, with massive deforestation, extensive fires, and biomass burning altering the biomes, on top of an ongoing necropolitical infodemic and COVID-19 pandemic. In the face of these multiple challenges, this MOOC offers a timely resource for health professionals and communities, encouraging them to address planetary challenges as fundamental health determinants.

Next Generation Influenza Vaccines: Looking into the Crystal Ball.

Influenza infections are responsible for significant number of deaths and overwhelming costs worldwide every year. Vaccination represents the only cost-efficient alternative to address this major problem in human health. However, current vaccines are fraught by many limitations, being far from optimal. Among them, the need to upgrade vaccines every year through a time-consuming process open to different caveats, and the critical fact that they exhibit poorer efficacy in individuals who are at high risk for severe infections. Where are we? How can knowledge and technologies contribute towards removing current roadblocks? What does the future offer in terms of next generation vaccines?

Seropositivity of selected chronic infections and different measures of obesity.

The impact of sex-specific body fat distribution on the susceptibility to five chronic infections, helicobacter pylori and human herpesviruses 3 to 6 (i.e. varicella-zoster, Epstein-Barr, cytomegalo- and human herpesvirus 6), has not previously been examined. In the present study, seropositivity was determined via multiplex serology in serum samples of study participants collected in 2006/08 and 2013/14 during the follow-up examinations F4 (n = 3080) and FF4 (n = 2279) of the German population-based baseline KORA S4 survey. We quantified the severity of overall and abdominal obesity by body mass index, body adiposity index, waist circumference, waist-to-hip ratio, and waist-to-height ratio. Using sex-specific logistic spline-models, cross-sectional and longitudinal associations between obesity measures and seropositivity of the previously mentioned infections were investigated. Overall and abdominal fat content were significantly associated with seropositivity of varicella-zoster virus in both cross-sectional and longitudinal analyses among women. In addition, a non-significant inverse relationship with Epstein-Barr virus seroprevalence in both sexes and a trend towards a positive association with human herpesvirus 6 seropositivity in women were observed. Therefore, in women total body fat may be associated with VZV-seropositivity and may influence the reactivation of the varicella-zoster virus, independent of adipose tissue distribution.

Self-Amplifying Replicon RNA Delivery to Dendritic Cells by Cationic Lipids.

Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA, termed replicons, RepRNA, or self-amplifying mRNA, with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication, without infectious progeny generation, providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells-critical for immune defense development. Several cationic lipids were assessed, with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells-important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that resulted in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly when combined with additional delivery elements.

Fatty Acid Profile, Phenolics and Flavonoids Contents in Olea europaea L. Callus Culture cv. cornicabra.

Olive trees are one of the most important oil crops in the world due to the sensorial and nutritional characteristics of olive oil, such as lipid composition and antioxidant content, and the medicinal properties of its leaves. In this paper, callus formation was induced using nodal segments of olive tree (Olea europaea cv. cornicabra) as explants. Fatty acid profile, total phenolic compounds and total flavonoid compounds were determined in callus culture after 15 weeks and compared with leaf and nodal segments tissues. There was no statistical difference in phenolic compounds among leaf, nodal segments and callus culture, whereas flavonoid compounds were higher in leaf. Fatty acid profile was similar in leaf, nodal segments and callus culture and was constituted by hexadecanoic acid, octadecanoic acid, cis-9-octadecenoic acid, cis-9,12-octadecadienoic acid, cis-9,12,15-octadecatrienoic acid. Hexadecanoic acid was the main fatty acid in callus, leaf and nodal segments with 35.0, 39.0 and 40.0% (w/w), of the lipid composition, respectively. With this paper, it is being reported for the first time the capacity of callus culture to accumulate fatty acids. Our results could serve to continue studying the production of fatty acids in callus cultivation as a biotechnological tool to improve different olive cultivars.

Blockade of Neutrophil's Chemokine Receptors CXCR1/2 Abrogate Liver Damage in Acute-on-Chronic Liver Failure.

BACKGROUND: Neutrophils serve as critical players in the pathogenesis of liver diseases. Chemokine receptors CXCR1 and CXCR2 are required for neutrophil chemotaxis to the site of inflammation/injury and are crucial in hepatic inflammatory response. However, key mechanism of neutrophil-mediated liver injury in acute-on-chronic liver failure (ACLF) remains highly elusive; which could be targeted for the development of new therapeutic interventions. METHODS: To demonstrate the role of CXCR1/CXCR2-expressing neutrophils in hepatic injury, we investigated CXCR1/CXCR2 receptor expression in 17 hepatitis B virus-related ACLF patients in comparison to 42 chronic hepatitis B and 18 healthy controls. Mechanism of neutrophil-mediated cell death was analyzed by in vitro coculture assays and correlated with the patient data. In addition, to find out any etiological-based variations in ACLF, 19 alcohol-related ACLF patients were also included. RESULTS: In ACLF, neutrophils have high expression of CXCR1/CXCR2 receptors, which potentially participate in hepatocyte death through early apoptosis and necrosis in contact-dependent and -independent mechanisms. Importantly, blockade of CXCR1/CXCR2 with SCH 527123 antagonist significantly reduced cell death by targeting both the mechanisms. No etiology-based differences were seen between ACLF groups. Importantly, absolute neutrophil count was particularly higher in clinically severe ACLF patients and non-survivors (p < 0.0001). Multivariate analysis demonstrated ANC and CXCL8/IL-8 as a predictor of mortality. Further, receiver operating characteristics curve confirmed the cutoff of ANC >73.5% (sensitivity: 76.5% and specificity: 76.5%) and CXCL8/IL-8 >27% (sensitivity: 70% and specificity: 73%) in prediction of mortality. CONCLUSION: Blockade of CXCR1/CXCR2 diminished the production of inflammatory mediators and reduced cell death; therefore, pharmacological neutralization of CXCR1/CXCR2 could provide novel therapeutic target in the management of ACLF.

Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

Dynamic changes in viral population structure and compartmentalization during chronic hepatitis C virus infection in children.

Classic phylogenetic and modern population-based clustering methods were used to analyze hepatitis C virus (HCV) evolution in plasma and to assess viral compartmentalization within peripheral blood mononuclear cells (PBMCs) in 6 children during 3.2-9.6yr of follow-up. Population structure analysis of cloned amplicons encompassing hypervariable region 1 led to the distinction of two evolutionary patterns, one highly divergent and another one genetically homogeneous. Viral adaptability was reflected by co-evolution of viral communities switching rapidly from one to another in the context of divergence and stability associated with highly homogeneous communities which were replaced by new ones after long periods. Additionally, viral compartmentalization of HCV in PBMCs was statistically demonstrated, suggesting their role as a pool of genetic variability. Our results support the idea of a community-based structure of HCV viral populations during chronic infection and highlight a role of the PBMC compartment in the persistence of such structure.

Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP.

Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 µg haemagglutinin; HA) in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥ 40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines.

Genetic immunization: bacteria as DNA vaccine delivery vehicles.

The so-called DNA vaccination represents one of the most notable tools under development in the field of vaccinology. The concept of administering the gene coding for any given protective antigen and make responsible vaccinee's own cells to produce the protein appeals as too simple to be true. Indeed, the implementation of this approach for mass vaccination should overcome several bottlenecks, such as need of high dosages and poor immunogenicity. In this context, the use of live attenuated bacteria as delivery system for plasmid DNA has emerged as a promising alternative to overcome many of those pitfalls. In addition, this approach is not only amenable for mucosal administration, but allows to specifically target professional antigen presenting cells. This results in their transfection, as well as in their activation and maturation, due to their built-in adjuvant properties resulting from the stimulation of pattern recognition receptors. This chapter discusses the specific features that should be taken into consideration when designing a plasmid vector, current candidate bacterial carriers for DNA delivery and main safety issues.

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit.

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.