Maria Guadalupe Vizoso-Pinto

Immunobiotic Lactobacillus rhamnosus improves resistance of infant mice against respiratory syncytial virus infection

Previously we showed that orally administered Lactobacillus rhamnosus CRL1505 beneficially regulated the balance between pro- and anti-inflammatory mediators in the lungs of poly(I:C)-challenged mice, allowing an effective inflammatory response against the TLR3/RIG-I agonist but at the same time reducing tissue damage. The aim of the present study was to investigate whether oral administration of the CRL1505 strain was able to improve resistance against respiratory syncytial virus (RSV) infection in infant mice and to evaluate the immunological mechanisms involved in the immunobiotic effect. We demonstrated that treatment of 3-week old BALB/c mice with L. rhamnosus CRL1505 significantly reduce lung viral loads and tissue injuries after the challenge with RSV. Moreover, we showed that the protective effect achieved by the CRL1505 strain is related to its capacity to differentially modulate respiratory antiviral immune response. Our results shows that IFN-γ and IL-10 secreted in response to L. rhamnosus CRL1505 oral stimulation would modulate the pulmonary innate immune microenvironment conducting to the activation of CD103(+) and CD11b(high) dendritic cells and the generation of CD3(+)CD4(+)IFN-γ(+) Th1 cells with the consequent attenuation of the strong and damaging Th2 reactions associated with RSV challenge. Our results indicate that modulation of the common mucosal immune system by immunobiotics could favor protective immunity against respiratory viral pathogens with a high attack rate in early infancy, such as RSV.

Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells

BackgroundPrevious findings suggested that Lactobacillus rhamnosus CRL1505 is able to increase resistance of children to intestinal viral infections. However, the intestinal cells, cytokines and receptors involved in the immunoregulatory effect of this probiotic strain have not been fully characterized.ResultsWe aimed to gain insight into the mechanisms involved in the immunomodulatory effect of the CRL1505 strain and therefore evaluated in vitro the crosstalk between L. rhamnosus CRL1505, porcine intestinal epithelial cells (IECs) and antigen presenting cells (APCs) from swine Peyer’s patches in order to deepen our knowledge about the mechanisms, through which this strain may help preventing viral diarrhoea episodes. L. rhamnosus CRL1505 was able to induce IFN–α and –β in IECs and improve the production of type I IFNs in response to poly(I:C) challenge independently of Toll-like receptor (TLR)-2 or TLR9 signalling. In addition, the CRL1505 strain induced mRNA expression of IL-6 and TNF-α via TLR2 in IECs. Furthermore, the strain significantly increased surface molecules expression and cytokine production in intestinal APCs. The improved Th1 response induced by L. rhamnosus CRL1505 was triggered by TLR2 signalling and included augmented expression of MHC-II and co-stimulatory molecules and expression of IL-1β, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs.ConclusionsIt was recently reviewed the emergence of TLR agonists as new ways to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies. The use of L. rhamnosus CRL1505 as modulator of innate immunity and inductor of antiviral type I IFNs, IFN-γ, and regulatory IL-10 clearly offers the potential to overcome this challenge.

Intestinal Innate Antiviral Immunity and Immunobiotics: Beneficial Effects against Rotavirus Infection

The mucosal tissues of the gastrointestinal tract are the main portal entry of pathogens such as rotavirus (RV), which is a leading cause of death due to diarrhea among young children across the globe and a major cause of severe acute intestinal infection in livestock animals. The interactions between intestinal epithelial cells (IECs) and immune cells with RVs have been studied for several years, and now, it is known that the innate immune responses triggered by this virus can have both beneficial and detrimental effects for the host. It was demonstrated that natural RV infection in infants and experimental challenges in mice result in the intestinal activation of pattern recognition receptors (PRRs) such as toll-like receptor 3 (TLR3) and striking secretion of proinflammatory mediators that can lead to increased local tissue damage and immunopathology. Therefore, modulating desregulated intestinal immune responses triggered by PRRs activation are a significant promise for reducing the burden of RV diseases. The ability of immunoregulatory probiotic microorganisms (immunobiotics) to protect against intestinal infections, such as those caused by RVs, is among the oldest effects studied for these important group of beneficial microbes. In this review, we provide an update of the current status on the modulation of intestinal antiviral innate immunity by immunobiotics and their beneficial impact on RV infection. In addition, we describe the research of our group that demonstrated the capacity of immunobiotic strains to beneficially modulated TLR3-triggered immune response in IECs, reduce the disruption of intestinal homeostasis caused by intraepithelial lymphocytes, and improve the resistance to RV infections.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation–coagulation interactions and reduces influenza virus-associated pulmonary damage

ObjectiveTo evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury.MethodsSix-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV.ResultsBoth viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4+IFN-γ+ lymphocytes, and lung CD11c+CD11blowCD103+ and CD11c+CD11bhighCD103− dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response.ConclusionsOur work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation–coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

Serologic analysis of the IgG antibody response in children with varicella zoster virus wild-type infection and vaccination.

Introduction: In contrast to varicella zoster virus (VZV) primary infection, VZV vaccination does not seem to provide lifelong immunity against varicella. Because more people get vaccinated every year, the development of sensitive serological test systems for the detection of protective anti-VZV IgG will become important in the future. Methods: We have previously developed a novel VZV line assay based on 5 different recombinant VZV antigens. In this study, we compared this novel assay with a commercially available glycoprotein enzyme immunoassay (RIDASCREEN VZV IgG) in detecting anti-VZV IgG of children with previous varicella infection and VZV vaccination. Results: One hundred twenty-five children were included in this study, 72 with a history of varicella infection and 53 with VZV vaccination. Both assays detected anti-VZV IgG antibodies in both study groups with similar sensitivities. The VZV line assay revealed striking differences in the anti-VZV IgG composition against the VZV open reading frames, 4, 14 and 49, between both study groups, indicating that wild-type varicella infection causes a more diverse immune response against VZV than does vaccination. The exploitation of these results enabled the discrimination of both study groups with a sensitivity of 0.93 and a specificity of 0.83, indicating that the serologic differentiation of children with previous varicella infection and VZV vaccination might be possible. Conclusion: The VZV line assay enables the detection of anti-VZV IgG with sensitivities comparable to glycoprotein enzyme immunoassays and might be suitable for the serologic discrimination between children with a history of varicella infection and VZV vaccination.

Respiratory Commensal Bacteria Corynebacterium pseudodiphtheriticum Improves Resistance of Infant Mice to Respiratory Syncytial Virus and Streptococcus pneumoniae Superinfection

Corynebacterium pseudodiphtheriticum is a Gram-positive bacterium found as a member of the normal microbiota of the upper respiratory tract. It was suggested that C. pseudodiphtheriticum may be potentially used as a next-generation probiotic for nasal application, although no deep studies were performed in this regard. We hypothesized that human isolate C. pseudodiphtheriticum strain 090104 is able to modulate the respiratory innate immune response and beneficially influence the resistance to viral and bacterial infections. Therefore, in the present study we investigated how the exposure of infant mice to nasal priming with viable or non-viable C. pseudodiphtheriticum 090104 influences the respiratory innate immune response triggered by Toll-like receptor (TLR)-3 activation, the susceptibility to primary Respiratory Synsytial Virus (RSV) infection, and the resistance to secondary Streptococcus pneumoniae pneumonia. We demonstrated that the nasal priming with viable C. pseudodiphtheriticum 090104 differentially modulated TLR3-mediated innate antiviral immune response in the respiratory tract of infant mice, improving their resistance to primary RSV infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that viable C. pseudodiphtheriticum improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-β+ alveolar macrophages. Of interest, non-viable bacteria did not have the same protective effect, suggesting that C. pseudodiphtheriticum colonization is needed for achieving its protective effect. In conclusion, we present evidence that nasal application of viable C. pseudodiphtheriticum could be thought as an alternative to boost defenses against RSV and secondary pneumococcal pneumonia, which should be further studied and validated in clinical trials. Due to the absence of a long-lasting immunity, re-infection with RSV throughout life is common. Thus, a possible perspective use could be a seasonal application of a nasal probiotic spray to boost respiratory innate immunity in immunocompetent subjects.

The Hepatitis E virus intraviral interactome.

Hepatitis E virus (HEV) is an emerging virus causing epidemic acute hepatitis in developing countries as well as sporadic cases in industrialized countries. The life cycle of HEV is still poorly understood and the lack of efficient cell culture systems and animal models are the principal limitations for a detailed study of the viral replication cycle. Here we exhaustively examine all possible intraviral protein-protein interactions (PPIs) of HEV by systematic Yeast two-hybrid (Y2H) and LuMPIS screens, providing a basis for studying the function of these proteins in the viral replication cycle. Key PPIs correlate with the already published HEV 3D structure. Furthermore, we report 20 novel PPIs including the homodimerization of the RNA dependent RNA polymerase (RdRp), the self-interaction of the papain like protease, and ORF3 interactions with the papain-like protease and putative replicase components: RdRp, methylase and helicase. Furthermore, we determined the dissociation constant (Kd) of ORF3 interactions with the viral helicase, papain-like protease and methylase, which suggest a regulatory function for ORF3 in orchestrating the formation of the replicase complex. These interactions may represent new targets for antiviral drugs.

A novel indirect immunofluorescence test for the detection of IgG and IgA antibodies for diagnosis of Hepatitis E Virus infections

Hepatitis E Virus (HEV) causes epidemic infections in regions of poor hygiene in the developing world. Over the last years, however, increasing numbers of autochthonous infections in industrialized countries have been described, leading to new interest in this pathogen. Currently available serological test formats to detect IgG and IgM antibodies are mainly based on bacterially expressed ORF2 and ORF3 antigens and often give ambiguous results. The objective of this study was the development of a different assay format for HEV diagnosis--a HEV immunofluorescence test (HEV-IFT) based on mammalian cells transiently expressing recombinant HEV ORF2 protein with a simple production and staining protocol and the investigation of its performance and methodical feasibility under diagnostic laboratory conditions. 31 sera of patients at different phases of HEV infection and 40 control sera from a non-endemic region were analyzed for anti-HEV IgG, IgM, and IgA antibodies. The HEV-IFT detected successfully anti-HEV IgG and IgA, but not anti-HEV IgM antibodies. In the study group the HEV-IFT was able to confirm HEV infections and to support diagnosis when ambiguous results were obtained by commercial assays. Signal localization and staining patterns helped to gather additional information about reactive antibodies present in patient sera. In conclusion the developed IFT for the detection of anti-HEV IgG and IgA antibodies can be used for diagnosis and for the serological confirmation of HEV infections.

The interactome of Streptococcus pneumoniae and its bacteriophages show highly specific patterns of interactions among bacteria and their phages

Although an abundance of bacteriophages exists, little is known about interactions between their proteins and those of their bacterial hosts. Here, we experimentally determined the phage-host interactomes of the phages Dp-1 and Cp-1 and their underlying protein interaction network in the host Streptococcus pneumoniae. We compared our results to the interaction patterns of E. coli phages lambda and T7. Dp-1 and Cp-1 target highly connected host proteins, occupy central network positions, and reach many protein clusters through the interactions of their targets. In turn, lambda and T7 targets cluster to conserved and essential proteins in E. coli, while such patterns were largely absent in S. pneumoniae. Furthermore, targets in E. coli were mutually strongly intertwined, while targets of Dp-1 and Cp-1 were strongly connected through essential and orthologous proteins in their immediate network vicinity. In both phage-host systems, the impact of phages on their protein targets appears to extend from their network neighbors, since proteins that interact with phage targets were located in central network positions, have a strong topologically disruptive effect and touch complexes with high functional heterogeneity. Such observations suggest that the phages, biological impact is accomplished through a surprisingly limited topological reach of their targets.

β-Carboline derivatives as novel antivirals for herpes simplex virus

Several commercial and novel synthetic β-carbolines (βCs) were evaluated for their antiviral activity against herpes simplex virus type 1 (HSV-1) using an adapted MTS assay. Of 21 drugs tested, although 11 exerted antiviral activity at non-cytotoxic concentrations, only 3 of them [9-methyl-norharmane (9-Me-nHo), 9-methyl-harmane (9-Me-Ho) and 6-methoxy-harmane (6-MeO-Ho)] completely avoided virus-driven cytopathic effects. Half-maximal effective concentrations (EC50 values) (4.9 ± 0.4, 5.9 ± 0.8 and 19.5 ± 0.3 µM, respectively) and selectivity indexes (88.8, 40.2 and 7.0, respectively) of the latter three βCs against HSV-1 were determined by MTS, flow cytometry and plaque reduction assays. The mode of action of these drugs was also evaluated. According to time-of-addition assays, the selected compounds were not virucidal and did not interfere with attachment or penetration of HSV-1, but interfered with later events of virus infection. Western blot studies showed that early and late protein expression was significantly delayed or even suppressed. Herpes simplex virus type 2 (HSV-2) was also inhibited by the selected substances in a similar manner. Interestingly, 6-MeO-Ho, 9-Me-Ho and 9-Me-nHo restricted HSV-1 ICP0 localisation to the nucleus during later stages of infection, possibly affecting its functionality in the cytoplasm where ICP0 normally inhibits antiviral signalling and promotes viral replication. In silico prediction of ADME (Absorption, Distribution, Metabolism and Excretion) properties showed that all compounds fulfilled Lipinskis rule and their calculated absorptions were >95%.