Uterine B cells exhibit regulatory properties during the peri-implantation stage of murine pregnancy

A successful outcome to pregnancy is dependent on the ability of the maternal uterine microenvironment to regulate inflammation processes and establish maternal tolerance. Recently, B cells have been shown to influence pregnancy outcomes as aberrations in their numbers and functions are associated with obstetric complications. In this study, we aimed to comprehensively examine the population frequency and phenotypic profile of B cells over the course of murine pregnancy. Our results demonstrated a significant expansion in B cells within the uterus during the peri-implantation period, accompanied by alterations in B cell phenotype. Functional evaluation of uterine B cells purified from pregnant mice at day 5.5 post-coitus established their regulatory capacity as evidenced by effective suppression of proliferation and activation of syngeneic CD4+ T cells. Flow cytometric analysis revealed that the uterine B cell population has an expanded pool of IL-10-producing B cells bearing upregulated expression of co-stimulatory molecules CD80 and CD86 and activation marker CD27. Our investigations herein demonstrate that during the critical stages surrounding implantation, uterine B cells are amplified and phenotypically modified to act in a regulatory manner that potentially contributes toward the establishment of maternal immunological tolerance in early pregnancy.Ruth Marian Guzman-Genuino, Preethi Eldi, Pablo Garcia-Valtanen, John D. Hayball and Kerrilyn R. Diene

Evaluation of trained immunity by beta-1, 3 (D)-glucan on murine monocytes in vitro and duration of response in vivo

The β-1, 3 (D)-glucan (β-glucan) present in the cell wall of Candida albicans induces epigenetic changes in human monocytes resulting in primed macrophages exhibiting increased cytokine responsiveness to reinfection. This phenomenon is referred to as trained immunity or innate immune memory. However, whether β-glucan can reprogramme murine monocytes in vitro or induce lasting effects in vivo has yet to be elucidated. Thus, purified murine spleen-derived monocytes were primed with β-glucan in vitro and assessed for markers of differentiation and survival. Important macrophage cell markers during monocyte-to-macrophage differentiation were downregulated and survival enhanced due to partial inhibition of apoptosis. Increased survival and not the β-glucan training effect explained the elevated production of tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) induced by subsequent lipopolysaccharide (LPS) challenge. In vivo, 4 days after systemic administration of β-glucan, mice were more responsive to LPS challenge as shown by the increased serum levels of TNFα, IL-6 and IL-10, an effect shown to be short lived as enhanced cytokine production was lost by day 20. Here, we have characterised murine macrophages derived from β-glucan-primed monocytes based on their surface marker expression and for the first time provide evidence that the training effect of β-glucan in vivo declines within a 3-week period.Pablo Garcia-Valtanen, Ruth Marian Guzman-Genuino, David L Williams, John D Hayball, and Kerrilyn R Diene

SARS-CoV-2 Omicron variant escapes neutralising antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents. Garcia-Valtanen et al

Raw data from experimental results featured in: SARS-CoV-2 Omicron variant escapes neutralising antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents by Garcia-Valtanen et alTHIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH), Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH), Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV) nonstructural protein 5 (NS5) contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds) to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol) were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular docking, high-throughput computing, AutoDock/Vina, ADMET, SuperNatural database, inhibitors, NS5 RNA-dependent RNA polymeras

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from Vaccinia virus

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.Liang Liu, Tamara Cooper, Preethi Eldi, Pablo Garcia- Valtanen, Kerrilyn R. Diener, Paul M. Howley, and John D. Haybal

Development of new therapeutical/adjuvant molecules by pepscan mapping of autophagy and IFN inducing determinants of rhabdoviral G proteins

Surface glycoproteins of enveloped virus are potent elicitors of both innate and adaptive host immune responses. Therefore, the identification of viral glycoprotein determinants directly implicated in the induction of these responses might be of special interest for designing new therapeutical/adjuvant molecules. In this work we review the contribution of the "pepscan" approach to the screening of viral functions in the sequence of glycoprotein G (gpG) of the fish rhabdovirus of viral hemorrhagic septicemia (VHSV). Among others, by scanning gpG peptides, it has been possible to identify and validate minimal determinants for gpG directly implicated in initiating the fish type I Interferon-associated immune responses as well as in the antiviral autophagy program. Further fine-tunning of the identified peptides in the gpG of VHSV has allowed designing novel adjuvants that decrease DNA vaccine requirements and identify possible innovative antiviral molecules. In addition, these results have also contributed to improve our knowledge on how to stimulate the fish immune system. © 2016

Maternal host responses to poly(I:C) during pregnancy leads to both dysfunctional immune profiles and altered behaviour in the offspring

Problem: Autism spectrum disorder (ASD)-like phenotypes in murine models are linked to elevated proinflammatory cytokine profiles caused by maternal immune activation (MIA) but whether MIA alters the immune response in the offspring remains unclear. Method of Study: Polyinosinic:polycytidylic acid (poly:[IC]) was used to induce MIA in immunocompetent and control TLR3-deficient pregnant mice and cytokine levels were measured in maternal and fetal organs. Furthermore, cytokines and behaviour responses were tested after challenge with lipopolysaccharide in 7-day-old and adult mice. Results: MIA induced on E12 resulted in changes in the cytokine expression profile in maternal and fetal organs and correlated with TNFα and IL-18 dysregulation in immune organs and brains from neonatal mice born to MIA-induced dams. Such changes further correlated with altered behavioural responses in adulthood. Conclusion: MIA induced by pathogens during pregnancy can interfere with the development of the fetal immune and nervous systems leading to dysfunctional immune responses and behaviour in offspring.Pablo Garcia-Valtanen, Bianca A. van Diermen, Nerissa Lakhan, Erin L. Lousberg, Sarah A. Robertson, John D. Hayball, Kerrilyn R. Diene

Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates

DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and GVHSV-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, GVHSV up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation

VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants

We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpGVHSV), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpGVHSV. In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31+p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1μg of the construct pMCV1.4-p31+p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31+p33 construct was assayed for molecular adjuvantcity's for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-GSVCV). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-Gsvcv without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system. © 2014 Elsevier Ltd