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The bacille Calmette-Guèrin (BCG) vaccine has been used for a century; nonetheless, tuberculosis (TB) remains one of the deadliest diseases in the world. Thus, new approaches to developing a new, more efficient vaccine are desirable. Mucosal vaccines are of particular interest, considering that Mycobacterium tuberculosis first enters the body through the mucosal membranes. We have previously demonstrated the immunogenicity of a recombinant Lactiplantibacillus plantarum delivery vector with TB hybrid antigen Ag85B-ESAT-6 anchored to the cell membrane. The goal of the present study was to analyze the impact of antigen localization in the immune response. Thus, we assessed two novel vaccine candidates, with the TB antigen either non-covalently anchored to the cell wall (LysMAgE6) or located intracellularly (CytAgE6). In addition, we compared two expression systems, using an inducible (LipoAgE6) or a constitutive promoter (cLipoAgE6) for expression of covalently anchored antigen to the cell membrane. Following administration to mice, antigen-specific CD4+ T-cell proliferation and IFN-γ and IL-17A secretion were analyzed for lung cell and splenocyte populations. Generally, the immune response in lung cells was stronger compared to splenocytes. The analyses showed that the type of expression system did not significantly affect the immunogenicity, while various antigen localizations resulted in markedly different responses. The immune response was considerably stronger for the surface-displaying candidate strains compared to the candidate with an intracellular antigen. These findings emphasize the significance of antigen exposure and further support the potential of L. plantarum as a mucosal vaccine delivery vehicle in the fight against TB.

International audience; Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

Table S1. RNA samples that were used in the study. RIN - RNA integrity number; RT-qPCR - reverse transcription-quantitative PCR; miRNAs seq - microRNA sequencing. X indicates which samples were used in each of the experiments. (DOCX 16 kb)

Figure S1. Experimental design of reverse transcription-quantitative PCR (RT-qPCR) and microRNA sequencing experiments. S. agal: samples of in vitro exposure of blood monocyte-derived macrophages with live Streptococcus agalactiae strain ST103 or strain ST12, respectively; Neg.con: negative control: sample of uninfected blood monocyte-derived macrophages; 6 h – 6 h infection with Streptococcus agalactiae, 2 h – 2 h exposure to LPS. For details on the experimental design, see the Materials and Methods section. (PDF 134 kb)

Figure S2. Principal component analysis of mapped sequence reads. Control represents sequences from non-infected bovine monocyte-derived macrophage libraries, and a) Streptococcus agalactiae strain ST103 infected; b) Streptococcus agalactiae strain ST12 infected; c) LPS-challenged; and d) Streptococcus agalactiae strain ST103 or strain ST12 infected macrophages isolated from the same animals. (PDF 264 kb)

Table S2. List of primers used for reverse transcription-quantitative PCR (RT-qPCR). (DOCX 14 kb)

The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

International audience; NKp46 has proven a reliable NK cell marker in both cattle and sheep. Four anti-bovine/ovine NKp46 mAbs reacted to caprine lymphocytes and specificity was confirmed as all mAbs reacted with caprine NKp46 transfectants. At young ages, goat NKp46+ cells were scarcer in circulation (~1% of PBMC) than in cattle and sheep, but the numbers increase with age. Similar to cattle and sheep, these cells are CD16+, CD8+, moderately perforin+, and CD2 marks a major negative and a minor positive subset. NKp46+ cells produce IFN-γ in response to cytokine stimulation, and following positive selection and culture they upregulate perforin and respond with NKp46-mediated cytotoxicity, thus confirming typical NK cell functions. NKp46 expression was non-overlapping with CD21+ B-cells, CD4+ T-cells and CD14+ monocytes. Most caprine-reactive gamma/delta TcR antibodies do not mark NKp46+ cells. However, in older goats, a significant proportion of NKp46+ cells express the TcR delta chain. This occurs at an age where the proportion of classical gamma-delta T-cells is in decline. NKp46+/TcRdelta+ cells were also present in cultures of IL-2 expanded caprine NKp46+ cells. Since NK cells in most species are CD3-/NKp46+, we stained permeabilized caprine PBMCs with a human cytoplasmatic CD3 epsilon mAb, which is cross-reactive in several species. Staining patterns corresponded well with expected T-cell populations, but this mAb also stained a subset of NKp46+ cells, the majority of which were also TcRdelta+. Thus, cytCD3e cannot be used straightforward for NK cell discrimination in goats, and these overlapping populations need closer investigation.

International audience