Immunization With Mycobacterium tuberculosis Antigens Encapsulated in Phosphatidylserine Liposomes Improves Protection Afforded by BCG.

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol Mycobacterium tuberculosis, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.

Photoperiod regulates hypothalamic miR-155 gene expression in female, but not male, Siberian hamsters (Phodopus sungorus).

In many species, seasonal changes in photoperiod regulate several behaviors and physiological systems, including reproduction, energy balance, and immune function. MicroRNAs (miRs) regulate numerous physiological processes and developmental transitions through translational repression and mRNA degradation. Their role in seasonal transitions has been vastly understudied, with only a few reports in animals. Furthermore, no study has assessed whether there are sex differences in seasonal regulation of miRs. miR-155 is a primary candidate for seasonal regulation because it influences immune responses, energetics, and reproductive function. In this study, we tested the hypothesis that photoperiod regulates miR-155 gene expression in Siberian hamsters and whether there were sex differences in this photoperiod regulation. miR-155 gene expression levels were measured in hypothalamus, hippocampus, and spleen of male and female Siberian hamsters reared in short days (SDs) or long days (LDs). As expected, SD-reared hamsters had significantly reduced body mass, lightened pelage color, and lower reproductive organ size than LD-reared hamsters. Notably, SDs increased hypothalamic miR-155 gene expression in females but not in males. No differences were observed in hippocampus and spleen of either sex. These findings demonstrate sex-specific photoperiod regulation of miR-155 gene expression. Future studies should consider possible sex differences in miR contributions to seasonal changes in physiology and behavior. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Antibody degradation in tobacco plants: a predominantly apoplastic process.

BACKGROUND: Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. RESULTS: In this study, we investigated the dynamics of the assembly and breakdown of a human IgG(1)κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space. CONCLUSIONS: The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.

Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate.

Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1-20 microM). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression was determined in seedlings expressing Escherichia coli otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1 by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues.

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1.

Transgenic potato (Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene (SnRK1) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%-167% compared with that in the wild-type. Glucose levels were decreased, at 17%-56% of the levels of the wild-type, and the starch content showed an increase of 23%-30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, alpha-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%-60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.

Molecular cloning of an arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation.

DNA homologous to the yeast ( Saccharomyces cerevisiae) protein kinase gene, GCN2, was amplified from arabidopsis [ Arabidopsis thaliana (L.) Heynh.] RNA and given the name AtGCN2. The AtGCN2 peptide sequence included adjacent protein kinase and histidyl tRNA synthetase-like domains and showed 45% sequence identity with the GCN2 peptide sequence in the protein kinase domain. AtGCN2 transcripts were detectable in RNA from roots, leaves, stems, buds, flowers, siliques and seedlings. GCN2 is required for yeast cells to respond to amino acid starvation. Expression of AtGCN2 in yeast gcn2 mutants complemented the mutation, enabling growth in the presence of sulfometuron methyl, an inhibitor of branched-chain amino acid biosynthesis, and 3-aminotriazole, an inhibitor of histidine biosynthesis.