Evaluation of viral peptide targeting to porcine sialoadhesin using a porcine reproductive and respiratory syndrome virus vaccination-challenge model.

Targeting antigens to professional antigen presenting cells resident at the sites where effective immune responses are generated is a promising vaccination strategy. As such, targeting sialoadhesin (Sn)-expressing macrophages, abundantly present in spleen and lymph nodes where they appear to be strategically placed for antigen capture and processing, is recently gaining increased attention. Previously, we have shown that humoral immune responses to the model antigen human serum albumin can be enhanced by using a porcine Sn-specific monoclonal antibody to target the model antigen to Sn-expressing macrophages. To date however, no studies have been performed to evaluate whether targeted delivery of a pathogen-derived antigen can enhance the pathogen-specific immune response. Therefore, we selected a linear epitope on glycoprotein 4 of porcine reproductive and respiratory syndrome virus (PRRSV), which is known to be a target of virus-neutralizing antibodies. This paper reports on the targeted delivery of this viral peptide to porcine Sn-expressing macrophages and the evaluation of the subsequent immune response in a vaccination-challenge set-up. Four copies of the selected PRRSV epitope were genetically fused to a previously developed porcine Sn-targeting recombinant antibody or an irrelevant isotype control. Fusion proteins were shown to be efficiently purified from HEK293T cell supernatants and subsequently, only Sn-specific fusion proteins were shown to bind to and to be internalized into Sn-expressing cells. Subsequent immunizations with a single dose of the fusion proteins showed that peptide-specific immune responses and neutralizing antibody responses after PRRSV challenge were enhanced in animals receiving a single 500 µg intramuscular dose of the Sn-targeting fusion protein, although correlations between the two read-outs were hard to effectuate. Furthermore, a minor beneficial effect on viral clearance was observed. Together, these data show that viral peptide targeting to porcine Sn-expressing macrophages can improve the anti-viral immune response, although more research will be needed to further explore vaccination potential.

Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages.

BACKGROUND: Sialoadhesin (Sn)-expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. This makes Sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. Furthermore, since Sn was shown to be an endocytic receptor, the conjugation of effector molecules to an Sn-specific ligand should allow intracellular delivery of these conjugates. Previously, we developed functional Sn-specific immunoconjugates that were generated via chemical coupling. Although successful, the system requires significant optimization for each immunoconjugate to be made. To generate a more flexible and controlled system, we developed a recombinant antibody vector allowing the creation of genetic antibody fusion constructs. This paper reports on the characterization of the recombinant antibody and the evaluation of its use for Sn-directed targeting. RESULTS: The variable domains of the porcine Sn-specific monoclonal antibody 41D3 were sequenced and cloned in frame with a mouse IgG1 backbone. Transfection of HEK293T cells with the resulting plasmid led to the secretion of fully assembled IgG into the culture medium. This recombinant antibody rec41D3 was shown to specifically bind to porcine Sn with a comparable affinity as the native monoclonal antibody. In addition, rec41D3 also induced Sn endocytosis in primary macrophages and resided for prolonged times in early/late endosomes. To allow the generation of antibody fusion constructs, a multiple cloning site was introduced at the C-terminus of the heavy chain. Two fusion constructs were generated, one containing a V5 peptide tag and one containing an eGFP molecule. Both constructs were shown to be efficiently produced in HEK293T cells and easily purified using standard protein G chromatography. In addition, both V5 and eGFP were shown to be co-internalized together with rec41D3 into Sn-expressing primary macrophages. CONCLUSIONS: A recombinant antibody allowing targeted delivery of peptides and proteins to Sn-expressing macrophages was developed. Production and purification of antibody fusion constructs was possible without major optimization and with batch to batch consistency, confirming the development of a versatile antibody vector to evaluate Sn-directed targeting strategies in a porcine animal model.

AtWRKY15 perturbation abolishes the mitochondrial stress response that steers osmotic stress tolerance in Arabidopsis.

Environmental stresses adversely affect plant growth and development. A common theme within these adverse conditions is the perturbation of reactive oxygen species (ROS) homeostasis. Here, we demonstrate that the ROS-inducible Arabidopsis thaliana WRKY15 transcription factor (AtWRKY15) modulates plant growth and salt/osmotic stress responses. By transcriptome profiling, a divergent stress response was identified in transgenic WRKY15-overexpressing plants that linked a stimulated endoplasmic reticulum-to-nucleus communication to a disrupted mitochondrial stress response under salt-stress conditions. We show that mitochondrial calcium-flux sensing might be important for regulating an active mitochondrial retrograde signaling and launching an appropriate defense response to confer salt-stress tolerance.

A subcellular localization compendium of hydrogen peroxide-induced proteins.

The signal transduction mechanisms of the oxidative stress response in plants remain largely unexplored. Previously, increased levels of cellular hydrogen peroxide (H(2)O(2)) had been shown to drastically affect the plant transcriptome. Genome-wide transcriptome analyses allowed us to build a comprehensive inventory of H(2)O(2)-induced genes in plants. Here, the primary objective was to determine the subcellular localization of these genes and to assess potential trafficking during oxidative stress. After high-throughput cloning in Gateway-derived vectors, the subcellular localization of 49 proteins fused to the green fluorescent protein (GFP) was identified in a transient assay in tobacco (Nicotiana benthamiana) by means of agro-infiltration and confirmed for a selection of genes in transgenic Arabidopsis thaliana plants. Whereas eight of the GFP-tagged proteins are exclusively localized in the nucleus, 23 reside both in the nucleus and cytosol, in which several classes of known transcription factors and proteins of unknown function can be recognized. In this study, the mapping of the subcellular localization of H(2)O(2) -induced proteins paves the way for future research to unravel the H(2)O(2) responses in plants. Furthermore, the effect of increased H(2)O(2) levels on the subcellular localization of a subset of proteins was assessed.