Search

Vaccine biomarkers are critical to many aspects of vaccine development and licensure, including bridging findings in pre-clinical studies to clinical studies, predicting potential adverse events, and predicting vaccine efficacy. Despite advances in our understanding of various biological pathways, and advances in systems analyses of the immune response, there remains much to learn about qualitative and quantitative aspects of the human host response to vaccination. To stimulate discussion and identify opportunities for collaborative ways to advance the field of vaccine biomarkers, A Next Generation Vaccine Biomarker workshop was held in Ottawa. The two day workshop, sponsored by the National Research Council Canada, Canadian Institutes of Health Research, Public Health Agency of Canada, Pfizer, and Medicago, brought together stakeholders from Canadian and international industry, government and academia. The workshop was grouped in themes, covering vaccine biomarker challenges in the pre-clinical and clinical spaces, veterinary vaccines, regulatory challenges, and development of biomarkers for adjuvants and cancer vaccines. The use of case studies allowed participants to identify the needs and gaps requiring innovation. The workshop concluded with a discussion on opportunities for vaccine biomarker discovery, the Canadian context, and approaches for moving forward. This article provides a synopsis of these discussions and identifies steps forward for advancing vaccine biomarker research in Canada.

The anti-tumor function of Stat1 as a regulator of innate immunity and tumor immune surveillance has been long studied and is well understood; however, less clear is its tumor-site specific role. Although Stat1 phosphorylated at tyrosine (Y) 701 and serine (S) 727 is essential for interferon (IFN) signalling, its function in signalling induced in breast cancer cells is not understood. Herein, we show that Stat1 Y701 phosphorylation is increased in human breast tumor cells with elevated levels of ErbB2/HER-2 and in cells transfected with ErbB2/Neu. However, pharmacological inhibition of ErbB2/HER-2 results in the inhibition of Stat1 Y701 phosphorylation indicating an atypical role of phosphorylated Stat1 in the inhibition of ErbB2/HER-2 signalling. Consistent with this notion, we found that Stat1 suppresses tumor development by an activated form of ErbB2/Neu in mouse embryonic fibroblasts in xenograft tumor assays; however, this anti-tumor function of Stat1 does not rely on Y701 and S727 phosphorylation. Experiments with transgenic mice demonstrated that Stat1 acts to suppress Neu-mediated breast tumorigenesis through immune regulatory and tumor-site specific mechanisms. Our data reveal a previous uncharacterized anti-tumor activity of Stat1 in ErbB2/Neu-mediated cell transformation and breast oncogenesis with possible implications in the diagnosis and treatment of ErbB2-positive breast cancers.

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1α and 1β), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1α or -1β increased the synthesis and secretion of [3H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL1(Sf> 100) and VLDL2(Sf20–100). Expression of lipin-1α or -1β increased secretion efficiency and decreased intracellular degradation of [35S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [3H]glycerolipids and [35S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1α not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1α mutant failed to promote [35S]apoB100 synthesis or secretion, and showed compromised stimulation in [3H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

Global control of mRNA translation plays key roles in cell regulation, including growth, differentiation and apoptosis. Human monocyte-like U937 cells differentiate into macrophage-like cells upon 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, a process which is known to be accompanied with a large decrease in general protein synthesis. Here, we found that protein levels of AMAP1 (also called ASAP1 or DDEF1), a GTPase-activating protein for Arf GTPases, increase several fold during U937 cell differentiation. This increase was not accompanied with a notable increase in the AMAP1 gene transcript, nor seemed to be due to 5’-Cap-dependent mRNA translational activities in differentiated U937 cells. We identified the 5’-untranslated region (5’-UTR) of AMAP1 mRNA, and found that this 5’-UTR exhibits significant internal ribosome entry site (IRES)-dependent translational activity in differentiated U937 cells, but not in undifferentiated cells. Our results indicate that monocyte differentiation involves enhancement of IRES activity, by which protein levels of AMAP1 are primarily upregulated.

Regulating gene expression is an effective way for cells to deal with various stresses. The outcome of this regulation differs with the type of stress, and can promote either cell survival or cell death depending on the severity of the injury incurred. Gene expression can be controlled at several steps, including transcription, translation and degradation. An extensively studied protein involved in translational control is the eukaryotic translation initiation factor 2 (eIF2). When eIF2 becomes phosphorylated on a specific serine residue located within the alpha (α) subunit, global protein synthesis is halted. This phosphorylation occurs following periods of environmental stress, and plays a significant role in the cellular response to these events. The eIF2α kinase family consists of four members, which are each activated in response to different stimuli. Our group has recently discovered that two members of this family, the protein kinase activated by double-stranded RNA (PKR) and the PKR-like endoplasmic reticulum (ER) kinase (PERK) can also regulate the expression of specific proteins by promoting their degradation by the 26S proteasome. Specifically, we demonstrated that degradation of the cell cycle regulator cyclin D1, and the tumour suppressor p53 was promoted by PERK and PKR during periods of ER stress. This novel function may allow the eIF2α kinases to affect a larger number of cellular processes than previously believed.

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the α subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2α kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR−/−or PERK−/−mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2α kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2α phosphorylation as demonstrated by the inactivation of endogenous eIF2α by small interfering RNA or utilization of MEFs bearing the eIF2α Ser51Ala mutation. Our data reveal a novel property of eIF2α kinases as activators of PI3K signaling and cell survival.