The Evolution of Medical Countermeasures for Ebola Virus Disease: Lessons Learned and Next Steps.

The Ebola virus disease outbreak that occurred in Western Africa from 2013-2016, and subsequent smaller but increasingly frequent outbreaks of Ebola virus disease in recent years, spurred an unprecedented effort to develop and deploy effective vaccines, therapeutics, and diagnostics. This effort led to the U.S.

A U.S. Government-Coordinated Effort to Leverage Non-Human Primate Data to Facilitate Ebolavirus Vaccine Development.

A United States Government (USG) interagency group, the Filovirus Animal Non-Clinical Group (FANG), has been established to support the development of biodefense medical countermeasures (MCMs). As both vaccines and therapeutics are licensed using "non-traditional pathways", such as the U.S.

Selection of Filovirus Isolates for Vaccine Development Programs.

The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for . However, effective medical countermeasures, such as vaccines for other filoviruses such as and the Marburg virus, are presently in early stages of development and, in the absence of a large outbreak, would require regulatory approval via the U.

Classical Immunoproteomics: Serological Proteome Analysis (SERPA) for Antigen Identification.

The study of the humoral immune response to infectious and chronic diseases is important for understanding the disease progression, identification of protective antigens, vaccine development, and discovery of biomarkers for early diagnosis. Proteomic approaches, including serological proteome analysis (SERPA), have been used to identify the repertoire of immunoreactive proteins in various diseases. In this chapter, we provide an outline of the SERPA approach, using the analysis of sera from mice vaccinated with a live attenuated tularemia vaccine as an example.

The Cynomolgus Macaque Natural History Model of Pneumonic Tularemia for Predicting Clinical Efficacy Under the Animal Rule.

is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans and a Tier 1 select agent. The natural incidence of pneumonic tularemia worldwide is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCM) in human populations. Development and licensure of tularemia therapeutics and vaccines need to occur under the Food and Drug Administration's (FDA's) Animal Rule under which efficacy studies are conducted in well-characterized animal models that reflect the pathophysiology of human disease.

Methods and applications of serological proteome analysis.

The study of the humoral response to infectious diseases and chronic diseases, such as cancer, is important for many reasons, including understanding the host response to disease, identification of protective antigens, vaccine development, and discovery of biomarkers for early diagnosis. During the past decade, proteomic approaches, such as serological proteome analysis (SERPA), have been used to identify the repertoire of immunoreactive proteins in various diseases. In this chapter, we provide an outline of the SERPA approach, using the analysis of sera from mice vaccinated with a live attenuated tularemia vaccine as an example.

Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL.

Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation.

Signatures of T cells as correlates of immunity to Francisella tularensis.

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e.

Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination.

Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection.

Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis.

The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here, lymphocyte proliferation and cytokine production in response to F.

Expression of chimeric antigen receptors in natural killer cells with a regulatory-compliant non-viral method.

Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors.

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation.

Unlabelled: Oval cells are hepatocytic precursors that proliferate in late-stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor-beta (TGF-beta) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo.

Cutting Edge: TCR-induced NAB2 enhances T cell function by coactivating IL-2 transcription.

TCR engagement leads to the up-regulation of genetic programs that can both activate and inhibit T cell function. The early growth receptor (Egr) proteins Egr-2 and Egr-3 have recently been identified as TCR-induced negative regulators of T cell function. NAB2 (NGFI-A-binding protein 2) is both a coactivator and a corepressor of Egr-mediated transcription and has been implicated in regulating Schwann cell myelination.

Smad4 signalling in T cells is required for suppression of gastrointestinal cancer.

SMAD4 (MAD homologue 4 (Drosophila)), also known as DPC4 (deleted in pancreatic cancer), is a tumour suppressor gene that encodes a central mediator of transforming growth factor-beta signalling. Germline mutations in SMAD4 are found in over 50% of patients with familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to hamartomatous polyps and gastrointestinal cancer. Dense inflammatory cell infiltrates underlay grossly normal appearing, non-polypoid colonic and gastric mucosa of patients with familial juvenile polyposis.

Treating autoimmune diseases through restoration of antigen-specific immune tolerance.

The first line of treatment for many human autoimmune diseases involves the use of anti-inflammatory or immunosuppressive drugs such as prednisone or other steroids that not only suppress the underlying autoimmune disease, but lead to global suppression of the immune system. The sequelae of this approach include increased risk of infection, carcinogenesis, and osteoporosis. Moreover, such broad spectrum immunosuppression tends to have transient therapeutic benefit, as in many cases the disease becomes refractory to these drugs.

Bone marrow transplantation combined with gene therapy to induce antigen-specific tolerance and ameliorate EAE.

Hematopoietic stem cell (HSC) transplantation is a potential therapy that can offer multiple sclerosis patients a radical, potentially curative treatment. Using experimental autoimmune encephalomyelitis (EAE) as a model, we previously reported that retrovirally transduced B cells expressing myelin basic protein (MBP), MBP Ac1-11, or myelin oligodendrocyte glycoprotein p35-55 induced tolerance and reduced symptoms. Here, we extend our tolerance approach using bone marrow (BM) cells expressing full-length phospholipid protein (PLP) in a model for relapsing, remitting EAE.

Cutting edge: p27Kip1 deficiency reduces the requirement for CD28-mediated costimulation in naive CD8+ but not CD4+ T lymphocytes.

Cell cycle re-entry of quiescent T cells is dependent upon cyclin-dependent kinase 2. Inhibition of cyclin-dependent kinase 2 by p27(Kip1) is believed to be the principal constraint on S-phase entry in T cells. We report that deficiency for p27(Kip1) has a more pronounced effect on the expansion of murine naive CD8(+) T cells and that this disparity is due to a reduced requirement for CD28-mediated costimulation in CD8(+) but not CD4(+) T cells lacking p27(Kip1).

p21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-beta-mediated G1 arrest through modulation of IL-2 responsiveness.

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation.

Loss of Smad3 in acute T-cell lymphoblastic leukemia.

Background: The receptors for transforming growth factor beta (TGF-beta) and their signaling intermediates make up an important tumor-suppressor pathway. The role of one of these intermediates--Smad3--in the pathogenesis of lymphoid neoplasia is unknown.

Methods: We measured Smad3 messenger RNA (mRNA) and protein in leukemia cells obtained at diagnosis from 19 children with acute leukemia, including 10 with T-cell acute lymphoblastic leukemia (ALL), 7 with pre-B-cell ALL, and 2 with acute nonlymphoblastic leukemia (ANLL).

Development and application of fully functional epitope-tagged forms of transforming growth factor-beta.

Administration of transforming growth factor-beta (TGF-beta) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-beta and related TGF-beta isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-beta1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-beta signaling intermediate, smad 2.

The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF.

Characterization of the TATA-less core promoter of the cell cycle-regulated cdc25C gene.

The TATA- and Inr-less promoter of the human cdc25C gene is regulated during the cell cycle through binding of a repressor to two contiguous promoter-proximal elements, the CDE and CHR. In this study we have characterized in detail the region of the cdc25C promoter immediately downstream of these elements. Several lines of evidence suggest that this region of approximately 60 bp acts as the core promoter.

CDF-1, a novel E2F-unrelated factor, interacts with cell cycle-regulated repressor elements in multiple promoters.

The cdc25C , cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study we have identified a factor, CDF-1, which interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro , in a manner similar to that seen by genomic footprinting.

Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression.

The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element ('cell cycle-dependent element'; CDE) located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. We now show that CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes.

Differential expression of S1 and elongation factor-1 alpha during rat development.

Elongation factor-1 alpha (EF-1 alpha) is a highly conserved protein functioning in peptide elongation during translation. A cDNA, S1, was isolated; its deduced amino acid sequence shares high similarity with mammalian EF-1 alpha s (92%). While EF-1 alpha is present in all tissues, S1 mRNA can only be detected in brain, heart, and muscle.