Matthew R. Collinson-Pautz

Rotavirus Disrupts Calcium Homeostasis by NSP4 Viroporin Activity

ABSTRACT Many viruses alter intracellular calcium homeostasis. The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmic Ca2+ ([Ca2+]cyto) through a phospholipase C-independent pathway, which is required for virus replication and morphogenesis. However, the NSP4 domain and mechanism that increases [Ca2+]cyto are unknown. We identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca2+ by 3.7-fold, but NSP4 viroporin mutants maintained low levels of [Ca2+]cyto, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca2+]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca2+]cyto, a critical event that regulates rotavirus replication and virion assembly. IMPORTANCE Rotavirus is the leading cause of viral gastroenteritis in children and young animals. Rotavirus infection and expression of nonstructural protein 4 (NSP4) alone dramatically increase cytosolic calcium, which is essential for replication and assembly of infectious virions. This work identifies the intracellular mechanism by which NSP4 disrupts calcium homeostasis by showing that NSP4 is a viroporin, a class of virus-encoded transmembrane pores. Mutational analyses identified residues critical for viroporin activity. Viroporin mutants did not elevate the levels of cytoplasmic calcium in mammalian cells and were maintained in the endoplasmic reticulum rather than forming punctate vesicular structures that are critical for virus replication and morphogenesis. Pharmacological elevation of cytoplasmic calcium levels rescued puncta formation in viroporin mutants, demonstrating that elevation of calcium levels and puncta formation are distinct NSP4 functions. While viroporins typically function in virus entry or release, elevation of calcium levels by NSP4 viroporin activity may serve as a regulatory function to facilitate virus replication and assembly. Rotavirus is the leading cause of viral gastroenteritis in children and young animals. Rotavirus infection and expression of nonstructural protein 4 (NSP4) alone dramatically increase cytosolic calcium, which is essential for replication and assembly of infectious virions. This work identifies the intracellular mechanism by which NSP4 disrupts calcium homeostasis by showing that NSP4 is a viroporin, a class of virus-encoded transmembrane pores. Mutational analyses identified residues critical for viroporin activity. Viroporin mutants did not elevate the levels of cytoplasmic calcium in mammalian cells and were maintained in the endoplasmic reticulum rather than forming punctate vesicular structures that are critical for virus replication and morphogenesis. Pharmacological elevation of cytoplasmic calcium levels rescued puncta formation in viroporin mutants, demonstrating that elevation of calcium levels and puncta formation are distinct NSP4 functions. While viroporins typically function in virus entry or release, elevation of calcium levels by NSP4 viroporin activity may serve as a regulatory function to facilitate virus replication and assembly.

MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity.

Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways.

Abstract A057: Uni-CIDeCAR-T cells: MyD88/CD40-enhanced, Ab-directed CAR incorporating the CaspaCIDe® safety switch

Background: While chimeric antigen receptor (CAR)-T immunotherapies are remarkably effective against a subset of leukemias and lymphomas, three current hurdles for broad deployment include lack of regulation once administered to the patient, modest efficacy against solid tumors, and the necessity to make separate GMP vectors for each tumor target. Methods: We developed two methods that utilize chemical induction of protein dimerization (CID) to regulate the activity of engineered T cells containing a CAR of broad utility. “Uni-iC9CAR” combines Bellicum9s caspase-9-based, rimiducid-inducible safety switch, CaspaCIDe, with a first generation CD16/FCGR3A–CAR. Antigen receptor specificity relies on the interaction of the Fc-binding domain of CD16 with various tumor-targeted antibodies. In the “Uni-GoCAR” strategy, signaling domains from MyD88 and CD40 are fused to two copies of FKBPv36 to generate iMC, which is co-expressed with the CD16-based CAR. In each strategy, the same dimerizer, rimiducid, binds to FKBPv36 with sub-nanomolar affinity to cause the activation of signaling molecules with distinct functions and outcomes. Lastly, we generated the “Uni-CIDeCAR” vector that combines the iCaspase-9 and CD16-CAR activities from Uni-iC9CAR with augmented ligand-independent MyD88/CD40 costimulation to generate a potent universal CAR with a rapid and effective suicide gene, activated by the normally bio-inert ligand, rimiducid. Results: Both Uni-GoCAR and Uni-iC9CAR constructs demonstrated rapid, effective and durable, rituximab-dependent antitumor activity when expressed in human T cells and mixed with Raji B cells as early as 7 days after T cell co-culture at a 1:1 ratio. Additionally, rimiducid induced robust cytokine production, including IL-2 and IL-6, and proliferation in T cells transduced with the Uni-GoCAR vector, which expresses the iMC activation switch. In contrast, co-expression of iC9 in the Uni-iC9CAR vector demonstrated robust rimiducid-dependent T cell apoptosis, thus providing a valuable safety mechanism for clinical applications. Finally, in the enhanced, but regulated, Uni-CIDeCAR vector, iC9 maintains safety in a CD16-CAR that is functionally enhanced by rimiducid-independent, basal MC activity. Conclusion: We report an improved “universal” CAR-T technology that employs a CD16-based CAR (described by Kudo et al (14) Cancer Res) coupled with Bellicum9s costimulatory and safety switches to effectively target tumor cells while providing a broad clinical safety net. Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Uni-CIDeCAR-T cells: MyD88/CD40-enhanced, Ab-directed CAR incorporating the CaspaCIDe® safety switch. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A057.

Abstract 1568: Developing a novel in vivo electroporation-based DNA vaccine utilizing a small molecule regulated immune switch

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Dendritic cell (DC) vaccines are a rapidly progressing area of translational research intended for developing new cancer treatment modalities. Although ex vivo DC-based vaccines have been extensively studied and clinically tested for the treatment of cancer, they are inherently limited by short DC lifespan after activation, labor-intensive production (requiring ex vivo manipulation of autologous cells), high cost and consequent limited accessibility to the patient. Therefore, we have attempted to develop portable, regulatable adjuvants that could be targeted to DCs and activated in situ. Development by our lab of an inducible variant of the costimulatory molecule, CD40 (iCD40), and a composite, inducible MyD88/CD40 (iMC) adjuvant, that also incorporates the universal Toll like receptor adapter, MyD88, have been shown to increase the potency and lifespan of DC vaccines. These inducible receptors act as in vivo DC “switches” that lead to the priming and robust expansion of antigen (Ag)-specific T-cells capable of eliminating pre-established tumors in mice. Despite the success of these DC “switch” systems, practicality and scalability of patient-tailored ex vivo DC vaccines remains a major hindrance to their widespread applicability. Therefore, the future of DC vaccines lies in the development of “off-the-shelf” methodologies, such as viral or non-viral vectors that can deliver adjuvants along with tumor antigens. DNA vaccines are attractive for this purpose owing to their elegant simplicity, ease of production, and lack of anti-vector immune responses. In order to successfully vaccinate patients with DNA, one must be able to deliver plasmid encoding therapeutic genes to target cells efficiently. In vivo electroporation is a relatively new technology just starting to be tested in clinical trials, and provides a safe, simple, and effective means by which to administer DNA vaccines. Data from our preliminary studies of the electroporative delivery of plasmids encoding the model antigen β-galactosidase (LacZ) in mice suggests that LacZ-specific T-cell responses are induced, and that upon tumor challenge, mice receiving prophylaxis via electroporation had slower tumor growth kinetics when compared to controls. Together, this preliminary data supports the notion that DNA vaccination with tumor Ag by electroporation may be a simple and effective “off-the-shelf” cancer vaccine strategy. Additionally, this vaccination strategy may be enhanced by the addition of the iMC adjuvant. Therefore, further investigation of intradermal electroporation is warranted by these findings, to better characterize the induced immune responses and better optimize vaccination Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1568. doi:1538-7445.AM2012-1568