Next generation self-replicating RNA vectors for vaccines and immunotherapies.

RNA technology has recently come to the forefront of innovative medicines and is being explored for a wide range of therapies, including prophylactic and therapeutic vaccines, biotherapeutic protein expression and gene therapy. In addition to conventional mRNA platforms now approved for prophylactic SARS-CoV2 vaccines, synthetic self-replicating RNA vaccines are currently being evaluated in the clinic for infectious disease and oncology. The prototypical srRNA vectors in clinical development are derived from alphaviruses, specifically Venezuelan Equine Encephalitis Virus (VEEV). While non-VEEV alphaviral strains have been explored as single cycle viral particles, their use as synthetic vectors largely remains under-utilized in clinical applications. Here we describe the potential commonalities and differences in synthetic alphaviral srRNA vectors in host cell interactions, immunogenicity, cellular delivery, and cargo expression. Thus, unlike the current thinking that VEEV-based srRNA is a one-size-fits-all platform, we argue that a new drug development approach leveraging panels of customizable, synthetic srRNA vectors will be required for clinical success.

Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach.

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4+ and CD8+ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine.

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.

The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor.

Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage-restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

TOX3 is expressed in mammary ER(+) epithelial cells and regulates ER target genes in luminal breast cancer.

BACKGROUND: A breast cancer susceptibility locus has been mapped to the gene encoding TOX3. Little is known regarding the expression pattern or biological role of TOX3 in breast cancer or in the mammary gland. Here we analyzed TOX3 expression in murine and human mammary glands and in molecular subtypes of breast cancer, and assessed its ability to alter the biology of breast cancer cells. METHODS: We used a cell sorting strategy, followed by quantitative real-time PCR, to study TOX3 gene expression in the mouse mammary gland. To study the expression of this nuclear protein in human mammary glands and breast tumors, we generated a rabbit monoclonal antibody specific for human TOX3. In vitro studies were performed on MCF7, BT474 and MDA-MB-231 cell lines to study the effects of TOX3 modulation on gene expression in the context of breast cancer cells. RESULTS: We found TOX3 expression in estrogen receptor-positive mammary epithelial cells, including progenitor cells. A subset of breast tumors also highly expresses TOX3, with poor outcome associated with high expression of TOX3 in luminal B breast cancers. We also demonstrate the ability of TOX3 to alter gene expression in MCF7 luminal breast cancer cells, including cancer relevant genes TFF1 and CXCR4. Knockdown of TOX3 in a luminal B breast cancer cell line that highly expresses TOX3 is associated with slower growth. Surprisingly, TOX3 is also shown to regulate TFF1 in an estrogen-independent and tamoxifen-insensitive manner. CONCLUSIONS: These results demonstrate that high expression of this protein likely plays a crucial role in breast cancer progression. This is in sharp contrast to previous studies that indicated breast cancer susceptibility is associated with lower expression of TOX3. Together, these results suggest two different roles for TOX3, one in the initiation of breast cancer, potentially related to expression of TOX3 in mammary epithelial cell progenitors, and another role for this nuclear protein in the progression of cancer. In addition, these results can begin to shed light on the reported association of TOX3 expression and breast cancer metastasis to the bone, and point to TOX3 as a novel regulator of estrogen receptor-mediated gene expression.

The many roles of TOX in the immune system.

TOX is a member of an evolutionarily conserved DNA-binding protein family and is expressed in several immune-relevant cell subsets. Here, we review the key role of TOX in regulating development of CD4 T cells, natural killer cells and lymphoid tissue inducer cells, the latter responsible for the generation of lymph nodes. Although the exact molecular mechanism of action of TOX remains to be elucidated, the role of TOX in establishment of gene programs in the thymus and the potential of TOX as a regulator of E protein activity are discussed.

TOX is required for development of the CD4 T cell lineage gene program.

The factors that regulate thymic development of the CD4(+) T cell gene program remain poorly defined. The transcriptional regulator ThPOK is a dominant factor in CD4(+) T cell development, which functions primarily to repress the CD8 lineage fate. Previously, we showed that nuclear protein TOX is also required for murine CD4(+) T cell development. In this study, we sought to investigate whether the requirement for TOX was solely due to a role in ThPOK induction. In apparent support of this proposition, ThPOK upregulation and CD8 lineage repression were compromised in the absence of TOX, and enforced ThPOK expression could restore some CD4 development. However, these "rescued" CD4 cells were defective in many aspects of the CD4(+) T cell gene program, including expression of Id2, Foxo1, and endogenous Thpok, among others. Thus, TOX is necessary to establish the CD4(+) T cell lineage gene program, independent of its influence on ThPOK expression.

Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages.

TOX is a DNA-binding factor required for development of CD4(+) T cells, natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells, a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis, required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow, consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus, many cell lineages of the immune system share a TOX-dependent step for development.

Development of all CD4 T lineages requires nuclear factor TOX.

CD8(+) cytotoxic and CD4(+) helper/inducer T cells develop from common thymocyte precursors that express both CD4 and CD8 molecules. Upon T cell receptor signaling, these cells initiate a differentiation program that includes complex changes in CD4 and CD8 expression, allowing identification of transitional intermediates in this developmental pathway. Little is known about regulation of these early transitions or their specific importance to CD4 and CD8 T cell development. Here, we show a severe block at the CD4(lo)CD8(lo) transitional stage of positive selection caused by loss of the nuclear HMG box protein TOX. As a result, CD4 lineage T cells, including regulatory T and CD1d-dependent natural killer T cells, fail to develop. In contrast, functional CD8(+) T cells develop in TOX-deficient mice. Our data suggest that TOX-dependent transition to the CD4(+)CD8(lo) stage is required for continued development of class II major histocompatibility complex-specific T cells, regardless of ultimate lineage fate.

Commitment issues: linking positive selection signals and lineage diversification in the thymus.

The thymus is responsible for the production of CD4+ helper and CD8+ cytotoxic T cells, which constitute the cellular arm of the immune system. These cell types derive from common precursors that interact with thymic stroma in a T-cell receptor (TCR)-specific fashion, generating intracellular signals that are translated into function-specific changes in gene expression. This overall process is termed positive selection, but it encompasses a number of temporally distinct and possibly mechanistically distinct cellular changes, including rescue from apoptosis, initiation of cell differentiation, and commitment to the CD4+ or CD8+ T-cell lineage. One of the puzzling features of positive selection is how specificity of the TCR controls lineage commitment, as both helper and cytolytic T cells utilize the same antigen-receptor components, with the exception of the CD4 or CD8 coreceptors themselves. In this review, we focus on the signals required for positive selection, particularly as they relate to lineage commitment. Identification of genes encoding transcriptional regulators that play a role in T-cell development has led to significant recent advances in the field. We also provide an overview of nuclear factors in this context and, where known, how their regulation is linked to the same TCR signals that have been implicated in initiating and regulating positive selection.

Cell surface immunoglobulin regulated checkpoints in chicken B cell development.

The bursa of Fabricius is critical for the normal development of B lymphocytes in avian species. Productive colonization of bursal follicles by B cell precursors requires surface immunoglobulin expression. We have shown using retroviral gene transfer that expression of chimeric receptors containing the extracellular and transmembrane domains of murine CD8alpha and CD8beta fused to the cytoplasmic domains of chicken Igalpha and Igbeta can support productive bursal colonization in the chicken embryo in bursal cells lacking the expression of endogenous sIgM. We show here that chimeric receptor expression does not support continued bursal cell development after hatch. However intrabursal administration of anti-CD8 antibodies that ligate the CD8alpha:Igalpha chimeric receptor results in maintained numbers of bursal cells that express the chimeric receptor in the absence of endogenous sIgM. These results support a model in which sIgM receptor expression is required for productive bursal colonization in the chick embryo but sIgM receptor ligation is required to support later B cell development after hatch.

TOX provides a link between calcineurin activation and CD8 lineage commitment.

T cell development is dependent on the integration of multiple signaling pathways, although few links between signaling cascades and downstream nuclear factors that play a role in thymocyte differentiation have been identified. We show here that expression of the HMG box protein TOX is sufficient to induce changes in coreceptor gene expression associated with beta-selection, including CD8 gene demethylation. TOX expression is also sufficient to initiate positive selection to the CD8 lineage in the absence of MHC-TCR interactions. TOX-mediated positive selection is associated with up-regulation of Runx3, implicating CD4 silencing in the process. Interestingly, a strong T cell receptor-mediated signal can modify this cell fate. We further demonstrate that up-regulation of TOX in double positive thymocytes is calcineurin dependent, linking this critical signaling pathway to nuclear changes during positive selection.