Viral delivery of a peptide-based immunomodulator enhances T cell priming during vaccination.

Modern, subunit-based vaccines have so far failed to induce significant T cell responses, contributing to ineffective vaccination against many pathogens. Importantly, while today's adjuvants are designed to trigger innate and non-specific immune responses, they fail to directly stimulate the adaptive immune compartment. Programmed cell death 1 (PD-1) partly regulates naïve-to-antigen-specific effector T cell transition and differentiation by suppressing the magnitude of activation. Indeed, we previously reported on a microbial-derived, peptide-based PD-1 checkpoint inhibitor, LD01, which showed potent T cell-stimulating activity when combined with a vaccine. Here we sought to improve the potency of LD01 by designing and testing new LD01 derivatives. Accordingly, we found that a modified version of an 18-amino acid metabolite of LD01, LD10da, improved T cell activation capability in a malaria vaccine model. Specifically, LD10da demonstrates improved antigen-specific CD8+ T cell expansion when combined prophylactically with an adenovirus-based malaria vaccine. A single dose of LD10da at the time of vaccination is sufficient to increase antigen-specific CD8+ T cell expansion in wild-type mice. Further, we show that LD10 can be encoded and delivered by a Modified Vaccinia Ankara viral vector and can enhance antigen-specific CD8+ T cell expansion comparable to that of synthetic peptide administration. Therefore, LD10da represents a promising biologic-based immunomodulator that can be genetically encoded and delivered, along with the antigen, by viral or other nucleic acid vectors to improve the efficacy and delivery of vaccines for ineradicable and emerging infectious diseases.

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting.

Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.

A Peptide-Based Checkpoint Immunomodulator Alleviates Immune Dysfunction in Murine Polymicrobial Sepsis.

ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8+ T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3+Tbet+CD4+ regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically.

Novel Peptide-Based PD1 Immunomodulators Demonstrate Efficacy in Infectious Disease Vaccines and Therapeutics.

Many pathogens use the same immune evasion mechanisms as cancer cells. Patients with chronic infections have elevated levels of checkpoint receptors (e.g., programed cell death 1, PD1) on T cells. Monoclonal antibody (mAb)-based inhibitors to checkpoint receptors have also been shown to enhance T-cell responses in models of chronic infection. Therefore, inhibitors have the potential to act as a vaccine "adjuvant" by facilitating the expansion of vaccine antigen-specific T-cell repertoires. Here, we report the discovery and characterization of a peptide-based class of PD1 checkpoint inhibitors, which have a potent adaptive immunity adjuvant capability for vaccines against infectious diseases. Briefly, after identifying peptides that bind to the recombinant human PD1, we screened for in vitro efficacy in reporter assays and human peripheral blood mononuclear cells (PBMC) readouts. We first found the baseline in vivo performance of the peptides in a standard mouse oncology model that demonstrated equivalent efficacy compared to mAbs against the PD1 checkpoint. Subsequently, two strategies were used to demonstrate the utility of our peptides in infectious disease indications: (1) as a therapeutic in a bacteria-induced lethal sepsis model in which our peptides were found to increase survival with enhanced bacterial clearance and increased macrophage function; and (2) as an adjuvant in combination with a prophylactic malaria vaccine in which our peptides increased T-cell immunogenicity and the protective efficacy of the vaccine. Therefore, our peptides are promising as both a therapeutic agent and a vaccine adjuvant for infectious disease with a potentially safer and more cost-effective target product profile compared to mAbs. These findings are essential for deploying a new immunomodulatory regimen in infectious disease primary and clinical care settings.

Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas.

Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals.

SHEP1 partners with CasL to promote marginal zone B-cell maturation.

The marginal zone is a cellular niche bordering the marginal sinus of the spleen that contains specialized B-cell and macrophage subsets poised to capture bloodborne antigens. Marginal zone B cells are retained in this niche by integrin-mediated signaling induced by G protein-coupled receptors (GPCRs) and, likely, the B-cell receptor (BCR). Sphingosine-1-phosphate (S1P) signaling via the S1P family of GPCRs is known to be essential for B-cell localization in the marginal zone, but little is known about the downstream signaling events involved. Here, we demonstrate that the adaptor protein SHEP1 is required for marginal zone B-cell maturation. SHEP1 functions in concert with the scaffolding protein CasL, because we show that SHEP1 and CasL are constitutively associated in B cells. SHEP1 association is required for the BCR or S1P receptor(s) to induce the conversion of CasL into its serine/threonine hyperphosphorylated form, which is important for lymphocyte adhesion and motility. Thus, SHEP1 orchestrates marginal zone B-cell movement and retention as a key downstream effector of the BCR and S1P receptors.

Suppression of phosphatidylinositol 3,4,5-trisphosphate production is a key determinant of B cell anergy.

Anergy is a critical physiologic mechanism to sensor self-reactive B cells. However, a biochemical understanding of how anergy is achieved and maintained is lacking. Herein, we investigated the role of the phosphoinositide 3-kinase (PI3K) lipid product PI(3,4,5)P(3) in B cell anergy. We found reduced generation of PI(3,4,5)P(3) in anergic B cells, which was attributable to reduced phosphorylation of the PI3K membrane adaptor CD19, as well as increased expression of the inositol phosphatase PTEN. Sustained production of PI(3,4,5)P(3) in B cells, achieved through conditional deletion of Pten, resulted in failed tolerance induction and abundant autoantibody production. In contrast to wild-type immature B cells, B cell receptor engagement of PTEN-deficient immature B cells resulted in activation and proliferation, indicating a central defect in early B cell responsiveness. These findings establish repression of the PI3K signaling pathway as a necessary condition to avert the generation, activation, and persistence of self-reactive B cells.

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.

Inhibition of endothelial cell proliferation and angiogenesis by orlistat, a fatty acid synthase inhibitor.

Orlistat, an antiobesity drug, is cytostatic and cytotoxic to tumor cells. The antitumor activity of orlistat can be attributed to its ability to inhibit the thioesterase domain of fatty acid synthase (FAS). The objective of the present study was to test the effect of orlistat on endothelial cell proliferation and angiogenesis. Orlistat inhibits endothelial cell FAS, blocks the synthesis of fatty acids, and prevents endothelial cell proliferation. More significantly, orlistat inhibits human neovascularization in an ex vivo assay, which suggests that it may be useful as an antiangiogenic drug. The mechanism of these effects can be traced to the fact that orlistat prevents the display of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR/Flk1) on the endothelial cell surface. Thus, orlistat is an antiangiogenic agent with a novel mechanism of action.

A fatty acid synthase blockade induces tumor cell-cycle arrest by down-regulating Skp2.

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins.

Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor.

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.