RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

mRNA aggregates harness danger response for potent cancer immunotherapy.

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

Protective immunity enhanced Salmonella vaccine vectors delivering Helicobacter pylori antigens reduce H. pylori stomach colonization in mice.

Helicobacter pylori is a major cause of gastric mucosal inflammation, peptic ulcers, and gastric cancer. Emerging antimicrobial-resistant H. pylori has hampered the effective eradication of frequent chronic infections. Moreover, a safe vaccine is highly demanded due to the absence of effective vaccines against H. pylori. In this study, we employed a new innovative Protective Immunity Enhanced Salmonella Vaccine (PIESV) vector strain to deliver and express multiple H. pylori antigen genes. Immunization of mice with our vaccine delivering the HpaA, Hp-NAP, UreA and UreB antigens, provided sterile protection against H. pylori SS1 infection in 7 out of 10 tested mice. In comparison to the control groups that had received PBS or a PIESV carrying an empty vector, immunized mice exhibited specific and significant cellular recall responses and antigen-specific serum IgG1, IgG2c, total IgG and gastric IgA antibody titers. In conclusion, an improved S. Typhimurium-based live vaccine delivering four antigens shows promise as a safe and effective vaccine against H. pylori infection.

Novel application of single-cell next-generation sequencing for determination of intratumoral heterogeneity of canine osteosarcoma cell lines.

Osteosarcoma (OSA) is a highly aggressive and metastatic neoplasm of both the canine and human patient and is the leading form of osseous neoplasia in both species worldwide. To gain deeper insight into the heterogeneous and genetically chaotic nature of OSA, we applied single-cell transcriptome (scRNA-seq) analysis to 4 canine OSA cell lines. This novel application of scRNA-seq technology to the canine genome required uploading the CanFam3.1 reference genome into an analysis pipeline (10X Genomics Cell Ranger); this methodology has not been reported previously in the canine species, to our knowledge. The scRNA-seq outputs were validated by comparing them to cDNA expression from reverse-transcription PCR (RT-PCR) and Sanger sequencing bulk analysis of 4 canine OSA cell lines (COS31, DOUG, POS, and HMPOS) for 11 genes implicated in the pathogenesis of canine OSA. The scRNA-seq outputs revealed the significant heterogeneity of gene transcription expression patterns within the cell lines investigated (COS31 and DOUG). The scRNA-seq data showed 10 distinct clusters of similarly shared transcriptomic expression patterns in COS31; 12 clusters were identified in DOUG. In addition, cRNA-seq analysis provided data for integration into the Qiagen Ingenuity Pathway Analysis software for canonical pathway analysis. Of the 81 distinct pathways identified within the clusters, 33 had been implicated in the pathogenesis of OSA, of which 18 had not been reported previously in canine OSA.

Canine osteosarcoma checkpoint expression correlates with metastasis and T-cell infiltrate.

BACKGROUND: Immune-targeted therapies are being successfully implemented into cancer clinical practice. In particular checkpoint inhibitors are employed to modulate the immune microenvironment of solid tumors. We sought to determine the expression of PD-L1, HVEM, and B7H3 in human and canine osteosarcoma, and correlate expression with clinical features and tumor infiltrating lymphocytes in naturally-occurring canine osteosarcoma. METHODS: Flow cytometry was used to measure ligand surface expression of five human and three canine cell lines. Immunohistochemistry was utilized for expression of ligands and lymphocyte markers in thirty-seven treatment-naïve canine osteosarcoma patients. RESULTS: All cell lines expressed all three ligands at variable levels in both species. Metastatic lesions were associated with higher expression of all three ligands in patient tumor samples. PD-L1 expression strongly correlated with B7H3 and HVEM expression, while HVEM and B7H3 were weakly correlated. Whereas peritumoral T-cell expression positively correlated with PD-L1 and HVEM tumor expression, the presence of T-cells intratumorally were rare. Furthermore, intratumor penetration by T-cells was greatest in metastatic lesions, despite log-fold increases in peritumoral T-cells. In summary, PD-L1, HVEM, and B7H3 are expressed in osteosarcoma, with metastatic disease lesions expressing higher levels. We show for the first time that these ligands expressed on osteosarcoma cells positively correlate with each other and the presence of peritumoral T cell infiltration. Furthermore, osteosarcoma appears to be an intratumoral immune desert with significant resistance to effector T cells. Multiple agents targeting checkpoints are in clinical practice, and may have immune modulating benefit in osteosarcoma.

Immunogenicity and Efficacy of a Novel Multi-Antigenic Peptide Vaccine Based on Cross-Reactivity between Feline and Human Immunodeficiency Viruses.

For the development of an effective HIV-1 vaccine, evolutionarily conserved epitopes between feline and human immunodeficiency viruses (FIV and HIV-1) were determined by analyzing overlapping peptides from retroviral genomes that induced both anti-FIV/HIV T cell-immunity in the peripheral blood mononuclear cells from the FIV-vaccinated cats and the HIV-infected humans. The conserved T-cell epitopes on p24 and reverse transcriptase were selected based on their robust FIV/HIV-specific CD8⁺ cytotoxic T lymphocyte (CTL), CD4⁺ CTL, and polyfunctional T-cell activities. Four such evolutionarily conserved epitopes were formulated into four multiple antigen peptides (MAPs), mixed with an adjuvant, to be tested as FIV vaccine in cats. The immunogenicity and protective efficacy were evaluated against a pathogenic FIV. More MAP/peptide-specific CD4⁺ than CD8⁺ T-cell responses were initially observed. By post-third vaccination, half of the MAP/peptide-specific CD8⁺ T-cell responses were higher or equivalent to those of CD4⁺ T-cell responses. Upon challenge, 15/19 (78.9%) vaccinated cats were protected, whereas 6/16 (37.5%) control cats remained uninfected, resulting in a protection rate of 66.3% preventable fraction (p = 0.0180). Thus, the selection method used to identify the protective FIV peptides should be useful in identifying protective HIV-1 peptides needed for a highly protective HIV-1 vaccine in humans.

Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy.

Translation of nanoparticles (NPs) into human clinical trials for patients with refractory cancers has lagged due to unknown biologic reactivities of novel NP designs. To overcome these limitations, simple well-characterized mRNA lipid-NPs have been developed as cancer immunotherapeutic vaccines. While the preponderance of RNA lipid-NPs encoding for tumor-associated antigens or neoepitopes have been designed to target lymphoid organs, they remain encumbered by the profound intratumoral and systemic immunosuppression that may stymie an activated T cell response. Herein, we show that systemic localization of untargeted tumor RNA (derived from whole transcriptome) encapsulated in lipid-NPs, with excess positive charge, primes the peripheral and intratumoral milieu for response to immunotherapy. In immunologically resistant tumor models, these RNA-NPs activate the preponderance of systemic and intratumoral myeloid cells (characterized by coexpression of PD-L1 and CD86). Addition of immune checkpoint inhibitors (ICIs) (to animals primed with RNA-NPs) augments peripheral/intratumoral PD-1+CD8+ cells and mediates synergistic antitumor efficacy in settings where ICIs alone do not confer therapeutic benefit. These synergistic effects are mediated by type I interferon released from plasmacytoid dendritic cells (pDCs). In translational studies, personalized mRNA-NPs were safe and active in a client-owned canine with a spontaneous malignant glioma. In summary, we demonstrate widespread immune activation from tumor loaded RNA-NPs concomitant with inducible PD-L1 expression that can be therapeutically exploited. While immunotherapy remains effective for only a subset of cancer patients, combination therapy with systemic immunomodulating RNA-NPs may broaden its therapeutic potency.

Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling.

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacterium Borrelia burgdorferi C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated with B. burgdorferi produce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences of B. burgdorferi-induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.

Dual-route targeted vaccine protects efficiently against botulinum neurotoxin A complex.

Clostridium botulinum readily persists in the soil and secretes life-threatening botulinum neurotoxins (BoNTs) that are categorized into serotypes A to H, of which, serotype A (BoNT/A) is the most commonly occurring in nature. An efficacious vaccine with high longevity against BoNT intoxication is urgent. Herein, we developed a dual-route vaccine administered over four consecutive weeks by mucosal and parenteral routes, consisting of the heavy chain (Hc) of BoNT/A targeting dendritic cell peptide (DCpep) expressed by Lactobacillus acidophilus as a secretory immunogenic protein. The administered dual-route vaccine elicited robust and long-lasting memory B cell responses comprising germinal center (GC) B cells and follicular T cells (Tfh) that fully protected mice from lethal oral BoNT/A fatal intoxication. Additionally, passively transferring neutralizing antibodies against BoNT/A into naïve mice induced robust protection against BoNT/A lethal intoxication. Together, a targeted vaccine employing local and systemic administrative routes may represent a novel formulation eliciting protective B cell responses with remarkable longevity against threatening biologic agents such as BoNTs.

The role of the calcium-sensing receptor in gastrointestinal inflammation.

The gastrointestinal (GI) tract must balance the extraction of energy and metabolic end-products from ingested nutrition and resident gut microbes and the maintenance of a symbiotic relationship with this microbiota, with the ability to mount functional immune responses to pathogenic organisms to maintain GI health. The gut epithelium is equipped with bacteria-sensing mechanisms that discriminate between pathogenic and commensal microorganisms and regulate host responses between immunity and tolerance. The epithelium also expresses numerous nutrient-sensing receptors, but their importance in the preservation of the gut microbiota and immune homeostasis remains largely unexplored. Observations that a deficiency in the extracellular calcium-sensing receptor (CaSR) using intestinal epithelium-specific receptor knockout mice resulted in diminished intestinal barrier integrity, altered composition of the gut microbiota, modified expression of intestinal pattern recognition receptors, and a skewing of local and systemic innate responses from regulatory to stimulatory, may change the way that this receptor is considered as a potential immunotherapeutic target in gut homeostasis. These findings suggest that pharmacologic CaSR activators and CaSR-based nutrients such as calcium, polyamines, phenylalanine, tryptophan, and oligo-peptides might be useful in conditioning the gut microenvironment, and thus, in the prevention and treatment of disorders such as inflammatory bowel disease (IBD), infectious enterocolitis, and other inflammatory and secretory diarrheal diseases. Here, we review the emerging roles of the CaSR in intestinal homeostasis and its therapeutic potential for gut pathology.

New generation of oral mucosal vaccines targeting dendritic cells.

As most infectious organisms gain entry at mucosal surfaces, there is a great deal of interest in developing vaccines that elicit effective mucosal immune responses against pathogen challenge. Targeted vaccination is one of the most effective methods available to prevent and control infectious diseases. Mucosal vaccines can offer lower costs, better accessibility, needle free delivery, and a higher capacity for mass immunizations during pandemics. Both local mucosal immunity and robust systemic responses can be achieved through mucosal vaccination. Recent progress in understanding the molecular and cellular components of the mucosal immune system have allowed for the development of a novel mucosal vaccine platform utilizing specific dendritic cell-targeting peptides and orally administered lactobacilli to elicit efficient antigen specific immune responses against infections, including Bacillus anthracis in experimental models of disease.

Colonic immune stimulation by targeted oral vaccine.

BACKGROUND: Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that Lactobacillus gasseri expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against Bacillus anthracis Sterne challenge. METHODOLOGY/PRINCIPAL FINDING: In the present study, we investigated the effects of a dose dependent treatment of mice with L. gasseri expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of L. gasseri expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4(+)Foxp3(+) and CD8(+)Foxp3(+) T cell immune responses. Notably, high doses of L. gasseri expressing PA-DCpep (10(12) CFU) were not toxic to the mice. Treatment of mice with L. gasseri expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with L. gasseri expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG(1), IgG(2b), IgG(2c) and IgG(3). L. gasseri expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors. CONCLUSION/SIGNIFICANCE: These findings suggest that L. gasseri expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.

Targeting aberrant colon cancer-specific DNA methylation with lipoteichoic acid-deficient Lactobacillus acidophilus.

Pathogenic autoinflammatory responses triggered by dysregulated microbial interactions may lead to intestinal disorders and malignancies. Previously, we demonstrated that a lipoteichoic acid (LTA)-deficient Lactobacillus acidophilus strain, NCK2025, ameliorated inflammation-induced colitis, significantly reduced the number of polyps in a colonic polyposis cancer model and restored physiological homeostasis in both cases. Nonetheless, the regulatory signals delivered by NCK2025 to reprogram the gastrointestinal microenvironment, and thus resist colonic cancer progression, remain unknown. Accumulating evidence suggest that epigenetic changes, in the presence and absence of pathogenic inflammation, can result in colorectal cancer (CRC). To test possible epigenetic modifications induced by NCK2025, the expression of epigenetically regulated, CRC-associated genes was measured with and without bacterial treatment. In vivo and in vitro, NCK2025 enhanced the expression of tumor suppressor genes that may regulate CRC development. Therefore, differential epigenetic regulation of CRC-related genes by NCK2025 represents a potential therapy against colitis-associated and sporadic CRC.

Development of tolerogenic dendritic cells and regulatory T cells favors exponential bacterial growth and survival during early respiratory tularemia.

Tularemia is a vector-borne zoonosis caused by Ft, a Gram-negative, facultative intracellular bacterium. Ft exists in two clinically relevant forms, the European biovar B (holarctica), which produces acute, although mild, self-limiting infections, and the more virulent United States biovar A (tularensis), which is often associated with pneumonic tularemia and more severe disease. In a mouse model of tularemia, respiratory infection with the virulence-attenuated Type B (LVS) or highly virulent Type A (SchuS4) strain engenders peribronchiolar and perivascular inflammation. Paradoxically, despite an intense neutrophilic infiltrate and high bacterial burden, T(h)1-type proinflammatory cytokines (e.g., TNF, IL-1ß, IL-6, and IL-12) are absent within the first ∼72 h of pulmonary infection. It has been suggested that the bacterium has the capacity to actively suppress or block NF-κB signaling, thus causing an initial delay in up-regulation of inflammatory mediators. However, our previously published findings and those presented herein contradict this paradigm and instead, strongly support an alternative hypothesis. Rather than blocking NF-κB, Ft actually triggers TLR2-dependent NF-κB signaling, resulting in the development and activation of tDCs and the release of anti-inflammatory cytokines (e.g., IL-10 and TGF-ß). In turn, these cytokines stimulate development and proliferation of T(regs) that may restrain T(h)1-type proinflammatory cytokine release early during tularemic infection. The highly regulated and overall anti-inflammatory milieu established in the lung is permissive for unfettered growth and survival of Ft. The capacity of Ft to evoke such a response represents an important immune-evasive strategy.

CD14 signaling restrains chronic inflammation through induction of p38-MAPK/SOCS-dependent tolerance.

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes.

Toll-like receptor 2 is required for control of pulmonary infection with Francisella tularensis.

Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2-/- mice. Collectively, these findings suggest that TLR2 plays an important role in tempering the host response to pneumonic tularemia.