DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Lipidation of pneumococcal proteins enables activation of human antigen-presenting cells and initiation of an adaptive immune response.

Streptococcus pneumoniae remains a significant global threat, with existing vaccines having important limitations such as restricted serotype coverage and high manufacturing costs. Pneumococcal lipoproteins are emerging as promising vaccine candidates due to their surface exposure and conservation across various serotypes. While prior studies have explored their potential in mice, data in a human context and insights into the impact of the lipid moiety remain limited. In the present study, we examined the immunogenicity of two pneumococcal lipoproteins, DacB and MetQ, both in lipidated and non-lipidated versions, by stimulation of primary human immune cells. Immune responses were assessed by the expression of common surface markers for activation and maturation as well as cytokines released into the supernatant. Our findings indicate that in the case of MetQ lipidation was crucial for activation of human antigen-presenting cells such as dendritic cells and macrophages, while non-lipidated DacB demonstrated an intrinsic potential to induce an innate immune response. Nevertheless, immune responses to both proteins were enhanced by lipidation. Interestingly, following stimulation of dendritic cells with DacB, LipDacB and LipMetQ, cytokine levels of IL-6 and IL-23 were significantly increased, which are implicated in triggering potentially important Th17 cell responses. Furthermore, LipDacB and LipMetQ were able to induce proliferation of CD4+ T cells indicating their potential to induce an adaptive immune response. These findings contribute valuable insights into the immunogenic properties of pneumococcal lipoproteins, emphasizing their potential role in vaccine development against pneumococcal infections.

SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity.

Carrier-free naked mRNA vaccines may reduce the reactogenicity associated with delivery carriers; however, their effectiveness against infectious diseases has been suboptimal. To boost efficacy, we targeted the skin layer rich in antigen-presenting cells (APCs) and utilized a jet injector. The jet injection efficiently introduced naked mRNA into skin cells, including APCs in mice. Further analyses indicated that APCs, after taking up antigen mRNA in the skin, migrated to the lymph nodes (LNs) for antigen presentation. Additionally, the jet injection provoked localized lymphocyte infiltration in the skin, serving as a physical adjuvant for vaccination. Without a delivery carrier, our approach confined mRNA distribution to the injection site, preventing systemic mRNA leakage and associated systemic proinflammatory reactions. In mouse vaccination, the naked mRNA jet injection elicited robust antigen-specific antibody production over 6 months, along with germinal center formation in LNs and the induction of both CD4- and CD8-positive T cells. By targeting the SARS-CoV-2 spike protein, this approach provided protection against viral challenge. Furthermore, our approach generated neutralizing antibodies against SARS-CoV-2 in non-human primates at levels comparable to those observed in mice. In conclusion, our approach offers a safe and effective option for mRNA vaccines targeting infectious diseases.

Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.

Programming peptide-oligonucleotide nano-assembly for engineering of neoantigen vaccine with potent immunogenicity.

Background: Neoantigen nanovaccine has been recognized as a promising treatment modality for personalized cancer immunotherapy. However, most current nanovaccines are carrier-dependent and the manufacturing process is complicated, resulting in potential safety concerns and suboptimal codelivery of neoantigens and adjuvants to antigen-presenting cells (APCs). Methods: Here we report a facile and general methodology for nanoassembly of peptide and oligonucleotide by programming neoantigen peptide with a short cationic module at N-terminus to prepare nanovaccine. The programmed peptide can co-assemble with CpG oligonucleotide (TLR9 agonist) into monodispersed nanostructures without the introduction of artificial carrier. Results: We demonstrate that the engineered nanovaccine promoted the codelivery of neoantigen peptides and adjuvants to lymph node-residing APCs and instigated potent neoantigen-specific T-cell responses, eliciting neoantigen-specific antitumor immune responses with negligible systemic toxicity. Furthermore, the antitumor T-cell immunity is profoundly potentiated when combined with anti-PD-1 therapy, leading to significant inhibition or even complete regression of established melanoma and MC-38 colon tumors. Conclusions: Collectively, this work demonstrates the feasibility and effectiveness of personalized cancer nanovaccine preparation with high immunogenicity and good biosafety by programming neoantigen peptide for nanoassembly with oligonucleotides without the aid of artificial carrier.

DNA nanodevice as a multi-module co-delivery platform for combination cancer immunotherapy.

A major challenge in combining cancer immunotherapy is the efficient delivery of multiple types of immunological stimulators to elicit a robust anti-tumor immune response and reprogram the immunosuppressive tumor microenvironment (TME). Here, we developed a DNA nanodevice that was generated by precisely assembling three types of immunological stimulators. The doxorubicin (Dox) component induced immunogenic cell death (ICD) in tumor cells and enhanced phagocytosis of antigen-presenting cells (APCs). Exogenous double-stranded DNA (dsDNA) could act as a molecular adjuvant to activate the stimulator of interferon genes (STING) signaling in APCs by engulfing dying tumor cells. Interleukin (IL)-12 and small hairpin programmed cell death-ligand 1 (shPD-L1) transcription templates were designed to regulate TME. Additionally, for targeted drug delivery, multiple cyclo[Arg-Gly-Asp-(d-Phe)-Cys] (cRGD) peptide units on DNA origami were employed. The incorporation of disulfide bonds allowed the release of multiple modules in response to intracellular glutathione (GSH) in tumors. The nanodevice promoted the infiltration of CD8+ and CD4+ cells into the tumor and generated a highly inflamed TME, thereby enhancing the effectiveness of cancer immunotherapy. Our research results indicate that the nanodevice we constructed can effectively inhibit tumor growth and prevent lung metastasis without obvious systemic toxicity, providing a promising strategy for cancer combination treatment.

Early life imprinting of intestinal immune tolerance and tissue homeostasis.

Besides its canonical role in protecting the host from pathogens, the immune system plays an arguably equally important role in maintaining tissue homeostasis. Within barrier tissues that interface with the external microenvironment, induction of immune tolerance to innocuous antigens, such as commensal, dietary, and environmental antigens, is key to establishing immune homeostasis. The early postnatal period represents a critical window of opportunity in which parallel development of the tissue, immune cells, and microbiota allows for reciprocal regulation that shapes the long-term immunological tone of the tissue and subsequent risk of immune-mediated diseases. During early infancy, the immune system appears to sacrifice pro-inflammatory functions, prioritizing the establishment of tissue tolerance. In this review, we discuss mechanisms underlying early life windows for intestinal tolerance with a focus on newly identified RORγt+ antigen-presenting cells-Thetis cells-and highlight the role of the intestinal microenvironment in shaping intestinal immune system development and tolerance.

Local administration of myeloid-derived suppressor cells prevents progression of immune-mediated dry eye disease.

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.

Tsyn-Seq: a T-cell Synapse-Based Antigen Identification Platform.

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

Multimodal probing of T-cell recognition with hexapod heterostructures.

Studies using antigen-presenting systems at the single-cell and ensemble levels can provide complementary insights into T-cell signaling and activation. Although crucial for advancing basic immunology and immunotherapy, there is a notable absence of synthetic material toolkits that examine T cells at both levels, and especially those capable of single-molecule-level manipulation. Here we devise a biomimetic antigen-presenting system (bAPS) for single-cell stimulation and ensemble modulation of T-cell recognition. Our bAPS uses hexapod heterostructures composed of a submicrometer cubic hematite core (α-Fe2O3) and nanostructured silica branches with diverse surface modifications. At single-molecule resolution, we show T-cell activation by a single agonist peptide-loaded major histocompatibility complex; distinct T-cell receptor (TCR) responses to structurally similar peptides that differ by only one amino acid; and the superior antigen recognition sensitivity of TCRs compared with that of chimeric antigen receptors (CARs). We also demonstrate how the magnetic field-induced rotation of hexapods amplifies the immune responses in suspended T and CAR-T cells. In addition, we establish our bAPS as a precise and scalable method for identifying stimulatory antigen-specific TCRs at the single-cell level. Thus, our multimodal bAPS represents a unique biointerface tool for investigating T-cell recognition, signaling and function.

Restoring tolerance with antigen delivery.

Strategies that modulate antigen delivery are being tested to reverse autoimmunity.

Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization.

The COVID-19 pandemic has fostered major advances in vaccination technologies1-4; however, there are urgent needs for vaccines that induce mucosal immune responses and for single-dose, non-invasive administration4-6. Here we develop an inhalable, single-dose, dry powder aerosol SARS-CoV-2 vaccine that induces potent systemic and mucosal immune responses. The vaccine encapsulates assembled nanoparticles comprising proteinaceous cholera toxin B subunits displaying the SARS-CoV-2 RBD antigen within microcapsules of optimal aerodynamic size, and this unique nano-micro coupled structure supports efficient alveoli delivery, sustained antigen release and antigen-presenting cell uptake, which are favourable features for the induction of immune responses. Moreover, this vaccine induces strong production of IgG and IgA, as well as a local T cell response, collectively conferring effective protection against SARS-CoV-2 in mice, hamsters and nonhuman primates. Finally, we also demonstrate a mosaic iteration of the vaccine that co-displays ancestral and Omicron antigens, extending the breadth of antibody response against co-circulating strains and transmission of the Omicron variant. These findings support the use of this inhaled vaccine as a promising multivalent platform for fighting COVID-19 and other respiratory infectious diseases.

CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours.

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.

Trimeric, APC-Targeted Subunit Vaccines Protect Mice against Seasonal and Pandemic Influenza.

Viral subunit vaccines contain the specific antigen deemed most important for development of protective immune responses. Typically, the chosen antigen is a surface protein involved in cellular entry of the virus, and neutralizing antibodies may prevent this. For influenza, hemagglutinin (HA) is thus a preferred antigen. However, the natural trimeric form of HA is often not considered during subunit vaccine development. Here, we have designed a vaccine format that maintains the trimeric HA conformation while targeting antigen toward major histocompatibility complex class II (MHCII) molecules or chemokine receptors on antigen-presenting cells (APC) for enhanced immunogenicity. Results demonstrated that a single DNA vaccination induced strong antibody and T-cell responses in mice. Importantly, a single DNA vaccination also protected mice from lethal challenges with influenza viruses H1N1 and H5N1. To further evaluate the versatility of the format, we developed MHCII-targeted HA from influenza A/California/04/2009(H1N1) as a protein vaccine and benchmarked this against Pandemrix and Flublok. These vaccine formats are different, but similar immune responses obtained with lower vaccine doses indicated that the MHCII-targeted subunit vaccine has an immunogenicity and efficacy that warrants progression to larger animals and humans. IMPORTANCE Subunit vaccines present only selected viral proteins to the immune system and allow for safe and easy production. Here, we have developed a novel vaccine where influenza hemagglutinin is presented in the natural trimeric form and then steered toward antigen-presenting cells for increased immunogenicity. We demonstrate efficient induction of antibodies and T-cell responses, and demonstrate that the vaccine format can protect mice against influenza subtypes H1N1, H5N1, and H7N1.

Novel antigen-presenting cell imparts Treg-dependent tolerance to gut microbiota.

Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development1-4. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota5-8, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells have not been identified. Here we describe the identification of a class of RORγt+ antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pTreg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pTreg generation, including the TGF-ß-activating integrin αvß8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pTreg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pTreg generation, further implicating TC IV as the tolerogenic RORγt+ antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

A Barrier to Defend - Models of Pulmonary Barrier to Study Acute Inflammatory Diseases.

Pulmonary diseases represent four out of ten most common causes for worldwide mortality. Thus, pulmonary infections with subsequent inflammatory responses represent a major public health concern. The pulmonary barrier is a vulnerable entry site for several stress factors, including pathogens such as viruses, and bacteria, but also environmental factors e.g. toxins, air pollutants, as well as allergens. These pathogens or pathogen-associated molecular pattern and inflammatory agents e.g. damage-associated molecular pattern cause significant disturbances in the pulmonary barrier. The physiological and biological functions, as well as the architecture and homeostatic maintenance of the pulmonary barrier are highly complex. The airway epithelium, denoting the first pulmonary barrier, encompasses cells releasing a plethora of chemokines and cytokines, and is further covered with a mucus layer containing antimicrobial peptides, which are responsible for the pathogen clearance. Submucosal antigen-presenting cells and neutrophilic granulocytes are also involved in the defense mechanisms and counterregulation of pulmonary infections, and thus may directly affect the pulmonary barrier function. The detailed understanding of the pulmonary barrier including its architecture and functions is crucial for the diagnosis, prognosis, and therapeutic treatment strategies of pulmonary diseases. Thus, considering multiple side effects and limited efficacy of current therapeutic treatment strategies in patients with inflammatory diseases make experimental in vitro and in vivo models necessary to improving clinical therapy options. This review describes existing models for studyying the pulmonary barrier function under acute inflammatory conditions, which are meant to improve the translational approaches for outcome predictions, patient monitoring, and treatment decision-making.

Orf Virus-Based Vectors Preferentially Target Professional Antigen-Presenting Cells, Activate the STING Pathway and Induce Strong Antigen-Specific T Cell Responses.

Background: Orf virus (ORFV)-based vectors are attractive for vaccine development as they enable the induction of potent immune responses against specific transgenes. Nevertheless, the precise mechanisms of immune activation remain unknown. This study therefore aimed to characterize underlying mechanisms in human immune cells. Methods: Peripheral blood mononuclear cells were infected with attenuated ORFV strain D1701-VrV and analyzed for ORFV infection and activation markers. ORFV entry in susceptible cells was examined using established pharmacological inhibitors. Using the THP1-Dual™ reporter cell line, activation of nuclear factor-κB and interferon regulatory factor pathways were simultaneously evaluated. Infection with an ORFV recombinant encoding immunogenic peptides (PepTrio-ORFV) was used to assess the induction of antigen-specific CD8+ T cells. Results: ORFV was found to preferentially target professional antigen-presenting cells (APCs) in vitro, with ORFV uptake mediated primarily by macropinocytosis. ORFV-infected APCs exhibited an activated phenotype, required for subsequent lymphocyte activation. Reporter cells revealed that the stimulator of interferon genes pathway is a prerequisite for ORFV-mediated cellular activation. PepTrio-ORFV efficiently induced antigen-specific CD8+ T cell recall responses in a dose-dependent manner. Further, activation and expansion of naïve antigen-specific CD8+ T cells was observed in response. Discussion: Our findings confirm that ORFV induces a strong antigen-specific immune response dependent on APC uptake and activation. These data support the notion that ORFV D1701-VrV is a promising vector for vaccine development and the design of innovative immunotherapeutic applications.