Preclinical evaluation of two phylogenetically distant arenavirus vectors for the development of novel immunotherapeutic combination strategies for cancer treatment.

BACKGROUND: Engineered arenavirus vectors have recently been developed to leverage the body's immune system in the fight against chronic viral infections and cancer. Vectors based on Pichinde virus (artPICV) and lymphocytic choriomeningitis virus (artLCMV) encoding a non-oncogenic fusion protein of human papillomavirus (HPV)16 E6 and E7 are currently being tested in patients with HPV16+ cancer, showing a favorable safety and tolerability profile and unprecedented expansion of tumor-specific CD8+ T cells. Although the strong antigen-specific immune response elicited by artLCMV vectors has been demonstrated in several preclinical models, PICV-based vectors are much less characterized. METHODS: To advance our understanding of the immunobiology of these two vectors, we analyzed and compared their individual properties in preclinical in vivo and in vitro systems. Immunogenicity and antitumor effect of intratumoral or intravenous administration of both vectors, as well as combination with NKG2A blockade, were evaluated in naïve or TC-1 mouse tumor models. Flow cytometry, Nanostring, and histology analysis were performed to characterize the tumor microenvironment (TME) and T-cell infiltrate following treatment. RESULTS: Despite being phylogenetically distant, both vectors shared many properties, including preferential infection and activation of professional antigen-presenting cells, and induction of potent tumor-specific CD8+ T-cell responses. Systemic as well as localized treatment induced a proinflammatory shift in the TME, promoting the infiltration of inducible T cell costimulator (ICOS)+CD8+ T cells capable of mediating tumor regression and prolonging survival in a TC-1 mouse tumor model. Still, there was evidence of immunosuppression built-up over time, and increased expression of H2-T23 (ligand for NKG2A T cell inhibitory receptor) following treatment was identified as a potential contributing factor. NKG2A blockade improved the antitumor efficacy of artARENA vectors, suggesting a promising new combination approach. This demonstrates how detailed characterization of arenavirus vector-induced immune responses and TME modulation can inform novel combination therapies. CONCLUSIONS: The artARENA platform represents a strong therapeutic vaccine approach for the treatment of cancer. The induced antitumor immune response builds the backbone for novel combination therapies, which warrant further investigation.

Local immunotherapy with the RNA-based immune stimulator CV8102 induces substantial anti-tumor responses and enhances checkpoint inhibitor activity.

Immunotherapy has revolutionized cancer treatment in recent years. Although currently approved checkpoint inhibitors (CPIs) yield remarkable anti-tumoral responses in several cancer types, a substantial proportion of patients do not benefit from such therapies. Local activation of innate immune signaling pathways is a promising approach to overcome the immunosuppressive tumor microenvironment, induce anti-tumor immunity, and improve the efficacy of CPI therapies. Here, we assessed the mode of action and efficacy of the RNA-based innate immune stimulator CV8102 for local immunotherapy in preclinical models. Intratumoral (i.t.) administration of CV8102 activated innate immune responses in the tumor microenvironment and draining lymph nodes, resulting in a dose-dependent anti-tumoral response. Combining i.t. CV8102 with systemic anti-programmed death protein 1 (PD-1) treatment further enhanced anti-tumoral responses, inducing tumor infiltration and activation of CD8+ T cells. The resulting memory response prevented tumor growth in rechallenged animals and impaired the growth of non-injected distal tumors. Therefore, i.t. CV8102 delivery is a promising approach for local cancer immunotherapy, especially in combination with CPIs. Clinical testing of CV8102 is ongoing (NCT03291002).

Live-attenuated lymphocytic choriomeningitis virus-based vaccines for active immunotherapy of HPV16-positive cancer.

Infection with human papillomavirus (HPV) is associated with a variety of cancer types and limited therapy options. Therapeutic cancer vaccines targeting the HPV16 oncoproteins E6 and E7 have recently been extensively explored as a promising immunotherapy approach to drive durable antitumor T cell immunity and induce effective tumor control. With the goal to achieve potent and lasting antitumor T cell responses, we generated a novel lymphocytic choriomeningitis virus (LCMV)-based vaccine, TT1-E7E6, targeting HPV16 E6 and E7. This replication-competent vector was stably attenuated using a three-segmented viral genome packaging strategy. Compared to wild-type LCMV, TT1-E7E6 demonstrated significantly reduced viremia and CNS immunopathology. Intravenous vaccination of mice with TT1-E7E6 induced robust expansion of HPV16-specific CD8+ T cells producing IFN-γ, TNF-α and IL-2. In the HPV16 E6 and E7-expressing TC-1 tumor model, mice immunized with TT1-E7E6 showed significantly delayed tumor growth or complete tumor clearance accompanied with prolonged survival. Tumor control by TT1-E7E6 was also achieved in established large-sized tumors in this model. Furthermore, a combination of TT1-E7E6 with anti-PD-1 therapy led to enhanced antitumor efficacy with complete tumor regression in the majority of tumor-bearing mice that were resistant to anti-PD-1 treatment alone. TT1-E7E6 vector itself did not exhibit oncolytic properties in TC-1 cells, while the antitumor effect was associated with the accumulation of HPV16-specific CD8+ T cells with reduced PD-1 expression in the tumor tissues. Together, our results suggest that TT1-E7E6 is a promising therapeutic vaccine for HPV-positive cancers.

Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines.

mRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.

Receptor Activator of NF-κB Orchestrates Activation of Antiviral Memory CD8 T Cells in the Spleen Marginal Zone.

The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.

Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of 'soluble CD163' in plasma.

CD163 is the macrophage receptor for uptake of hemoglobin-haptoglobin complexes. The human receptor can be shed from the macrophage surface owing to a cleavage site for the inflammation-inducible TACE/ADAM17 enzyme. Accordingly, plasma 'soluble CD163' (sCD163) has become a biomarker for macrophage activity and inflammation. The present study disclosed that 10% of sCD163 in healthy persons is actually extracellular vesicle (EV)-associated CD163 not being cleaved and shed. Endotoxin injection of human volunteers caused a selective increase in the ectodomain CD163, while septic patients exhibited high levels of both soluble ectodomain CD163 and extracellular vesicle (EV) CD163, the latter representing up 60% of total plasma CD163. A poor prognosis of septic patients measured as the sequential organ failure assessment (SOFA) score correlated with the increase in membrane-associated CD163. Our results show that soluble ectodomain CD163 and EV CD163 in plasma are part of separate macrophage response in the context of systemic inflammation. While that soluble ectodomain CD163 is released during the acute systemic inflammatory response, this is not the case for EV CD163 that instead may be released during a later phase of the inflammatory response. A separate measurement of the two forms of CD163 constituting 'soluble CD163' in plasma may therefore add to the diagnostic and prognostic value.

Homeostatic NF-κB Signaling in Steady-State Migratory Dendritic Cells Regulates Immune Homeostasis and Tolerance.

Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKß in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.

Leukocyte population dynamics and detection of IL-9 as a major cytokine at the mouse fetal-maternal interface.

Despite much interest in the mechanisms regulating fetal-maternal interactions, information on leukocyte populations and major cytokines present in uterus and placenta remains fragmentary. This report presents a detailed and quantitative study of leukocyte populations at the mouse fetal-maternal interface, including a comparison between pregnancies from syngeneic and allogeneic crosses. Our results provide evidence for drastic differences not only in the composition of leukocyte populations in the uterus during pregnancy, but also between uterine and placental tissues. Interestingly, we have observed a significant decrease in the number of myeloid Gr1+ cells including monocytes, and myeloid CD11c+ cells including DCs in placenta from an allogeneic pregnancy. In addition, we have compared the expression levels of a panel of cytokines in non-pregnant (NP) or pregnant mouse uterus, in placenta, or in their isolated resident leukocytes. Qualitative and quantitative differences have emerged between NP, pregnant uterus and placenta. Unexpectedly, IL-9 was the major cytokine in NP uterus, and was maintained at high levels during pregnancy both in uterus and placenta. Moreover, we have found that pregnancy is associated with an increase in uterine IL-1a and a significant decrease in uterine G-CSF and GM-CSF. Comparing allogeneic versus syngeneic pregnancy, less allogeneic placental pro-inflammatory cytokines CCL2 (MCP-1), CXCL10 (IP-10) and more IL1-α in whole uterus was reproducibly observed. To our knowledge, this is the first report showing a detailed overview of the leukocyte and cytokine repertoire in the uterus of virgin females and at the fetal-maternal interface, including a comparison between syngeneic and allogeneic pregnancy. This is also the first evidence for the presence of IL-9 in NP uterus and at the maternal-fetal interface, suggesting a major role in the regulation of local inflammatory or immune responses potentially detrimental to the conceptus.

Bone marrow-specific knock-in of a non-activatable Ikkα kinase mutant influences haematopoiesis but not atherosclerosis in Apoe-deficient mice.

BACKGROUND: The Ikkα kinase, a subunit of the NF-κB-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikkα mutant knock-in on haematopoiesis and atherosclerosis in mice. METHODS AND RESULTS: Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (Ikkα(AA/AA)Apoe(-/-) ) or with Ikkα(+/+)Apoe(-/-) BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in Ikkα(AA/AA)Apoe(-/-) BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of Ikkα(AA/AA)Apoe(-/-) vs Ikkα(+/+)Apoe(-/-) BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from Ikkα(AA/AA)Apoe(-/-) vs Ikkα(+/+)Apoe(-/-) mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. CONCLUSION: Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of Ikkα(AA) mutant BM did not affect atherosclerosis in Apoe(-/-) mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach.

Penicillin kills Chlamydia following the fusion of bacteria with lysosomes and prevents genital inflammatory lesions in C. muridarum-infected mice.

The obligate intracellular bacterium Chlamydia exists as two distinct forms. Elementary bodies (EBs) are infectious and extra-cellular, whereas reticulate bodies (RBs) replicate within a specialized intracellular compartment termed an 'inclusion'. Alternative persistent intra-cellular forms can be induced in culture by diverse stimuli such as IFNγ or adenosine/EHNA. They do not grow or divide but revive upon withdrawal of the stimulus and are implicated in several widespread human diseases through ill-defined in vivo mechanisms. ß-Lactam antibiotics have also been claimed to induce persistence in vitro. The present report shows that upon penicillin G (pG) treatment, inclusions grow as fast as those in infected control cells. After removal of pG, Chlamydia do not revert to RBs. These effects are independent of host cell type, serovar, biovar and species of Chlamydia. Time-course experiments demonstrated that only RBs were susceptible to pG. pG-treated bacteria lost their control over host cell apoptotic pathways and no longer expressed pre-16S rRNA, in contrast to persistent bacteria induced with adenosine/EHNA. Confocal and live-video microscopy showed that bacteria within the inclusion fused with lysosomal compartments in pG-treated cells. That leads to recruitment of cathepsin D as early as 3 h post pG treatment, an event preceding bacterial death by several hours. These data demonstrate that pG treatment of cultured cells infected with Chlamydia results in the degradation of the bacteria. In addition we show that pG is significantly more efficient than doxycycline at preventing genital inflammatory lesions in C. muridarum-C57Bl/6 infected mice. These in vivo results support the physiological relevance of our findings and their potential therapeutic applications.

CD1d-restricted NKT cells modulate placental and uterine leukocyte populations during chlamydial infection in mice.

Invariant CD1d-restricted natural killer T cells play an important immunoregulatory role and can influence a broad spectrum of immunological responses including against bacterial infections. They are present at the fetal-maternal interface and although it has been reported that experimental systemic iNKT cell activation can induce mouse abortion, their role during pregnancy remain poorly understood. In the present work, using a physiological Chlamydia muridarum infection model, we have shown that, in vaginally infected pregnant mice, C. muridarum is cleared similarly in C57BL/6 wild type (WT) and CD1d(-/-) mice. We have also shown that infected- as well as uninfected-CD1d(-/-) mice have the same litter size as WT counterparts. Thus, CD1d-restricted cells are required neither for the resolution of chlamydial infection of the lower-genital tract, nor for the maintenance of reproductive capacity. However, unexpected differences in T cell populations were observed in uninfected pregnant females, as CD1d(-/-) placentas contained significantly higher percentages of CD4(+) and CD8(+) T cells than WT counterparts. However, infection triggered a significant decrease in the percentages of CD4(+) T cells in CD1d(-/-) mice. In infected WT pregnant mice, the numbers of uterine CD4(+) and CD8(+) T cells, monocytes and granulocytes were greatly increased, changes not observed in infected CD1d(-/-) mice. An increase in the percentage of CD8(+) T cells seems independent of CD1d-restricted cells as it occurred in both WT and CD1d(-/-) mice. Thus, in the steady state, the lack of CD1d-restricted NKT cells affects leukocyte populations only in the placenta. In Chlamydia-infected pregnant mice, the immune response against Chlamydia is dampened in the uterus. Our results suggest that CD1d-restricted NKT cells play a role in the recruitment or homeostasis of leukocyte populations at the maternal-fetal interface in the presence or absence of Chlamydia infection.

I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection.

Dendritic cells (DC) are required for priming antigen-specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto-reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti-inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen-specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.