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Background and Purpose: Castration-resistant prostate cancer (CRPC) is a common male malignancy that requires new therapeutic strategies due to acquired resistance to its first-line treatment, docetaxel. The benefits of vitamin D on prostate cancer (PCa) progression have been previously reported. This study aimed to investigate the effects of vitamin D on chemoresistance in CRPC., Experimental Approach: Structure function relationships of potent vitamin D analogues were determined. The combination of the most potent analogue and docetaxel was explored in chemoresistant primary PCa spheroids and in a xenograft mouse model derived from a patient with a chemoresistant CRPC., Key Results: Here, we show that Xe4MeCF3 is more potent than the natural ligand to induce vitamin D receptor (VDR) transcriptional activities and that it has a larger therapeutic window. Moreover, we demonstrate that VDR agonists restore docetaxel sensitivity in PCa spheroids. Importantly, Xe4MeCF3 reduces tumour growth in a chemoresistant CRPC patient-derived xenograft. In addition, this treatment targets signalling pathways associated with cancer progression in the remaining cells., Conclusion and Implications: Taken together, these results unravel the potency of VDR agonists to overcome chemoresistance in CRPC and open new avenues for the clinical management of PCa., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Extracellular vesicles (EVs) are mediators of intercellular communication in the tumor microenvironment. Tumor EVs are commonly associated with metastasis, immunosuppression or drug resistance. Viral infections usually increase EV secretion, but little is known about the effect of oncolytic viruses (OVs) on tumor EVs. Here, we investigated the impact of oncolytic vesicular stomatitis virus (VSV) and vaccinia virus on EVs secreted by human melanoma and thoracic cancer cells. We found that OV infection increases the production of EVs by tumor cells. These EVs contain proteins of viral origin, such as VSV-G, thus creating a continuum of particles sharing markers of both canonical EVs and viruses. As such, the presence of VSV-G on EVs improves the transfer of their protein content to cell types commonly found in the tumor microenvironment. A proteomic analysis also revealed that EVs-OV secreted during VSV infection are enriched in immunity-related proteins. Finally, CD8 + T cells incubated with EVs-OV from infected cells display slightly enhanced cytotoxic functions. Taken together, these data suggest that OVs enhance the communication mediated by tumor EVs, which could participate in the therapeutic efficacy of OVs. These results also provide rationale for engineering OVs to exploit EVs and disseminate therapeutic proteins within the tumor microenvironment., Competing Interests: P.E. is an employee of Transgene SA. Transgene SA is a member of the Institut Mérieux Group, a publicly traded French biopharmaceutical company., (© 2024 The Author(s).)

Background: TG6050 was designed as an improved oncolytic vector, combining the intrinsic properties of vaccinia virus to selectively replicate in tumors with the tumor-restricted expression of recombinant immune effectors to modify the tumor immune phenotype. These properties might be of particular interest for "cold" tumors, either poorly infiltrated or infiltrated with anergic T cells., Methods:  TG6050, an oncolytic vaccinia virus encodes single-chain human interleukin-12 (hIL-12) and full-length anti-cytotoxic T-lymphocyte-associated antigen-4 (@CTLA-4) monoclonal antibody. The relevant properties of TG6050 (replication, cytopathy, transgenes expression and functionality) were extensively characterized in vitro . The biodistribution and pharmacokinetics of the viral vector, @CTLA-4 and IL-12, as well as antitumoral activities (alone or combined with immune checkpoint inhibitors) were investigated in several "hot" (highly infiltrated) and "cold" (poorly infiltrated) syngeneic murine tumor models. The mechanism of action was deciphered by monitoring both systemic and intratumoral immune responses, and by tumor transcriptome analysis. The safety of TG6050 after repeated intravenous administrations was evaluated in cynomolgus monkeys, with a focus on the level of circulating IL-12., Results: Multiplication and propagation of TG6050 in tumor cells in vitro and in vivo were associated with local expression of functional IL-12 and @CTLA-4. This dual mechanism translated into a strong antitumoral activity in both "cold" and "hot" tumor models (B16F10, LLC1 or EMT6, CT26, respectively) that was further amplified when combined with anti-programmed cell death protein-1. Analysis of changes in the tumor microenvironment (TME) after treatment with TG6050 showed increases in interferon-gamma, of CD8+T cells, and of M1/M2 macrophages ratio, as well as a drastic decrease of regulatory T cells. These local modifications were observed alongside bolstering a systemic and specific antitumor adaptive immune response. In toxicology studies, TG6050 did not display any observable adverse effects in cynomolgus monkeys., Conclusions: TG6050 effectively delivers functional IL-12 and @CTLA-4 into the tumor, resulting in strong antitumor activity. The shift towards an inflamed TME correlated with a boost in systemic antitumor T cells. The solid preclinical data and favorable benefit/risk ratio paved the way for the clinical evaluation of TG6050 in metastatic non-small cell lung cancer (NCT05788926 trial in progress)., Competing Interests: Competing interests: All authors were employees and shareholders of Transgene SA., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The oncolytic virus represents a promising therapeutic strategy involving the targeted replication of viruses to eliminate cancer cells, while preserving healthy ones. Despite ongoing clinical trials, this approach encounters significant challenges. This study delves into the interaction between an oncolytic virus and extracellular matrix mimics (ECM mimics). A three-dimensional colorectal cancer model, enriched with ECM mimics through bioprinting, was subjected to infection by an oncolytic virus derived from the vaccinia virus (oVV). The investigation revealed prolonged expression and sustained oVV production. However, the absence of a significant antitumor effect suggested that the virus's progression toward non-infected tumoral clusters was hindered by the ECM mimics. Effective elimination of tumoral cells was achieved by introducing an oVV expressing FCU1 (an enzyme converting the prodrug 5-FC into the chemotherapeutic compound 5-FU) alongside 5-FC. Notably, this efficacy was absent when using a non-replicative vaccinia virus expressing FCU1. Our findings underscore then the crucial role of oVV proliferation in a complex ECM mimics. Its proliferation facilitates payload expression and generates a bystander effect to eradicate tumors. Additionally, this study emphasizes the utility of 3D bioprinting for assessing ECM mimics impact on oVV and demonstrates how enhancing oVV capabilities allows overcoming these barriers. This showcases the potential of 3D bioprinting technology in designing purpose-fit models for such investigations., Competing Interests: Author AS, CE, MN, BM, PE, J-MB, EQ and CZ were employed by company Transgene SA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Marquette, Petiot, Spindler, Ebel, Nzepa, Moreau, Erbs, Balloul, Quemeneur and Zaupa.)

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors., Competing Interests: At the time this study was conducted, C.K., S.A., A.M., E.G., S.B., A.V., R.R., K.S., N.R.M., P.S., C.B., S.A.H., E.J.K., J.L., N.M.D., M.O., and M.A.S.B. were employees of AstraZeneca, with stock ownership and/or stock options or interests in the company; and J.F., N.S., E.Q., C.D., and P.K. were employees and stockholders of Transgene SA. N.R.M is an employee of Ratio Therapeutics (Boston, MA)., (© 2024 The Author(s).)

Arming oncolytic viruses with transgenes encoding immunomodulators improves their therapeutic efficacy by enhancing and/or sustaining the innate and adaptive anti-tumoral immune responses. We report here the isolation, selection, and vectorization of a blocking anti-human PDL1 single-domain antibody (sdAb) isolated from PDL1-immunized alpacas. Several formats of this sdAb were vectorized into the vaccinia virus (VV) and evaluated for their programmed cell death protein 1 (PD1)/PD1 ligand (PDL1) blocking activity in the culture medium of tumor cells infected in vitro . In those conditions, VV-encoded homodimeric sdAb generated superior PDL1 blocking activity compared to a benchmark virus encoding full-length avelumab. The sdAb was further used to design simple, secreted, and small tumor necrosis factor superfamily (TNFSF) fusions with the ability to engage their cognate receptors (TNFRSF) only in the presence of PDL1-positive cells. Finally, PDL1-independent alternatives of TNFRSF agonists were also constructed by fusing different variants of surfactant protein-D (SP-D) oligomerization domains with TNFSF ectodomains. An optimal SP-D-CD40L fusion with an SP-D collagen domain reduced by 80% was identified by screening with a transfection/infection method where poxvirus transfer plasmids and vaccinia virus were successively introduced into the same cell. However, once vectorized in VV, this construct had a much lower CD40 agonist activity compared to the SP-D-CD40L construct, which is completely devoid of the collagen domain that was finally selected. This latest result highlights the importance of working with recombinant viruses early in the payload selection process. Altogether, these results bring several complementary solutions to arm oncolytic vectors with powerful immunomodulators to improve their immune-based anti-tumoral activity., Competing Interests: Authors CR, EP, PK, HR, LF, RB, MG, CR, DS, EW, NS, EQ, and J-BM were employed by the company Transgene SA. MD, SS, CH, and PF were employed by the company Randox Laboratories Ltd., (Copyright © 2023 Remy, Pintado, Dunlop, Schön, Kleinpeter, Rozanes, Fend, Brandely, Geist, Suhner, Winter, Silvestre, Huguet, Fitzgerald, Quéméneur and Marchand.)

Radiation therapy and platinum-based chemotherapy are common treatments for lung cancer patients. Several factors are considered for the low overall survival rate of lung cancer, such as the patient's physical state and the complex heterogeneity of the tumor, which leads to resistance to the treatment. Consequently, precision medicines are needed for the patients to improve their survival and their quality of life. Until now, no patient-derived tumoroid model has been reported to predict the efficiency of radiation therapy in non-small-cell lung cancer. Using our patient-derived tumoroid model, we report that this model could be used to evaluate the efficiency of radiation therapy and cisplatin-based chemotherapy in non-small-cell lung cancer. In addition, these results can be correlated to clinical outcomes of patients, indicating that this patient-derived tumoroid model can predict the response to radiotherapy and chemotherapy in non-small-cell lung cancer.

Persistence of an immunosuppression, affecting both the innate and adaptive arms of the immune system, plays a role in sepsis patients' morbidity and late mortality pointing to the need for broad and effective immune interventions. MVA-hIL-7-Fc is a non-replicative recombinant Modified Vaccinia virus Ankara encoding the human interleukin-7 fused to human IgG2 Fc fragment. We have shown in murine sepsis models the capacity of this new virotherapy to stimulate both arms of the immune system and increase survival. Herein, an exploratory study in nonhuman primates was performed following a single intravenous injection of the MVA-hIL-7-Fc used at the clinical dose to assess its safety and biological activities. Four cynomolgus macaques were followed for 3 weeks post-injection (p.i), without observed acute adverse reactions. Circulating hIL-7-Fc was detected during the first 3-5 days p.i with a detection peaking at 12 h p.i. IL-7 receptor engagement and downstream signal transduction were detected in T cells demonstrating functionality of the expressed IL-7. Expansion of blood lymphocytes, mainly CD4 and CD8 naïve and central memory T cells, was observed on day 7 p.i. together with a transient increase of Ki67 expression on T lymphocytes. In addition, we observed an increase in circulating B and NK cells as well as monocytes were albeit with different kinetics and levels. This study indicates that a vectorized IL-7-Fc, injected by intravenous route at a relevant clinical dose in a large animal model, is active without adverse reactions supporting the clinical development of this novel virotherapy for treatment of sepsis patients.

Organ-on-chip and tumor-on-chip microfluidic cell cultures represent a fast-growing research field for modelling organ functions and diseases, for drug development, and for promising applications in personalized medicine. Still, one of the bottlenecks of this technology is the analysis of the huge amount of bio-images acquired in these dynamic 3D microenvironments, a task that we propose to achieve by exploiting the interdisciplinary contributions of computer science and electronic engineering. In this work, we apply this strategy to the study of oncolytic vaccinia virus (OVV), an emerging agent in cancer immunotherapy. Infection and killing of cancer cells by OVV were recapitulated and directly imaged in tumor-on-chip. By developing and applying appropriate image analysis strategies and advanced automatic algorithms, we uncovered synergistic cooperation of OVV and immune cells to kill cancer cells. Moreover, we observed that the kinetics of immune cells were modified in presence of OVV and that these immune modulations varied during the course of infection. A correlation between cancer cell infection and cancer-immune interaction time was pointed out, strongly supporting a cause-effect relationship between infection of cancer cells and their recognition by the immune cells. These results shed new light on the mode of action of OVV, and suggest new clinical avenues for immunotherapy developments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A majority of patients with sepsis surviving the first days in intensive care units (ICU) enter a state of immunosuppression contributing to their worsening. A novel virotherapy based on the non-propagative Modified Virus Ankara (MVA) expressing the human interleukin-7 (hIL-7) cytokine fused to an Fc fragment, MVA-hIL-7-Fc, was developed and shown to enhance innate and adaptive immunity and confer survival advantages in murine sepsis models. Here, we assessed the capacity of hIL-7-Fc produced by the MVA-hIL-7-Fc to improve ex vivo T lymphocyte functions from ICU patients with sepsis. Primary hepatocytes were transduced with the MVA-hIL-7-Fc or an empty MVA, and cell supernatants containing the secreted hIL-7-Fc were harvested for in vitro and ex vivo studies. Whole blood from ICU patients [septic shock = 15, coronavirus disease 2019 (COVID-19) = 30] and healthy donors (n = 36) was collected. STAT5 phosphorylation, cytokine production, and cell proliferation were assessed upon T cell receptor (TCR) stimulation in presence of MVA-hIL-7-Fc-infected cell supernatants. Cells infected by MVA-hIL-7-Fc produced a dimeric, glycosylated, and biologically active hIL-7-Fc. Cell supernatants containing the expressed hIL-7-Fc triggered the IL-7 pathway in T lymphocytes as evidenced by the increased STAT5 phosphorylation in CD3+ cells from patients and healthy donors. The secreted hIL-7-Fc improved Interferon-γ (IFN-γ) and/or Tumor necrosis factor-α (TNF-α) productions and CD4+ and CD8+ T lymphocyte proliferation after TCR stimulation in patients with bacterial and viral sepsis. This study demonstrates the capacity of the novel MVA-hIL-7-Fc-based virotherapy to restore ex vivo T cells immune functions in ICU patients with sepsis and COVID-19, further supporting its clinical development., Competing Interests: MC, J-BM, PM, and GI were employees of Transgene SA when the work was performed. Transgene SA is a publicly traded French biopharmaceutical company, with Institut Merieux as the major shareholder. MC, GM, FC and A-CL work at EA7426, a joint unit including University, Hospital and bioMérieux, but are not employed by bioMérieux. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received f​​​​unding from Transgene SA. The funder had the following involvement with the study: collection of data, analysis, interpretation of data and writing of the article., (Copyright © 2022 Crausaz, Monneret, Conti, Lukaszewicz, Marchand, Martin, Inchauspé and Venet.)

Persistence of an immunosuppressive state plays a role in septic patient morbidity and late mortality. Both innate and adaptive pathways are impaired, pointing toward the need for immune interventions targeting both arms of the immune system. We developed a virotherapy using the nonpropagative modified vaccinia virus Ankara (MVA), which harbors the intrinsic capacity to stimulate innate immunity, to deliver IL-7, a potent activator of adaptive immunity. The rMVA-human IL-7 (hIL-7)-Fc encoding the hIL-7 fused to the human IgG2-Fc was engineered and shown to express a dimeric, glycosylated, and biologically active cytokine. Following a single i.v. injection in naive mice, the MVA-hIL-7-Fc increased the number of total and activated B, T, and NK cells but also myeloid subpopulations (Ly6C high , Ly6C int , and Ly6C neg cells) in both lung and spleen. It triggered differentiation of T cells in central memory, effector memory, and acute effector phenotypes and enhanced polyfunctionality of T cells, notably the number of IFN-γ-producing cells. The MVA vector contributed significantly to immune cell activation, particularly of NK cells. The MVA-hIL-7-Fc conferred a significant survival advantage in the cecal ligation and puncture (CLP) and Candida albicans sepsis models. It significantly increased cell numbers and activation in both spleen and lung of CLP mice. Comparatively, in naive and CLP mice, the rhIL-7-Fc soluble counterpart overall induced less vigorous, shorter lasting, and narrower immune activities than did the MVA-hIL-7-Fc and favored TNF-α-producing cells. The MVA-hIL-7-Fc represents a novel class of immunotherapeutic with clinical potential for treatment of septic patients., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Patient-derived tumoroid (PDT) has been developed and used for anti-drug screening in the last decade. As compared to other existing drug screening models, a PDT-based in vitro 3D cell culture model could preserve the histological and mutational characteristics of their corresponding tumors and mimic the tumor microenvironment. However, few studies have been carried out to improve the microvascular network connecting the PDT and its surrounding microenvironment, knowing that poor tumor-selective drug transport and delivery is one of the major reasons for both the failure of anti-cancer drug screens and resistance in clinical treatment. In this study, we formed vascularized PDTs in six days using multiple cell types which maintain the histopathological features of the original cancer tissue. Furthermore, our results demonstrated a vascular network connecting PDT and its surrounding microenvironment. This fast and promising PDT model opens new perspectives for personalized medicine: this model could easily be used to test all therapeutic treatments and could be connected with a microfluidic device for more accurate drug screening.

Objective: Antitumor viral vaccines, and more particularly poxviral vaccines, represent an active field for clinical development and translational research. To improve the efficacy and treatment outcome, new viral vectors are sought, with emphasis on their abilities to stimulate innate immunity, to display tumor antigens and to induce a specific T-cell response., Methods: We screened for a new poxviral backbone with improved innate and adaptive immune stimulation using IFN-α secretion levels in infected PBMC cultures as selection criteria. Assessment of virus effectiveness was made in vitro and in vivo ., Results: The bovine pseudocowpox virus (PCPV) stood out among several poxviruses for its ability to induce significant secretion of IFN-α. PCPV produced efficient activation of human monocytes and dendritic cells, degranulation of NK cells and reversed MDSC-induced T-cell suppression, without being offensive to activated T cells. A PCPV-based vaccine, encoding the HPV16 E7 protein (PCPV-E7), stimulated strong antigen-specific T-cell responses in TC1 tumor-bearing mice. Complete regression of tumors was obtained in a CD8 + T-cell-dependent manner after intratumoral injection of PCPV-E7, followed by intravenous injection of the cancer vaccine MVA-E7. PCPV also proved active when injected repeatedly intratumorally in MC38 tumor-bearing mice, generating tumor-specific T-cell responses without encoding a specific MC38 antigen. From a translational perspective, we demonstrated that PCPV-E7 effectively stimulated IFN-γ production by T cells from tumor-draining lymph nodes of HPV + -infected cancer patients., Conclusion: We propose PCPV as a viral vector suitable for vaccination in the field of personalised cancer vaccines, in particular for heterologous prime-boost regimens., Competing Interests: All authors except RR, FK, CS, CLT, CaH and EP are employees of Transgene. Institute Curie employees RR, FK and EP receive funding from Transgene., (© 2022 Transgene SA. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Background: Immune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of patients with cancer including those with poorly immune infiltrated 'cold' tumors are resistant to currently available ICB therapies. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved αCTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for αCTLA-4; ICB and regulatory T cell (Treg) depletion are both thought to contribute efficacy and toxicity in available, systemic, αCTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe αCTLA-4 therapies have been lacking. Here we assess and identify spatially restricted exposure to a novel strongly Treg-depleting, checkpoint-blocking, vectorized αCTLA-4, as a highly efficacious and potentially safe strategy to target CTLA-4., Methods: A novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for monoclonal antibodies (mAbs) and targets associated with superior Treg-depleting activity. A tumor-selective oncolytic vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, αCTLA-4 antibody or a matching surrogate antibody, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (VV GM -αCTLA-4)., Results: The identified 4-E03 antibody showed significantly stronger Treg depletion, but equipotent checkpoint blockade, compared with clinically validated αCTLA-4 ipilimumab against CTLA-4-expressing Treg cells in a humanized mouse model in vivo. Intratumoral administration of VV GM -αCTLA-4 achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8 + T cells and antitumor immunity compared with systemic αCTLA-4 antibody therapy. Efficacy correlated with FcγR-mediated intratumoral Treg depletion. Remarkably, in a clinically relevant mouse model resistant to systemic ICB, intratumoral VV GM -αCTLA-4 synergized with αPD-1 to reject cold tumors., Conclusion: Our findings demonstrate in vivo proof of concept for spatial restriction of Treg depletion-optimized immune checkpoint blocking, vectorized αCTLA-4 as a highly effective and safe strategy to target CTLA-4. A clinical trial evaluating intratumoral VV GM -αhCTLA-4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has commenced., Competing Interests: Competing interests: MS, MR, PH, LM, CS, FJ, MB, IT, and BF are employees, MS, MR, LM, FJ, MB, IT, and BF are shareholders of BioInvent International. KLC received funding from BioInvent. J-BM, LF, CR, NS, PK, JD, JF, and EQ are employees and shareholders of Transgene., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Human papillomavirus 16 (HPV16) is the main cause of oropharyngeal squamous cell carcinoma (OPSCC). To date, the links between HPV16 gene expression and adaptive immune responses have not been investigated. We evaluated the correlation of HPV16 DNA, RNA transcripts and features of adaptive immune response by evaluating antibody isotypes against E2, E7 antigens and density of tumor-infiltrating lymphocytes (TIL)., Material and Methods: FFPE-tissue from 27/77 p16-positive OPSCC patients was available. DNA and RNA were extracted and quantified using qPCR for all HPV16 genes. The TIL status was assessed. Immune responses against E2 and E7 were quantified by ELISA (IgG, IgA, and IgM; 77 serum samples pre-treatment, 36 matched post-treatment)., Results: Amounts of HPV16 genes were highly correlated at DNA and RNA levels. RNA co-expression of all genes was detected in 37% (7/19). E7 qPCR results were correlated with higher anti-E7 antibody (IgG, IgA) level in the blood. Patients with high anti-E2 IgG antibody (>median) had better overall survival (p=0.0311); anti-E2 and anti-E7 IgA levels had no detectable effect. During the first 6 months after treatment, IgA but not IgG increased significantly, and >6 months both antibody classes declined over time. Patients with immune cell-rich tumors had higher levels of circulating antibodies against HPV antigens., Conclusion: We describe an HPV16 qPCR assay to quantify genomic and transcriptomic expression and correlate this with serum antibody levels against HPV16 oncoproteins. Understanding DNA/RNA expression, relationship to the antibody response in patients regarding treatment and outcome offers an attractive tool to improve patient care., Competing Interests: Author KB was employed by company Transgene SA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 von Witzleben, Currall, Wood, Chudley, Akinyegun, Thomas, Bendjama, Thomas, Friedmann, King, Laban and Ottensmeier.)

As a live biologic agent, oncolytic vaccinia virus has the ability to target and selectively amplify at tumor sites. We have previously reported that deletion of thymidine kinase and ribonucleotide reductase genes in vaccinia virus can increase the safety and efficacy of the virus. Here, to allow direct visualization of the viral genome in living cells, we incorporated the ANCH target sequence and the OR3-Santaka gene in the double-deleted vaccinia virus. Infection of human tumor cells with ANCHOR3-tagged vaccinia virus enables visualization and quantification of viral genome dynamics in living cells. The results show that the ANCHOR technology permits the measurement of the oncolytic potential of the double deleted vaccinia virus. Quantitative analysis of infection kinetics and of viral DNA replication allow rapid and efficient identification of inhibitors and activators of oncolytic activity. Our results highlight the potential application of the ANCHOR technology to track vaccinia virus and virtually any kind of poxvirus in living cells.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination., Competing Interests: Competing interestsThe authors from the BPRC, University of Pittsburgh, and NIH have no competing interests. IAVI (transferred from Aeras 2018) holds the patent to the classical 5Ag cassette. A.B., R.A., D.J.L., and T.G.E. are listed as an inventor on a patent application originally filed by Aeras. GlaxoSmithKline Biologicals SA and Transgene are the respective developers of the investigational vaccines in this study. N.O. is an employee and shareholder of the GSK group of companies and is listed as an inventor on a patent application filed by the GSK group of companies. Remaining authors of Aeras and Transgene have no competing interests., (© The Author(s) 2020.)

Treatment of chronic hepatitis B (CHB) typically requires life-long administration of drugs. Cohort and pre-clinical studies have established the link between a functional T-cell-mounted immunity and resolution of infection. TG1050 is an adenovirus 5-based vaccine that expresses HBV polymerase and domains of core and surface antigen and has shown immunogenicity and antiviral effects in mice. We performed a phase 1 clinical trial to assess safety and explore immunogenicity and early efficacy of TG1050 in CHB patients. This randomized, double blind, placebo-controlled study included two sequential phases: one single dose cohort (SD, n = 12) and one multiple (3) doses cohort (MD, n = 36). Patients, virally suppressed under nucleoside(d)tide analog NUC therapy, were randomized 1:1:1 across 3 dose levels (DL) and assigned to receive 10 9 , 10 10 , 10 11 virus particles (vp) of TG1050 and then randomized within each DL to placebo (3:1 and 9:3 vaccines/placebo in each DL, respectively, for the SD and MD cohorts). Cellular (ELISPOT) and antibody responses (anti-Adenovirus), as well as evolution of circulating HBsAg and HBcrAg, were monitored. All doses were well tolerated in both cohorts, without severe adverse event. TG1050 was capable to induce IFN-γ producing T-cells targeting 1 to 3 encoded antigens, in particular at the 10 10 vp dose. Overall, minor decreases of HBsAg were observed while a number of vaccinees reached unquantifiable HBcrAg by end of the study. In CHB patients under NUC, TG1050 exhibited a good safety profile and was capable to induce HBV-specific cellular immune response. These data support further clinical evaluation, especially in combination studies.

Background: While prophylactic human papillomavirus (HPV) vaccination exists, women are still developing cervical intraepithelial neoplasia (CIN) grade 2 or 3 for which an immunotherapeutic, non-surgical, approach may be effective. The primary aim was to assess the efficacy of tipapkinogen sovacivec (TS) vaccine in achieving histologic resolution of CIN2/3 associated with high risk (HR) HPV types., Methods: Women 18 years and older who had confirmed CIN2/3 were enrolled in a randomized, double blind, placebo-controlled phase II trial and assigned to drug in a 2:1 ratio (vaccine:placebo). The primary endpoint occurred at month 6 when the excisional therapy was performed; cytology and HR HPV typing were performed at months 3, 6 and every six months through month 30. The safety population included all patients who received at least one dose of study drug., Results: Of the 129 women randomized to vaccine and 63 to placebo, complete resolution was significantly higher in the vaccine group than placebo for CIN 2/3 regardless of the 13 HR HPV types assayed (24% vs. 10%, p < 0.05); as well as for only CIN 3 also regardless of HR HPV type (21% vs. 0%, p < 0.01). Irrespective of baseline HPV infection, viral DNA clearance was higher in the vaccine group compared to placebo (p < 0.01). The vaccine was well tolerated with the most common adverse events being injection site reactions., Conclusions: The TS vaccine provides histologic clearance of CIN 2/3 irrespective of HR HPV type in one third of subjects and is generally safe through 30 months., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Vaccinia virus, a member of the Poxviridae family, has been extensively used as an oncolytic agent and has entered late stage clinical development. In this study, we evaluated the potential oncolytic properties of other members of the Poxviridae family. Numerous tumor cell lines were infected with ten non-vaccinia poxviruses to identify which virus displayed the most potential as an oncolytic agent. Cell viability indicated that tumor cell lines were differentially susceptible to each virus. Raccoonpox virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all tumor cell lines. To investigate further the oncolytic capacity of the Raccoonpox virus, we have generated a thymidine kinase (TK)-deleted recombinant Raccoonpox virus expressing the suicide gene FCU1 . This TK-deleted Raccoonpox virus was notably attenuated in normal primary cells but replicated efficiently in numerous tumor cell lines. In human colon cancer xenograft model, a single intratumoral inoculation of the recombinant Raccoonpox virus, in combination with 5-fluorocytosine administration, produced relevant tumor growth control. The results demonstrated significant antitumoral activity of this new modified Raccoonpox virus armed with FCU1 and this virus could be considered to be included into the growing armamentarium of oncolytic virotherapy for cancer., Competing Interests: CONFLICTS OF INTEREST All authors except C. Camus-Bouclainville and S. Bertagnoli were employees of Transgene SA when the work was performed. Transgene SA is a member of the Institut Merieux Group, a publicly traded French biopharmaceutical company. The authors report no other conflicts of interest in this work.

Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1 . This TK-deleted virus, deVV5- fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.

Pre-clinical models mimicking persistent hepatitis B virus (HBV) expression are seldom, do not capture all features of a human chronic infection and due to their complexity, are subject to variability. We report a meta-analysis of seven experiments performed with TG1050, an HBV-targeted immunotherapeutic, 1 in an HBV-persistent mouse model based on the transduction of mice by an adeno-associated virus coding for an infectious HBV genome (AAV-HBV). To mimic the clinical diversity seen in HBV chronically infected patients, AAV-HBV transduced mice displaying variable HBsAg levels were treated with TG1050. Overall mean percentages of responder mice, displaying decrease in important clinical parameters i.e. HBV-DNA (viremia) and HBsAg levels, were 52% and 51% in TG1050 treated mice, compared with 8% and 22%, respectively, in untreated mice. No significant impact of HBsAg level at baseline on response to TG1050 treatment was found. TG1050-treated mice displayed a significant shorter Time to Response (decline in viral parameters) with an Hazard Ratio (HR) of 8.3 for viremia and 2.6 for serum HBsAg. The mean predicted decrease for TG1050-treated mice was 0.5 log for viremia and 0.8 log for HBsAg, at the end of mice follow-up, compared to no decrease for viremia and 0.3 log HBsAg decrease for untreated mice. For mice receiving TG1050, a higher decline of circulating viremia and serum HBsAg level over time was detected by interaction term meta-analysis with a significant treatment effect (p = 0.002 and p<0.001 respectively). This meta-analysis confirms the therapeutic value of TG1050, capable of exerting potent antiviral effects in an HBV-persistent model mimicking clinical situations.

Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by Mycobacterium tuberculosis (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host's immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. In vitro analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, i.e. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.

The treatment of cancer using nanomedicines is limited by the poor penetration of these potentially powerful agents into and throughout solid tumors. Externally controlled mechanical stimuli, such as the generation of cavitation-induced microstreaming using ultrasound (US), can provide a means of improving nanomedicine delivery. Notably, it has been demonstrated that by focusing, monitoring and controlling the US exposure, delivery can be achieved without damage to surrounding tissue or vasculature. However, there is a risk that such stimuli may disrupt the structure and thereby diminish the activity of the delivered drugs, especially complex antibody and viral-based nanomedicines. In this study, we characterize the impact of cavitation on four different agents, doxorubicin (Dox), cetuximab, adenovirus (Ad) and vaccinia virus (VV), representing a scale of sophistication from a simple small-molecule drug to complex biological agents. To achieve tight regulation of the level and duration of cavitation exposure, a "cavitation test rig" was designed and built. The activity of each agent was assessed with and without exposure to a defined cavitation regime which has previously been shown to provide effective and safe delivery of agents to tumors in preclinical studies. The fluorescence profile of Dox remained unchanged after exposure to cavitation, and the efficacy of this drug in killing a cancer cell line remained the same. Similarly, the ability of cetuximab to bind its epidermal growth factor receptor target was not diminished following exposure to cavitation. The encoding of the reporter gene luciferase within the Ad and VV constructs tested here allowed the infectivity of these viruses to be easily quantified. Exposure to cavitation did not impact on the activity of either virus. These data provide compelling evidence that the US parameters used to safely and successfully delivery nanomedicines to tumors in preclinical models do not detrimentally impact on the structure or activity of these nanomedicines., Competing Interests: Disclosure Robert Carlisle, Constantin Coussios, Colin Story and Christian M Coviello are founders and shareholders in OxSonics Ltd which holds intellectual property relating to the polymeric cups and US technology used in these experiments. The other authors report no conflicts of interest in this work.

Background: Advanced non-small cell lung cancer patients receiving TG4010, a therapeutic viral vaccine encoding human Mucin 1 and interleukin-2 in addition to standard chemotherapy, displayed longer overall survival in comparison to that of patients treated with standard chemotherapy alone. Our study intended to establish the association between overall survival and vaccine-induced T cell responses against tumor associated antigens (TAA) targeted by the vaccine., Method: The TIME trial was a placebo-controlled, randomized phase II study aimed at assessing efficacy of TG4010 with chemotherapy in NSCLC. 78 patients from the TIME study carrying the HLA-A02*01 haplotype were analyzed using combinatorial encoding of MHC multimers to detect low frequencies of cellular immune responses to TG4010 and other unrelated TAA., Results: We report that improvement of survival under TG4010 treatment correlated with development of T cell responses against MUC1. Interestingly, responses against MUC1 were associated with broadening of CD8 responses against non-targeted TAA, thus demonstrating induction of epitope spreading., Conclusion: Our results support the causality of specific T-cell response in improved survival in NSCLC. Additionally, vaccine induced epitope spreading to other TAA participates to the enrichment of the diversity of the anti-tumor response. Hence, TG4010 appears as a useful therapeutic option to maximize response rate and clinical benefit in association with other targeted immuno-modulators., Trial Registration: Registered on ClinicalTrials.gov under identifier NCT01383148 on June 23rd, 2011.

While interest in immunotherapies is renewed by the successful introduction of immune checkpoint blocking agent in the clinic, advances in genome sequencing are opening new possibilities in the design of increasingly personalized vaccines. Personalization of medicine can now be realistically contemplated at the single patient level. Unlike the previous generation of cancer vaccines, neoantigen directed vaccines would target truly specific tumor antigens resulting from acquired tumor genome mutations. Immune response induced by this next generation vaccine would not be subject to self-tolerance and will likely result to enhanced efficacy. Nevertheless, this new technologies can hold to their promises only if sponsors manage to meet several scientific, technical, logistical and regulatory challenges. In particular manufacturers will have to design, manufacture, and deliver to the patient a new pharmaceutical grade in a matters of weeks. In this paper, we briefly review current technologies currently tried at the translation of personalized vaccines and explore the possibilities offered by the Modified Vaccinia virus Ankara in this next wave of cancer vaccines.

Background: The success of immunotherapeutics in oncology and the search for further improvements has prompted revisiting the use of cancer vaccines. In this context, knowledge of the immunogenic epitopes and the monitoring of the immune response cancer vaccines generate are essential. MUC1 has been considered one of the most important tumor associated antigen for decades., Methods: To identify HLA-restricted MUC1 peptides we used eight human MHC class I transgenic mouse lines, together covering more than 80% of the human population. MUC1 peptides were identified by vaccinating each line with full length MUC1 coding sequences and using an IFNγ ELIspot restimulation assay. Relevant peptides were tested in a flow cytometry-based tetramer assay and for their capacity to serve as target in an in vivo killing assay., Results: Four previously identified MUC1 peptides were confirmed and five are described here for the first time. These nine peptide-MHC combinations were further characterized. Six gave above-background tetramer staining of splenocytes from immunized animals and three peptides were induced more than 5% specific in vivo killing., Conclusions: These data describe for the first time five new HLA class I-restricted peptides and revisit some that were previously described. They also emphasize the importance of using in vivo/ex vivo models to screen for immunogenic peptides and define the functions for individual peptide-HLA combinations.

Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP) and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) or transforming 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs) and the interaction with the autologous cytotoxic T lymphocyte (CTL) clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1), markers of immunogenic cell death (ICD), could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse melanoma cells and induce additional immunostimulatory effects to promote antitumor immune response. Further investigation in vivo is needed to consolidate the data., Competing Interests: Disclosure M Lusky, P Erbs and X Preville were employees of Trans-gene when the work was performed. Transgene is a member of the Institut Merieux Group, a publicly traded French biopharmaceutical company. The authors report no other conflicts of interest in this work.

Background: We previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. To investigate whether tumor irradiation augments the immune response to MUC1 tumor antigen, we have tested the efficacy of tumor irradiation combined with an MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for murine renal adenocarcinoma (Renca) cells transfected with MUC1., Methods: Established subcutaneous Renca-MUC1 tumors were treated with 8 Gy radiation on day 11 and peritumoral injections of MVA-MUC1-IL2 vector on day 12 and 17, or using a reverse sequence of vaccine followed by radiation. Growth delays were monitored by tumor measurements and histological responses were evaluated by immunohistochemistry. Specific immunity was assessed by challenge with Renca-MUC1 cells. Generation of tumor-specific T cells was detected by IFN-γ production from splenocytes stimulated in vitro with tumor lysates using ELISPOT assays., Results: Tumor growth delays observed by tumor irradiation combined with MVA-MUC1-IL-2 vaccine were significantly more prolonged than those observed by vaccine, radiation, or radiation with MVA empty vector. The sequence of cancer vaccine followed by radiation two days later resulted in 55-58% complete responders and 60% mouse long-term survival. This sequence was more effective than that of radiation followed by vaccine leading to 24-30% complete responders and 30% mouse survival. Responding mice were immune to challenge with Renca-MUC1 cells, indicating the induction of specific tumor immunity. Histology studies of regressing tumors at 1 week after therapy, revealed extensive tumor destruction and a heavy infiltration of CD45 + leukocytes including F4/80 + macrophages, CD8 + cytotoxic T cells and CD4 + helper T cells. The generation of tumor-specific T cells by combined therapy was confirmed by IFN-γ secretion in tumor-stimulated splenocytes. An abscopal effect was measured by rejection of an untreated tumor on the contralateral flank to the tumor treated with radiation and vaccine., Conclusions: These findings suggest that cancer vaccine given prior to local tumor irradiation augments an immune response targeted at tumor antigens that results in specific anti-tumor immunity. These findings support further exploration of the combination of radiotherapy with cancer vaccines for the treatment of cancer.

Several approaches to antigen-specific immunotherapy of cancer antigen-specific immunotherapy of cancer have been tested clinically. In this chapter, we will describe studies done with the antigen MUC1. Tested MUC1 therapeutic vaccines include the following: monoclonal antibodies (MAbs) specific for MUC1; synthetic and recombinant polypeptides from the protein sequence of MUC1; dendritic cells carrying MUC1; RNA and DNA vaccinations; and recombinant viruses carrying the MUC1 DNA sequence. Chemotherapy of cancer aims to be toxic to the cancer cells with manageable side effects to the patient. In contrast, antigen-specific immunotherapy of cancer aims to treat the patient, such that the patient is then able to control and eventually eliminate their cancer cells. It is therefore important to know the immune status of each cancer patient prior to therapy.

Background: TG4023 is a modified vaccinia virus Ankara (MVA) containing the yeast-originated transgene FCU1, expressing cytosine deaminase and uracil phosphoribosyltransferase enzymes that transform the prodrug flucytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5′-monophosphate, respectively. This first-in-human study aimed to assess the maximum tolerated dose (MTD) of intratumoral (IT) TG4023 and the safety, feasibility, and proof-of-concept (PoC) of TG4023/5-FC combination to deliver high 5-FU concentrations in tumors., Patients and Methods: Cancer patients without further therapeutic option and with at least one injectable primary or metastatic liver tumor underwent on day 1 a percutaneous IT injection of TG4023 at doses of 107, 108, or 4.108 plaque forming units (p.f.u.) using ultrasound imaging guidance, after a dose-limiting toxicities (DLTs)-driven 3 + 3 dose-escalating design. On day 2, patients were given intravenous and/or oral 5-FC at a dose of 200 mg/kg/day for 14 days and were followed for safety through day 43. Tumor response was assessed at week 6, according to RECIST. Plasma and tumor 5-FU concentrations were measured to establish the PoC., Results: In total, 16 patients completed treatment with TG4023 and 5-FC. One DLT/7 patients (ALT/aspartate aminotransferase transient increase) was observed at 4 × 108 p.f.u.; MTD was therefore not reached. The most frequent adverse events were pyrexia, asthenia, vomiting, and decreased appetite. Eight of 16 patients had stable disease. Mean 5-FU concentrations in plasma were 1.9 ± 2.6 ng/ml and 56 ± 30 ng/g in tumors. Seroconversion for anti-FCU1 antibodies was found for one patient from each cohort (16%, overall)., Conclusions: This phase I study demonstrated that IT injections of TG4023 were feasible and well tolerated; MTD was defined as 4 × 108 p.f.u. Therapeutic 5-FU concentrations in tumors established the virus-directed enzyme-prodrug therapy PoC., Clinicaltrials.gov Number: NCT00978107.

The selection of a cell substrate is a critical step for the development and manufacturing of a viral vaccine candidate. Several parameters such as cell susceptibility and permissiveness to the viral pathogens but also performance in terms of viral antigens quality and production yields are important considerations when identifying the ideal match between a viral vaccine and cell substrate. The modified vaccinia virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform, however only limited cell substrates have been tested or are available for industrialization. Here we evaluate the duck embryo-derived EB66® cell line as potential cell substrate for MVA production. To this end, we used two recombinant MVA constructs and demonstrated that EB66® cells are propagating the tested MVA viruses very efficiently, while preserving viral attenuation and transgene expression for up to 20 serial passages. Furthermore we developed upstream and downstream processes that enable industrialization of the virus production. In conclusion, we showed that EB66® cells can be used as potent cell substrate for MVA-based vaccines and represent therefore an attractive alternative for vaccine production., (Copyright © 2016. Published by Elsevier Ltd.)

Oncolytic viruses (OV) could become the most powerful and selective cancer therapies. However, the limited transport of OV into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. Application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. In particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as inertial cavitation can propel OV hundreds of microns. We have recently developed a polymeric cup formulation which, when delivered intravenously, provides the nuclei for instigation of sustained inertial cavitation events within tumors. Here we report that exposure of tumors to focused ultrasound after intravenous coinjection of cups and oncolytic vaccinia virus , leads to substantial and significant increases in activity. When cavitation was instigated within SKOV-3 or HepG2 xenografts, reporter gene expression from vaccinia virus was enhanced 1,000-fold (P < 0.0001) or 10,000-fold (P < 0.001), respectively. Similar increases in the number of vaccinia virus genomes recovered from tumors were also observed. In survival studies, the application of cup mediated cavitation to a vaccinia virus expressing a prodrug converting enzyme provided significant (P < 0.05) retardation of tumor growth. This technology could improve the clinical utility of all biological therapeutics including OV.

Background: MUC1 is a tumour-associated antigen expressed by many solid tumours, including non-small-cell lung cancer. TG4010 is a modified vaccinia Ankara expressing MUC1 and interleukin 2. In a previous study, TG4010 combined with chemotherapy showed activity in non-small-cell lung cancer and the baseline value of CD16, CD56, CD69 triple-positive activated lymphocytes (TrPAL) was shown to be potentially predictive of TG4010 efficacy. In this phase 2b part of the phase 2b/3 TIME trial, we further assess TG4010 in combination with first-line chemotherapy and use of the TrPAL biomarker in this setting., Methods: In this phase 2b part of a randomised, double-blind, placebo-controlled, phase 2b/3 trial, we recruited previously untreated patients aged 18 years or older with stage IV non-small-cell lung cancer without a known activating EGFR mutation and with MUC1 expression in at least 50% of tumoural cells. Patients were randomly allocated (1:1) by an external service provider to subcutaneous injections of 10(8) plaque-forming units of TG4010 or placebo from the beginning of chemotherapy every week for 6 weeks and then every 3 weeks up to progression, discontinuation for any reason, or toxic effects, stratified according to baseline value of TrPAL (≤ or > the upper limit of normal [ULN]) and, in addition, a dynamic minimisation procedure was used, taking into account chemotherapy regimen, histology, addition or not of bevacizumab, performance status, and centre. Patients, site staff, monitors, the study funder, data managers, and the statistician were masked to treatment identity. The primary endpoint was progression-free survival, assessed every 6 weeks, to validate the predictive value of the TrPAL biomarker. If patients with TrPAL values of less than or equal to the ULN had a Bayesian probability of more than 95% that the true hazard ratio (HR) for progression-free survival was less than 1, and if those with TrPAL values of greater than the ULN had a probability of more than 80% that the true HR for progression-free survival was more than 1, the TrPAL biomarker would be validated. We did primary analyses in the intention-to-treat population and safety analyses in those who had received at least one dose of study drug and had at least one valid post-baseline safety assessment. Monitors, site staff, and patients are still masked to treatment assignment. This trial is registered with ClinicalTrials.gov, number NCT01383148., Findings: Between April 10, 2012, and Sept 12, 2014, we randomly allocated 222 patients (TG4010 and chemotherapy 111 [50%]; placebo and chemotherapy 111 [50%]). In the whole population, median progression-free survival was 5·9 months (95% CI 5·4-6·7) in the TG4010 group and 5·1 months (4·2-5·9) in the placebo group (HR 0·74 [95% CI 0·55-0·98]; one-sided p=0·019). In patients with TrPAL values of less than or equal to the ULN, the HR for progression-free survival was 0·75 (0·54-1·03); the posterior probability of the HR being less than 1 was 98·4%, and thus the primary endpoint was met. In patients with TrPAL values of greater than the ULN, the HR for progression-free survival was 0·77 (0·42-1·40); the posterior probability of the HR being greater than 1 was 31·3%, and the primary endpoint was not met. We noted grade 1-2 injection-site reactions in 36 (33%) of 110 patients in the TG4010 group versus four (4%) of 107 patients in the placebo group. We noted no grade 3 or 4 nor serious adverse events deemed to be related to TG4010 only. Four (4%) patients presented grade 3 or 4 adverse events related to TG4010 and other study treatments (chemotherapy or bevacizumab) versus 11 (10%) in the placebo group. No serious adverse event was related to the combination of TG4010 with other study treatments. The most frequent severe adverse events were neutropenia (grade 3 29 [26%], grade 4 13 [12%] in the TG4010 group vs grade 3 22 [21%], grade 4 11 [10%] in the placebo group), anaemia (grade 3 12 [11%] vs grade 3 16 [15%]), and fatigue (grade 3 12 [11%], grade 5 one [1%] vs grade 3 13 [12%]; no grade 4 events)., Interpretation: TG4010 plus chemotherapy seems to improve progression-free survival relative to placebo plus chemotherapy. These data support the clinical value of the TrPAL biomarker in this clinical setting; because the primary endpoint was met, the trial is to continue into the phase 3 part., Funding: Transgene, Avancées Diagnostiques pour de Nouvelles Approches Thérapeutiques (ADNA), and OSEO., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB)., Methods: TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status., Results: In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (i.e., capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range., Conclusions: Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Hepatitis B virus (HBV) infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Current therapies based on nucleos(t)ide analogs or pegylated-interferon-α lead to control of viral replication in most patients but rarely achieve cure. A potential strategy to control chronic hepatitis B is to restore or induce functional anti-HBV T-cell immune responses using HBV-specific immunotherapeutics. However, viral diversity is a challenge to the development of this class of products as HBV genotypes display a sequence diversity of up to 8%. We have developed a novel HBV-targeted immunotherapeutic, TG1050, based on a non-replicative Adenovirus vector encoding a unique and large fusion protein composed of multiple antigenic regions derived from a HBV genotype D sequence. Using peripheral blood mononuclear cells from 23 patients chronically infected by five distinct genotypes (gt A, B, C, D and E) and various sets of peptides encompassing conserved versus divergent regions of HBV core we have measured ability of TG1050 genotype D core-derived peptides to be recognized by T-cells from patients infected by various genotypes. Overall, PBMCs from 78% of genotype B or C- and 100% genotype A or E-infected patients lead to detection of HBV core-specific T-cells recognizing genotype D antigenic domains located both in conserved and variable regions. This proof-of-concept study supports the clinical development of TG1050 in large patient populations independently of infecting genotypes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: This study describes the effect of TG4010 vaccine on Health related Quality of Life (HRQOL) in patients with stage IIIb and IV non-small-cell lung cancer (NSCLC)., Methods: 148 patients with advanced NSCLC expressing MUC1 were randomly assigned to receive TG4010 plus chemotherapy or chemotherapy alone. HRQOL was assessed with the Functional Assessment of Cancer Therapy-Lung (FACT-L) at baseline and every 6 weeks until disease progression. Time until definitive deterioration (TUDD) of the four well-being dimensions of the FACT-L physical (PWB), functional (FWB), emotional (EWB) and social well-being (SWB) and the Lung Cancer Subscale (LCS) domains were analyzed for a 5-point minimal clinically important difference., Results: No difference of TUDD of HRQOL has been found between treatment arms. No prognostic factors have been found to have a significant impact on the TUDD of PWB, SWB and LCS domains. The gender, the performance status and the smoking habits seemed to be associated with a shorter TUDD of EWB domain. The smokers and the former smokers seemed to present a shorter TUDD of FWB domain., Conclusion: This study suggests that adding therapeutic vaccination with TG4010 to standard chemotherapy in patients with advanced NSCLC is associated with a similar evolution in HRQOL compared to chemotherapy alone.

Objective: Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti-CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA., Methods: CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis., Results: CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1- monocytes., Conclusion: CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA., (Copyright © 2014 by the American College of Rheumatology.)

Experimental evidence shows the importance of the immune system in controlling tumor appearance and growth. Immunotherapy is defined as the treatment of a disease by inducing, enhancing or suppressing an immune response. In the context of cancer treatment, it involves breaking tolerance to a cancer-specific self-antigen and/or enhancing the existing anti-tumor immune response, be it specific or not. Part of the complexity in developing such treatment is that cancers are selected to escape adaptive or innate immune responses. These escape mechanisms are numerous and they may cumulate in one cancer. Moreover, different cancers of a same type may present different combinations of escape mechanisms. The limited success of immunotherapeutics in the clinic as stand-alone products may in part be explained by the fact that most of them only activate one facet of the immune response. It is important to identify novel methods to broaden the efficacy of immunotherapeutics. Calcium signaling is central to numerous cellular processes, leading to immune responses, cancer growth and apoptosis induced by cancer treatments. Calcium signaling in cancer therapy and control will be integrated to current cancer immunotherapy approaches. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Development of active targeted immunotherapeutics is a rapid developing field in the arena of chronic infectious diseases. The question of repeated, closely spaced administration of immunotherapeutics to achieve a rapid impact on the replicating agent is an important one. We analyzed here, using a prototype adenovirus-based immunotherapeutic encoding Core and Polymerase from the hepatitis B virus (Ad-HBV), the influence of closely spaced repeated immunizations on the level and quality of induced HBV-specific and vector-specific immune responses in various mouse models. Ad-HBV, whether injected once or multiple times, was able to induce HBV- and adeno-specific T cells both in HBV-free mice and in a HBV tolerant mouse model. Adenovirus-specific T cell responses and titers of neutralizing anti-Ad5 antibodies increased from time of the 3rd injection. Interestingly, single or multiple Ad-HBV injections resulted in detection of Polymerase-specific functional T cells in HBV tolerant mice. Overall no modulation of the levels of HBV-specific cytokine-producing (IFNγ/TNFα) and cytolytic T cells was observed following repeated administrations (3 or 6 weekly injections) when compared with levels detected after a single injection with the exception of two markers: 1. the proportion of HBV-specific IFNγ-producing cells bearing the CD27+/CD43+ phenotype appeared to be sustained in C57BL/6J mice following 6 weekly injections; 2. the percentage of IFNγ/TNFα Core-specific producing cells observed in spleens of HLA-A2 mice as well as of that specific of Polymerase observed in livers of HBV tolerant mice was maintained. In addition, percentage of HBV-specific T cells expressing PD-1 was not increased by multiple injections. Overall these data show that, under experimental conditions used, rapid, closely spaced administrations of an adenovirus-based HBV immunotherapeutics does not inhibit induced T-cell responses including in a HBV-tolerant environment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Telaprevir-based therapy is associated with rapid decline in HCV RNA, enabling the application of early futility rules., Objectives: To familiarize physicians with this paradigm, a comprehensive analysis of the most frequent HCV viral load profiles observed during treatment with telaprevir/Peg-IFN/RBV in Phase III trials is provided., Design: HCV RNA profiles were analyzed from 320 HCV genotype 1 treatment-naïve patients enrolled in the ADVANCE study, and 225 prior Peg-IFN/RBV treatment-experienced patients enrolled in the REALIZE study. Patients received 12 weeks of telaprevir with either 24 or 48 weeks of Peg-IFN alfa-2a/RBV. Patients with missing SVR assessments during follow-up, detectable HCV RNA at end of treatment but who did not have viral breakthrough (vBT), or with early vBT who discontinued telaprevir before time of failure were excluded., Results: All analyzed patients experienced a rapid decline in HCV RNA (>2.0 log(10)) by Day 14, irrespective of baseline characteristics and/or prior response to Peg-IFN/RBV (relapse, partial response and null response). Subsequently, HCV RNA continued to decline to undetectable levels in most patients. These patients went on to have one of the following outcomes: sustained virologic response, late vBT (after Week 12, i.e. during the Peg-IFN/RBV phase), or relapse. In the small subset of patients with early vBT or meeting a futility rule before Week 12, HCV RNA usually never became undetectable and/or increased rapidly after reaching the nadir., Conclusions: HCV RNA profiles with telaprevir/Peg-IFN/RBV are different from those with Peg-IFN/RBV alone. It is important that clinicians understand these HCV RNA profiles and monitor patient viral load in order to apply futility rules correctly., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: We explored the concept of heterologous prime/boost vaccination using 2 therapeutic vaccines currently in clinical development aimed at treating chronically infected hepatitis C virus (HCV) patients: prime with a DNA-based vaccine expressing HCV genotype 1a NS3/4A proteins (ChronVac-C) and boost with a modified vaccinia virus Ankara vaccine expressing genotype 1b NS3/4/5B proteins (MVATG16643)., Methods: Two ChronVac-C immunizations 4 weeks apart were delivered intramuscularly in combination with in vivo electroporation and subsequently 5 or 12 weeks later boosted by 3 weekly subcutaneous injections of MVATG16643. Two mouse strains were used, and we evaluated quality, magnitude, and functionality of the T cells induced., Results: DNA prime/MVA boost regimen induced significantly higher levels of interferon γ (IFN-γ) or interleukin 2 (IL-2) ELISpot responses compared with each vaccine alone, independent of the time of analysis and the time interval between vaccinations. Both CD8⁺ and CD4⁺ T-cell responses as well as the spectrum of epitopes recognized was improved. A significant increase in polyfunctional IFN-γ/tumor necrosis factor α (TNF-α)/CD107a⁺ CD8⁺ T cells was detected following ChronVac-C/MVATG16643 vaccination (from 3% to 25%), and prime/boost was the only regimen that activated quadrifunctional T cells (IFN-γ/TNF-α/CD107a/IL-2). In vivo functional protective capacity of DNA prime/MVA boost was demonstrated in a Listeria-NS3-1a challenge model., Conclusions: We provide a proof-of-concept that immunogenicity of 2 HCV therapeutic vaccines can be improved using their combination, which merits further clinical development.

Therapeutic vaccines are different from the well-known prophylactic vaccines in that they are designed to treat patients already suffering from a disease instead of preventing the disease in healthy individuals. Several therapeutic vaccines are today in late-stage clinical development for non-small-cell lung cancer. These vaccines use different approaches including peptides, cell lines and viral vectors, and explore different settings within the pathology. Some are given in monotherapy while others are combined with the classic therapies used with non-small-cell lung cancer. This review gives a summary of the therapeutic vaccines currently in late-stage clinical development for non-small-cell lung cancer.

TG4010 is a therapeutic cancer vaccine based on a viral vector, a modified vaccinia of Ankara (MVA), expressing MUC1 as well as interleukine 2. Today the clinical development is focused on advanced non-small cell lung cancer in combination with first line chemotherapy. Potential biomarkers predictive of activity have been identified.

We have generated a thymidine kinase gene-deleted vaccinia virus (VV) (Copenhagen strain) that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. Intratumoral inoculation of this thymidine kinase gene-deleted VV encoding FCU1 (VV-FCU1) in the presence of systemically administered prodrug 5-fluorocytosine (5-FC) produced statistically significant reductions in the growth of subcutaneous human colon cancer in nude mice compared with thymidine kinase gene-deleted VV treatments or with control 5-fluorouracil alone. A limitation of prodrug therapies has often been the requirement for the direct injection of the virus into relatively large, accessible tumors. Here we demonstrate vector targeting of tumors growing subcutaneously following systemic administration of VV-FCU1. More importantly we also demonstrate that the systemic injection of VV-FCU1 in nude mice bearing orthotopic liver metastasis of a human colon cancer, with concomitant administration of 5-FC, leads to substantial tumor growth retardation. In conclusion, the insertion of the fusion FCU1 suicide gene potentiates the oncolytic efficiency of the thymidine kinase gene-deleted VV and represents a potentially efficient means for gene therapy of distant metastasis from colon and other cancers.

Tumor-associated antigens (TAAs) have been formulated into vaccines that, combined with adjuvants, cytokines or other strategies to boost the immune response, are now in clinical development. Both humoral and cellular immune responses to TAAs have been generated using these vaccines. This approach relies on the patient's own immune system generating an effective anti-tumor immune response. The advantage of this over therapy with monoclonal antibodies for the treatment of cancer is that multiple antigenic epitopes can be involved and the immune system is able to adapt to the most effective antigenic specificity for tumor growth control and rejection. In this article, we describe the clinical use of vaccinia virus, in particular modified vaccinia virus Ankara (MVA), to express TAAs in vivo and to stimulate an effective immune response to the cancer antigens.

Multiepitope-based vaccines against hepatitis C virus (HCV) were designed in the form of three minigenes encompassing four domains of the NS3, NS4 and NS5B proteins that contain multiple class I/II restricted epitopes. The polyEp-WT minigene encodes all four domains in fusion, the polyEp-C minigene encodes the same fusion but optimised for mammalian translation and the polyEp-E3 minigene has an additional endoplasmic reticulum targeting sequence. Whereas the minigenes vectorised by DNA were poorly immunogenic, adenovirus vectorisation induced strong and broader IFNgamma-ELISpot and CTL responses in HLA-A2 transgenic mice. In addition, polyEp-WT and polyEp-E3 responses were found cross-reactive in a recombinant Listeria-NS3-based surrogate challenge. This study illustrates the potency of vectorised minigenes in the field of HCV vaccine development.

Both hepatitis B and hepatitis C viruses (HBV and HCV) cause chronic infections worldwide that are associated with development of liver diseases ranging from mild liver inflammation to hepatocellular carcinomas. While efficient preventive vaccines are available for HBV, efforts are ongoing to develop one in case of HCV. Yet, both infections share the fact that therapeutic agents available to treat already established infections are yet poorly efficient, toxic or associated with development of resistance. Thus, novel immune-based therapies are actively being developed to complement or replace standard antiviral treatments. Among those, development of therapeutic vaccines represents a major effort. Peptide-, recombinant protein- or viral vector-based vaccines have been engineered and tested at preclinical and clinical levels. Means to adjuvant these vaccines are being pursued, including approaches based on combining vaccines of different nature. This review will outline major advances in the field of both HBV and HCV therapeutic vaccine development with a particular focus on candidates presented at the 12th International Symposium on Viral Hepatitis and Liver Disease (July 2006, Paris, France).