Nathalie Silvestre

TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice

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 (ie, 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.

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

Immunological Characterization of a Modified Vaccinia Virus Ankara Vector Expressing the Human Papillomavirus 16 E1 Protein

ABSTRACT Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8+ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate.

A Novel MVA-Based Multiphasic Vaccine for Prevention or Treatment of Tuberculosis Induces Broad and Multifunctional Cell-Mediated Immunity in Mice and Primates

Bacille Calmette-Guérin (BCG) vaccination of new born babies can protect children against tuberculosis (TB), but fails to protect adults consistently against pulmonary TB underlying the urgent need to develop novel TB vaccines. Majority of first generation TB vaccine candidates have relied on a very limited number of antigens typically belonging to the active phase of infection. We have designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara virus (MVA). Up to fourteen antigens representative of the three phases of TB infection (active, latent and resuscitation) were inserted into MVA. Using three different strains of mouse (BALB/c, C57BL/6 and C3H/HeN), we show that a single vaccination results in induction of both CD4 and CD8 T cells, displaying capacity to produce multiple cytokines together with cytolytic activity targeting a large array of epitopes. As expected, dominance of responses was linked to the mouse haplotype although for a given haplotype, responses specific of at least one antigen per phase could always be detected. Vaccination of non-human primates with the 14 antigens MVA-TB candidate resulted in broad and potent cellular-based immunogenicity. The remarkable plasticity of MVA opens the road to development of a novel class of highly complex recombinant TB vaccines to be evaluated in both prophylactic and therapeutic settings.

Comparative analysis of immunization schedules using a novel adenovirus-based immunotherapeutic targeting hepatitis B in naïve and tolerant mouse models

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.

A multi-antigenic MVA vaccine increases efficacy of combination chemotherapy against Mycobacterium tuberculosis

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.

A meta-analysis of the antiviral activity of the HBV-specific immunotherapeutic TG1050 confirms its value over a wide range of HBsAg levels in a persistent HBV pre-clinical model

ABSTRACT 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.

Abstract LB-287: Pseudocowpox: A next generation viral vector for cancer immunotherapy

Engineered viral vectors are effective approaches to stimulate anti-tumor immunity, and change the tumor immune environment. Several viruses and strains have been developed to express tumor antigens and cytokines, and corresponding products are in advanced clinical trials. However, novel viral strains with improved immunogenic properties are sought. In this perspective, we screened a variety of poxviridae potentially usable in humans: Cowpox (CPX), Pseudocowpox (PCPV), Parapoxvirus Ovis (ORF), Myxoma virus (MYX), Swinepox (SWP), Yaba-like disease virus (YLDV), Raccoonpox (RCN), Cotia virus (CTV), and compared them to the well-established vaccine strain Modified Virus Ankara (MVA), and oncolytic Vaccinia Virus, strain Copenhagen (VV). Both in vitro with human primary immune cells, and in vivo with syngeneic mouse tumor models, PCPV proved to be a very promising vector for immunotherapy. Compared with MVA, PCPV induced a 1000-fold higher expression of IFN-alpha in human PBMCs, whereas SWPV and ORF displayed a lower 10 to 100-fold induction. Other viruses (i.e. VV, RCN, CTV, or MYX) did not raise the IFN-alpha level. PCPV was also shown to increase the level of GM-CSF, and to be safe for PBMCs, in contrast to ORF which displayed some cytotoxic effects. When tested for its capacity to trigger the expression of CD86, a co-stimulatory factor for T-cell activation, PCPV was shown to be superior than MVA in primary moDCs. Furthermore, PCPV treatment increased CD86 expression in human CD163+CD206+ “M2”-type macrophages derived from CD14+ monocytes, suggesting a shift to an antigen-presenting phenotype. In these cells, PCPV increased significantly the secretion of IL-18, IL-6 and IP-10, signing a conversion towards a less suppressive macrophage phenotype. Last, incubation of PCPV in a co-culture model overcame the immunosuppressive effect of MDSCs on human autologous CD8+ T. A GFP-encoding recombinant PCPV vector was generated, and we could demonstrate that PCPV was capable of infecting human primary immune cells, comparably to recombinant MVA vectors, except for activated T cells. A recombinant PCPV encoding for the HPV E7 protein was generated to assess the anti-tumor activity and immunogenicity in a syngeneic murine tumor model. Like MVA-E7, PCPV-E7 induced a strong cellular response (ELISPOT on splenocytes, and frequency of antigen-specific short-lived effector cells), but PCPV-E7 displayed a different cytokine/chemokine profile at the site of injection, with increased levels of pro-immune cytokines including IP-10, IFN-gamma, GM-CSF, IL-18, MIP-1 alpha, MIP-1 beta, IL-12 and IL-6. When injected intratumorally into fast growing MC-38 tumors, PCPV led to tumor control. Analysis of tumors infiltrates showed that PCPV treatment led to higher levels of neutrophils and decreased the frequency of MHCIIlo TAMs. Our data demonstrate that PCPV might display better properties than current viral vectors, in terms of local response and priming activity, of ability to induce effector T cells and to reshape the tumor infiltration profiles. It has the same capacity as other poxviruses to encode and deliver large genetic payload, which will be useful for designing advanced anti-tumor vaccines. Citation Format: Karola Rittner, Marine Ricordel, Caroline Tosch, Christine Thioudellet, Christelle Remy-Ziller, Marie-Christine Claudepierre, Chantal Hoffmann, Doris Schmitt, Benoit Sansas, Johann Foloppe, Philippe Erbs, Nathalie Silvestre, Kaidre Bendjama, Eric Quemeneur. Pseudocowpox: A next generation viral vector for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-287.

Abstract 2352: Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted in the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The 3 purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e. without tumor) confirming the virus tropism for tumoral cells and/or that tumoral microenvironment allows virus escape from immune surveillance. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mPD-1, than after IT administration of 10 μg of J43. Interestingly, in the MCA 205 tumor model, WR-mPD-1 (both mAb and scFv) induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that provided by an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. New generation of oncolytic vaccinia virus that will express several transgenes simultaneously may also be designed with the goal of providing to the patients enhanced therapeutic/toxicity ratio. Citation Format: Jean-Baptiste Marchand, Patricia Kleinpeter, Laetitia Fend, Christine Thioudellet, Michel Geist, Nathalie Sfrontato, Veronique Koerper, Renee Brandely, Dominique Villeval, Karola Rittner, Nathalie Silvestre, Philippe Erbs, Laurence Zitvogel, Eric Quemeneur, Xavier Preville. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2352.