Replication-competent Virus Vectors Research Articles

A replication-competent smallpox vaccine LC16m8Δ-based COVID-19 vaccine

ABSTRACT Viral vectors are a potent vaccine platform for inducing humoral and T-cell immune responses. Among the various viral vectors, replication-competent ones are less commonly used for coronavirus disease 2019 (COVID-19) vaccine development compared with replication-deficient ones. Here, we show the availability of a smallpox vaccine LC16m8Δ (m8Δ) as a replication-competent viral vector for a COVID-19 vaccine. M8Δ is a genetically stable variant of the licensed and highly effective Japanese smallpox vaccine LC16m8. Here, we generated two m8Δ recombinants: one harbouring a gene cassette encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, named m8Δ-SARS2(P7.5-S)-HA; and one encoding the S protein with a highly polybasic motif at the S1/S2 cleavage site, named m8Δ-SARS2(P7.5-SHN)-HA. M8Δ-SARS2(P7.5-S)-HA induced S-specific antibodies in mice that persisted for at least six weeks after a homologous boost immunization. All eight analysed serum samples displayed neutralizing activity against an S-pseudotyped virus at a level similar to that of serum samples from patients with COVID-19, and more than half (5/8) also had neutralizing activity against the Delta/B.1.617.2 variant of concern. Importantly, most serum samples also neutralized the infectious SARS-CoV-2 Wuhan and Delta/B.1.617.2 strains. In contrast, immunization with m8Δ-SARS2(P7.5-SHN)-HA elicited significantly lower antibody titres, and the induced antibodies had less neutralizing activity. Regarding T-cell immunity, both m8Δ recombinants elicited S-specific multifunctional CD8+ and CD4+ T-cell responses even after just a primary immunization. Thus, m8Δ provides an alternative method for developing a novel COVID-19 vaccine.

Abstract 74: Engineering tertiary lymphoid structures for glioblastoma: A novel gene combination promotes therapeutic TLS formation in an immune-competent mouse model of GBM

Abstract The absence of conventional lymphatic access to the brain parenchyma restricts the capacity of the peripheral immune system to recognize and elicit durable responses against glioma antigens. In some peripheral solid tumor types and autoimmune diseases such as multiple sclerosis, the spontaneous development of tertiary lymphoid structures (TLS) with varying degrees of organization have been observed in human patients and mice experiencing chronic inflammation. The presence of TLS in the context of cancer is generally associated with improved prognosis, particularly when these TLS are characterized by intratumoral infiltration of CD8+ T-cells and B-cells. We sought to engineer the development of TLS in the brain tumor setting, utilizing our SB28 glioblastoma (GBM) model which is sparsely infiltrated by lymphocytes and characterized by a significant myeloid-derived suppressor cell population. To determine the most critical chemokines and cytokines required to elicit TLS formation in the murine GBM context, we stably transduced SB28 with a combination of TLS-stimulating factors that we identified and injected these cells into the brain parenchyma of syngeneic C57BL/6J mice. A combination of the chemoattractant and lymphoid follicle-stimulating cytokines interleukin (IL)-7, LIGHT (TNFSF14), CCL21, and IL-17 promoted infiltration of CD8+CD3+ T-cells into the tumor and nearby parenchyma, but it also promoted enhanced tumor proliferation. A modified gene combination including IL-7, LIGHT, and CCL21 promoted CD8+ T-cell infiltration and clustering by immunofluorescence analysis. This combination also significantly decreased tumor burden as measured by bioluminescent imaging, resulting in complete tumor rejection in a subset of mice. Finally, modifying this combination to include CXCL13 promoted the infiltration and clustering of B-cells along with the observed CD8+ T-cell infiltration. This combination elicited the formation of vascular structures that stained for PNAd (MECA-79) and LYVE-1, markers for high endothelial venules (HEV) and lymphatic endothelium, respectively. Taken together, these analyses suggest that these factors are sufficient to stimulate the formation of TLS in the GBM setting. Future studies will evaluate a replication competent viral vector based approach for gene delivery and whether TLS formation can promote tumor specific T-cell memory and persistence in GBM. Ultimately, we aim to promote therapeutic TLS in a manner that is highly translational to brain tumor patients and complementary to existing T-cell therapies. Citation Format: Kira Morgan Downey, Bindu Hegde, Zinal Chheda, Jason Zhang, Hideho Okada. Engineering tertiary lymphoid structures for glioblastoma: A novel gene combination promotes therapeutic TLS formation in an immune-competent mouse model of GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 74.

A Protein Subunit Filovirus Vaccine Facilitates Maternal Immunization in Mice

Abstract Ebola virus disease (EVD) is highly pathogenic and virulent and has caused large scale outbreaks in Central and West Africa. To date there are no known correlates of protection however, recently the FDA and EMA have approved a vaccine, Ervebo. This vaccine utilizes a replication competent viral vector which may be contraindicated in immunocompromised individuals, pregnant women, and infants. Our lab has developed a recombinant protein subunit vaccine, a platform that is generally considered safe for immunocompromised, pregnant women, and infants. Our vaccine, when properly adjuvanted, demonstrates protection in non-human primates and may also induce maternal immunization. Maternal Immunization is the phenomena of inducing passive immunity via maternal antibody. This occurs via transfer of IgG using the FcRn. This receptor binds the Fc portion of IgG in a pH dependent manner which facilitates transcytosis of IgG across the placental, mammary tissue, as well as gut epithelial layer. In rodents, maternal IgG is transferred across the gut epithelia throughout the nursing period. This project demonstrates in mice the immunogenicity of a protein subunit vaccine comprised of Ebola glycoprotein and adjuvant administered during gestation as well as the transfer of antigen specific maternal antibody to offspring through milk. To investigate maternal immunization we vaccinated female Swiss Webster mice during gestation and measured antigen specific IgG levels in both the mothers and pups as well as in the milk. Antigen specific maternal antibody titers found in pup sera mimic those found in fully immunized adult females weeks after weaning, indicating the potential for maintaining an antigen specific humoral response in neonates.

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization.

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

The Science is There: Key Considerations for Stabilizing Viral Vector-Based Covid-19 Vaccines.

Once Covid-19 vaccines become available, 5–10 billion vaccine doses should be globally distributed, stored and administered. In this commentary, we discuss how this enormous challenge could be addressed for viral vector-based Covid-19 vaccines by learning from the wealth of formulation development experience gained over the years on stability issues related to live attenuated virus vaccines and viral vector vaccines for other diseases. This experience has led –over time– to major improvements on storage temperature, shelf-life and in-use stability requirements. First, we will cover work on ‘classical’ live attenuated virus vaccines as well as replication competent viral vector vaccines. Subsequently, we address replication deficient viral vector vaccines. Freeze drying and storage at 2–8 °C with a shelf life of years has become the norm. In the case of pandemics with incredibly high and urgent product demands, however, the desire for rapid and convenient distribution chains combined with short end-user storage times require that liquid formulations with shelf lives of months stored at 2–8 °C be considered. In confronting this “perfect storm” of Covid-19 vaccine stability challenges, understanding the many lessons learned from decades of development and manufacturing of live virus-based vaccines is the shortest path for finding promising and rapid solutions.

Abstract 4058: TheraT - a highly versatile arenavirus based vector platform for intravenous and intratumoral cancer immunotherapy

Abstract The unprecedented success of checkpoint blockade therapies clearly demonstrates the power of the immune system to fight cancer. Yet, only a minority of cancer patients respond with long-term control of the tumor or even cure, necessitating the development of other treatment modalities. Hookipa Pharma developed a novel attenuated, replication-competent viral vector platform (TheraT) that induces powerful cytotoxic T lymphocyte responses against foreign and self-antigens. In a preclinical model for human papilloma virus 16 (HPV16) associated cancer (TC-1), we evaluated immunogenicity and efficacy upon systemic and intratumoral application of TheraT vectors based on the arenaviruses lymphocytic choriomeningitis virus (LCMV) and pichinde virus (PICV). Both HB-201 (LCMV) and HB-202 (PICV) product candidates encode a non-oncogenic but highly antigenic E6/E7 fusion protein from HPV16. Independent of the route of administration, single administration of HB-201 or HB-202 induced potent peripheral E7-specific CD8+ T cell responses and led to efficient tumor growth control. Survival of TheraT treated animals was significantly longer compared to buffer treated animals. Similarly, single systemic as well as intratumoral application of HB-201 or HB-202 induced major infiltration of CD8+ T cells into the tumor microenvironment. Combination of intravenous HB-201 and anti-PD1 was well tolerated but did not further enhance efficacy in this model, implicating the presence of other immune evasion factors. In conclusion, replication-attenuated TheraT is safe, highly immunogenic and shows excellent therapeutic efficacy after single intravenous and intratumoral application. These data underline the potential and versatility of this novel vector platform. Mechanistic studies in various mouse tumor models are underway. Phase 1/2 clinical trial initiation of HB-201 monotherapy is planned for end of 2019 and preparations for an IND filing for a combination trial of HB-201 and HB-202 in H1 2020 have been initiated. Citation Format: Josipa Raguz, Sarah Schmidt, Theresa Kleisner, Manuel Zerbs, Goran Bekic, Sonja Feher, Daniel Oeler, Felix Stemeseder, Ursula Berka, Bettina Kiefmann, Sophie Schulha, Igor Matushansky, Henning Lauterbach, Klaus Orlinger. TheraT - a highly versatile arenavirus based vector platform for intravenous and intratumoral cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4058.

Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.

Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

Abstract B37: VSV-GP: A vaccine vector and oncolytic virus

Abstract Oncolytic viruses have recently shown significant clinical benefits in several cancers. Interestingly, in addition to debulking the tumor by direct oncolysis, they were found to act as a cancer vaccine by setting free tumor antigen in the presence of virus-induced danger signals, which have an adjuvant effect. However, as oncolytic viruses induce neutralizing antibodies that inactivate the virus, oncolytic viruses are not delivered efficiently to cancer tissue upon repeated systemic application. Thus, repeated applications of oncolytic viruses have been restricted to intratumoral injections, which clinically has not been convincingly effective in metastatic disease. We generated the oncolytic virus VSV-GP (Muik et al., Cancer Res, 2014) that is highly potent, lacks toxicity even at high doses (so far tested in mice and rabbits), can be produced at high titers and, above all, does not induce neutralizing antibodies readily. Thus, VSV-GP is unique among the oncolytic viruses in that it can be applied repeatedly without loosing efficacy. VSV-GP is highly effective leading to cure in several cancer mouse models, i.e. malignant glioma, melanoma, prostate cancer and ovarian cancer. In addition to oncolysis, VSV-GP induced a sound anti-tumor immune response and acted synergistically with other cancer immunotherapeutics. Moreover, VSV-GP was shown to be a highly promising vaccine vector and is the only replication competent viral vaccine vector that efficiently boosts the immune response upon repeated application. VSV-GP is thus a highly versatile replication competent viral vector platform for vaccination and cancer therapy. Citation Format: Alexander Muik, Janine Kimpel, Reinhard Tober, Carles Urbiola, Dorothee von Laer. VSV-GP: A vaccine vector and oncolytic virus. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B37.

VSV-GP: a potent viral vaccine vector that boosts the immune response upon repeated applications.

Antivector immunity limits the response to homologous boosting for viral vector vaccines. Here, we describe a new, potent vaccine vector based on replication-competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP), which we previously showed to be safe in mice. In mice, VSV and VSV-GP encoding ovalbumin (OVA) as a model antigen (VSV-OVA and VSV-GP-OVA) induced equal levels of OVA-specific humoral and cellular immune responses upon a single immunization. However, boosting with the same vector was possible only for VSV-GP-OVA as neutralizing antibodies to VSV limited the immunogenicity of the VSV-OVA boost. OVA-specific cytotoxic T-lymphocyte (CTL) responses induced by VSV-GP-OVA were at least as potent as those induced by an adenoviral state-of-the-art vaccine vector and completely protected mice in a Listeria monocytogenes challenge model. VSV-GP is so far the only replication-competent vaccine vector that does not lose efficacy upon repeated application. Although there has been great progress in treatment and prevention of infectious diseases in the past several years, effective vaccines against some of the most serious infections, e.g., AIDS, malaria, hepatitis C, or tuberculosis, are urgently needed. Here, several approaches based on viral vector vaccines are under development. However, for all viral vaccine vectors currently in clinical testing, repeated application is limited by neutralizing antibodies to the vector itself. Here, we have exploited the potential of vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) as a vaccine platform. VSV-GP is the first replication-competent viral vector vaccine that does not induce vector-specific humoral immunity, i.e., neutralizing antibodies, and therefore can boost immune responses against a foreign antigen by repeated applications. The vector allows introduction of various antigens and therefore can serve as a platform technology for the development of novel vaccines against a broad spectrum of diseases.

Safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for H5N1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study

SummaryBackgroundReplication-competent virus vector vaccines might have advantages compared with non-replicating vector vaccines. We tested the safety and immunogenicity of an oral adenovirus serotype 4 vector vaccine candidate (Ad4-H5-Vtn) expressing the haemagglutinin from an avian influenza A H5N1 virus.MethodsWe did this phase 1 study at four sites in the USA. We used a computer-generated randomisation list (block size eight, stratified by site) to assign healthy volunteers aged 18–40 years to receive one of five doses of Ad4-H5-Vtn (107 viral particles [VP], 108 VP, 109 VP, 1010 VP, 1011 VP) or placebo (3:1). Vaccine or placebo was given on three occasions, about 56 days apart. Participants, investigators, and study-site personnel were masked to assignment throughout the study. Subsequently, volunteers received a boost dose with 90 μg of an inactivated parenteral H5N1 vaccine. Primary immunogenicity endpoints were seroconversion by haemagglutination-inhibition (HAI), defined as a four-times rise compared with baseline titre, and HAI geometric mean titre (GMT). We solicited symptoms of reactogenicity daily for 7 days after each vaccination and recorded symptoms that persisted beyond 7 days as adverse events. Primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01006798.FindingsWe enrolled 166 participants (125 vaccine; 41 placebo) between Oct 19, 2009, and Sept 9, 2010. HAI responses were low: 13 of 123 vaccinees (11%, 95% CI 6–17) and three of 41 placebo recipients (7%, 2–20) seroconverted. HAI GMT was 6 (95% CI 5–7) for vaccinees, and 5 (5–6) for placebo recipients. However, when inactivated H5N1 vaccine became available, one H5N1 boost was offered to all participants. In this substudy, HAI seroconversion occurred in 19 of 19 participants in the 1011 VP cohort (100%; 95% CI 82–100) and eight of 22 placebo recipients (36%; 17–59); 17 of 19 participants in the 1011 VP cohort (89%; 67–99) achieved seroprotection compared with four of 22 placebo recipients (18%; 5–40); GMT was 135 (89–205) with 1011 VP, compared with 13 (7–21) with placebo. The cumulative frequency of abdominal pain, diarrhoea, and nasal congestion after all three vaccinations was significantly higher in vaccinees than placebo recipients (21 [16·8%] of 125 vs one [2·4%] of 41, p=0·017; 24 [19·2%] of 125 vs two [4·9%] of 41, p=0·027; 41 [32·8%] of 125 vs six [14·6%] of 41, p=0·028; respectively). No serious treatment-related adverse events occurred.InterpretationOral Ad4 vector priming might enhance the efficacy of poorly immunogenic vaccines such as H5N1.FundingWellcome Trust Foundation, PaxVax.

Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein

Neutrophil-activating protein (NAP) is a major virulence factor expressed by Helicobacter pylori isolates associated with severe chronic gastroduodenal inflammation and peptic ulcers. NAP is one of the main protective antigens and a target for vaccine development against Helicobacter infection. In addition, NAP is a potent immune stimulator with potential application as a general vaccine adjuvant and in treatment of allergic diseases and cancer immunotherapy. NAP-specific immunoassays are needed for both H. pylori diagnostics and characterization of NAP-based vaccines and immunomodulatory preparations. We generated a panel of NAP-specific monoclonal antibodies (MAbs) by immunization of BALB/c mice with synthetic NAP peptides. The antibody reactivity against recombinant or native NAP antigen was characterized by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence. A sensitive capture ELISA was developed using MAbs 23C8 and 16F4 (directed against different NAP epitopes) for detection of native or measles virus (MV) vector-expressed recombinant NAP in a concentration range of 4ng/ml to 2000ng/ml. MAb 23C8 antigen-binding depends on Tyr101 in a variable amino acid sequence of the NAP molecule, indicating the existence of antigenic variants among H. pylori strains. MAb 16F4 reacted with NAP from different H. pylori strains and was a sensitive tool for detection of small amounts of isolated NAP antigen or whole bacteria by immunoblotting or immunofluorescence. In conclusion, MAb-based immunoassays are highly specific and sensitive for detection of native NAP antigen and recombinant NAP immunostimulatory transgenes expressed by replication competent virus vectors.

Abstract C185: Head and neck squamous carcinoma cells are susceptible to the attenuated vesicular stomatitis virus expressing suicide genes

Abstract Background: The predominant loco-regional progression of Head and Neck Squamous Cell Carcinoma (HNSCC) makes it suitable for testing oncolytic viruses. Recent work from several laboratories including ours has focused on developing vesicular stomatitis virus (VSV) as a replication-competent anticancer viral vector. Attenuated strains, such as M protein mutant VSV (M51R) are more likely to be promoted in clinical research. The major shortcoming of viral therapy is related to the large proportion of tumor cells remaining uninfected. We are addressing this limitation in two ways: the expression of the cytosine deaminase-uracil phosphoribosyltransferase (C:U) suicide gene by the rVSV vector, expected to produce a strong bystander effect and the exploitation of the radiosensitizing potential of the 5-fluorouracil (5-FU) chemo-drug produced intratumorally, as well as of the VSV virus itself. Methods: We determined the susceptibility to oncolytic VSV in a collection of HNSCC cell lines and in patient HN primary tumor cells by measuring: 1) the level of GFP expression after infection with rVSV-GFP and M51R-GFP; 2) the viral production by plaque assay and 3) cell death by Trypan blue exclusion. The extent of defectiveness in interferon pathways was tested by employing pre-treatment with IFN and INF-blocking antibodies before the infection with the oncolytic VSV. We have generated the rVSV-C:U and M51R-C:U viruses and we tested our collection of HNSCC cell models to infection with these viruses in the presence and absence of 5-fluorocytosine. SQ20B cell line is a model of radiation resistant HNSCC. Regular SQ20B and SQ20B infected with low-dose M51R-VSV were evaluated by clonogenic assay. Results: 1) All HNSCC cell models tested are susceptible to the infection with the rVSV but many present a degree of resistance to the attenuated M51R-VSV. 2) Human Primary HNSCC cells are more resistant than the HNSCC cell lines to the M51R-VSV; 3) The activation of IFN pathways is contributing to the resistance to M51R-VSV. 4) Addition of the suicide gene C:U to the attenuated VSV model significantly increased the oncolytic effect in vitro; 5) HNSCC cells infected with M51R-VSV become more radiation sensitive. Conclusions: The decreased oncolytic effect of the attenuated M51R-VSV can be overcome by the expression of suicide genes such as the yeast C:U fusion model. The demonstrated radiosensiting potential of the M51R-VSV virus as well as the known radiosensitizing effect of the generated chemotherapeutic 5-FU can be further exploited against the loco-regional HNSCC tumors. Finally, we have important new information, both from cell lines and patient tissues, for proof-of-principle that cancers can down-regulate antiviral responses. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C185.

Stable expression of a foreign protein by a replication-competent rubella viral vector

Live, attenuated rubella vaccine has been used successfully for many years. By expressing additional viral antigens in rubella, we could expand its range and utility as a live, replicating viral vector. Previously, limitations on insert size and stability restricted rubella's ability to express exogenous antigens and immunize against other viruses. In this study, we have overcome this problem by creating a deletion in non-structural protein P150 that makes room for the insert. The resulting rubella hybrid stably expressed a model protein for over 10 passages, while replicating and expressing rubella proteins normally. The foreign protein, GFP, was as large as many important viral antigens, and the virus grew to sufficiently high titers for vaccine use. Further progress in expressing exogenous viral antigens in rubella may produce live viral vectors capable of immunizing against viruses for which attenuation is not currently feasible.

Susceptibility of head and neck cancer to an attenuated model of the oncolytic vesicular stomatitis virus expressing suicide genes

e14604 Background: The predominant loco-regional progression of head and neck squamous cell carcinoma (HNSCC) makes it suitable for testing oncolytic viruses. Recent work from several laboratories including ours has focused on developing vesicular stomatitis virus (VSV) as a replication-competent anticancer viral vector. Attenuated strains, such as M protein mutant VSV (M51R) are more likely to be promoted in clinical research. The major shortcoming of viral therapy is related to the large proportion of tumor cells remaining uninfected. We are addressing this limitation in two ways: the expression of the cytosine deaminase-uracil phosphoribosyltransferase (C:U) suicide gene by the rVSV vector, expected to produce a strong bystander effect and the exploitation of the radiosensitizing potential of the 5-FU chemo-drug produced intratumorally, as well as of the VSV virus itself. Methods: We determined the extent of defectiveness in interferon pathways and susceptibility to oncolytic VSV in a collection of HNSCC cell lines and in patient HN primary tumor cells. We employed gene array to screen for differences in gene expression in sensitive and resistant models. We have generated the rVSV-C:U and M51R-C:U viruses and we tested our collection of HNSCC cell models to infection with these viruses in the presence and absence of 5-FC. In vivo experiments were performed on a localized and on a metastatic SQ-20B model of HNSCC in nude mice. Results: 1) the primary patient HNC cells are more resistant than the HNC cell lines to the M51R-VSV; 2) the activation of IFN pathways and /or alterations of AKT pathway is contributing to resistance to VSVs. 3) Addition of the suicide gene to the attenuated VSV provided significantly more oncolytic effect in vitro and in vivo. The efficacy and the radiosensitizing potential of M51R- C:U on a nude mice model of HNSCC (SQ-20B) will be presented. Conclusions: M51R-VSV has increased efficiency and radiosensitizing effect on HNSCC models. We can define an optimal approach to testing VSV vectors for treatment of HNSCC, with a clinical trial in HNSCC patients as our next goal. Finally, we have important new information, both from cell lines and patient tissues, for proof-of-principle that cancers can down-regulate antiviral responses. No significant financial relationships to disclose.

Controlled propagation of replication-competent Sindbis viral vector using suicide gene strategy

A major concern of using viral gene therapy is the potential for uncontrolled vector propagation and infection that might result in serious deleterious effects. To enhance the safety, several viral vectors, including vectors based on Sindbis virus, were engineered to lose their capability to replicate and spread after transduction of target cells. Such designs, however, could dramatically reduce the therapeutic potency of the viral vectors, resulting in the need for multiple dosages to achieve treatment goals. Earlier, we showed that a replication-defective (RD) Sindbis vector achieved specific tumor targeting without any adverse effects in vivo. Here, we present a replication-competent Sindbis viral vector that has an hsvtk suicide gene incorporated into ns3, an indispensable non-structural gene for viral survival. The capability of viral propagation significantly increases tumor-specific infection and enhances growth suppression of tumor compared with the conventional RD vectors. Furthermore, in the presence of the prodrug ganciclovir, the hsvtk suicide gene serves as a safety mechanism to prevent uncontrolled vector propagation. In addition to suppressing vector propagation, toxic metabolites, generated by prodrug activation, could spread to neighboring uninfected tumor cells to further enhance tumor killing.

Antibody-mediated targeting of replication-competent retroviral vectors.

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

Replication-Competent Herpes Simplex Virus Vector G207 and Cisplatin Combination Therapy for Head and Neck Squamous Cell Carcinoma

Replication-competent virus vectors are attractive therapeutic agents for cancer. G207, a second-generation, multimutated herpes simplex virus type 1 (HSV-1), is one such vector that is safe in primates and efficacious against human tumors in athymic mice. Squamous cell carcinoma is the most frequently encountered malignancy of the head and neck, and the chemotherapeutic agent cisplatin is a standard treatment for recurrent head and neck cancer. In this study we examine the therapeutic potential of G207, alone and in combination with cisplatin, against squamous cell carcinoma. Human squamous cell carcinoma cell lines are sensitive to G207 replication and cytotoxicity in vitro at a multiplicity of infection of 0.01, including cisplatin sensitive (UMSCC-22A), moderately sensitive (UMSCC-38), and weakly sensitive (SQ20B) cell lines. Cisplatin did not inhibit the cytopathic effect of G207. G207 inhibited the growth of established subcutaneous head and neck tumors in athymic mice. The therapeutic effects of cisplatin and G207 in vivo were independent. However, in cisplatin-sensitive tumors (UMSCC-38), combination therapy resulted in 100% cures in contrast to 42% with G207 or 14% with cisplatin alone. We conclude that G207 should be considered for the treatment of head and neck cancer and that combination with chemotherapeutic agents may improve efficacy.

Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity.

The success of cancer gene therapy is likely to require the targeting of multiple antitumor mechanisms. One strategy involves the use of attenuated, replication-competent virus vectors, such as herpes simplex virus type 1 (HSV-1) mutant G207, which is able to replicate in human tumor cells with resultant cell death and tumor growth inhibition, yet is nonpathogenic in normal tissue. In this study, we demonstrate that infection of established tumors with G207 also induces a highly specific systemic anti-tumor immune response. In a syngeneic, bilateral established subcutaneous tumor model, with mouse CT26 colorectal carcinoma cells in BALB/c mice or M3 melanoma cells in DBA/2 mice, unilateral intratumoral inoculation with G207 caused a significant reduction in the growth of both the inoculated and contralateral noninoculated tumors. This elicited anti-tumor response is dependent on viral infection of the tumor, as intradermal inoculation of G207 in BALB/c mice had no effect on CT26 tumor growth. Treatment of subcutaneous CT26 tumors by intratumoral inoculation of G207 induced a tumor-specific T cell response. CD8+ cytotoxic T lymphocyte (CTL) activity was generated that recognized a dominant "tumor-specific" major histocompatibility complex (MHC) class I-restricted epitope (AH1) from CT26 cells. In immune-competent animals, G207 is acting as an in situ tumor vaccine. Therefore, intratumoral G207 inoculation is able to inhibit tumor growth both by local cytotoxic viral replication in tumor cells and induction of a systemic anti-tumor immune response.

Gene therapy and viral vaccination: the interface

Live viral vaccines have had a major impact on the incidence of acute virus infections world-wide. Virus infections recognised as future vaccine targets will require a modified approach based on a detailed understanding of the immunobiology of specific infections combined with the application of new technologies designed to generate specific and appropriate protective immunity. A similar vector technology directed at in vivo gene delivery is currently being exploited both for gene therapy and vaccination. The induction of an immune response to an expressed transgene represents a potential hazard for a gene therapy protocol but is the object of a vaccine strategy. In vivo gene delivery using replication-competent or replication-deficient viral vector systems and by direct transfer of naked DNA can generate an effective humoral, secretory and cell-mediated immune response to expressed transgenes.