Development of improved therapeutic mesothelin-based vaccines for pancreatic cancer.

Pancreatic cancer is the 5th leading cause of cancer deaths, and there are no effective treatments. We developed a poxvirus platform vaccine with improved immunogenicity and inserted the mesothelin gene to create an anti-mesothelin cancer vaccine. Mesothelin expression is mostly restricted to tumors in adult mammals and thus may be a good target for cancer treatment. We show here that the modified vaccinia virus Ankara (MVA) virus expressing mesothelin and the enhanced MVA virus missing the immunosuppressive A35 gene and expressing mesothelin were both safe in mice and were able to induce IFN-gamma secreting T cells in response to mesothelin expressing tumor cells. In addition, the MVA virus has oncolytic properties in vitro as it can replicate in and kill Panc02 pancreatic adenocarcinoma cell line tumor cells, even though it is unable to replicate in most mammalian cells. Deletion of the A35 gene in MVA improved T cell responses as expected. However, we were unable to demonstrate inhibition of Panc02 tumor growth in immunocompetent mice with pre-vaccination of mice, boosts, or even intratumoral injections of the recombinant viruses. Vaccine efficacy may be limited by shedding of mesothelin from tumor cells thus creating a protective screen from the immune system.

The effects of diets enriched in omega-3 polyunsaturated fatty acids on systemic vaccinia virus infection.

Omega-3 polyunsaturated fatty acids (PUFA, n-3 fatty acids), the key components of fish and flaxseed oils, are increasingly consumed by the public because of their potential health benefits and are available by prescription for hypertriglyceridemia. However, numerous studies have shown that these compounds are immunoregulatory and immunosuppressive and thus may increase susceptibility to infection. In this study, we tested the effects of the amount of fat and the types of fatty acid in the diet on infection by vaccinia virus, an acute infection that begins in the respiratory tract and spreads by viremia to internal organs. Male C57Bl6 mice (~5 week old) were fed for 3 weeks prior to infection and continuing during infection and recovery one of the following: 1) a normal low fat (13% kcal) diet, 2) a low fat diet containing n-3 PUFAs, 3) a high fat (41% kcal) diet rich in n-3 PUFAs, 4) a high fat n-6 PUFA diet, or 5) a high fat monounsaturated diet. We found no statistically significant differences in the susceptibility of mice to viral infection, morbidity, viral organ titers, recovery time, or mortality with these diets, indicating that, over this approximately 6-week time period, dietary fats did not substantially affect responses to poxviral infection.

Murine mesothelin: characterization, expression, and inhibition of tumor growth in a murine model of pancreatic cancer.

BACKGROUND: Mesothelin has attracted much interest as a tumor specific antigen; it has been reported to promote tumor development and to be a good target for cancer treatment. Most studies to date have used human mesothelin in immunocompromised mice. Since these models do not allow for study of the natural immune response to mesothelin expressing tumors, we have undertaken the characterization of mouse mesothelin so the effects of this protein can be assessed in immunocompetent mouse strains. METHODS: We analyzed mouse mesothelin expression, tissue distribution, shedding and biochemistry. In addition we constructed stable mesothelin overexpressing lines of the pancreatic cancer line Panc02 by two methods and tested them for growth and tumorigencity in vitro and in vivo. RESULTS: We show here that mouse mesothelin is similar to human mesothelin in biochemical characteristics, tumor expression and tissue distribution, suggesting the mouse may be a suitable model for study of mesothelin. Stable overexpression of mesothelin in a pancreatic cancer cell line did not increase cell proliferation or anchorage-independent growth in vitro, suggesting that mesothelin is not necessarily a tumor progression factor. Surprisingly overexpression of mesothelin inhibited tumor formation in vivo in immunocompetent mice. CONCLUSION: The mouse may be a good model for studying mesothelin in the context of an intact immune response. Mesothelin is not necessarily a tumor progression factor, and indeed mesothelin overexpression inhibited tumor growth in immunocompetent mice.

Genome sequence and comparative virulence of raccoonpox virus: the first North American poxvirus sequence.

We report here the complete genome sequence of raccoonpox virus (RCNV), a naturally occurring North American poxvirus. This is the first such North American sequence to the best of our knowledge, and the data showed that RCNV forms a new phylogenetic branch between orthopoxviruses and Yoka poxvirus. RCNV shared overall similarity in genome organization with orthopoxviruses, and the proteins in the central conserved region shared approximately 90  % amino acid identity with orthopoxviruses. RCNV proteins shared approximately 81  % amino acid identity with Yokapox virus proteins. RCNV is missing 10 genes normally conserved in orthopoxviruses, most of which are implicated in virulence. These gene deletions may explain the attenuated phenotype of RCNV in mammals. RCNV contained one unique genome region containing approximately 1 kb of DNA sequence that is not present in any reported poxvirus. It contained a unique ORF predicted to encode a protein with a transmembrane domain. RCNV replicates well in mammalian cells, is naturally attenuated and has been shown to be effective as a vaccine vector platform, so we further tested its safety. We showed here that RCNV is substantially more attenuated than even the highly attenuated VACV-A35Del mutant virus in pregnant, nude and severe combined immunodeficient (SCID) mouse models. RCNV was much safer in pregnant mice and was cleared rapidly from tissues, even in immunocompromised animals, whereas the VACV-A35Del mutant retains virulence and persists in tissues. Thus, RCNV is expected to be a superior vaccine vector for infectious diseases and cancer due to its excellent safety profile, reported vaccine efficacy and ability to replicate in mammalian cells.

Raccoonpoxvirus safety in immunocompromised and pregnant mouse models.

Numerous poxviruses infect humans and animal hosts, and a poxvirus vaccine with an improved safety profile is needed as the current vaccinia virus vaccine is contraindicated in individuals that have a history of eczema or heart disease, or are immunocompromised or pregnant. In addition, poxviruses make excellent vaccine vectors for other infectious diseases and cancer. Raccoonpoxvirus is a naturally occurring attenuated North American poxvirus, and thus it is of interest as a vaccine vector platform. This study explores the effects of raccoonpoxvirus in SCID and Nude immunocompromised and pregnant mouse models to assess its virulence and probable safety for human and animal populations. We also analyzed the safety of recombinant raccoonpox carrying a gene expressing a foreign antigen, rabies virus glycoprotein, designed for heterologous vaccine protection. Our data show that recombinant raccoonpoxviruses are avirulent in many cases and are much safer than vaccinia virus (strain WR). Raccoonpoxviruses also have the advantage of being able to replicate in mammalian cells. This allows increased immunogenicity and production efficiency, giving an advantage over non replicating vectors such as Modified Vaccinia Ankara MVA or canarypoxvirus.

Vaccinia virus A35R inhibits MHC class II antigen presentation.

The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation.

The poxvirus A35 protein is an immunoregulator.

It was shown previously that the highly conserved vaccinia virus A35 gene is an important virulence factor in respiratory infection of mice. We show here that A35 is also required for full virulence by the intraperitoneal route of infection. A virus mutant in which the A35 gene has been removed replicated normally and elicited improved antibody, gamma interferon-secreting cell, and cytotoxic T-lymphocyte responses compared to wild-type virus, suggesting that A35 increases poxvirus virulence by immunomodulation. The enhanced immune response correlated with an improved control of viral titers in target organs after the development of the specific immune response. Finally, the A35 deletion mutant virus also provided protection from lethal challenge (1,000 50% lethal doses) equal to that of the wild-type virus. Together, these data suggest that A35 deletion viruses will make safer and more efficacious vaccines for poxviruses. In addition, the A35 deletion viruses will serve as improved platform vectors for other infectious diseases and cancer and will be superior vaccine choices for postexposure poxvirus vaccination, as they also provide improved kinetics of the immune response.

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II.

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-gamma, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC.