Targeting FOXP3 Tumor-Intrinsic Effects Using Adenoviral Vectors in Experimental Breast Cancer.

The regulatory T cell master transcription factor, Forkhead box P3 (Foxp3), has been detected in cancer cells; however, its role in breast tumor pathogenesis remains controversial. Here we assessed Foxp3 tumor intrinsic effects in experimental breast cancer using a Foxp3 binder peptide (P60) that impairs Foxp3 nuclear translocation. Cisplatin upregulated Foxp3 expression in HER2+ and triple-negative breast cancer (TNBC) cells. Foxp3 inhibition with P60 enhanced chemosensitivity and reduced cell survival and migration in human and murine breast tumor cells. We also developed an adenoviral vector encoding P60 (Ad.P60) that efficiently transduced breast tumor cells, reduced cell viability and migration, and improved the cytotoxic response to cisplatin. Conditioned medium from transduced breast tumor cells contained lower levels of IL-10 and improved the activation of splenic lymphocytes. Intratumoral administration of Ad.P60 in breast-tumor-bearing mice significantly reduced tumor infiltration of Tregs, delayed tumor growth, and inhibited the development of spontaneous lung metastases. Our results suggest that Foxp3 exerts protumoral intrinsic effects in breast cancer cells and that gene-therapy-mediated blockade of Foxp3 could constitute a therapeutic strategy to improve the response of these tumors to standard treatment.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Current status of virus-vectored vaccines against pathogens that affect poultry.

The most effective strategies for the control of disease in poultry are vaccination and biosecurity. Vaccines useful against pathogens affecting poultry must be safe, effective with a single dose, inexpensive, applicable by mass vaccination methods, and able to induce a protective immune response in the presence of maternal antibodies. Viral vector meet some of these characteristics and if the attenuated virus used as vector infects birds, the vaccine will have the advantage of being bivalent. Thus, viral vectors are currently a tool of choice for the development of new poultry vaccines. This review describes the main viruses used as vectors for the delivery and in vivo expression of antigens of poultry pathogens. It also presents the methodologies most frequently used to obtain recombinant viral vectors and summarizes the state-of-the-art related to vectored vaccines in poultry (some of them currently licensed), the pathogens targeted and their antigens, and the ability of these vaccines to induce an effective immune response. Finally, the review discusses the results of a few studies comparing recombinant viral vector vaccines and live-attenuated vaccines in vaccine matching challenges, and mentions strategies and future researches that can help to improve the efficacy of vectored vaccines in poultry birds.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Viral gene therapy for breast cancer: progress and challenges.

INTRODUCTION: Breast cancer is the most common cancer in women all over the world. Furthermore, up to one third of breast tumors develop metastases that are resistant to standard therapies. Gene therapeutic strategies have been developed in order to specifically target cancer cells either directly or through the stimulation of antitumor immunity. Areas covered: This review describes the therapeutic strategies that are currently under development to treat this disease using engineered viral vectors including: adenovirus, adeno-associated virus, lentivirus, poxvirus, reovirus, baculovirus, herpesvirus and oncolytic viruses. Advantages and disadvantages of these multiple gene therapy platforms are discussed in detail. Expert opinion: Metastatic breast cancer is a perfect candidate for gene therapy approaches due to the presence of several tumor antigens and the aberrant expression of many molecular pathways. Oncolytic vectors are able to attack tumor cells while sparing normal cells and their activity is often enhanced by the administration of chemotherapy. However, more efforts are needed in order to reduce toxicity and to achieve better transduction efficiency. Improved preclinical models and a more critical patient selection for clinical trials, along with advances in gene therapy regulations, will surely facilitate the evolution of gene therapy for the treatment of metastatic breast cancer.

Induction of Both Local Immune Response in Mice and Protection in a Rabbit Model by Intranasal Immunization with Modified Vaccinia Ankara Virus Expressing a Secreted Form of Bovine Herpesvirus 1 Glycoprotein D.

In this study, we evaluated the immunogenicity and efficacy of mucosal delivery of a recombinant modified vaccinia Ankara virus (MVA) expressing the secreted version of bovine herpesvirus type 1 (BoHV-1) glycoprotein D (MVA-gDs) without addition of adjuvant in two animal models. First, we demonstrated the capability of MVA-gDs of inducing both local and systemic anti-gD humoral immune response after intranasal immunization of mice. Then, we confirmed that two doses of MVA-gDs administered intranasally to rabbits induced systemic anti-gD antibodies and conferred protection against BoHV-1 challenge. Our results show the potential of using MVA as a vector for the rational design of veterinary vaccines capable of inducing specific and protective immune responses both at local and systemic level.

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins.

In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ(+) CD4(+) and CD8(+) specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens.

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

[Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate]. / Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica.

Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate. In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 IU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 IU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary interest.

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens.

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.

Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica / Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate

En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.

In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.

Recombinant MVA expressing secreted glycoprotein D of BoHV-1 induces systemic and mucosal immunity in animal models.

Bovine herpesvirus-1 (BoHV-1) infection is distributed worldwide and the development of new tools to fight against this pathogen has become extremely important. In this work a recombinant modified vaccinia virus Ankara (MVA) vector expressing the secreted version of glycoprotein D, MVA-gDs, was obtained and evaluated as a candidate vaccine. First, the correct expression, antigenicity, and N-glycosylation of glycoprotein D were confirmed by molecular techniques. Then MVA-gDs was used as parenteral immunogen in BALB/C mice in which a specific anti-gD humoral immune response was induced and maintained for 7 mo. Two doses of MVA-gDs supplemented with cholera toxin delivered by intranasal immunization induced IgA anti-gD humoral immune responses in nasal and bronchopulmonary washes, as well as IgG anti-gD antibodies in serum samples. In order to evaluate the protection conferred by MVA-gDs immunization, a rabbit BoHV-1 challenge assay was performed. A shorter viral excretion period and a reduction in the number of animals shedding BoHV-1 was observed in the group immunized with recombinant MVA-gDs. In conclusion our data encourage further studies to evaluate MVA-gDs, alone or combined with other immunogens, as a candidate vaccine for BoHV-1.

Effect of host selective pressure on Newcastle disease virus virulence.

Newcastle disease virus (NDV) causes an economically important disease that can vary from clinically inapparent to highly virulent forms. Generally, NDV strains isolated from wild birds are non-pathogenic for chicken. However, there are evidences supporting the fact that avirulent viruses maintained in feral birds could have caused outbreaks of virulent NDV in poultry. The strain-specific difference in virulence is determined by structural variations on the fusion glycoprotein (F). More basic amino acids are present in the F cleavage site of virulent strains. Nevertheless, other regions have been involved in virulence determination. When we subjected an avirulent NDV isolated from a wild bird to a host change we found that the virus arose was virulent for chicken. Nucleotide changes in the F protein cleavage site amino acid sequence and in the hemagglutinin-neuraminidase protein sequence are reported.

Mucosal and systemic immunization elicited by Newcastle disease virus (NDV) transgenic plants as antigens.

As a step towards developing a safe and effective edible vaccine against Newcastle disease virus (NDV), we have explored the use of plants genetically engineered to express viral proteins. We report the construction of transgenic potato plants expressing the genes coding for immunogenic proteins of NDV under the regulation of CaMV 35S promoter and its immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund adjuvant produced specific anti-NDV antibodies. Animals fed with transgenic leaves showed a specific response against NDV. Detection of IgA released from in vitro-cultured intestinal tissue fragments indicated the presence of IgA-secreting cells in the gut.

Molecular characterization and phylogenetic analysis of Newcastle disease virus isolates from healthy wild birds.

Wild waterfowl is considered a natural reservoir of potentially infectious agents and a source of pathogenic viruses like avian paramyxoviruses type 1 (APMV 1). In 1997, commercial poultry in Argentina had reached the status of being free from virulent Newcastle disease virus (NDV) infections. Vaccination and biosecurity measures are actively performed to maintain this preferential sanitary condition. However, the risk of reintroduction of pathogenic viruses is always present. In this context, we conducted a study to describe the status of wild healthy birds in a geographic region relevant for the poultry industry. The presence of anti-NDV antibodies was determined in different species in all areas sampled suggesting previous contact with NDV. Seven ND viruses were isolated and characterized as apathogenic strains by biological and molecular methods. The phylogenetic analysis revealed that the majority of the Argentinian isolates form a subgroup related to viruses of genotype II. The results presented here highlight the importance of maintaining strict biosecurity measures and vaccination programs in poultry industries in order to preserve the virulent NDV-free status for commercial flocks in the country.

Matrix protein gene sequence analysis of avian paramyxovirus 1 isolates obtained from pigeons.

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.

Efficacy of Helicobacter pylori eradication in raising platelet count in adult patients with idiopathic thrombocytopenic purpura.

BACKGROUND AND OBJECTIVES: There are data consistent with an association between idiopathic thrombocytopenic purpura (ITP) and Helicobacter pylori (HP) infection. In addition, a significant increase of platelet count following HP eradication has been reported in a proportion of ITP patients. We describe here our experience on the efficacy of anti-HP treatment in ITP patients. DESIGN AND METHODS: Between December 1998 and May 2001 sixteen adult patients with ITP and documented HP infection were treated with standard antibiotic therapy for HP eradication (amoxicillin and clarithromycin plus pantoprazole combination). Of these patients, 7 had untreated ITP with mild/moderate thrombocytopenia (median platelet count 70x10(9)/L, range 41-91), 5 had relapsed following a previous steroid treatment (median platelet count 39x10(9)/L, range 30-90) and 4 were refractory to steroids (median platelet count 18.5x10(9)/L, range 9-30). RESULTS: An improvement of platelet count was observed in 11/15 patients (73.3%) who achieved HP eradication. The difference between the mean platelet count SD before and after HP eradication was statistically significant (51.6 28.2x10(9)/L vs. 143.3 131.1x10(9)/L; p=0.01). Complete or partial responses were obtained in 11/16 treated patients (68.7%). This result still persisted after a median follow-up of 11.7 months. INTERPRETATION AND CONCLUSIONS: Our data confirm the efficacy of Helicobacter pylori eradication in increasing platelet count in adult ITP patients.