CONSTRUCTION AND EVALUATION OF RECOMBINANT IMMUNOGENS AS THERAPEUTIC VACCINES AGAINST HPV-RELATED CANCERS

Background Considering the high number of new cases of cervical cancer each year caused by human papilloma viruses (HPVs), the development of an effective vaccine for the prevention and therapy of HPV-associated cancers, and in particular against the high-risk HPV-16 genotype, remains a priority. Vaccines expressing the E6 and E7 proteins, which are detectable in all HPV-positive pre-cancerous and cancer cells, might support the treatment of HPV-related lesions and clear already established tumors. Methods In this study, DNA and fowlpox virus recombinants expressing the E6F47R and E7GGG mutated forms of the HPV-16 E6 and E7 oncoproteins were generated, and their correct expression verified by RT-PCR, Western blotting and immunofluorescence. The immune responses were determined by ELISA and ELISPOT assays and the therapeutic efficacy was evaluated In mice, as a pre-clinical model of HPV-16 tumorigenicity, using heterologous (DNA/FP) or homologous (DNA/DNA and FP/FP) prime/ boost regimens after challenge with syngeneic TC-1* cells. Results The analyses of the different recombinants showed the correct expression of the inserted heterologous genes. After mice immunization, while specific anti-E6 and anti-E7 humoral responses were just detectable, specific T-cell responses were elicited. In the therapeutic protocols, after the challenge and the subsequent immunizations, a delay in cancer appearance was shown, thus confirming the pivotal role of the T-cell response in the control of tumor growth also in the absence of E6- and E7-specific antibodies. These in-vivo experiments resulted in higher numbers of tumor-free mice after either the homologous or heterologous immunizations compared to the controls. Conclusions These data establish a preliminary indication for the prevention and treatment of HPV-related tumors by the use of DNA and avipox constructs as safe and effective immunogens administered by the prime/boost strategy. The combined use of the recombinants expressing both the E6F47R and E7GGG proteins should improve the antitumor efficacy and represent an important approach to control/clear HPV-associated cancers

Removal of enteric viruses and Escherichia coli from municipal treated effluent by zebra mussels

Dreissena polymorpha is a widespread filter-feeder species, resistant to a broad range of environmental conditions and different types of pollutants, which has recently colonized Italian freshwaters. Although widely used to monitor pollution in freshwater environments, this species is also an important food source for some fish and water birds. It can also be used to concentrate or remove particulate organic matter to interrupt avian-to-human transmission of pollutants and control health risks for animals and humans. In this study, the accumulation/inactivation in D. polymorpha of human health-related spiked enteric viruses was described. The removal of endogenous Escherichia coli, the classical indicator of fecal contamination, was tested as well. Our preliminary lab-scale results demonstrate that zebra mussels can reduce significantly poliovirus titer after 24 h and rotavirus titer after 8 h. E. coli counts were also reduced in the presence of zebra mussels by about 1.5 log after 4 h and nearly completely after 24 h. The fate of the two enteric viruses after concentration by zebra mussels was also investigated after mechanical disruption of the tissues. To our knowledge, the accumulation from water and inactivation of human health-related enteric viruses by zebra mussels has never been reported

L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines

Background: The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity. Methods: Four novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence. Results and Conclusions: Using immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non\u2013cross-reactive with vaccinia virus. These recombinants might therefore represent safer and more promising immunogens that can circumvent neutralisation by vector-generated immunity in smallpox-vaccine-experienced humans

Prime-boost therapeutic vaccination in mice with DNA/DNA or DNA/Fowlpox virus recombinants expressing the Human Papilloma Virus type 16 E6 and E7 mutated proteins fused to the coat protein of Potato virus X

The therapeutic antitumor potency of a prime-boost vaccination strategy was explored, based on the mutated, nontransforming forms of the E6 (E6F47R) and E7 (E7GGG) oncogenes of Human Papilloma Virus type 16 (HPV16), fused to the Potato virus X (PVX) coat protein (CP) sequence. Previous data showed that CP fusion improves the immunogenicity of tumor-associated antigens and may thus increase their efficacy. After verifying the correct expression of E6F47RCP and E7GGGCP inserted into DNA and Fowlpox virus recombinants by Western blotting and immunofluorescence, their combined use was evaluated for therapy in a pre-clinical mouse model of HPV16-related tumorigenicity. Immunization protocols were applied using homologous (DNA/DNA) or heterologous (DNA/Fowlpox) prime-boost vaccine regimens. The humoral immune responses were determined by ELISA, and the therapeutic efficacy evaluated by the delay in tumor appearance and reduced tumor volume after inoculation of syngeneic TC-1* tumor cells. Homologous DNA/DNA genetic vaccines were able to better delay tumor appearance and inhibit tumor growth when DNAE6F47RCP and DNAE7GGGCP were administered in combination. However, the heterologous DNA/Fowlpox vaccination strategy was able to delay tumor appearance in a higher number of animals when E6F47RCP and in particular E7GGGCP were administered alone

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs

Mammalian homologs of the yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains to be defined. Recently, syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was shown to bind mAtg8s. Here we broadened the analysis of potential mAtg8-SNARE interactions and identified LC3-interacting regions (LIRs) in several SNAREs. Syntaxin 16 (Stx16), and its cognate SNARE partners all have LIR motifs and bind mAtg8s. A knockout in STX16 caused defects in lysosome biogenesis whereas a double STX16 and STX17 knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 maintained several aspects of lysosomal compartments including their functionality as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.</p

A prime/boost strategy using DNA/fowlpox recombinants expressing the genetically attenuated E6 protein as a putative vaccine against HPV-16-associated cancers

Background: Considering the high number of new cases of cervical cancer each year that are caused by human papilloma viruses (HPVs), the development of an effective vaccine for prevention and therapy of HPV-associated cancers, and in particular against the high-risk HPV-16 genotype, remains a priority. Vaccines expressing the E6 and E7 proteins that are detectable in all HPV-positive pre-cancerous and cancer cells might support the treatment of HPV-related lesions and clear already established tumors. Methods: In this study, DNA and fowlpox virus recombinants expressing the E6F47R mutant of the HPV-16 E6 oncoprotein were generated, and their correct expression verified by RT-PCR, Western blotting and immunofluorescence. Immunization protocols were tested in a preventive or therapeutic pre-clinical mouse model of HPV-16 tumorigenicity using heterologous (DNA/FP) or homologous (DNA/DNA and FP/FP) prime/boost regimens. The immune responses and therapeutic efficacy were evaluated by ELISA, ELISPOT assays, and challenge with TC-1* cells. Results: In the preventive protocol, while an anti-E6-specific humoral response was just detectable, a specific CD8+ cytotoxic T-cell response was elicited in immunized mice. After the challenge, there was a delay in cancer appearance and a significant reduction of tumor volume in the two groups of E6-immunized mice, thus confirming the pivotal role of the CD8+ T-cell response in the control of tumor growth in the absence of E6-specific antibodies. In the therapeutic protocol, in-vivo experiments resulted in a higher number of tumor-free mice after the homologous DNA/DNA or heterologous DNA/FP immunization. Conclusions: These data establish a preliminary indication for the prevention and treatment of HPV-related tumors by the use of DNA and avipox constructs as safe and effective immunogens following a prime/boost strategy. The combined use of recombinants expressing both E6 and E7 proteins might improve the antitumor efficacy, and should represent an important approach to control HPV-associated cancers