Massimiliano Bissa

Benzodioxane-benzamides as new bacterial cell division inhibitors.

A SAR study was performed on 3-substituted 2,6-difluorobenzamides, known inhibitors of the essential bacterial cell division protein FtsZ, through a series of modifications first of 2,6-difluoro-3-nonyloxybenzamide and then of its 3-pyridothiazolylmethoxy analogue PC190723. The study led to the identification of chiral 2,6-difluorobenzamides bearing 1,4-benzodioxane-2-methyl residue at the 3-position as potent antistaphylococcal compounds.

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

BackgroundConsidering 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.MethodsIn 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.ResultsIn 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.ConclusionsThese 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.

A prime/boost strategy by DNA/fowlpox recombinants expressing a mutant E7 protein for the immunotherapy of HPV-associated cancers.

Development of effective therapeutic vaccines against human papilloma virus (HPV) infections remains a priority, considering the high number of new cases of cervical cancer each year by high-risk HPVs, in particular by HPV-16. Vaccines expressing the E7 oncoprotein, which is detectable in all HPV-positive pre-cancerous and cancer cells, might clear already established tumors and support the treatment of HPV-related lesions. In this study, DNA or fowlpox virus recombinants expressing the harmless variant E7GGG of the HPV-16 E7 oncoprotein (DNA(E7GGG) and FP(E7GGG)) were generated. Two immunization regimens were tested in a pre-clinical mouse model by homologous (FP/FP) or heterologous (DNA/FP) prime-boost protocols to evaluate the immune response and therapeutic efficacy of the proposed HPV-16 vaccine. Low levels of anti-E7-specific antibodies were elicited after immunization, and in vivo experiments resulted in a higher number of tumor-free mice after the heterologous immunization. These results establish a preliminary indication for therapy of HPV-related tumors by the combined use of DNA and avipox recombinants, which might represent safer immunogens than vaccinia-based vaccines.

Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

BackgroundHuman papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP)-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1) have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species.MethodsA new fowlpox virus recombinant encoding HPV-L1 (FPL1) was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays.ResultsThe FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector.ConclusionThis FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

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

BackgroundThe 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.MethodsFour 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 conclusionsUsing 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–cross-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.

Anti-tumor effects of genetic vaccines against HPV major oncogenes

Expression of HPV E5, E6 and E7 oncogenes are likely to overcome the regulation of cell proliferation and to escape immunological control, allowing uncontrolled growth and providing the potential for malignant transformation. Thus, their three oncogenic products may represent ideal target antigens for immunotherapeutic strategies. In previous attempts, we demonstrated that genetic vaccines against recombinant HPV16 E7 antigen were able to affect the tumor growth in a pre-clinical mouse model. To improve this anti-HPV strategy we developed a novel approach in which we explored the effects of E5-based genetic immunization. We designed novel HPV16 E5 genetic vaccines based on two different gene versions: whole E5 gene and E5Multi. The last one is a long multi epitope gene designed as a harmless E5 version. Both E5 genes were codon optimized for mammalian expression. In addition, we demonstrated that HPV 16 E5 oncogene is expressed in C3 mouse cell line making it an elective model for the study of E5 based vaccine. In this mouse model the immunological and biological activity of the E5 vaccines were assessed in parallel with the activity of anti-E7 and anti-E6 vaccines already reported to be effective in an immunotherapeutic setting. These E7 and E6 vaccines were made with mutated oncogenes, the E7GGG mutant that does not bind pRb and the E6F47R mutant that is less effective in inhibiting p53, respectively. Results confirmed the immunological activity of genetic formulations based on attenuated HPV16 oncogenes and showed that E5-based genetic immunization provided notable anti-tumor effects.

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.

Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain.

The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected.

GFP co-expression reduces the A33R gene expression driven by a fowlpox vector in replication permissive and non-permissive cell lines

The development of an effective prophylactic vaccine is still necessary to improve the safety of the conventional although-discontinued smallpox vaccine, and to protect from the threat of deliberate release of variola virus. This need also arises from the number of new cases of animal orthopoxvirus infections each year, and to reduce the risk to animal handlers. Fowlpox (FP) recombinants only replicate in avian species and have been developed against human infectious diseases, as they can elicit an effective immune response, are not cross-reactive immunologically with vaccinia, and represent safer and more promising immunogens for immunocompromised individuals. The aim of this study was the characterisation of two new fowlpox recombinants expressing the A33R vaccinia virus gene either alone (FP(A33R)) or with the green fluorescent protein (FP(A33R-GFP)) to verify whether GFP can affect the expression of the transgene. The results show that both FP(A33R) and FP(A33R-GFP) can express A33R correctly, but A33R mRNA and protein synthesis are higher by FP(A33R) than by FP(A33R-GFP). Therefore, GFP co-expression does not prevent, but can reduce the level of a vaccine protein, and may affect the protective efficacy of the immune response.

Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses

ABSTRACT An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen. IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.