Antonia Radaelli

Protective properties of non-nucleoside reverse transcriptase inhibitor (MC1220) incorporated into liposome against intravaginal challenge of Rhesus Macaques with RT-SHIV

In the absence of an effective vaccine against HIV, it is urgent to develop an effective alternative such as a microbicide. Single and repeated applications of MC1220 microbicide were evaluated in macaques. First, animals were given a single application of 0.5% or 1.5% MC1220-containing liposomal gel. A second group were treated with 0.5% MC1220 once a day for 4 days. The control groups were treated by liposomal gel alone. Thirty minutes after the last application, animals were challenged with RT-SHIV. In the first protocol, 2 of 4 animals treated by 0.5% of the MC1220 and 2 of 5 treated by 1.5% were protected. In the second protocol, 3 of 5 treated animals were protected and 5 of 5 controls were infected. The RNA viral load at necropsy was significantly lower (p=0.05) in treated-infected animals than in controls. In both protocols, the number of CD4+ T cells was lower at viremia peak in infected than in protected animals.

Expression of HIV-1 envelope gene by recombinant avipox viruses

Recombinant canarypox (CP) and fowlpox (FP) viruses that contained two forms of the HIV-1 (SF2 strain) env gene were engineered and their expression analysed in chick, simian and human cells. These vectors can efficiently replicate in avian but not in mammalian cells, in which infection is abortive. The two forms, consisting of the entire env open reading frame (IS+) or of the same gene lacking the putative immunosuppressive (IS-) region (amino acids 583-599), were individually inserted into the two virus vector backgrounds. In order to avoid premature transcription termination of the foreign gene and to improve protein expression, a mutagenesis was also performed within the T5NT motif without altering the amino acid sequence. By immunoprecipitation analyses, cells infected with CP and FP recombinants expressed HIV-1 env polypeptides of the appropriate molecular weight. We observed that the gp160 precursor was proteolytically cleaved except in MRC-5 cells infected with the IS- recombinants and that these polypeptides were glycosylated. Further analysis of these recombinant viruses by indirect immunofluorescence and syncytia inhibition assays indicated that the gp120 gp41 complex was present on the surface of infected cells, the number of syncytia being significantly lower when cells were infected by the CPIS- or FPIS- recombinants. Moreover, sera of immunized rabbits revealed the presence of specific antibodies in animals inoculated either with CP or with FP recombinants. These new constructs, which are unable to support a productive infection in human cells, might therefore also be a good anti-HIV-1 candidate vaccine in seropositive hosts.

Mycological and ultrastructural studies to evaluate biodeterioration of mural paintings. Detection of fungi and mites in Frescos of the monastery of St Damian in Assisi

Abstract The role of fungi in the biodeterioration of frescos has been investigated by microbiological and ultrastructural techniques. With this aim, the mycoflora present on samples taken from deteriorated indoor wall paintings (frescos) in the Monastery of St Damian in Assisi was isolated and identified. The results showed that the fungal colonization of the two paintings ‘Crocifisso con Francesco giovane’ and the ‘Mensa di S. Chiara’, located inside St Clares Refectory, was mainly due to Cladosporium, Aspergillus, Alternaria, Penicillium and Fusarium genera. Although considered not uncommon in frescos, their direct involvement in enzymatic degradation of paints was not confirmed. Interestingly, the presence of mites in proximity to actively growing fungal mycelium, as revealed by scanning electron microscopy, although considered an example of parasitic nutritional relation, could also suggest a metabolic interaction resulting in the synthesis of biodeteriogenic substances.

Canarypox and fowlpox viruses as recombinant vaccine vectors: A biological and immunological comparison

Canarypox and fowlpox viruses represent alternative vaccine vectors due to their natural host-range restriction to avian species. Although they cannot replicate in mammals, they correctly express transgenes in human cells and elicit a complete immune response in vaccinated subjects. Several studies have evaluated their genomic differences and protective efficacy in preclinical trials, but detailed information is not available for their transgene expression, cytokine modulation and abortive replication in mammals. This study demonstrates that the heterologous HIV gag/pol and env genes are more efficiently expressed by fowlpox in non-immune and immune cells. The production of retrovirus-like particles, the longer transgene expression, and a balanced cytokine induction may confer to fowlpox-based recombinants the ability to elicit a better immune response.

Expression and immunogenicity of V3 loop epitopes of HIV-1, isolates SC and WMJ2, inserted in Salmonella flagellin.

Synthetic oligonucleotides corresponding to specific V3 loop portions of two HIV-1 isolates, SC and WMJ2, were expressed in the flagella of a Salmonella live-vaccine strain. Expression of the inserted epitopes in flagellin and their exposure at the surface of flagellar filaments were shown by immunoblotting and immunogold labeling with anti-flagellin (Salmonella d) and anti-HIV-1(IIIB) V3 loop peptide sera. Live recombinant Salmonella strains expressing either one of the two V3 loop inserts were administered intraperitoneally to BALB/c mice. All these animals developed antibodies specific for the heterologous glycoprotein 120 (gp120) of HIV-1 MN strain, as detected by enzyme-linked immunosorbent assays (ELISA), two of the sera had neutralizing activity against the heterologous HIV-1 MN strain. Moreover, oral administration of the live Salmonella recombinant strains to mice evoked specific IgA directed against gp120.

Comparative analysis of immune responses and cytokine profiles elicited in rabbits by the combined use of recombinant fowlpox viruses, plasmids and virus-like particles in prime-boost vaccination protocols against SHIV.

Three different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of identifying a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic chimera simian/human immunodeficiency virus (SHIV)(89.6P). Protocols were based on priming with two fowlpox (FP) recombinant vectors and two expression plasmids, which express either the simian immunodeficiency virus (SIV)mac(239) gag/pol or the human immunodeficiency virus (HIV-1)env(89.6P) genes, followed by boosting with virus-like particles (VLP). All protocols were effective in eliciting homologous neutralizing Ab and highlighted the efficacy of VLP boosting. The FP vector was less efficient than plasmid DNA in inducing Ab against the gag core proteins. Analysis of cytokine expression 5 months after last immunization indicated that priming with pcDNA3gag/pol(SIV) and FPenv(89.6P) followed by VLP boosting generated a T helper (Th0) profile and a good Ab titer, suggesting a potential protocol to be tested in the SHIV-macaque model of HIV-1 infection.

Construction and characterization of recombinant fowlpox viruses expressing human papilloma virus E6 and E7 oncoproteins.

Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype and the expression of the E6 and E7 proteins, which can bind to the p53 and p105Rb host cell-cycle regulatory proteins, is related to its tumorigenicity. Virus-like-particle (VLP)-based immunogens developed recently are successful as prophylactic HPV vaccines. However, given the high number of individuals infected already with HPV and the absence of expression of the L1 structural protein in HPV-infected or HPV-transformed cells, an efficient therapeutic vaccine targeting the non-structural E6 and E7 oncoproteins is required. In this study, two new fowlpox virus (FPV) recombinants encoding the HPV-16 E6 and E7 proteins were engineered and evaluated for their correct expression in vitro, with the final aim of developing a therapeutic vaccine against HPV-related cervical tumors. Although vaccinia viruses expressing the HPV-16 and HPV-18 E6 and E7 oncoproteins have already been studied, due to their natural host-range restriction to avian species and their ability to elicit a complete immune response, FPV recombinants may represent efficient and safer vectors also for immunocompromised hosts. The results indicate that FPV recombinants can express correctly the E6 and E7 oncoproteins, and they should represent appropriate vectors for the expression of these oncoproteins in human cells.

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.

Humoral and cell-mediated immunity in rabbits immunized with live non-replicating avipox recombinants expressing the HIV-1SF2env gene

Abstract The canarypox (CP) and fowlpox (FP) viruses, which are unable to replicate productively in non-avian species, have been utilized as live vectors carrying the HIV-1 SF2 env gene with the putative immunosuppressive (IS) region complete (CPIS + and FPIS + ) or deleted (CPIS − and FPIS − ). To determine if these avipox- env recombinants could be utilized to elicit a specific immune response against HIV-1, six groups of rabbits were immunized with CPIS + , CPIS − , FPIS + , FPIS − constructs or their non-engineered wild-type CP wt or FP wt counterparts. After a primary inoculation and successive boosters, env -specific humoral and cell-mediated immunity were demonstrated by ELISA, immunoblots and lymphoproliferation assays. Antibody titres and neutralization activities were higher in CP- than FP-inoculated rabbits, the CPIS + always showing a similar immunogenic capacity to CPIS − . Evidence is also presented indicating that rabbit sera possess group-specific antibodies, which were, however, unable to cross-neutralize divergent HIV-1 strains. Although the protective capacity against HIV-1 experimental infection has not yet been determined in these animals, our results suggest that these recombinants might represent promising and safer candidate vaccines against HIV-1.

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.