Pier Giorgio Balboni

Inhibition of HIV-1 replication by a Tat transdominant negative mutant in human peripheral blood lymphocytes from healthy donors and HIV-1-infected patients

It was previously shown that a tat mutant (tat22) where cysteine 22 is substituted by glycine behaves as a transdominant negative mutant in Jurkat T cells lytically or latently infected by HIV-1. In this study we demonstrate that tat22 controls HIV-1 replication in primary cells. This effect was observed both after in vitro infection of peripheral blood mononuclear cells (PBMCs) from normal donors and after reactivation of the latent infection in PBMCs from seropositive patients. The antiviral effect of tat22 was limited to conditions of low virus production. The use of tat22 may be promising for a gene therapy approach to AIDS during the asymptomatic phase of the disease allowing control of virus replication in infected cells and inhibition of virus spread to uninfected cells.

Local and systemic inoculation of DNA or protein gB1s-based vaccines induce a protective immunity against rabbit ocular HSV-1 infection

A secreted form of gB1 (gB1s), previously shown to protect rabbits against HSV-1 ocular infection when inoculated systemically, was delivered to rabbit periocular area to evaluate its vaccine efficacy upon local administration. The efficacy of local or systemic inoculation of a gB1s-DNA-based vaccine in the rabbit model of ocular HSV-1 infection was assessed in parallel flow. Rabbits received four inoculations of the different immunogens, then immune responses and clinical symptoms were evaluated. Both the local protein and the systemic DNA administration elicited a neutralizing antibody response, reduced ocular symptoms with respect to controls (P<0.01), and completely prevented the death of rabbits from encephalitis. Conversely, local DNA vaccination did not induce any detectable antibody response, and could only partially protect rabbits from the development of encephalitis and severe ocular infection.

Studies on the effect of the combined expression of anti-tat and anti-rev genes on HIV-1 replication

A series of retroviral vectors with potential anti-tat and anti-rev activity was developed. Vectors containing a tat transdominant negative mutant (tat22/37) and an RRE decoy in different positions, directed by the same promoter or by different promoters, were generated. Retroviral vectors containing tat22/37 and the RevM10 transdominant negative mutant were also constructed. Jurkat cells were transduced with the recombinant retroviruses to produce monoclonal and polyclonal cultures. In these cell lines the recombinant proviruses were correctly integrated and expression of the inserted genes was detected by Northern blot or RT-PCR analysis. However, infection of these cell lines with HIV-1 showed that none of these recombinant constructs inhibited virus replication at a high multiplicity of infection (MOI). At a low MOI, two cell clones containing tat22/37 and the RRE decoy in 3′ position showed a long lasting protection against virus replication, in comparison to control cultures expressing tat22/37 or RRE alone. Combination of tat and rev mutants was ineffective in inhibiting HIV-1 replication at both low and high MOIs. At a low MOI, HIV-1 replication was efficiently blocked in two cell clones expressing the RevM10 mutant alone. These results show a synergic effect of anti-tat and anti-rev molecules when the RRE sequence is cloned 3′ to tat22/37, suggesting the possibility of using this vector design to control HIV-1 replication.

New BK virus episomal vector for complementary DNA expression in human cells

SummaryThe properties of pRP-c, a new vector for complementary DNA (cDNA) expression, are described. The vector contains the early region and replication origin of BK virus (BKV), a human papovavirus. Due to the presence of these BKV sequences, pRP-c replicates in human cells allowing amplification of inserted cDNAs. The promoter, intron and polyadenylation region for cDNA expression are separated by unique restriction sites and can therefore be individually excised and substituted with different transcription signals. Coding sequences of the bacterial genes for chloramphenicol-acetyl transferase (CAT) or neomycin phosphotransferase (neo) were inserted into the cDNA cloning site of pRP-c and expressed in human cells in transient assays or stable clones. In both cases expression of the inserted sequences was significantly more efficient than by using the integration vectors pSV2CAT and pSV2neo, demonstrating the advantages of episomal expression vectors in human cells. Possible uses of pRP-c to express viral and cellular cDNAs in human cells are discussed.

Impact of a Probiotic-Based Cleaning Intervention on the Microbiota Ecosystem of the Hospital Surfaces: Focus on the Resistome Remodulation.

Background Contamination of hospital surfaces by clinically-relevant pathogens represents a major concern in healthcare facilities, due to its impact on transmission of healthcare-associated infections (HAIs) and to the growing drug resistance of HAI-associated pathogens. Routinely used chemical disinfectants show limitations in controlling pathogen contamination, due to their inefficacy in preventing recontamination and selection of resistant strains. Recently we observed that an innovative approach, based on a cleanser added with spores of non-pathogenic probiotic Bacilli, was effective in stably counteracting the growth of several pathogens contaminating hospital surfaces. Methods Here, we wanted to study the impact of the Bacillus-based cleanser on the drug-resistance features of the healthcare pathogens population. In parallel, the ability of cleanser-derived Bacilli to infect hospitalized patients was also investigated. Results Collected data showed that Bacilli spores can germinate on dry inanimate surfaces, generating the bacterial vegetative forms which counteract the growth of pathogens and effectively substitute for them on treated surfaces. Strikingly, this procedure did not select resistant species, but conversely induced an evident decrease of antibiotic resistance genes in the contaminating microbial population. Also importantly, all the six HAI-positive patients hosted in the treated areas resulted negative for probiotic Bacilli, thus adding evidences to their safety-to-use. Conclusions These results indicate that this probiotic-based procedure is active not only in controlling surface microbial contamination but also in lowering drug-resistant species, suggesting that it may have relevant clinical and therapeutical implications for the management of HAIs.

Hard surface biocontrol in hospitals using microbial-based cleaning products.

Background Healthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcare-associated pathogens, thus indicating the need for new and sustainable strategies. Aim This study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting. Methods The effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected. Results Microbial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3–4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m2 when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities. Conclusions This study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAI-related microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.

Mice Genetic Immunization with Plasmid DNA Encoding a Secreted Form of HSV-1 gB Induces a Protective Immune Response against Herpes simplex Virus Type 1 Infection

Intramuscularly (i.m.) delivered plasmid DNA encoding a secreted form of glycoprotein B of herpes simplex virus type 1 (HSV-1 gB1s) was evaluated for the ability to elicit a protective immune response in Balb/c mice. Animals received three i.m. injections of a gB1s expression plasmid (pRP-RSV-gB1s) or of a wild-type transmembrane gB1 coding plasmid (pRP-RSV-gB1), while control mice were injected with the vector alone (pRP-RSV). A specific antibody response was observed in almost all immunized animals, and in most cases antibodies were also detected after 1 month in the absence of further vaccine boosts. Serum antibodies mostly displayed neutralizing activity against HSV-1. Glycoprotein B1s DNA immunization was also effective in protecting animals against the primary infection induced by a subsequent HSV-1 challenge and limited HSV-1 infection of sensitive ganglia.

Panbacterial real-time PCR to evaluate bacterial burden in chronic wounds treated with Cutimed™ Sorbact™

The impact of polymicrobial bacterial infection on chronic wounds has been studied extensively, but standard bacteriological analysis is not always sensitive enough. Molecular approaches represent a promising alternative to the standard bacteriological analysis. This work aimed to assess the usefulness of a panbacterial quantitative real-time PCR reaction to quantitate the total bacterial load in chronic wounds treated with Cutimed™ Sorbact™, a novel therapeutic approach based on hydrophobic binding of bacteria to a membrane. The results obtained by panbacterial real-time PCR on conserved sequences of the bacterial 16S gene show that the bacterial burden significantly decreased in 10 out of 15 healing chronic wounds, and did not change in 5 out of 5 non-healing chronic wounds. On the contrary, classical culture for S. aureus and P. aeruginosa, and real-time PCR for Bacteroides and Fusobacterium did not show any correlation with the clinical outcome. Our study also shows that quantification of chronic wounds by panbacterial real-time PCR is to be performed on biopsies and not on swabs. These results show that panbacterial real-time PCR is a promising and quick method of determining the total bacterial load in chronic wounds, and suggest that it might be an important biomarker for the prognosis of chronic wounds under treatment.

Factors affecting amplification of BK virus episomal vectors in human cells

SummaryAnalysis of factors determining replication of BK virus (BKV) episomal vectors in human cells showed that vector copy number was related to the level of BKV T antigen expression. T antigen was synthesized efficiently, as assessed by indirect immunofluorescence, in vector-transfected primary embryonic fibroblasts undergoing neoplastic transformation. Surprisingly, transfected continuous cell lines (143B, HeLa and KB), kept under biochemical selection or tested in transient assays, produced negligible amounts or no T antigen, revealed only by a sensitive ELISA test, suggesting that in these cells vector amplification was under the control of cellular factors. Presence or absence of BKV late region sequences, BKV strain, orientation of the inserted genes and presence or absence of selection were not relevant for vector replication. Type of biochemical selection, however, was important, since BKV vectors containing the thymidine kinase gene replicated better than those containing theneo gene. Despite great variability, vector copy number increased in transfected clones of adenovirus 5-transformed 293 cells, in the absence of immunofluorescence detectable T antigen. These cells express adenovirus immediate early proteins E 1 A and E 1 B which may directly or indirectly activate BKV origin of replication.