The human papillomavirus 16 (HPV16) E7 oncoprotein can be considered a ‘tumor-specific antigen’ and, therefore, it represents a promising target for a therapeutic vaccine against HPV-associated tumors. Efficient production of E7 protein with a plant-based transient expression system has been already described and it was demonstrated that E7-containing crude plant extracts confer partial protection against tumor challenge in a mouse model system. Before adopting the plant-based system as a cost-effective method for the production of an E7-based anti-cancer vaccine, some aspects, such as the oncoprotein yield, need further investigation. In the present study, we report the transient expression, mediated by a potato virus X (PVX)-derived vector, of the E7 protein targeted to the secretory system of Nicotiana benthamiana plants by using a plant-derived signal sequence. Targeting the antigen to the secretory pathway enhanced the E7 protein expression levels about five-fold. Mice immunized by s.c. administration with crude foliar extracts containing E7 showed strong stimulation of cell-mediated immune response after five boosters, as detected by ELISPOT. After challenging with the E7-expressing C3 tumor cells, tumor growth was completely inhibited in 80% of the vaccinated animals and a drastic reduction of tumor burden was observed in the remaining tumor-affected mice. These data demonstrate that, by enhancing E7 yield, it is possible to improve the anti-cancer activity of the plant-based experimental vaccine and open the way for a large-scale production of the E7 protein which could be purified or used as ‘in planta’ formulation, also suitable for oral therapeutic vaccination.

Severe acute respiratory syndrome (SARS) is a dangerous infection with pandemic potential. It emerged in 2002 and its aetiological agent, the SARS Coronavirus (SARS-CoV), crossed the species barrier to infect humans, showing high morbidity and mortality rates. No vaccines are currently licensed for SARS-CoV and important efforts have been performed during the first outbreak to develop diagnostic tools. Here we demonstrate the transient expression in Nicotiana benthamiana of two important antigenic determinants of the SARS-CoV, the nucleocapsid protein (N) and the membrane protein (M) using a virus-derived vector or agro-infiltration, respectively. For the M protein, this is the first description of production in plants, while for plant-derived N protein we demonstrate that it is recognized by sera of patients from the SARS outbreak in Hong Kong in 2003. The availability of recombinant N and M proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious SARS-CoV outbreaks.

41 42The emergence and diffusion of antibiotic-resistant bacteria has been a major public health 43 problem for many years now. In this study, antibiotic-resistance of coliforms and Escherichia 44 coli were investigated after their isolation from samples collected in a municipal wastewater 45 treatment plant in the Milan area (Italy) along different points of the treatment sequence: 46 inflow to biological treatment; outflow from biological treatment following rapid sand 47 filtration; and outflow from peracetic acid disinfection. The presence of E. coli that showed 48 resistance to ampicillin (AMP) and chloramphenicol (CAF), used as representative antibiotics 49 for the efficacy against Gram-positive and Gram-negative bacteria, was evaluated. After 50 determining E. coli survival using increasing AMP and CAF concentrations, specific single-51 resistant (AMP R or CAF R ) and double-resistant (AMP R /CAF R ) strains were identified among 52 E. coli colonies, through amplification of the β-lactamase Tem-1 (bla) and acetyl-transferase 53 catA1 (cat) gene sequences. While a limited number of CAF R bacteria was observed, most 54 AMP R colonies showed the specific resistance genes to both antibiotics, which was mainly 55 due to the presence of the bla gene sequence. The peracetic acid, used as disinfection agent, 56showed to be very effective in reducing bacteria at the negligible levels of less than 10 57 CFU/100 ml, compatible with those admitted for the irrigation use of treated waters. 58 59

This study reports the immunomodulatory activity on human monocyte derived dendritic cells (MDDCs) of a vaccine preparation shown to be effective against an HPV16-related tumour in an animal model. The vaccine is composed of extract from Nicotiana benthamiana leaves containing HPV16 E7 protein expressed by a potato virus X-derived vector (NbPVX-E7). The effect of the extract was evaluated on MDDC differentiation and maturation by monitoring the phenotypic expression of specific markers. The results show that NbPVX-E7 does not induce monocyte differentiation to dendritic cells, but does induce MDDC maturation. Plant extract does not influence MDDC-uptake of E7-FITC while it significantly improves the Ovalbumin-FITC uptake, considered as a model antigen. Importantly, NbPVX-E7-pulsed MDDCs/PBMCs are able to prime human blood-derived lymphocytes from healthy individuals to induce HPV16 E7-specific cytotoxic activity. This is a propaedeutic study for a possible use of E7-containing plant extract in human immunotherapy of HPV-related lesions.