Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens.

Nucleic acid vaccines represent an attractive approach to vaccination, combining the positive attributes of both viral vectors and live-attenuated vaccines, without the inherent limitations of each technology. We have developed a novel technology, the Self-Amplifying mRNA (SAM) platform, which is based on the synthesis of self-amplifying mRNA formulated and delivered as a vaccine. SAM vaccines have been shown to stimulate robust innate and adaptive immune responses in small animals and non-human primates against a variety of viral antigens, thus representing a safe and versatile tool against viral infections. To assess whether the SAM technology could be used for a broader range of targets, we investigated the immunogenicity and efficacy of SAM vaccines expressing antigens from Group A (GAS) and Group B (GBS) Streptococci, as models of bacterial pathogens. Two prototype bacterial antigens (the double-mutated GAS Streptolysin-O (SLOdm) and the GBS pilus 2a backbone protein (BP-2a)) were successfully expressed by SAM vectors. Mice immunized with both vaccines produced significant amounts of fully functional serum antibodies. The antibody responses generated by SAM vaccines were capable of conferring consistent protection in murine models of GAS and GBS infections. Inclusion of a eukaryotic secretion signal or boosting with the recombinant protein resulted in higher specific-antibody levels and protection. Our results support the concept of using SAM vaccines as potential solution for a wide range of both viral and bacterial pathogens, due to the versatility of the manufacturing processes and the broad spectrum of elicited protective immune response.

Immune Responses to Invasive Group B Streptococcal Disease in Adults.

Immunization of nonpregnant adults could help prevent invasive group B Streptococcus (GBS) infections, but adult immune responses have not been investigated. We defined capsular polysaccharide (CPS) and pilus island (PI) surface antigen distribution and expression and immune responses to GBS infection in nonpregnant adults. Prospective surveillance from 7 hospitals in Houston, Texas, USA, identified 102 adults with GBS bacteremia; 43% had skin/soft tissue infection, 16% bacteremia without focus, and 12% osteomyelitis. CPS-specific IgG was determined by ELISA and pilus-specific IgG by multiplex immunoassay. CPS types were Ia (24.5%), Ib (12.7%), II (9.8%), III (16.7%), IV (13.7%), and V (12.7%); 9.8% were nontypeable by serologic methods. Pili, expressed by 89%, were most often PI-2a. CPS and pilus-specific IgG increased during convalescence among patients with strains expressing CPS or PI. All GBS expressed CPS or PI; 79% expressed both. Increased antibodies to CPS and PI during recovery suggests that GBS bacteremia in adults is potentially vaccine preventable.

Successful completion of a semi-automated enzyme-free cloning method.

Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94). Here we show that we successfully set up a platform able to complete, with high efficiency, a wide range of molecular biology and biochemical steps. 329 gene targets (99 %) were effectively amplified using the automated procedure and 286 (87 %) of these PCR products were successfully cloned in expression vectors, with cloning success rates being higher for the automated protocols respect to the manual procedure (93.6 and 74.5 %, respectively).

Seasonal screening of AChE, GSH and gonad histology, in European sea bass Dicentrarchus labrax L. reared in three different fish farms.

The aim of this work was to do a preliminary seasonal screening of ecotoxicological biomarkers in European sea bass Dicentrarchus labrax in three different fish farms, to know if the different location and typology can discriminate them. A set of selected biomarkers of xenobiotic exposure, such as acetylcholinesterase (AChE) activity, Glutathione (GSH) and gonad morphology were investigated seasonally in male European sea bass D. labrax (L.) reared in three different intensive farms: a land-based farm of cement tanks (T), an in-shore sea cages farm (C1) and an off-shore sea cages farm (C2). The results showed that both location and typology can discriminate AChE activity, GSH content and gonad morphology. Further investigation is needed to propose these biomarkers in the protocol of fish farm quality control.

Screening of ecotoxicological, qualitative and reproductive variables in male European sea bass Dicentrarchus labrax (L.) reared in three different fish farms: Facility location and typology.

The aim of this study was to investigate the influence of both facility location and typology of fish farm on some ecotoxicological, qualitative and reproductive variables in European sea bass Dicentrarchus labrax L. Several variables were investigated: gonado-somatic index (GSI), liver-somatic index (LSI); 7-ethoxyresorufin-O-deethylase (EROD), benzo(a)pyrene monooxygenase and acetylcholinesterase activities; glutathione (GSH), testosterone, 17ß-estradiol, total lipid, phospholipid (PL) and triglyceride contents. In addition, the histological sections of gonads were examined. Results suggest that LSI, EROD activity, GSI, GSH, PL, hormone levels and gonad morphology were influenced by different facility locations and typologies of fish farm.

Capturing host-pathogen interactions by protein microarrays: identification of novel streptococcal proteins binding to human fibronectin, fibrinogen, and C4BP.

Microbial pathogen entry and survival in the host is mediated by a network of molecular interactions between the two partners, which has been the subject of many research efforts. A complex picture is emerging in which host-pathogen crosstalk involves a high number of proteins, often with redundant functions. In the present study, we investigated the potential of protein microarrays to simultaneously scan interactions between surface proteins from two main human streptococcal pathogens, Streptococcus pyogenes and Streptococcus agalactiae, and three human ligands, fibronectin, fibrinogen, and C4 binding protein, known to play an important role in streptococcal pathogenesis. By using this technology, we confirmed interactions described in the literature and detected a novel set of streptococcal proteins with binding capacities for the human ligands. The observations were validated by Western blot and ELISA techniques. Three of the newly identified proteins were isoforms of a group B streptococcus-secreted component named Fib and displayed differential binding capacities for fibronectin, fibrinogen, and C4BP. The protein regions involved in the interaction with each ligand were identified by constructing fragments of one of the Fib variants. The approach proved valuable for the acquisition of novel insights into the complex network of protein-protein interactions occurring during microbial infection.

Cholinesterases in the Antarctic scallop Adamussium colbecki: characterization and sensitivity to pollutants.

Antarctica is affected by man-made contamination and development of sensitive ecotoxicological tools for impact assessment is a priority task. The aims of the present study were to characterize cholinesterase (ChE) activities in an Antarctic key species, the scallop Adamussium colbecki, and to investigate their sensitivity as biological markers (biomarkers) of exposure to pollutants and of their effects. Our results show that ChEs in gills share most characteristics with true acetylcholinesterase. The present results show that ChE activities in A. colbecki are significantly inhibited by organophosphates (OPs) and somehow affected by in vitro exposure to mixtures of marine contaminants such as polycyclic aromatic hydrocarbons (PAHs), even if no concentration-dependent pattern of response was observed and no effect was elicited by polychlorinated biphenyls (PCBs). The present results do not demonstrate ChEs in A. colbecki as sensitive tools to measure exposure to the above chemicals, but they may be worthy of further study considering the importance of the scallop in Antarctic marine ecosystems and its suitability as a sentinel species.

Biomonitoring aquatic environmental quality in a marine protected area: a biomarker approach.

The main aims of the present study, conducted in the framework of the MONIQUA-Egadi Scientific Project, were twofold: first, to make the first step in the development and validation of an ecotoxicological approach for the assessment of marine pollution in coastal environments on the basis of a set of biomarker responses in new sentinel species; and second, to obtain preliminary information on environmental quality in an Italian marine protected area, the Egadi Islands (Sicily). Several cytochrome P450-dependent mixed-function oxidase activities were measured in the following sentinel species: rainbow wrasse Coris julis, gastropod limpet Patella caerulea, and sea urchin Paracentrotus lividus. The results suggest that specimens from the Favignana Harbor may be exposed to P450 inducers, whereas most of the other sites seem to share similar environmental quality. The proposed approach has potential for assessment of environmental quality in marine protected areas.

Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus.

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.

Esterase activities in the bivalve mollusc Adamussium colbecki as a biomarker for pollution monitoring in the Antarctic marine environment.

Marine environments are continuously being threatened by a large number of xenobiotics from anthropogenic sources. Even in sparsely populated and relatively pristine areas, such as Antarctica, hazardous chemicals can pose a serious environmental problem. The main aims of the present study were to (1) validate and optimize an analytical technique utilizing a microtitre-plate photometer to ascertain background levels of esterase activities in the Antarctic bivalve Adamussium colbecki, (2) carry out in situ monitoring of esterase activities to assess any potential environmental impacts of the Italian Scientific Antarctic Base "Terra Nova Bay" on the surrounding marine area. Results showed the presence of organophosphorous-sensitive cholinesterase (ChE) and carboxylesterase (CbE) activities in the gills of A. colbecki and optimal assay conditions were comparable with those found for bivalve species from temperate areas. A higher sensitivity of ChE versus acetylthiocholine activity in A. colbecki to chlorpyrifos compared to species from temperate areas may also be inferred. The in situ study indicated no differences in the environmental quality of the three study sites located around the Italian Base.