Detection of adenovirus in the waters of the Seine River estuary by nested-PCR.

Several systems for isolating viruses from environmental samples have been tested. The most promising method is based on genomic amplification. The authors attempted to detect adenovirus in nucleic-acid extracts from the Seine River estuary by a two-step amplification of a 220-bp segment of the conserved coding region of type 2 adenovirus hexon protein L3. The primers used in this study detected the most prevalent adenovirus serotypes in human disease in France, but not other virus strains or bacteria. The sensitivity of the nested polymerase chain reaction (PCR) amplification was estimated to be 10(2) copies of the adenovirus target sequence per ml of Seine River water. Nucleic-acid extracts from Seine River estuary waters were analysed and some tested positive for the presence of adenoviruses.

A new family of carriers (biovectors) enhances the immunogenicity of rabies antigens.

Biovectors (BV) are a new family of protein carriers. They are nanoparticles of polymerized polysaccharides substituted with phosphate residues and surrounded by covalently bound lipid molecules (palmitic acid). The effect of BV was tested on the immunogenicity of rabies antigens. Biovectors enhanced the production of antibody induced by both rabies glycoprotein and ribonucleoprotein. Moreover, they enhanced the protective activity of an experimental rabies vaccine composed of inactivated and purified virus. The isotype profile of antibody produced in vivo was not modified when BV were mixed with rabies antigens. To clarify the mechanism of the adjuvant/ immunostimulation effect of BV, two types of approach were used: (1) analysis of the antibody response when antigen and BV were injected separately; (2) determination of the nature of cells involved in the proliferation in vitro of murine splenocytes in the presence of BV. The enhancing effect of BV on antibody production was highest when mixed with antigens. In vitro BV induced the proliferation of B cells. These findings suggest that BV have immunostimulating properties in addition to their probable depot and/or antigen-presentation effect which explain in part their adjuvant activity.

Stabilization and enhancement of interleukin-2 in vitro bioactivity by new carriers: supramolecular biovectors.

Human recombinant interleukin-2 can be associated and released from supramolecular biovectors (SMBVs), consisting of particles made of polymerized polysaccharides. The particles are substituted with phosphate residues and contain bound lipid molecules (palmitic acid) buried near their surfaces. The association of IL-2 with SMBVs modifies its in vitro bioactivity. SMBVs prolong the growth of IL-2-dependent cells, enhance IL-2 proliferative activity and restore the activity of impaired IL-2. These properties mainly depend on the presence of lipids linked to the SMBV and on both the degree of acylation and the SMBV: IL-2 ratio. SMBVs are therefore good candidates for the stabilization and enhancement of the biological activity of IL-2.

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: use of various cell lines.

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2-3 x 10(6) to 6-12 x 10(6) cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium. A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3-4-fold in comparison to serum-containing medium. For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.