Cytokines as fast indicator of infectious virus titer during process development.

Measuring infectious titer is the most time-consuming method during the production and process development of live viruses. Conventionally, it is done by measuring the tissue culture infectious dose (TCID50) or plaque forming units (pfu) in cell-based assays. Such assays require a time span of more than a week to the readout and significantly slow down process development. In this study, we utilized the pro-inflammatory cytokine response of a Vero production cell line to a recombinant measles vaccine virus (MVV) as model system for rapidly determining infectious virus titer within several hours after infection instead of one week. Cytokines are immunostimulatory proteins contributing to the first line of defence against virus infection. The probed cytokines in this study were MCP-1 and RANTES, which are secreted in a virus dose as well as time dependent manner and correlate to TCID50 over a concentration range of several logarithmic levels with R2 = 0.86 and R2 = 0.83, respectively. Furthermore, the pro-inflammatory cytokine response of the cells was specific for infectious virus particles and not evoked with filtered virus seed. We also discovered that individual cytokine candidates may be more suitable for off- or at-line analysis, depending on the secretion profile as well as their sensitivity towards changing process conditions. Furthermore, the method can be applied to follow a purification procedure and is therefore suited for process development and control.

Multispecies biofilm behavior and host interaction support the association of Tannerella serpentiformis with periodontal health.

The recently identified bacterium Tannerella serpentiformis is the closest phylogenetic relative of Tannerella forsythia, whose presence in oral biofilms is associated with periodontitis. Conversely, T. serpentiformis is considered health-associated. This discrepancy was investigated in a comparative study of the two Tannerella species. The biofilm behavior was analyzed upon their addition and of Porphyromonas gingivalis-each bacterium separately or in combinations-to an in vitro five-species oral model biofilm. Biofilm composition and architecture was analyzed quantitatively using real-time PCR and qualitatively by fluorescence in situ hybridization/confocal laser scanning microscopy, and by scanning electron microscopy. The presence of T. serpentiformis led to a decrease of the total cell number of biofilm bacteria, while P. gingivalis was growth-promoting. This effect was mitigated by T. serpentiformis when added to the biofilm together with P. gingivalis. Notably, T. serpentiformis outcompeted T. forsythia numbers when the two species were simultaneously added to the biofilm compared to biofilms containing T. forsythia alone. Tannerella serpentiformis appeared evenly distributed throughout the multispecies biofilm, while T. forsythia was surface-located. Adhesion and invasion assays revealed that T. serpentiformis was significantly less effective in invading human gingival epithelial cells than T. forsythia. Furthermore, compared to T. forsythia, a higher immunostimulatory potential of human gingival fibroblasts and macrophages was revealed for T. serpentiformis, based on mRNA expression levels of the inflammatory mediators interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein-1 and tumor necrosis factor α, and production of the corresponding proteins. Collectively, these data support the potential of T. serpentiformis to interfere with biological processes relevant to the establishment of periodontitis.