Conjugation of Toll Like Receptor 7 agonist through conjugation to immunogenic proteins. Synthesis, characterization, formulation and pre-clinical evaluation

The next generation of vaccine adjuvant are represented by a wide ranging set of molecules called Toll like agonists (TLR’s). Although many of these molecules are complex structures extracted from microorganisms, small molecule TLR agonists have also been identified. However, delivery systems have not been optimized to allow their effective delivery in conjunction with antigens. Here we describe a novel approach in which a small molecule TLR agonist has been conjugated directly to antigens to ensure effective co delivery. We describe the conjugation of a relevant protein, a recombinant protective antigen from S.pneumoniae (RrgB), which is linked to a TLR7 agonist. Following thorough characterization to ensure there was no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended animals’ survival after lethal challenge with S.pneumoniae. Comparable results were obtained with a 10 fold lower dose than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and is was more effective than simple co-administration. Likely, this approach can be adopted to reduce the dose of antigen required to induce protective immunity, and potentially increase the safety of a broad variety of vaccine candidates

A corpus-assisted approach to apology-like expressions in Japanese computer-mediated discourse

This thesis provides a corpus-assisted pragmatic investigation of three Japanese expressions commonly signalled as apologetic, namely gomen, su(m)imasen and mōshiwake arimasen, which can be roughly translated in English with ‘(I’m) sorry’. The analysis is based on a web corpus of 306,670 tokens collected from the Q&A website Yahoo! Chiebukuro, which is examined combining quantitative (statistical) and qualitative (traditional close reading) methods. By adopting a form-to-function approach, the aim of the study is to shed light on three main topics of interest: the pragmatic functions of apology-like expressions, the discursive strategies they co-occur with, and the behaviours that warrant them. The overall findings reveal that apology-like expressions are multifunctional devices whose meanings extend well beyond ‘apology’ alone. These meanings are affected by a number of discursive strategies that can either increase or decrease the perceived (im)politeness level of the speech act to serve interactants’ face needs and communicative goals. The study also identifies a variety of behaviours that people frame as violations, not necessarily because they are actually face-threatening to the receiver, but because doing so is functional to the projection of the apologiser as a moral persona. An additional finding that emerged from the analysis is the pervasiveness of reflexive usages of apology-like expressions, which are often employed metadiscursively to convey, negotiate and challenge opinions on how language should be used. To conclude, the study provides a unique insight into the use of three expressions whose pragmatic meanings are more varied than anticipated. The findings reflect the use of (im)politeness in an online and non-Western context and, hopefully, represent a step towards a more inclusive notion of ‘apologies’ and related speech acts.