Influenza and Respiratory Syncytial Virus: three approaches of methods Validation

Influenza and Respiratory Syncytial viruses are the most common pathogens able to infect humans causing respiratory illness. They both have a high impact on the morbidity and mortality worldwide in particular for high-risk population. Influenza A and B viruses can have an impact in all the population, regardless the age of people, while respiratory syncytial virus is well known for its role in paediatric and geriatric respiratory infections. Given their high transmissibility, vaccination represents the most important tool to prevent repeated infections. Candidate vaccines are administered during clinical trials phases in order to assess their immunogenicity and efficacy before to obtain the license. During this period, biological samples are collected from clinical trial participants and tested through different bioanalytical methods evaluating different, but required, parameters. Before to start clinical testing, the methods must be validated following ICH (International Conference on Harmonisation), FDA (Food and Drug Administration) or any other guideline required for regulatory submission. The validation process allows to define the method valid for its purpose and to guarantee the solidity of the analytical results. Parameters and acceptance criteria to be evaluated are different based on the applied technique and are defined by the different regulatory bodies. The present work has been divided in three main tasks. The first one reports the validation process for flow cytometry for the evaluation of the immune response in human samples. The second one refers to the validation process of Real Time RT-PCR technique by using TaqMan technology for the detection and characterization of Influenza viruses by using viral RNA as starting template. The application of this technique allows to have a prediction of the efficacy of the candidate vaccine formulation. The third task includes the development and validation of the Microneutralization ELISA-based method for the detection and quantization of neutralizing antibodies against respiratory syncytial virus A and B subtype. The presence or absence of neutralizing antibodies allows to evaluate the immunogenicity of the new vaccines. In general, acceptance criteria were fulfilled for each method analysed in the three tasks, demonstrating that the assays can be considered validated and subsequently applied for future clinical studies.