Preparedness and surveillance of public health emergency pathogens through authentic virus and pseudotype based neutralisation assays

Climate, technological and demographic change of the last decades has led to multiple infectious disease outbreaks of international concern. The main strategy of WHO to control newly epidemics and pandemics, is to define specific countermeasures and enhance the preparedness against emerging pathogens. This can be achieved with international cooperation, that could help in fasten the research and development of responses against future epidemics and pandemics. This thesis focused on the study and optimisation of Neutralisation assays, which have historically demonstrated their efficacy to evaluate pathogens’ transmissibility, perform vaccine assessment and epidemiology studies. One of the first emergence response that could be applied at the beginning of an outbreak to prevent a rapid spread of the virus, is to use vaccines that are already commercially available but designed for a virus that belongs to the same family as the one of interest. This strategy was investigated in the first task, were a complement-based neutralisation assay was used to evaluate whether the smallpox vaccination is able to provide cross-protection against the MonkeyPox disease. The use of viruses which require specific high-containment laboratories is one of the major drawbacks of neutralisation assays, because of the limited number of BSL3/4 worldwide and the high cost, slowing down emergency responses. In the second task of this work, it was investigated the possibility to replace the authentic virus with a pseudotyped one, which is able to undergo only one round of replication and, by this means, can be handled in BSL2 laboratories. Here, a neutralisation assay based on the use of a VSV pseudotype for the Rift Valley Fever Virus was optimised and qualified following the ICH Q2 guidelines, demonstrating to fit for a subsequent validation and the future use in clinical trials. A further optimisation of the developed pseudotype-neutralisation assays was performed in the third task, where the commonly used firefly luciferase reporter was replaced with a newly produced one (HRP-TM). With the aim of making the detection assay more accessible to perform in the Global south, the new reporter gene was designed to reduce the detection cost and avoid the requirement of specialised instruments or particularly low storage temperatures. The HRP-TM resulted to have a lower sensitivity than the luciferase platform but further optimisation experiments could provide promising results, especially in the application of pseudoviruses that are usually produced at high yields. In conclusion, in this thesis it was possible to confirm the utility of neutralising assays in the field of preparedness and serosurveillance against emerging viruses. The use of pseudotypes could help the international cooperation by reducing the biosafety requirement and making the assay accessible to more facilities. The pseudotype platform was also identified as a flexible tool for further improvements and facilitate the inclusion of middle/low-income countries to study high priority pathogens. The second and third task were carried out at the South Campus of the Medicines and Healthcare Products Regulatory Agency (MHRA), UK.