Study and development of a microneutralisation assay to evaluate the immunological efficacy of vaccines against recent pandemic and epidemic viruses

Epidemics and pandemics caused by the emergence of new viruses have not been so rare in human history. The latest in timeline are the COVID-19 pandemic caused by the Sars-CoV-2 virus and the outbreak in Europe and America of monkeypox (MPXV). These new health emergencies have highlighted the crucial role vaccines play in preventing viral-borne diseases, especially for individuals considered immunologically weak. In order for new vaccines against emerging viruses to be approved and marketed, they have to undergo several phases of clinical trials to evaluate the vaccine-induced immune response and, consequently, efficacy. To do this, clinical trials are organised in which participants undergo vaccination and subsequent biological sampling. The use of serological tests to analyse these samples makes it possible to assess antibody components in immunised and non-immunised subjects, to highlight differences in immunological responses. In antibody quantification and evaluation, the differentiation between neutralising and non-neutralising antibodies is very useful. The Microneutralisation (MN) test makes it possible to derive neutralising antibody titres in human serum samples by observing the suppression of the cytopathic effect (CPE) in a cell substrate incubated with a standardised dose of live virus and serial dilutions of the serum sample. This thesis work is divided into two tasks. In the first study, the MN-CPE test is used to analyse, in a population of healthcare workers (HWs) vaccinated with a double dose of BNT162b2 mRNA vaccine, the decline of the immune response 180 days after the second administration. In the second task, the MN-CPE test protocol was developed to be able to effectively assess the presence of neutralising anti-MPXV antibodies and examine possible cross-reactions in subjects previously vaccinated with the smallpox vaccine. To do this, a panel of human sera containing MPXV convalescent subjects and subjects vaccinated against smallpox virus was tested. The CPE-based microneutralisation test proved reliable and effective in both studies to examine the presence of neutralising antibodies in serum samples from convalescent or vaccinated subjects.