Alsteens, DavidCotin, FlorianFlorianCotin2025-05-142025-05-142025-05-142019https://hdl.handle.net/2078.2/12477Herpesviruses (HVs) are ubiquitous enveloped viruses that establish long-term infections and are responsible for a variety of diseases, especially in immuno-compromised hosts. The first stage of the virus life cycle is the attachment to the cell surface, followed by entry of the virus inside the cell. HVs attachment and entry process is more complex than that of other viruses as it requires the concerted effort of multiple glycoproteins and involve multiple host receptors. In the past three decades, the atomic force microscope (AFM) has become a multifunctional tool allowing the exploration of complex biological samples in physiological-like conditions, enabling to decipher cells-virus interactions at high resolution. The recent developments of new modes of operation has transformed AFM into a versatile technique that allows to image live cells while simultaneously quantifying the interactions that guide the virus entry. In particular, the recently developed single-virus force spectroscopy (SVFS) has made it possible to study single virus attachment to mammalian cells during the very first steps of infection. After characterization of the molecular contributions in HV binding to its cognate attachment factors, the next step is to probe the effect of molecules interfering with viral attachment. As they are the key molecules involved in host immune response, antibodies are great potential candidates for AFM investigation of virus-cell binding inhibitors. In this work, we demonstrated that the effect of antibodies on single-herpesviruses particles can be investigated using atomic force microscopy. The model herpesvirus we used for our experiments was the murine gamma-herpesvirus 4 (MuHV-4). The MuHV-4 surface glycoproteins gH/gL and gp70 are known to bind cell surface glycosaminoglycans (GAGs) in the very first step of infection, giving MuHV-4 virions a first vital foothold, with gH/gL playing the major binding role. We observed that an anti-gH/gL (T2C12) antibody injection significantly decreased the MuHV-4 specific binding frequency on GAG-coated surfaces. Furthermore, we performed dynamic force spectroscopy analysis of virion- GAG bonds to extract kinetic parameters of the interaction. We introduced a new quantitative approach to compare the contribution of individual glycoproteins before and after antibody injection. Thanks to this approach, we concluded that anti-gH/gL antibodies strongly decreased the multivalency of the interaction. We showed that T2C12-treated virions could still bind GAGs after T2C12 antibody injection, but with much weaker strength and at lower frequency. Therefore, T2C12 might not completely neutralize the virion at the attachment stage, but probably help to reduce the attachment efficiency, which facilitate neutralization at a subsequent stage. While experiments on model surfaces were reliable and well-defined, similar experiments on live cells were difficult to interpret, highlighting the complexity of cell-virus interplay. Validation of our results on live cells is certainly the next key step for further investigations of virus binding blockade by antibodies. This work aims to prove that nanoscale investigations with the AFM are promising for the screening of drugs specifically targeting the early stages of viral infection and offers new perspectives towards neutralization of viruses in the future.AFMHerpesvirusSingle-virus force spectroscopyNeutralizing antibodiesGlycosaminoglycansMultivalencyGlycoproteinsAttachmenttext::thesis::master thesisthesis:19585