Periodic Reporting for period 1 - FILM-HIV (Super-resolution quantitative imaging of HIV fusion and its neutralisation by antibodies) Reporting period: 2021-01-01 to 2022-12-31 Summary of the context and overall objectives of the project Viruses are small biological particles that can infect humans and cause major health complications. In the case of the Human Immunodeficiency Virus (HIV), that causes the acquired immunodeficiency syndrome (AIDS) and is responsible for almost one million deaths every year worldwide, the viral particle is just 120 nm big. However, conventional microscopes are diffraction-limited, as described by the German physicist Ernst Abbe in the XIX century. This means that we are not able to resolve objects that are smaller than 250 nm, thus preventing the study of small viruses. It is however imperative that we are able to understand the molecular mechanisms governing their infection cycle and the natural responses developed by our immune system to be able to efficiently treat viral infections in the clinic, through vaccination or immune therapies.The goal of the FILM-HIV project is to develop and apply advanced microscopy methods that can overcome the diffraction limit and observe viruses with nanometre precision. This multidisciplinary biophysics project spans from the optics fields to immunology and cell biology, and has been developed at the Leibniz Institute of Photonic Technology in Jena, Germany. The university of Jena was the alma matter of Ernst Abbe, and the city is internationally known for its long tradition on microscopy development and manufacturing. The project aims to apply these new methodologies to record the entry of HIV virions in immune cells, and how a specific type of broadly neutralising antibodies isolated from HIV patients can stop this process.One of the main challenges when developing microscopy techniques is to make them compatible with the observation of biological samples, such as human cells, that are highly sensitive to the high laser powers required in modern microscopy proceedings. To overcome this limitation, we have applied a new methodology that consists in the constant recycling of the fluorescent molecules that allow us to visualise cells. These novel dyes, known as exchangeable dyes, not only allow us to observe cells with nanometre resolution, but also allow as to quantify their biophysical properties, such as the diffusion of single molecules or their order, which in turn offers us valuable information about key biological processes such as membrane fusion, which HIV requires to infect cells.Finally, we have applied novel super-resolution techniques to the study of the binding of potent neutralising antibodies to HIV. We have identified key features of these antibodies responsible for their potency and found that we can naturally enhance their potency by creating engineered versions that increase their binding capacity to the HIV membrane. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far At the beginning of the project, we set up virus and cell models that allowed us to investigate HIV in biosafe environments. We built a biosafe (non-infectious) virus collection that allowed us to study it using different microscopy techniques. Among them, we would like to emphasise the use of the super-resolution methodologies STED and MINFLUX, which we used to investigate the HIV replication cycle. During this process certain lipids are known to play key roles, as they get enriched in the viral membrane when the virus is released from infected cells. In collaboration with the Karolinska Institutet in Sweden, we investigated this process in a non-infectious model, and studied how HIV can alter the biophysical properties of human cell membranes.Interestingly, lipids also play a key role during the immune response to HIV, as a specific type of antibodies produced by our immune system, albeit rarely, are able to interact with them, enhancing their capacity to bind virions and neutralise them. In a collaboration with the University of the Basque Country in Spain, we have built a library of fluorescent versions of these antibodies, so that we can observe them under our advanced microscopes. We investigated the ability of the antibodies to bind to HIV viruses with our super-resolution microscopes, that have the sensitivity to observe individual antibody molecules, which are just 2-3 nanometres big. We found that we could tune the neutralisation ability of these antibodies by changing their lipid-interacting capacity. Thus, we designed new antibody versions that could bind to viral lipids more efficiently, increasing their blocking activity.These results have been published in renowned scientific journals openly accessible worldwide and also communicated in important international conferences. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We expect our results to have a profound impact in the fields of microscopy, cell biology and immunology. The methodology developed for the observation of membrane fusion events, that is compatible with simultaneous quantitative measurements of their biophysical properties, has the potential to be applied to the study of numerous biological processes, as membrane fusion is a ubiquitous mechanism not only used by many viruses, but also by all the cells found in nature to incorporate nutrients or uptake messaging signals.We have also pushed the limits of what we can measure with super-resolution microscopy. By enhancing its compatibility with imaging biological samples, such as viruses and living cells, now we are able to measure them for longer periods of time and with greater detail. Moreover, we have applied new microscopy techniques to the study of antibodies, nanometric molecules that are key to our natural response to viruses and other pathogens.Europe is a world leader in microscopy. By continue working together, breaking the boundaries between different scientific disciplines, life scientists and technology developers will allow us to study relevant biomedical issues with the highest possible detail, allowing us to understand how life works, and apply this knowledge to improve our health.