Periodic Reporting for period 2 - FUSION (Single Molecule Imaging-based design of HIV-1 vaccines)
Okres sprawozdawczy: 2021-10-01 do 2023-03-31
In HIV-1 research the identification of antibodies that neutralize many circulating strains represent a big hope to produce a vaccine. The mechanistic mode of action of most broadly neutralizing antibodies is not well understood. The use of new and radical technologies on single molecule light microscopy will shed light into these processes to understand with single molecule sensitivity how different families of broadly neutralizing antibodies (bNabs) interact with the envelope glycoprotein of HIV-1 (Env). To identify the mechanistic changes in HIV-1 receptor stoichiometry (inter and intramolecular dynamics) associated to Env binding we have developed a new approach that combines lifetime imaging (FLIM) and single molecule FRET (smFRET).
We have also developed a new technique able to detect single molecules and single antibodies as they interact with single viruses based on fluorescence correlation spectroscopy. We have invested considerable time and effort to validate this approach, and we are convinced that this new technology will be very useful to characterize not only HIV-1 viruses as they interact with bNabs but any virus (for example SARS-CoV 2). We have partnered up with a company to produce a compact prototype and have applied to ERC proof of concept (PoC) to get funding to being able to translate these ideas to the market. The main aim of this ERC PoC is to translate this knowledge into a more compact and easy-to-use system to make it available to the scientific community (virologists and pharma). This system will be able to render quantitative information and will be crucial to better characterize and test bNAbs for many enveloped viruses utilizing a compact apparatus that can be easily installed in hoods of high containment labs. Moreover, we will simplify the acquisition software and analysis so that users do not necessarily need to have previous experience in single molecule biophysics. Such a system has the potential to revolutionize virology and infection research, rendering new quantitative knowledge to the clinic almost instantly.
In the second part of our ERC CoG, we will start producing relevant data on the interactions between HIV-1 relevant strains and bNAbs. We will characterize how virus maturation affects Env dynamics and the importance of Env clusters in the process of neutralization. We will also produce data on the different stoichiometries between Env primary isolates and different batteries of bNabs (also labelled and tested for functionality). This approach will render important results that will, for the first time, relate structure-based approaches, virus maturation and functional assays on infectivity. The overall objective for this second part of the ERC will be to fully characterise a number pf bNAbs as they interact with relevant Env strains to understand their common mechanistic traits.
We have also developed a new technique able to detect single molecules and single antibodies as they interact with single viruses based on fluorescence correlation spectroscopy. We have invested considerable time and effort to validate this approach, and we are convinced that this new technology will be very useful to characterize not only HIV-1 viruses as they interact with bNabs but any virus (for example SARS-CoV 2). We have partnered up with a company to produce a compact prototype and have applied to ERC proof of concept (PoC) to get funding to being able to translate these ideas to the market. The main aim of this ERC PoC is to translate this knowledge into a more compact and easy-to-use system to make it available to the scientific community (virologists and pharma). This system will be able to render quantitative information and will be crucial to better characterize and test bNAbs for many enveloped viruses utilizing a compact apparatus that can be easily installed in hoods of high containment labs. Moreover, we will simplify the acquisition software and analysis so that users do not necessarily need to have previous experience in single molecule biophysics. Such a system has the potential to revolutionize virology and infection research, rendering new quantitative knowledge to the clinic almost instantly.
In the second part of our ERC CoG, we will start producing relevant data on the interactions between HIV-1 relevant strains and bNAbs. We will characterize how virus maturation affects Env dynamics and the importance of Env clusters in the process of neutralization. We will also produce data on the different stoichiometries between Env primary isolates and different batteries of bNabs (also labelled and tested for functionality). This approach will render important results that will, for the first time, relate structure-based approaches, virus maturation and functional assays on infectivity. The overall objective for this second part of the ERC will be to fully characterise a number pf bNAbs as they interact with relevant Env strains to understand their common mechanistic traits.
We have characterized both the importance of intra and intermolecular Env dynamics in real viruses. Moreover, we have characterized these interactions in the presence of different ligands (such as the receptor sCD4 and different families of broadly neutralizing antibodies). Importantly, we have combined FLIM with single virus tracking when real HIV-1 viruses engaged with T cells demonstrating the importance of intermolecular dynamics and the establishment of Env functional clusters. This work was recently published (Carlon-Andres et al., 2021. Commun Biol)).
We have also investigated the importance of in innate immunity for HIV-1 entry. We have shown that the interferon-inducible transmembrane (IFITM) proteins have an antiviral function which is strictly related to their oligomeric state, and therefore both their potential to cluster and the mechanical tension in the membrane are crucial to understand their antiviral activity in the host. (Rahman et al., 2020. eLife).
In the context of HIV-1 transinfection we have put together a novel imaging approach able to render up to 9 different colours simultaneously. This has allowed to characterize HIV-1 cell-cell fusion and which are the molecular determinants for this process, including the role of Dynamin 2 and the stoichiometry of these interactions (Starling et al., 2022. BioRxiv/ Under review).
One of the caveats of this work was that we worked with an Env lab strain (termed HXB2). Currently, we have developed new labelling strategies employing nanobodies using a primary isolate (JR-FL) that presents different dynamics and is more resistant to bNabs neutralization. Importantly, we have employed artificial intelligence to evaluate the impact on the 3D structure of our labelling strategy and shown also with fusion and infection experiments that these labelled Envs are viable and functional. Our next step is to test these Env decorating HIV-1 viruses to understand their inter and intramolecular dynamics as compared with our previous results using the lab strain (HXB2).
Finally we have also presented our work in international conferences (CSH-Retroviruses, New York or EMBO 8th National Biosensor Meeting, France) both in 2022.
We have also investigated the importance of in innate immunity for HIV-1 entry. We have shown that the interferon-inducible transmembrane (IFITM) proteins have an antiviral function which is strictly related to their oligomeric state, and therefore both their potential to cluster and the mechanical tension in the membrane are crucial to understand their antiviral activity in the host. (Rahman et al., 2020. eLife).
In the context of HIV-1 transinfection we have put together a novel imaging approach able to render up to 9 different colours simultaneously. This has allowed to characterize HIV-1 cell-cell fusion and which are the molecular determinants for this process, including the role of Dynamin 2 and the stoichiometry of these interactions (Starling et al., 2022. BioRxiv/ Under review).
One of the caveats of this work was that we worked with an Env lab strain (termed HXB2). Currently, we have developed new labelling strategies employing nanobodies using a primary isolate (JR-FL) that presents different dynamics and is more resistant to bNabs neutralization. Importantly, we have employed artificial intelligence to evaluate the impact on the 3D structure of our labelling strategy and shown also with fusion and infection experiments that these labelled Envs are viable and functional. Our next step is to test these Env decorating HIV-1 viruses to understand their inter and intramolecular dynamics as compared with our previous results using the lab strain (HXB2).
Finally we have also presented our work in international conferences (CSH-Retroviruses, New York or EMBO 8th National Biosensor Meeting, France) both in 2022.
The implementation of three different imaging approaches to better understand how HIV-1 Env intracts with bNabs are the key technological approaches beyond the state of the art:
1- Simultaneous Imaging of single HIV-1 viruses engaged with live T cells. We recovered single molecule Env dynamics of HIV-1 viruses engaged with T cells in the presence and absence of different families of broadly neutralizing antibodies.
2- Implementation of a new technique to detect single bNabs as they interact with single viruses in a femtoliter volume. With this technology we will be able to characterize both Env inter and intramolecular dynamics and the impact of bNAbs in single viruses.
3- Multicolor Imaging using FLIM. We have also implemented a new technique able to image up to 9 colors simultaneously. With this technique we are characterizing HIV-1 induced cell-cell fusion and which are the molecular determinants for these interactions.
We anticipate that these three approaches will be crucial to achieve the two aims of the action and they are already providing preliminary data to be presented in international conferences and other venues.
1- Simultaneous Imaging of single HIV-1 viruses engaged with live T cells. We recovered single molecule Env dynamics of HIV-1 viruses engaged with T cells in the presence and absence of different families of broadly neutralizing antibodies.
2- Implementation of a new technique to detect single bNabs as they interact with single viruses in a femtoliter volume. With this technology we will be able to characterize both Env inter and intramolecular dynamics and the impact of bNAbs in single viruses.
3- Multicolor Imaging using FLIM. We have also implemented a new technique able to image up to 9 colors simultaneously. With this technique we are characterizing HIV-1 induced cell-cell fusion and which are the molecular determinants for these interactions.
We anticipate that these three approaches will be crucial to achieve the two aims of the action and they are already providing preliminary data to be presented in international conferences and other venues.