Cryo-EM imaging and epitope mapping: we look at novel approaches by Dr Andrew Ward at Scripps Research
Olivia Siviter looks back at the work of Dr Andrew Ward from Scripps Research, which was presented at the Antibody Engineering & Therapeutics 2021 conference.
Cryo-EM imaging can be used to visualise the epitopes of a specific antigen, at a granular level for structural refinement of antigens. Epitope mapping via cryoEM can help give a better understanding of immune response to a given disease to inform therapeutic strategies.
As an example of the power of the cryoEM, Dr Andrew Ward from Scripps Research, describes his groups analysis of HIV Env elicited pAbs in NHPs (non-human primate model) which led to the discovery of structurally unique pAbs classes.
A total of 21 cryoEM maps featuring distinct pAbs were reconstructed from serum samples from 4 animals. 8 unique epitope clusters were identified which allowed for the antigen to be characterized in further detail.
They found that certain epitopes are immunodominant across individuals/species, which means that pAbs will be competing for binding with the epitope.
His lab is in the process of improving the sensitivity of cryoEM in order to improve throughput of the technology. In data not yet published, they have been able to construct 11 unique maps from a single dataset when mapping HIV Env trimer elicited pAbs in rabbits.
This allowed for the distinction of 5 different epitope clusters. They aim to continue this research longitudinally so they can understand whether there is synergy between or inhibition between epitopes.
This information can go into the molecular redesign of antigens to shift the immune response towards antigens that are more efficient.
Real power in cyroEM is that the structural data can be compared to databases Vh/Vl germline sequences to identify Vh/Vl genes for different polyclonal antibodies to infer the germline usage to look for convergence, divergence etc.
One of the main goals of this technology is to create databases so that epitope mapping can happen in a more automated fashion.
To achieve this, they have developed a sequence assignment system that categorises epitopes based on the structural feature of different amino acids.
This has allowed Charles Bowman at Scripps Research to create an algorithm to search databases specifically for sequences that fit these sequence assignments.
This helps to figure out which pieces of the antibody we need to get strong positive hits which removes the necessity to sequence the entire antibody.
The technology has also been used to compare monoclonal and polyclonal antibody complexes. It has been found that the structures of monoclonal and polyclonal antibody complexes are highly consistent.
In relation to SARS-CoV-2, the system has been used to understand cross-reactive responses in polyclonal antibodies between seasonal CoVs and SARS-CoV-2 to understand which antibodies against SARS-CoV-2 may be circulating in memory cells.
Dr Ward’s group have found that OC43 antibodies bind to the NTD receptor binding site and that OC43 CTD antibodies are immuno-focused on a single flexible loop. This information can be used for vaccine design to ensure that the antigens used are not ones with variable responses.
As there has been far less discovery of S2 antibodies, Dr Ward’s group is currently focusing on building these libraries to understand cross-reactive responses of SARS-related viruses.
By focusing on cryoEM, antibodies can be isolated directly from the sera in a less than two weeks. Dr Ward’s aim is to make the process even faster so they can be prepared for generating more monoclonals for emerging pathogens. CryoEMPEM data can be used in b-cell response analysis, vaccine redesign and antibody discovery.
