Vaccine Research with Cryo-EM

One of the major challenges to develop vaccines against infectious diseases such as RSV and HIV is the design of an effective antigen. Cryo-EM supports successful vaccine design by:

• High-resolution structural biology
  • High-resolution structural information of the pathogen (e.g., viruses, bacteria) enables the identification of antigenic regions on the pathogen.
  • Epitope mapping at a level sufficient to see the precise interaction between amino acid side chains allows the detailed characterization highly antigenic regions of the pathogen. Highly antigenic regions on the pathogen can be used to design effective immunogens to elicit an immune response. 
  • Structure guided rational design of the immunogen will help improve the immunogenicity and stability of the antigen.
  • Detailed structural analysis of the immune response against immunogens reveals precise binding sites of both neutralizing and non-neutralizing antibodies generated against the immunogen. This information helps to understand the immunogenicity potential of an antigen and enables engineering of immunogens with optimized immunogenicity.

• Particle characterization using image analysis    

Transmission electron microscopy is an important technique for characterizing attributes of all vaccine classes. At room temperature, imaging of inactivated and attenuated viruses and with adjuvant is common. For more advanced vaccine formats, such as liposomes, lipid nanoparticles (LNPs), VLPs and inorganic nanoparticles, cryo-EM based particle characterization ensures unbiased direct assessment of size, morphology, particle integrity, encapsulation state, and the stability of a vaccine formulation.

Recommended further reading:

1. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study, Lancet. (2017)
2. Cryo-EM structure of the nucleocapsid-like assembly of respiratory syncytial virus, Signal Transduction and Targeted Therapy (2023).
3. Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine, Science Translational Medicine (2023).
4. Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses, Immunity (2021).

Some of the vaccines format require a delivery vehicle to ensure safe and targeted delivery of the vaccine's antigen. Liposomes, lipid nanoparticles (LNPs), virus-like particles (VLPs), and inorganic nanoparticles are few such delivery vehicles. To read more about viral vector-based delivery vehicles, which are also extensively leveraged in cell and gene therapy, see more detailed information on cryo-EM for cell and gene therapy. Below we focus on LNPs, a non-viral vector-based vaccine delivery vehicle. Note that due to their ability to encapsulate a wide range of materials and their targetability, LNPs are also in the development for therapeutic applications like delivering vehicles for gene therapy and gene editing.

LNPs are small, spherical, lipid-based structures often ranging from 20 to 200 nanometers in size. They are of significant interest due to their ability to accommodate various types of genetic material to deliver it to target cells or tissue with specificity. Stimulated by the recent success of LNPs in COVID-related vaccines, there is wide interest and activity in developing LNP-based vaccines to target other infectious and non-infectious diseases, such as cancer and cardiovascular disease.

The successful design and application of LNPs depend heavily on their structural and morphological properties as they are directly related to their function. The safety and efficacy of LNPs are directly correlated to their size and morphology. Selection of the core structural components of LNPs can affect the structure and function of Lipid nanoparticles. Furthermore, external conditions such as buffer, temperature, and pH, the manufacturing process, storage conditions also affect the size and morphology of LNPs.

There are 5 major components of a therapeutic LNP: ionizable lipids, helper phospholipids, cholesterol, PEG lipids and, of course, the therapeutic (typically nucleic acid) to be delivered. Changes in any of these components, identify or ratio, will have a direct effect on LNP properties.

Due to their susceptibility to wide range of components as mentioned above, regulatory agencies around the world ask for comprehensive sample characterization to study the critical quality attributes (QCA) using multiple techniques to ensure the safety and effectiveness of a vaccine formulation. Cryo-EM is the only technology that offers direct visualization of LNP with nanometer-scale resolution. Furthermore, lipid nanoparticles are complex systems that require multiple techniques to characterize several parameters, cryo-EM offers to characterize several properties of LNP samples from a single experiment.

Multiple quality attributes of LNP formulation can be characterized from a single cryo-EM dataset.

Vaccine immunogenicity refers to a vaccine's potential to stimulate an immune response. To evaluate this potential, pre-clinical immunogenicity studies are carried out on animals such as mice, rabbits, and monkeys. These studies help identify the most promising candidates and determine the optimal doses and schedules for the vaccine.

• High-resolution cryo-EM studies enable immunogenicity characterization of vaccine candidates

Cryo-EM supports the study of the structural basis of the immune response against immunogens. Detailed structural analysis reveals precise binding sites of both neutralizing and non-neutralizing antibodies generated against the immunogen. This information not only helps to understand the immunogenicity potential of an antigen but also offers to engineer the immunogen to increase its immunogenicity.

• Cryo-EM Polyclonal Epitope Mapping (CryoEMPEM) studies

Cryo-EM PEM (or EMPEM) is growing in popularity to study the immune response (immunogenicity) against biologics such as antibodies and vaccines. This technology allows the study of the polyclonal antibody response against an immunogen/vaccine candidate. With cryo-EM, it is possible to unambiguously characterize the epitope-paratope interaction at a level at which amino acid side chains and their interactions are observable. With this information, it is possible to optimize desirable immunogenicity for vaccine design or reduce the immunogenicity for antibody therapeutics and gene therapy viral vectors.

Recommended further reading:

1)      Structural mapping of antibody landscapes to human betacoronavirus spike proteins, Science Advances (2022).

2)      Mapping Polyclonal Antibody Responses in Non-human Primates Vaccinated with HIV Env Trimer Subunit Vaccines, Cell Reports (2020).

3)      Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM, Nature Communications (2021).