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The value of structure in vaccine development

Vaccines are specialized preparations that prime the body’s immune response to respond rapidly to infectious diseases. They do so by exposing the body to components of the pathogen that elicit and remember antibodies that can neutralize the pathogen. The immune system recognizes the antigen as foreign and generates an immune response which ultimately results in the production of neutralizing antibodies against the antigen. This “memory” of the pathogen is developed and retained in the form of memory cells which accelerates the immune response during a future exposure of the body to the actual, disease-causing pathogen.

 

There are a wide variety of vaccine strategies, which have evolved in complexity over time. First vaccines were generated in the format of live attenuated pathogens (ex. MMR vaccine), inactivated viruses (ex. polio vaccine), subunit vaccines (ex. Hepatitis B vaccine), and virus-like particles (VLPs) (ex. Human papillomavirus VLP vaccine). Taking advantage of technologies developed over the first part of this century, new vaccines formats like viral vector vaccines, and mRNA vaccines (ex. Pfizer-BioNTech, Moderna Covid-19 vaccines) were matured into regular use during the SARS-Cov-2 pandemic. Cryo-electron microscopy is playing a crucial role in expediting the vaccine design and discovery process. 


Vaccine development supported by structure

To accelerate the development of vaccines that are both safe and effective, it is crucial to use imaging methods that offer comprehensive structural data throughout the vaccine discovery and development process. Cryo-electron microscopy (cryo-EM) is a powerful tool for determining 3D structures and is enabling the optimization of vaccine design and development pipelines. Cryo-EM can also be used effectively for lipid nanoparticle characterization throughout the vaccine development, formulation, and manufacturing process.

Antigen design for effective vaccine development

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.

 

Case study – Development of an antigen for the RSV vaccine

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection and death amongst infants and elderly. Worldwide, about 118,200 children die annually from RSV. Cryo-EM has played a crucial role in the development of RSV vaccines. The prefusion conformation of the RSV fusion (F) glycoprotein is a key immunogen for vaccine development. Cryo-EM based structural biology helped solve the structure of RSV nucleocapsid like assemblies, providing information on the virus architecture. Furthermore, the cryo-EM structure of a prefusion-stabilized RSV antigen (F protein) contributed to the design of effective RSV vaccine candidates.


Vaccine Delivery Vehicles

Design and characterization of vaccine delivery vehicles

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.

 

Lipid nanoparticle (LNP): delivery vehicles for both vaccines and therapeutics

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.

 

Characterization of lipid nanoparticles (LNPs) using cryo-EM

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.

 

Comprehensive characterization of LNP formulations using cryo-EM

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

Examples showing the application of cryo-EM for the characterization of lipid nanoparticles.

Direct visualization of shape and size of LNPs

Characterization of encapsulation state of mRNA inside lipid nanoparticles by cryo-EM. Images adopted from Brader M et al 2021.

Characterization of the effects of physical stress on the size, morphology, and internal structure of LNPs. Image adopted from Brader M et al 2021.


Vaccine Immunogenicity

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.

Cryo-EM Polyclonal Epitope mapping (cryoEMPEM) workflow


Cryo-EM solutions

Our full cryo-TEM portfolio features state-of-the-art technology with a range of automation features designed to extend accessibility, reduce the need for user intervention, and enable easy organization, viewing, and sharing data.

Glacios 2 Cryo-TEM

A complete solution for high-resolution structure determination with unparalleled automation and ease-of-use.

Krios G4 Cryo-TEM

Atomic resolution cryo-EM instrument with enhanced productivity and compact design.

Tundra Cryo-TEM

Dedicated structural analysis solution designed to bring cryo-EM to your laboratory.

Talos L120C

Versatile TEM and STEM microscope for 2D and 3D visualization of beam sensitive samples and materials using high contrast

Resources

Cryo-EM structure of AAV8 and epitope mapping of CaptureSelect AAVX

Leveraging cryo-electron microscopy for innovative vaccine development


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