Case Study: Using cryoEM to characterize 
virus-like particles in GARDASIL®

The challenge

Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections that result in cervical cancer and warts. The yeast-derived recombinant quadrivalent HPV L1 (Types 6, 11, 16, and 18) vaccine GARDASIL has played an important role in reducing cervical cancer since their introduction for human use in 2006.

The L1 proteins expressed in yeast self-assemble into virus-like particles (VLPs) that when properly adjuvanted, elicit protective immune responses by mimicking the authentic epitopes of virions. Routine biochemical techniques can be applied to confirm the primary structure of the proteins in these recombinant vaccines. However, other tools are needed to visualize critical morphological characteristics of these VLP vaccine intermediates.

The solution

Cryo transmission electron microscopy (cryoEM) provides an excellent visualization tool to directly determine morphological characteristics of VLP vaccine intermediates, including shape, particle integrity, and aggregation. It can also be used to observe the VLPs when absorbed onto aluminum adjuvants. 

These data can be combined with the results from orthogonal methods to provide information important for process development, process optimization, and comprehensive characterization of pivotal vaccine lots. One of the unique advantages of the cryoEM method is that a 3D map of the structure can be reconstructed by combining particles of the same morphology and conformation but in different relative orientations.

The results

VLP Morphology
CryoEM images for each of the four HPV L1 serotypes (L1 types 6, 11, 16 and 18) are shown in Figure 1. In the top left panel, the image is split to show type 16 particles on the left and type 16 particles decorated with an antibody fragment on the right. Scale bar is 200 nm.

Most of the VLPs appear to be fully assembled, are observed to have a range of sizes, and are predominantly spherical or ellipsoidal in shape, with no evidence of filaments or other large aggregates. 
image of VLP morphology
3D reconstruction of HPV VLP and VLP:Fab structure
CryoEM can provide a 3D map of the structure, reconstructed by combining particles of the same morphology and conformation but in different relative orientations. A set of particles of similar diameter (54 ± 3 nm) were selected from images of VLPs of type 11 and type 16, and single particle analysis methods were used to reconstruct 3D maps of each serotype, as shown in Figure 2. 

The three-dimensional reconstructions of HPV11 can be observed in image (A) while image HPV11, decorated with antibody fragment H11.B2, is shown in image (B). HPV16 is represented in image (C) while HPV16, decorated with antibody fragment H16.V5, is illustrated in image (D). The reconstructed volumes show that the capsids are constructed of 72 pentameric capsomers arranged in a T = 7 icosahedral lattice that closely resembles that of the native virions of human papillomavirus. 
3D reconstruction of HPV VLPs
VLPs maintain their spherical morphology on aluminum adjuvant
The quadrivalent HPV VLP vaccine, GARDASIL, is a sterile liquid suspension prepared by combining VLPs of each HPV type. All the VLPs were adsorbed onto an aluminum-containing adjuvant (Amorphous Aluminum Hydroxyphosphate Sulfate or AAHS) and suspended in the final formulation buffer.

To understand whether interaction with the aluminum adjuvant surface alters the VLP morphology, HPV VLPs adsorbed onto the aluminum adjuvant were directly visualized using cryoTEM in the vitrified state (see Fig. 3). There was no apparent change in shape upon adsorption onto the amorphous adjuvants and the VLPs retained their spherical shape.
image of VLPs on aluminum


CryoEM can be a highly informative complementary tool for VLP characterization. The characterization of a recombinant VLP-based vaccine requires the application of an extensive panel of analytical procedures to the product, and the characterization data set is included in regulatory filings to demonstrate an understanding of the product. 

A comprehensive characterization data set is important for supporting selection of the potency assay and justifying specifications for the product and the impurity profile. It is also important for characterizing the process and establishing the “historical” comparability and therapeutic/clinical equivalence of the product. 


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