Characterization of virus-like particles in GARDASIL® by cryo transmission electron microscopy

Zhao, Qinjian; Potter, Clinton S.; Carragher, Bridget; Lander, Gabriel C.; Sworen, Jaime; Towne, Victoria; Abraham, Dicky G.; Duncan, Paul; Washabaugh, Michael W.; Sitrin, Robert D.

doi:10.4161/hv.27316

Cited by 55 publications

(65 citation statements)

References 33 publications

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“…However, the Fab densities at the 5-fold were weaker, indicating a clash with the neighboring Fab bound to the hexavalent capsomer. This occupancy of Fab was inconsistent with a previous study that showed exclusive binding to the hexavalent capsomers (17). This discrepancy could be due to the different capsid forms used (L1-only VLP versus QV), different contour levels displayed for the maps, or other variations in experimental conditions, such as temperature and incubation time.…”

Section: Resultscontrasting

confidence: 54%

“…Atomic structures of HPV16 L1-pentamers and a Tϭ1 capsid have been solved by X-ray crystallography (12-14); however, the HPV Tϭ7 capsid has been visualized only by cryo-electron microscopy (cryo-EM) reconstructions (11,(15)(16)(17)(18). Twelve of the pentamers lie on the icosahedral 5-fold axes (pentavalent capsomers), whereas the other 60 pentamers are positioned at the pseudo 6-fold axes (hexavalent capsomers).…”

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confidence: 99%

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A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

Lee

Brendle

Bywaters

et al. 2015

J Virol

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Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5. Human papillomavirus (HPV) is a nonenveloped doublestranded DNA virus that can induce several epithelial cancers, especially cervical cancer (1-3). HPV16 is the most prevalent high-risk type of HPV (4, 5) and has been a primary target for the development of prophylactic vaccines (6, 7). HPV is epitheliotropic, and its replication is tightly associated with terminal differentiation of keratinocytes. This restricted tropism makes the production of high-titer preparations of authentic virion challenging. Alternative production methods have been developed to produce high-titer stocks of virus-like particles (VLP) (8), pseudovirions (PsV) (9), and quasivirions (QV) (10) while preserving the main attributes of the native capsid structure. These particles have been used successfully for vaccine development and for studies of antigenicity, receptor usage, entry mechanisms, and capsid structure.The infectious HPV has a Tϭ7 icosahedral capsid (55 to 60 nm in diameter), composed of 72 L1 capsid protein pentamers and up to 72 copies of L2 capsid protein located bene...

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Section: Resultscontrasting

confidence: 54%

mentioning

confidence: 99%

A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

Lee

Brendle

Bywaters

et al. 2015

J Virol

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“…Since the HPV life cycle depends on the differentiation of keratinocytes, it is difficult to purify high-titer virus stocks for structural studies. Virus-like particles (VLPs) that are devoid of viral genome (11) have been used successfully for structural studies (8,12,13), whereas both pseudovirus (PsV) and quasivirus (QV), which contain expression plasmid DNA (14,15), have been used for structural studies and infectivity assays (9,10). For the work presented here, quasivirus has been used throughout.…”

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confidence: 99%

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

Guan

Bywaters

Brendle

et al. 2015

J Virol

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The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T‫7؍‬ icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers. H uman papillomavirus (HPV) infections continue to be a significant health burden in patient populations (1, 2). Although commercial vaccines targeting the viral capsid proteins have been applied successfully to protect against high-risk HPV, the efficacy of vaccines is genotype specific, and vaccines provide little therapeutic benefit against existing infections (3). Understanding the antigenic nature of the HPV capsid offers an opportunity to discover structural features that are crucial to capsid integrity and conserved across species. Panels of monoclonal antibodies and mutational analyses have helped to define several antigenic epitopes (4-10); however, determining the conformational epitopes on the capsid surface requires structural analyses, which can be accomplished by cryo-electron microscopy (cryo-EM) technology. Since the HPV life cycle depends on the differentiation of keratinocytes, it is difficult to purify high-titer virus stocks for structural studies. Virus-like particles (VLPs) that are devoid of viral genome (11) have been used successfully for structural studies (8, 12, 13), whereas both pseudovirus (PsV) and quasivirus (QV), which contain expression plasmid DNA (14, 15), have been used for structural studies and infectivity assays (9, 10). For the work presented here, quasivirus has been used throughout.Papillomaviruses form a nonenveloped Tϭ7 icosahedral capsid that is ϳ55 to 60 nm in diameter and contains a circular double-stranded DNA (dsDNA) genome of 8 kb. The capsid is comprised of 360 copies of the L1 major structural protein and an uncertain number of the L2 minor structural protein (15,16). Five copies of the L1 protein intertwine to form each capsomer, and 72 capsomers interact to constitute a capsid. T...

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“…The VLPs in GARDASIL Ò were desorbed using a citrate phosphate buffer, and a full recovery of the antigenicity of mAb-based analysis was observed after adjuvant dissolution. 25,39 This technique enabled the development of an in vitro relative potency assay to replace the animal-based potency assay for product release and stability testing. 39 In contrast, Tleugabulova et al reported a denaturation of HBsAg particles after desorption from aluminum adjuvant under harsher conditions, which led to irreversible modifications of the particles likely as a result of much stronger interaction between phospholipids in HBsAg particles and adjuvants.…”

Section: Discussionmentioning

confidence: 99%

“…18,[22][23][24] Cryo-transmission electron microscopy (Cryo-EM) and binding analyses of VLP antigens in GARDASIL Ò , the quadrivalent human papillomavirus (HPV) VLP vaccine, showed no apparent changes in their spherical shape and key epitopes of VLP antigens when adsorbed onto adjuvants and after dissolution of amorphous adjuvants. 25,39 Comparable physiochemical properties of the recovered HBsAg antigen of Hepatitis B virus versus the antigen in aqueous solution were shown using multiple biophysical methods. 22 The purpose of the studies was to explore the interactions between proteins and the aluminumcontaining adjuvants and to provide guidance for the production of more consistent and stable vaccine formulations.…”

Section: Introductionmentioning

confidence: 99%

Comparable quality attributes of hepatitis E vaccine antigen with and without adjuvant adsorption-dissolution treatment

Zhang

Yang

et al. 2015

Human Vaccines & Immunotherapeutics

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Most vaccines require adjuvants for antigen stabilization and immune potentiation. Aluminum-based adjuvants are the most widely used adjuvants for human vaccines. Previous reports demonstrated the preservation of antigen conformation and other antigen characteristics after recovery from adjuvanted Hepatitis B and human papillomavirus vaccines. In this study, we used a combination of various physiochemical and immunochemical methods to analyze hepatitis E vaccine antigen quality attributes after recovery from adjuvants. All biochemical and biophysical methods showed similar characteristics of the p239 protein after recovery from adjuvanted vaccine formulation compared to the antigen in solution which never experienced adsorption/desorption process. Most importantly, we demonstrated full preservation of key antigen epitopes post-recovery from adjuvanted vaccine using a panel of murine monoclonal antibodies as exquisite probes. Antigenicity of p239 was probed with a panel of 9 mAbs using competition/blocking ELISA, surface plasmon resonance and sandwich ELISA methods. These multifaceted analyses demonstrated the preservation of antigen key epitopes and comparable protein thermal stability when adsorbed on adjuvants or of the recovered antigen post-dissolution treatment. A better understanding of the antigen conformation in adjuvanted vaccine will enhanced our knowledge of antigen-adjuvant interactions and facilitate an improved process control and development of stable vaccine formulation.

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Characterization of virus-like particles in GARDASIL® by cryo transmission electron microscopy

Cited by 55 publications

References 33 publications

A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

Comparable quality attributes of hepatitis E vaccine antigen with and without adjuvant adsorption-dissolution treatment

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