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

Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL®. CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant.

Hexavalent IPV-based combination vaccines for public-sector markets of low-resource countries.

In anticipation of the successful eradication of wild polio virus, alternative vaccination strategies for public-sector markets of low-resource countries are extremely important, but are still under development. Following polio eradication, inactivated polio vaccine (IPV) would be the only polio vaccine available, and would be needed for early childhood immunization for several years, as maintenance of herd immunity will be important for sustaining polio eradication. Low-cost combination vaccines containing IPV could provide reliable and continuous immunization in the post-polio eradication period.

Toolbox for non-intrusive structural and functional analysis of recombinant VLP based vaccines: a case study with hepatitis B vaccine.

Background: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

Background: Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs).

Disassembly and reassembly improves morphology and thermal stability of human papillomavirus type 16 virus-like particles.

Unlabelled: Recombinant human papillomavirus (HPV) 16 L1 protein self-assembles into virus-like particles (VLPs) with diameters of 40 to 60 nm, which are key components in prophylactic HPV vaccines. Marked improvement in morphology and thermal stability on VLP disassembly and reassembly was demonstrated at production scale. Differential scanning calorimetry showed enhanced conformational stability as indicated by the unfolding temperatures and peak heights/areas.

In-depth process understanding of RECOMBIVAX HB® maturation and potential epitope improvements with redox treatment: multifaceted biochemical and immunochemical characterization.

Recombinant Hepatitis B surface antigen virus-like particles (VLPs) produced in yeast undergo spontaneous maturation during the vaccine production process, and the biophysical characteristics of the particles with respect to maturation were described in Zhao et al. (2006) [13]. Here we report additional biochemical and immunochemical characterization by various techniques, including the use of a panel of monoclonal antibodies (mAbs) that differ in their selectivity and conformation-sensitivity, for probing surface epitope structures.

Real time monitoring of antigenicity development of HBsAg virus-like particles (VLPs) during heat- and redox-treatment.

The Hepatitis B virus major surface antigen (HBsAg) is a cysteine-rich, membrane-bound protein which self-assembles into 22-nm spherical virus-like particles (VLPs). While this VLP based human vaccine has been demonstrated to be safe and efficacious since 1986, the structural and exact molecular basis for its antigenic determinants has not been elucidated. Maturation of the yeast-derived purified VLPs was characterized for the changes in 37 their biophysical properties.

Relationship between tightness of binding and immunogenicity in an aluminum-containing adjuvant-adsorbed hepatitis B vaccine.

Phospholipid-containing antigens, such as Hepatitis B Surface Antigen (HBsAg), adsorb to aluminum-containing adjuvants by ligand exchange of a phosphate group for a hydroxyl group on the adjuvant surface. In this study, a tightness of binding (TOB) assay was developed to characterize the strength of binding between HBsAg and aluminum hydroxyphosphate sulfate adjuvant containing two levels of phosphate. Antigen desorption was induced using either fluoride or phosphate as a competing ion.

Effect of alternative aluminum adjuvants on the absorption and immunogenicity of HPV16 L1 VLPs in mice.

Aluminum adjuvants are commonly used in prophylactic vaccines to enhance antigen immunogenicity through induction of high-titer antibody responses. Three major forms of aluminum adjuvants with substantially different physical and chemical properties have been described: aluminum phosphate (AlPO(4)), aluminum hydroxide (AlOH) and amorphous aluminum hydroxyphosphate sulfate (AAHS). Here we describe the effect of these different aluminum adjuvants on the formulation and subsequent immunogenicity in mice of virus-like particles (VLPs) consisting of the L1 protein of Human Papillomavirus (HPV) Type 16.

Maturation of recombinant hepatitis B virus surface antigen particles.

The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation.

Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq).

A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24h.

Visualization of discrete L1 oligomers in human papillomavirus 16 virus-like particles by gel electrophoresis with Coomassie staining.

The recombinant major capsid protein (L1) of human papillomavirus (HPV) can self-assemble into virus-like particles (VLPs) with 360 L1 molecules per VLP. These tightly associated L1 oligomers in the assembled VLPs were disrupted in a pH-, denaturant-, time-, and temperature-dependent fashion. With non-reducing Laemmli-type SDS-PAGE, primarily the monomeric L1 protein ( approximately 55 kDa) is observed when analyzing VLP preparations.