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Thimerosal has been widely used as a preservative in drug and vaccine products for decades. Due to the strong propensity to modify thiols in proteins, conformational changes could occur due to covalent bond formation between ethylmercury (a degradant of thimerosal) and thiols. Such a conformational change could lead to partial or even complete loss of desirable protein function. This study aims to investigate the effects of thimerosal on the capsid stability and antigenicity of recombinant human papillomavirus (HPV) 18 virus-like particles (VLPs). Dramatic destabilization of the recombinant viral capsid upon thimerosal treatment was observed. Such a negative effect on the thermal stability of VLPs preserved with thimerosal was shown to be dependent on the thimerosal concentration. Two highly neutralizing antibodies, 13H12 and 3C3, were found to be the most sensitive to thimerosal treatment. The kinetics of antigenicity loss, when monitored with 13H12 or 3C3 as probes, yielded two distinctly different sets of kinetic parameters, while the data from both monoclonal antibodies (mAbs) followed a biphasic exponential decay model. The potential effect of thimerosal on protein function, particularly for thiol-containing proteinaceous active components, needs to be comprehensively characterized during formulation development when a preservative is necessary. [Display omitted] • Altered antigenicity of thimerosal-treated HPV VLPs was observed with antibodies. • Antigenicity reduction and capsid destabilization were concentration dependent. • The kinetics of epitope-specific antigenicity loss were monitored in real time. • The reduced antigenicity of adjuvant-adsorbed antigens was visualized. [ABSTRACT FROM AUTHOR]

• Different epitopes showed different levels of sensitivity to thimerosal treatment. • Reduced conformational stability of HPV antigens in the presence of thimerosal. • Reduced immunogenicity of HPV16 and HPV18 VLPs after thimerosal treatment. • Altered structure of the recombinant HPV18 capsids due to thimerosal treatment. • Thiol modification is likely the basis for structural and functional alterations. Thimerosal has been widely used as a preservative in human vaccines for decades. Thimerosal, a thiol capping agent with ethyl mercury being the active degradant, could have impacts on the vaccine potency due to potential thiol modification. The effects on the antigenicity and immunogenicity of human papillomavirus (HPV) virus-like particles (VLPs) in the presence of thimerosal was studied. In general, reduced binding activity was observed between HPV antigens and monoclonal antibodies (mAbs) upon thimerosal treatment, accompanied by reduced protein conformational stability. The immunogenicity of a pentavalent vaccine formulation (HPV6, HPV11, HPV16, HPV18 and hepatitis E virus) with or without thimerosal was studied in mice. The functional antibody titres, as well as the binding titres, were determined, showing a substantial decrease for vaccine formulations containing thimerosal for HPV16/18. Similarly, epitope-specific competition assays using specific and functional mAbs as tracers also showed a significant reduction in immunogenicity for HPV16/18 in the presence of thimerosal. Structural alterations in the capsid protein for HPV18 were observed with cryo-electron microscopy and 3-dimensional reconstruction in the comparative structural analysis. The results should alert scientists in formulation development field on the choice for vaccine preservatives, in particular for thiol-containing antigens. [ABSTRACT FROM AUTHOR]

Abstract Human papillomavirus (HPV) type 16 is the most common type implicated as the etiological agent that causes cervical cancer. The marketed prophylactic vaccines against HPV infection are composed of virus-like particles (VLPs) assembled from the recombinant major capsid protein L1. Elicitation of functional and neutralizing antibodies by vaccination is the mode of action by which the vaccines prevent the viral infection. In this study, a panel of murine mAbs against HPV16 L1 were generated and comprehensively characterized with respect to their mapping to the epitope spectrum on the viral capsid. These mAbs were categorized into five epitope bins by two different methods based on the pairwise cross-inhibition and competition with human polyclonal antibodies. In addition, a preliminary demonstration of the spatial relationship of the epitopes recognized by these mAbs was performed using a cross-blocking assay with a well-characterized human mAb, 26D1. Interestingly, two mAbs recognizing different epitopes were found to act synergistically in the pseudovirion-based neutralization assay (PBNA). To facilitate cross-lab and cross-study comparison, the international standard (IS) serum 05/134 was used to calibrate the mAbs as well as the human serum samples from the HPV16/18 vaccine recipients. The neutralizing mAbs, particularly those that recognizing immunodominant epitopes, would be useful in developing epitope-specific assays for monitoring the vaccine production process and for serological assessment. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) infection causes sporadic outbreaks of acute hepatitis worldwide. HEV was previously considered to be restricted to resource-limited countries with poor sanitary conditions, but increasing evidence implies that HEV is also a public health problem in developed countries and regions. Fortunately, several vaccine candidates based on virus-like particles (VLPs) have progressed into the clinical development stage, and one of them has been approved in China. This review provides an overview of the current HEV vaccine pipeline and future development with the emphasis on defining the critical quality attributes for the well-characterized vaccines. The presence of clinically relevant epitopes on the VLP surface is critical for eliciting functional antibodies against HEV infection, which is the key to the mechanism of action of the prophylactic vaccines against viral infections. Therefore, the epitope-specific immunochemical assays based on monoclonal antibodies (mAbs) for HEV vaccine antigen are critical methods in the toolbox for epitope characterization and for in vitro potency assessment. Moreover, serological evaluation methods after immunization are also discussed as biomarkers for clinical performance. The vaccine efficacy surrogate assays are critical in the preclinical and clinical stages of VLP-based vaccine development. [ABSTRACT FROM AUTHOR]