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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]

The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine—formed through the structural clustering of HPV epitopes—confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens. An effective vaccine for human papillomavirus (HPV) needs to protect from several genotypes. Here, Wang et al. provide a strategy to produce single capsomere-hybrid virus-like particle (chVLP) composed of capsomers of different genotypes and show that a nona-type chVLP induces similar levels of neutralizing antibodies as an approved HPV vaccine. [ABSTRACT FROM AUTHOR]

Background: Naturally occurring variants of human papillomavirus (HPV) 58 have been defined as lineages and sublineages but little is known about the impact of this diversity on protein function. We investigated the impact of variation within the major (L1) and minor (L2) capsid proteins of HPV58 on susceptibility to neutralizing antibodies.Methods: Pseudovirus (PsV) representing A1, A2, A3, B1, B2, C, D1, and D2 variants were evaluated for their susceptibility to antibodies elicited during natural infection, preclinical antisera generated against virus-like particles, and monoclonal antibodies (MAbs).Results: Lineage C PsV demonstrated a decreased sensitivity to antibodies raised against lineage A antigens. Exchange of the DE, FG, and/or HI loops between sublineage A1 and lineage C demonstrated that residues within all 3 loops were essential for the differential sensitivity to natural infection antibodies, with slightly different requirements for the animal antisera and MAbs. Comparison between the HPV58 A1 L1 pentamer crystal structure and an HPV58 C homology model indicated that these differences in neutralization sensitivity were likely due to subtle epitope sequence changes rather that major structural alterations.Conclusions: These data improve our understanding of the impact of natural variation on HPV58 capsid antigenicity and raise the possibility of lineage-specific serotypes. [ABSTRACT FROM AUTHOR]

Abstract: This paper describes an approach to surface plasmon resonance (SPR) based epitope mapping, also referred to as pairwise antibody footprinting, involving the direct immobilization of an antigen-specific primary mAb to the surface of an SPR interface. This technique offers a more straightforward approach than indirect capture (e.g., via rabbit anti-mouse Fc) as it does not require additional steps to block the unoccupied immobilized anti-Fc to prevent non-specific antibody binding. This is also an alternative to the direct immobilization of an antigen of interest, which may cause conformational changes in the antigen or epitope degradation upon chemical immobilization, particularly in successive regeneration cycles. It is particularly suitable for highly multivalent targets such as virus-like particles (VLPs). Using this technique, we assessed a panel of eight monoclonal antibodies against HPV (human papilloma virus) L1 protein VLPs expressed by Saccharomyces cerevisiae. In the antibody epitope screening studies, HPV16 L1-directed conformational mAbs were clearly distinguished from the linear mAbs and consistent with known epitope information. Additional studies using a linear mAb and a conformational mAb demonstrate the practical application of this technique for characterizing the result of process changes and the consistency of recombinant HPV16 VLPs. The method is readily extensible to other VLPs and VLP-based vaccines. [Copyright &y& Elsevier]

Abstract: 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. Cloud point studies indicated (1) a much lower propensity for post-reassembly VLPs to aggregate during a time course study and (2) much higher cloud point temperatures. In-solution atomic force microscopy showed more uniform size distribution and fully closed particles, with evidence of virion-like assembly revealed by the structural details from a single particle image. Similar approaches for the reassembly of other recombinant VLPs with intrinsic conformational switches would be expected to improve the particle properties and render nanoparticles more suitable for use as vaccines or therapeutics. From the Clinical Editor: The authors of this study demonstrated that recombinant human papillomavirus 16 L1 protein self-assembles into virus-like particles (VLPs) with marked improvement in morphology and thermal stability on VLP disassembly and reassembly at production scale. This is expected to render these nanoparticles more suitable for use as vaccines or therapeutics. [Copyright &y& Elsevier]

Abstract: 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 (∼55kDa) is observed when analyzing VLP preparations. When the pH was lowered to pH 7.0 in NuPAGE system and the gel temperature during electrophoresis was maintained at a lower temperature (∼7°C), a ladder of protein bands in ∼55kDa increments were detected above the monomeric p55 band. These discrete bands visualized as a ladder are likely the disulfide-linked L1 oligomers. In addition to the gel running conditions, an increase in pH, temperature, or SDS concentration during sample treatment was also shown to significantly reduce the amount of detectable oligomers, further corroborating the labile nature of these oligomers. Altogether, the results also implicate the redox-responsive nature of the HPV capsid comprising of >95% L1 protein. Molecular basis of the facile disulfide bond inter-change is discussed. This electrophoretic technique for trapping the disulfide-linked oligomers may be employed to detect the oligomeric status of other protein aggregates or assembled particles. [Copyright &y& Elsevier]

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]

Cervical cancer, the second leading cause of cancer-related deaths among women, is caused by human papillomavirus (HPV), a sexually transmitted virus. Vaccination is an effective preventive measure against viral infections and subsequent development of cervical cancer. Enzyme-linked immunosorbent assay (ELISA) is commonly used to measure specific binding antibody titers and assess the immunogenicity of test vaccines in preclinical models or clinical volunteers. Two methods of deriving titers, the endpoint titer (ET) and the effective dilution producing a median maximal effective fold of dilution (ED 50) with a cut-off value, are widely used. For HPV, a pseudovirion-based neutralization assay (PBNA) is used to measure functional antibody titers. The ELISA binding titers and functional PBNA titers were found to be well-correlated for all nine HPV types tested in the vaccine, consistent with previous studies on HPV 16/18. Comparing the PBNA results with the two titration methods, the ED 50 method showed higher precision and a closer correlation with PBNA results, both for individual types and pooled data analysis for all nine types. When comparing the titration results of the ET method based on a cut-off value with the ED 50 method using all the data points across the dilution series, the ED 50 method demonstrated better precision and a stronger correlation with PBNA results. • Mid-point vs. end point ELISA analysis showed improved precision of the former. • Higher coefficients were demonstrated with ED 50 method when correlating to PBNA. • The PBNA vs. ELISA correlation was confirmed for all 9 HPV vaccine types. • Mid-point method is a preferred method over the cut-off method in ELISA analysis. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4 ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as “a black box” due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples. [ABSTRACT FROM AUTHOR]

Summary Cervical cancer is the second most prevalent malignant tumor among women worldwide. High-risk human papillomaviruses (HPVs) are believed to be the major causative pathogens of mucosal epithelial cancers including cervical cancer. The HPV capsid is made up of 360 copies of major (L1) and 72 copies of minor (L2) capsid proteins. To date, limited high-resolution structural information about the HPV capsid has hindered attempts to understand details concerning the mechanisms by which HPV assembles and infects cells. In this study, we have constructed a pseudo-atomic model of the HPV59 L1-only capsid and demonstrate that the C-terminal arm of L1 participates in virus-host interactions. Moreover, when conjugated to a scaffold protein, keyhole limpet hemocyanin (KLH), this arm is immunogenic in vivo. These results provide new insights that will help elucidate HPV biology, and hence pave a way for the design of next-generation HPV vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly. [Copyright &y& Elsevier]

Abstract: Effective and safe novel adjuvants are of great interest to the vaccine research community. In this study, we describe our evaluation of adjuvant formulations containing a TLR9 agonist adjuvant (ISS1018) or ISCOMATRIX™ adjuvant for a two-dose regimen of hepatitis B virus surface antigen virus-like particle vaccine in mice and rhesus macaques. Our results show a 10–20 fold improvement in Ab binding titers determined in an antigen-sandwich assay for adjuvant formulations with ISCOMATRIX™ adjuvant, in comparison to routine aluminum formulation. Furthermore, we optimized a competition assay to evaluate a functional component of immune sera, using a conformation-dependent and protective mAb, RFHBs1, as the probe. Although good correlation was observed between Ab binding titers from the antigen-sandwich assay and functional titers from the in-solution competition against RFHBs1, the latter assessment provided a much more stringent ranking of adjuvant formulations than the former. These results indicate the importance of evaluating functional Abs when assessing and comparing novel adjuvant formulations, as it provides another angle to investigate the effects of change in adjuvant composition on antigenic integrity of the testing vaccines. [Copyright &y& Elsevier]