Capsid destabilization and epitope alterations of human papillomavirus 18 in the presence of thimerosal

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 thiolcontaining proteinaceous active components,needs to be comprehensively characterized during formulation development when a preservative is necessary.

N-terminusα-synuclein detection reveals new and more diverse aggregate morphologies in multiple system atrophy and Parkinson’s disease

Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are classified asα-synucleinopathies and are primarily differentiated by their clinical phenotypes.Delineating these diseases based on their specificα-synuclein(α-Syn)proteoform pathologies is crucial for accurate antemortem biomarker diagnosis.Newly identifiedα-Syn pathologies in PD raise questions about whether MSA exhibits a similar diversity.This prompted the need for a comparative study focusing onα-Syn epitope-specific immunoreactivities in both diseases,which could clarify the extent of pathological overlap and diversity,and guide more accurate biomarker development.Methods We utilised a multiplex immunohistochemical approach to detect multiple structural domains ofα-Syn proteoforms across multiple regions prone to pathological accumulation in MSA(n=10)and PD(n=10).Comparison of epitope-specificα-Syn proteoforms was performed in the MSA medulla,inferior olivary nucleus,substantia nigra,hippocampus,and cerebellum,and in the PD olfactory bulb,medulla,substantia nigra,hippocampus,and entorhinal cortex.Results N-terminus and C-terminus antibodies detected significantly moreα-Syn pathology in MSA than antibodies for phosphorylated(pS129)α-Syn,which are classically used to detectα-Syn.Importantly,C-terminus immunolabelling is more pronounced in MSA compared to PD.Meanwhile,N-terminus immunolabelling consistently detected the highest percentage ofα-Syn across pathologically burdened regions of both diseases,which could be of biological significance.As expected,oligodendroglial involvement distinguished MSA from PD,but in contrast to PD,no substantial astrocytic or microglialα-Syn accumulation in MSA occurred.These data confirm glial-specific changes between these diseases when immunolabelling the N-terminus epitope.In comparison,N-terminus neuronalα-Syn was present in PD and MSA,with most MSA neurons lacking pS129α-Syn proteoforms.This explains why characterisation of neuronal MSA pathologies is lacking and challenges the reliance on pS129 antibodies for the accurate quantification ofα-Syn pathological load acrossα-synucleinopathies.Conclusions These findings underscore the necessity of utilising a multiplex approach to detectα-Syn,most importantly including the N-terminus,to capture the entire spectrum ofα-Syn proteoforms inα-synucleinopathies.The data provide novel insights toward the biological differentiation of theseα-synucleinopathies and pave the way for more refined antemortem diagnostic methods to facilitate early identification and intervention of these neurodegenerative diseases.