Vaccination against tick-borne encephalitis (TBE) is based on the use of formalin-inactivated, culture-derived whole-virus vaccines. Immune response following vaccination is primarily directed to the viral envelope (E) protein, the major viral surface antigen. In Europe, two TBE vaccines are available in adult and pediatric formulations, namely FSME-IMMUN® (Pfizer) and Encepur® (GlaxoSmithKline). Herein, we analyzed the content of these vaccines using mass spectrometry (MS). The MS analysis revealed that the Encepur vaccine contains not only proteins of the whole virus particle, but also viral non-structural protein 1 (NS1). MS analysis of the FSME-IMMUN vaccine failed due to the high content of human serum albumin used as a stabilizer in the vaccine. However, the presence of NS1 in FSME-IMMUN was confirmed by immunization of mice with six doses of this vaccine, which led to a robust anti-NS1 antibody response. NS1-specific Western blot analysis also detected anti-NS1 antibodies in sera of humans who received multiple doses of either of these two vaccines; however, most vaccinees who received ≤3 doses were negative for NS1-specific antibodies. The contribution of NS1-specific antibodies to protection against TBE was demonstrated by immunization of mice with purified NS1 antigen, which led to a significant (p < 0.01) prolongation of the mean survival time after lethal virus challenge. This indicates that stimulation of anti-NS1 immunity by the TBE vaccines may increase their protective effect.

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.