Figure
4: Inactivation of VACV and MPXV with different methods. (A) VACV was
spiked into 20 % HSA and inactivated either with heat (30 min 56 °C),
protocol p1=0.3 %Triton/0.3 %Tween/1 %TNBP,
p2=0.5 %Triton/0.5 %Tween/1h60 °C or
p3=0.5 %Triton/0.5 %Tween/30min56 °C. The number of infectious
particles was then measured by TCID50. (B) MPXV was
spiked into 20 % HSA and inactivated with p3, and the number of
infectious particles was measured by TCID50. As a
reference non-inactivated virus spiked into 20 % HSA was used (w/o). n
= number of replicates. The titre of p2 and p3 are maximum titres since
residual toxicity of the inactivation reagents prohibited an evaluation
of the lowest dilution step.
Finally, serum was tested after inactivation with the p3 protocol in the
IFA and the ELISA. For comparison of native and inactivated IFA titres,
five sera from IMVANEX-vaccinated individuals were used which showed an
expected and easy-to-interpret cytoplasmic fluorescence of the cells
(plasma fluorescence, PF). Additionally, a panel of five sera was taken
that had been collected in the course of routine diagnostics and whose
fluorescence signals were more challenging to interpret. These sera
showed fluorescence of inclusion bodies (IBs; n=4) or fluorescence of
cytoplasm and IBs. Analysing these ten sera by IFA showed that the p3
inactivation step did not alter the results (Table 1).