Neutralization test
Micro-neutralization tests (NT) were set up with three different OPXV
strains: MPXV clade IIb (in-house isolate), VACV Lister-Elstree Bavarian
Nordic (LELS-2003-007, Bavarian Nordic GmbH) and CPXV strain HumGri07/1
(in-house isolate) [16]. For this purpose, Vero E6 cells
(#85020206, European Collection of Authenticated Cell Cultures (ECACC))
in DMEM (Gibco) with 10 % FCS (Sigma) were infected at MOI ⁓ 0.1. After
four to seven days — depending on the morphological cell status —
the infected cells were lysed by three freeze-thaw cycles and the virus
suspension was vortexed for 10 sec. Cell debris was pelleted for 10 min
at 1300×g, and from the supernatant a stock virus solution with a target
titre of 1000 TCID50/mL was prepared in cell culture
medium and stored in aliquots at -80 °C. The stock titre was confirmed
by triplicate titration. For detection of neutralising antibodies, the
patient sera were diluted in medium (DMEM) to six two-fold dilution
steps, resulting in dilutions of 1:10 to 1:320. 500 µL of virus stock
were added to 500 µL of each respective serum dilution and mixed by
pipetting up and down. Following incubation at RT for one hour, 100 µL
of each virus/serum dilution were added per well in eight replicates to
96-well plates containing Vero E6 cells seeded the previous day
(1.5×104 cells/well in 100 µL of medium) and incubated
at 37 °C, 5 % CO2, for 7 days. In each experiment, the
virus stock used was titrated as a control. For this purpose, 1 mL of
the virus dilution was mixed with 1 mL of medium and incubated at RT for
1 h. Decimal dilutions were prepared in medium, resulting in dilutions
of 1:101 to 1:109, then 100 µL of
each virus dilution were added per well in eight replicates to a 96-well
plate pre-seeded with Vero E6 cells (1.5×104cells/well in 100 µL of medium). As a negative control, 100 µL of
medium/well was added to eight wells. After 7 days the cells were
inspected by microscopy for CPE. The titre of the virus stock was
calculated according to the following formula: TCID50/mL
= 10^((n/8+0.5))/0.5 where n = number of wells with CPE. The titre of
the patient sera was calculated according to the following formula:
Titre (1:x) =10×2^((n/8+0.5)) where n = number of wells without CPE.
In case that not all wells in the last dilution step showed a CPE, the
titre was indicated as “≥”.