Figure 2: Micro-neutralization tests using different OPXVs. Five sera from MPXV-infected (M1-M5) and six sera from CPXV-infected persons (C1-C6) plus five sera from IMVANEX-vaccinated individuals (V1-V5) were analysed by using TCID50 assays with different viruses.
Since IFA and NT are both low-throughput methods to detect binding or neutralising antibodies, they are not suited to screen larger sample numbers, e.g., in an outbreak situation; therefore, for this purpose an ELISA was updated that had previously been established in the lab [11]. The ELISA uses VACV-infected HEp2 cells as the specific antigen and non-infected HEp2-cells as the negative control. Hereby, unspecific or background binding can be eliminated effectively by subtracting non-specific HEp2 signals from VACV-specific signals. To render the ELISA also useful for the detection of acute infections, IgM analysis was newly established. Initial experiments for IgM detection using serum dilutions in the casein blocking buffer (also used for serum dilution prior to IgG detection) led to high unspecific binding. Hence, different blocking buffers and commercially available diluents were tested (supporting figure 1). As LowCross buffer was most efficient in reducing high background signals while retaining specific binding of IgM-positive sera, it was used as the serum diluent in the final assay protocol.
To test the agreement between the established IFA and the modified or newly established IgG and IgM detection by ELISA, the quantified ELISA results for 28 sera were compared with the corresponding IFA titres (Figure 3). For IgG detection, a high level of correlation was found between IFA titres and ELISA results with higher titres, leading to higher ELISA results. This was also true for IgM detection, although differentiation between lower titres was not possible.
Figure 3: Comparison of IgG (A) and IgM (B) titres by IFA with results obtained by ELISA. IFA titres were determined by titration against CPXV- or VACV-infected HEp2 cells.
After demonstrating that IFA and ELISA, moreover, using less pathogenic OPXVs like VACV as antigens, can be used to detect (cross-reactive) antibodies against MPXV, the next aim was to further enhance the method’s biosafety with a robust inactivation method for the serum samples. A literature review resulted in two potential protocols using either 0.3 %Triton/0.3 %Tween/1 %TNBP (protocol 1, p1) [23] or 0.5 %Triton/0.5 %Tween/1h60 °C (protocol 2, p2) [22]. For comparison, standard heat inactivation was used for 30 min at 56 °C. First the protocols were tested with VACV, demonstrating that both protocols resulted in a depletion of infectious poxvirus particles (Figure 4A). However, the protocol p2 (Figure 4A) led to a better inactivation compared to protocol p1. Heat inactivation alone was least efficient in inactivating infectious virus particles (Figure 4A, heat). Since protocol p2 worked well the next aim was to reduce the heat inactivation time and reduce the incubation temperature to allow inactivation while still retaining the antibody-binding ability. For this purpose, p2 was tested with classical heat inactivation conditions (30 min 56 °C, protocol 3, p3) which led to a depletion comparable with the p2 protocol (Figure 4, p2 and p3). Next, three replicates of VACV inactivated with p3 were passaged three times in cell culture and checked for viral DNA by real-time-PCR in the cells and the supernatant. This resulted in verifying that in the 10-1 dilution of the TCID50 there are few infectious particles left, while in the 10-2 dilution no infectious particles could be detected in all three replicates. Taken together, using the p3 protocol, a depletion in infectious virus particles of about 5 log-steps could be shown. The inactivation potential of the p3 protocol was then also verified with MPXV (Figure 3B).