Discussion
To aid the establishment of MPXV serology in other laboratories, in the
present work methods are described for the detection of OPXV IgG and IgM
antibodies as well as neutralising antibodies. To this aim, a small
panel of sera from patients after MPXV or CPXV infection or VACV/IMVANEX
vaccination was used and IgG and IgM detection was tested by IFA and
ELISA and the presence of neutralising antibodies by NT. Furthermore,
MPXV, CPXV and VACV were employed as sources of antigens in the IFA and
the NT test. The aim was to test discrepancies or agreements between
different assays and different antigens for sera with different
infection or immunization backgrounds. It could be confirmed that IFAs
and NTs based on different OPXV species can be used for the detection of
antibodies in sera from individuals who had an infection with MPXV or
CPXV or were vaccinated against poxviruses, due to the well-known
antibody cross-reactivity between different OPXV species [1, 10].
Some sera (M3 and V3 to V5, Figure 1B) were not detected by all OPXV
IFAs as a result of their very low titre (≤20) at or below the limit of
detection of the IFA. Additionally, after MPXV infection some sera
showed higher titres on MPXV-infected slides, indicating higher
reactivity against homologue virus strains as previously described
[25].
The greatest differences in reactivity to the different OPXV were
observed in the NT. These titre differences could result from the
biological variation of the NTs since different OPXV species were used
and the tests were not optimised for each virus individually. Since cell
culture supernatant as well as virus particles from cells were used, the
virus stocks contained a mixture of the two poxvirus particle forms: the
extracellular enveloped virions (EEV) and the intracellular mature
virions (IMV). For EEV and IMV, different antibodies are needed for
neutralization since the epitopes on the virion surface differ. Hence,
the ratio of EEVs to IMVs could have been different in the different
OPXV stock preparations, leading to larger titre discrepancies.
Furthermore, although the amount of virus particles used was set to be
comparable between the different OPXV (1000 TCID50/mL),
fewer virus particles were detected in the back-titration of the VACV
stock (supporting table S1). This could explain the fact that the
observed neutralising antibody titres were highest in the VACV NT and
not in the NT corresponding to the “source virus” of the antibodies
contained in the different samples. However, except for this minor
technically induced variation, the NT results corresponded to the
expected results: CPXV sera showed the highest titres in the CPXV NT and
MPXV sera in MPXV NT. Finally, the larger differences between the titres
observed in the different OPXV NTs, as compared to the different OPXV
IFAs, could also be due to the generally much lower neutralising
antibody titres as compared to binding antibody titres and hence larger
variation between individual measurements.
Although serological assays have been described which are able to
discriminate between vaccination and infection with MPXV to some extent,
those assays rely on pre-absorption with viral antigen [25] or
combinations of specific peptides [17] while there is a large
overlap of cross-reactive epitopes between the closely related OPXVs. It
is known that individuals who received the first-generation poxvirus
vaccine against smallpox still have cross-reactive neutralising
antibodies against MPXV even 40 years post vaccination [26].
However, in accordance with a recent study, these neutralising antibody
titres in individuals with one or two vaccination doses are rather low
[12].
The newly optimised ELISA protocol is more suited for the detection of
acute infections due to its higher dynamic range which results from
quantification over standard curves and its ability to detect IgM
antibodies. The ELISA enables a higher throughput as compared to IFA and
NT, with 20 sera measured per ELISA plate if two serum dilutions are
tested. Testing two serum dilutions (1:100 and 1:1000) is advisable to
capture the higher dynamic range of acute infections in patients as
compared to seroprevalence studies for which the ELISA was initially
established at a single 1:100 dilution. One of the two sera with IFA
titres of 1:20, which gave higher ELISA signals than the sera with IFA
titres of 1:80, was highly positive for PPV, indicating a possible minor
cross-reactivity with the VACV-specific lysate used in the ELISA, while
the other serum was initially titrated to a titre of 1:320, indicating
some ambiguity regarding the exact titre (Figure 3). Some overlap
between ELISA results falling into different IFA titres indicates some
method-specific discrepancies; yet overall, both assays correlate well,
especially for samples with higher IFA titres. Differentiation between
lower IgM titres was not possible, which in part could be due to
cross-reactivity of the used detection antibodies, as one serum with a
high IgM reading by ELISA had an IFA IgM titre of only 1:20 but was
highly positive for IgG with a titre of 1:20,480.
It has been shown that during MPXV pathogenesis two viraemic phases
occurred that enabled virus dissemination in the infected individual
[3]. In contrast, viraemia in vaccinees seems to be rarer [27].
Viraemia in MPXV-infected individuals also seems to be rare [28] and
is potentially also clade specific as differences in dissemination are
seen between clade I and clade II MPXV. However, viraemia/DNAaemia has
been observed during MPXV infection in a prairie dog model, and in two
animals viable virus was also found in the blood [29]. Moreover,
DNAaemia was also shown to occur in CPXV-infected individuals [30].
This leads to the conclusion that viraemia cannot be excluded in
MPXV-infected individuals. Hence, a safe handling procedure was
established for BSL-2 laboratories with which at least a five log-level
depletion of infectious poxvirus particles could be accomplished in
simulated serum samples. The protocol using Triton, Tween and heat can
be used to inactivate sera for IFA and ELISA. Due to cytotoxicity, the
inactivation protocol is not applicable for lower dilution steps in NT,
but NTs based on VACV or other suitable OPXV may be performed in a BSL-2
laboratory under a class II biosafety cabinet.