Introduction
Monkeypox viruses (MPXV) belong to the family of Poxviridae , subfamily Chordopoxvirinae , and the genus of Orthopoxviruses (OPXV). OPXV comprise mostly animal-borne viruses with zoonotic potential such as cowpox virus (CPXV), vaccinia virus (VACV) and camelpox virus, amongst others [1]. However, the smallpox-causing Variola virus (VARV) also belongs to the OPXV genus, and infection with MPXV can potentially result in a smallpox-like disease. Until 2022 MPXV has historically been endemic in Central and Western Africa. However, between May 2022 and the writing of this manuscript in May 2023, WHO reports more than 87,000 laboratory-confirmed MPXV infections (Mpox) with about 140 fatalities reported internationally from 111 countries, especially from Europe and the Americas, with the Mpox outbreak being sustained by human-to-human transmission via close contact [2]. After entry through a mucous membrane or the skin, MPXV can replicate in the local endothelial tissue and spread via the blood stream to infect other tissues and organs. Following a second viraemia which enables further virus dissemination, the characteristic lesions appear on the skin [3, 4]. Typical symptoms of Mpox include, amongst others, the characteristic skin lesions, fever, headache, lethargy or exhaustion as well as more systemic respiratory and gastrointestinal symptoms [5]. While Mpox is typically self-limiting, some patients experience complications such as encephalitis and require hospitalization [6]. Whilst the case fatality rate during global outbreaks has remained relatively low (about 0.03 %), in previously endemic areas in Central and Western Africa, reported case fatality rates ranged from 3 to 6 % [7] which, combined with other factors, resulted in MPXV being classified as a risk group 3 biological agent. In contrast, CPXV is endemic in Europe with sporadic human cases being directly linked to contact with infected animals, with no proven human-to-human transmission. CPXV infection in humans results in localised lesions mostly on the fingers, hands and face, with complications mostly reported in immunocompromised individuals [8, 9]. The IMVANEX vaccine contains an attenuated version of VACV (modified vaccinia virus Ankara), and both CPXV and VACV are classified as risk group 2 biological agents.
Anti-OPXV antibodies have been shown to be cross-reactive against a broad range of different OPXV species, including VACV, CPXV and MPXV [10]. Antibodies against MPXV can be detected in serum approximately one to two weeks after the onset of symptoms [4]. Although detection of viral DNA is the gold standard to confirm an MPXV infection, detection of antibodies against MPXV by serological assays can aid diagnostics under certain circumstances. Furthermore, sero-epidemiological studies solely rely on the detection of antibodies in the post-acute phase to determine the overall burden of disease in an at-risk population or the presence and endurance of protective antibodies induced by immunization campaigns [11, 12].
For the detection of antibodies against MPXV, different laboratory tests can be used: IgM and IgG antibodies can be detected by immunofluorescence assay (IFA) [13, 14] or enzyme-linked immunosorbent assay (ELISA) [11, 15-17], while neutralising antibodies are commonly detected by neutralization tests (NT) [18-20]. Here, both the IFA and the ELISA detect antibodies binding to immobilised viral antigen. For the IFA, the viral antigens are present in infected and fixed cells, usually on multi-well slides to enable the testing of various serum dilutions for simultaneous antibody titration. Non-infected cells can also be included on the same slide to test for specificity, as well as a positive serum on infected cells to act as a positive control. Bound antibodies are detected by adding fluorophore-coupled human IgG- or IgM-specific secondary antibodies, and signals are read out on fluorescence microscopes. For OPXV ELISAs, different antigens have been employed, ranging from purified viral particles to lysed infected cells or recombinant viral proteins. Due to the antigen immobilization on microtitre ELISA plates, a greater number of sera can be tested simultaneously, leading to a higher throughput. Bound antibodies are usually detected by horseradish-peroxidase (HRP) or alkaline phosphatase (AP) labelled antibodies, leading to colorimetric chemical reactions which can be quantified by ELISA microplate readers. Finally, virus neutralising antibodies, which block virus uptake and replication in target cells, are quantified by NTs. Here, the assay read-out are plaques caused by the lysis of infected cells in a confluent cell monolayer which can be quantified as plaque-forming units (PFU). Alternatively, the assay can be simplified by determining cytopathic effects per well to determine tissue-culture infectious doses (TCID50). Currently, there are only very few commercially available ELISA kits for the detection of IgG and IgM antibodies against OPXV/MPXV. Furthermore, several commercial antibody rapid tests are available, but to date it is not known how these tests perform in comparison to in-house methods. Finally, there is no commercial assay for the detection of neutralising antibodies against MPXV, which is why these assays are generally established as in-house assays by specialist laboratories. Setting up such assays comes with different challenges and requirements with regard to equipment, reagents, facilities (biosafety level) and expertise needed. Furthermore, each of the assays has its merits and drawbacks and hence should be chosen according to its intended use.
In addition to technical limitations, one of the biggest hurdles when performing diagnostics for MPXV is the need for a dedicated BSL-3 facility when replication-competent virus must be handled, e.g., for antigen preparation or to perform NTs. Hence, safer protocols for MPXV serology, which still deliver meaningful results, enhance the overall biosafety while enabling broader applicability of diagnostic methods. Both are needed in the light of the ongoing MPXV circulation worldwide. Here different serological methods are described, the protocols provided and, moreover, options to perform safe antibody detection against MPXV under BSL-2 conditions are shown.