A method of quantifying and differentiating serological responses to mpox infection and vaccination

Abstract

An immunoassay is described that enables a precise evaluation of immune responses in individuals exposed to Mpox or vaccinated with MVA, with high sensitivity and specificity. The serological assay targets three Orthopoxvirus antigens, MPXV-B6R, MPXV-A27L, and VACV-B5R, which are sufficient to quantify antibody responses to both MVA-vaccination (VACV-B5R) and, separately, infection (MPXV-B6R). The three antigen titres can be combined in a simple function [sum of ((MPXV-B6R) / VACV-B5R) and (MPXV-A27L)] to provide a numerical test score that can be compared with a reference (threshold) value to differentiate between post infection and post vaccination immune responses.

Description

[0001] TITLE

[0002] A method of quantifying and differentiating serological responses to Mpox infection and vaccination

[0003] Field of the Disclosure

[0004] The disclosure relates to a method and assay that can be used to, for example, quantify and differentiate serological responses to Mpox infection and vaccination. Also described is an assay device and system to perform the method of the disclosure.

[0005] Technical Background

[0006] Until 2022, Mpox was considered a neglected tropical disease with limited data on the host immune response to vaccination or infection. Previous studies have largely inferred host immunity to Mpox from smallpox vaccination. While the Modified Vaccinia Ankara (MVA) smallpox vaccine was given emergency authorisation for prophylactic vaccination against Mpox, its real-world long-term efficacy remains uncertain. A meta-analysis has estimated the efficacy of two doses of MVA-BN vaccine to prevent Mpox at 81.8% (95% CI:65.0-89.1 %). However, emerging evidence has raised concerns regarding the durability of protection with clusters of cases reported in vaccinated individuals and reinfections. A recent study by Byrne et al. (Antibody Responses are Sustained 2 Years Post-Mpox Infection but not

[0007] Following Modified Vaccinia Ankara-Bavarian Nordic Vaccination, Open Forum

[0008] Infectious Diseases, Volume 12, Issue 9, September 2025, ofaf536, found that antibody responses were sustained for two years following Mpox infection, but not after vaccination with the Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) vaccine.

[0009] Furthermore, Orthopoxviruses are large dsDNA viruses with extensive antigenic cross-reactivity which have confounded immunoassay development. Additionally, there is no approved assay to differentiate between infection- and vaccination- induced immunity, highlighting a significant gap in the understanding of humoral responses to Mpox.

[0010] It is an object of the invention to overcome at least one of the above-referenced problems.

[0011] Summary of the Disclosure

[0012] The Applicant has developed an immunoassay that enables a precise evaluation of immune responses in individuals exposed to Mpox or vaccinated with MVA, with high sensitivity and specificity. The serological assay targets three Orthopoxvirus antigens, MPXV-B6R, MPXV-A27L, and VACV-B5R, which are sufficient to quantify antibody responses to both MVA-vaccination (VACV-B5R), infection (MPXV-B6R) and, importantly, to differentiate between post infection and post vaccination immune responses. The MPXV-A27L antigen is a distinct antigen specific to the Mpox virus and does not have a homolog within Vaccinia virus. The fact that antibody responses to just three antigens can differentiate between post infection and post vaccination immune responses in a test subject is surprising, given the high level of antigenic cross-reactivity amongst Orthopoxviruses.

[0013] The IgG titre values may be provided as normalised units (IgGnu) as described herein. In other embodiments, the antibody response to each target antigen may be calculated with reference to a standard curve generated using standardised controls. The antibody response to each of the three target antigens can be combined in a simple function / equation [sum of ((MPXV-B6R) / VACV-B5R) and (MPXV-A27L)] to provide a numerical test value that can be compared with a reference (threshold) value to differentiate between post infection and post vaccination immune responses. A reference (threshold) value derived from this function of 0.92 may be used where a test value of less than 0.92 is indicative of the subject exhibiting a vaccination immune response and a test value of greater than 0.92 is indicative of the subject exhibiting an infection immune response. The assay provides a practical, scalable assay that can be implemented in low- and middle-income countries (LMICs) like the DRC, where Mpox cases are rising and existing diagnostic tools are insufficient. Unlike labour-intensive and costly live virus neutralisation assays, this assay can be widely applied in large-scale vaccine trials and routine clinical settings. This is particularly crucial in resource-limited regions, where accurate serological surveillance can assist in controlling outbreaks. The assay provides a valuable tool for global public health efforts to monitor and respond to the ongoing Mpox public health emergency.

[0014] In one aspect, the invention provides a method comprising: providing a biological sample from a subject; and calculating an IgG titre value for an Orthopoxvirus antigen MPXV-A27L in the biological sample.

[0015] In another aspect, the invention provides a method comprising: providing a biological sample from a subject; and calculating an IgG titre value for each of a plurality of Orthopoxvirus antigens in the biological sample, wherein the plurality of Orthopoxvirus antigens comprise MPXV-B6R, VACV-B5R, and MPXV-A27L.

[0016] In any embodiment, the plurality of Orthopoxvirus antigens consist of MPXV-B6R, VACV-B5R, and MPXV-A27L.

[0017] In any embodiment, the method is a method of determining serological status of the subject. The serological status may be whether the serological response is (or the likelihood of it being) a post Mpox infection immune response, a post vaccination immune response (e.g. post MVA-vaccination immune response), or a result of Mpox infection or vaccination.

[0018] In any embodiment, the method comprises correlating the IgG titre levels for the three antigens with likelihood of the serological response in the subject being a post Mpox infection immune response or post Mpox vaccination immune response.

[0019] In any embodiment, the correlating comprises combining the three IgG titre values into a function to provide a test score, and comparing the test score with a threshold score.

[0020] In any embodiment, the correlating comprises calculating [((MPXV-B6R) IgG titre value / VACV-B5R IgG titre value) + (MPXV-A27L) IgG titre value] to provide a test score for the subject.

[0021] In any embodiment, the test score is compared with a reference (threshold) score, wherein a test score lower than the reference score is indicative of the serological response in the subject being a post-vaccination serological response and a test score higher than the reference score is indicative of the serological response in the subject being a post Mpox infection serological response.

[0022] In any embodiment, the IgG titre value is selected from a normalised value (e.g. an IgG normalised unit (“nu”)) or an IgG concentration. The IgG normalised unit may be calculated by dividing the test IgG titre by an average of control IgG titres for the same antigen obtained from positive (infection) subjects and I or positive (vaccination) subjects. When normalised units (nu) are used as IgG titre values, the value derived from the [((MPXV-B6R) IgG titre value / VACV-B5R IgG titre value) + (MPXV-A27L) IgG titre value] formula may be used as a test score, and a test score of 0.92 may be employed.

[0023] In another embodiment, the IgG concentration for a given target antibody is calculated against a reference IgG titre value for that antibody obtained from a standard (e.g. a plasma standard). The National Institute for Biological Standards and Control (NIBSC) in collaboration with the WHO, released the first Working Standard (Working reagent for anti-monkeypox antibodies), intended to be used for the development and evaluation of serological assays for the detection of antibodies against monkeypox virus (more as a positive control, than a purified calibrator).

[0024] In any embodiment, the method comprises using the standard to generate a multiple-point standard curve derived from different concentrations of a suitable standard.

[0025] In any embodiment, the target IgG concentration is determined using a four- parameter logistic (4PL) fit. Thus, when employing an electrochemiluminesence method, target IgG concentration are extrapolated from the electrochemiluminescent signal (ECL), based on the standard curve. For that calculation, the following formula / equation (taken directly from the instrument's manual) may be employed: y = b2 + ((b1-b2) / 1 + (x / b3) b4) y: the response (response / signal). b1 : the upper asymptote (the maximum response). b2: the lower asymptote (the minimum response). x: the concentration (or input value, in the case of International Units). b3: the concentration at which 50% of the response is observed (the EC50 or midpoint). b4: the slope factor, which controls the steepness of the curve

[0026] That equation above models the relationship between the known IgG concentration (from the standards) and the corresponding ECL signal, from which a standard curve can be derived. By fitting this curve to the multi-point standard, the IgG concentration of each test sample is extrapolated based on their ECL signal. Viewed graphically, the test sample’s ECL response will be on the Y-axis and will be compared to the curve, and the corresponding IgG concentration (X-axis) will be determined through inversion of the 4PL function.

[0027] In any embodiment, the method is a method of determining whether a serological response in the subject is a post Mpox infection serological response or a postvaccination serological response, wherein the method comprises: calculating [((MPXV-B6R) IgGnu I VACV-B5R IgGnu) + (MPXV-A27L) IgGnu] to provide a test score for the subject. comparing the test score with 0.92; and calculating whether the serological response in the subject, based on the test score, is a post-Mpox infection serological response or a postvaccination serological response, wherein a test score lower than 0.92 is indicative of the serological response in the subject being a post-vaccination serological response and a test score higher than 0.92 is indicative of the serological response in the subject being a post-Mpox infection serological response.

[0028] In any embodiment, the step of quantifying IgG in the sample employs an immunoassay.

[0029] In any embodiment, the step of quantifying IgG in the sample employs an electrochemiluminescence immunoassay.

[0030] In any embodiment, the immunoassay employs a multiwell assay plate comprising at least one well coated with MPXV-A27L antigen.

[0031] In any embodiment, the immunoassay employs a multiwell assay plate comprising: at least one well coated with MPXV-B6R antigen; at least one well coated with VACV-B5R antigen; and at least one well coated with MPXV-A27L antigen.

[0032] In any embodiment, each well is coated with 1 to 10 picomoles, 3-7 picomoles, or about 5 picomoles, of antigen.

[0033] In any embodiment, the multiwell assay plate comprises at least 3, 5, 10, 20 or 30 wells coated with MPXV-A27L antigen.

[0034] In any embodiment, the multiwell assay plate comprises: at least 3, 5, 10, 20 or 30 wells coated with MPXV-B6R antigen; at least 3, 5, 10, 20 or 30 wells coated with VACV-B5R antigen; and at least 3, 5, 10, 20 or 30 wells coated with MPXV-A27L antigen.

[0035] In any embodiment, the biological sample is diluted plasma. Whole blood or other blood fractions may also be used.

[0036] In any embodiment, the plasma is diluted 1 :200 to 1 :1000, typically about 1 :400 to 1 :600.

[0037] In any embodiment, the immunoassay comprises the steps of: adding the diluted plasma to each well of the multiwell plate; incubating the diluted plasma in the wells for a first incubation period of time; washing the wells; adding labelled anti-human IgG secondary antibody to each well; incubating the labelled anti-human IgG secondary antibody in the wells for a second incubation period of time; and analysing the wells with a plate reader to quantify the labelled anti-human IgG secondary antibody in each well.

[0038] In any embodiment, the method comprises a step of washing the wells after the second incubation period.

[0039] In any embodiment, the method comprises adding an electrochemoluminesence read buffer after the second incubation period.

[0040] In any embodiment, the step of quantifying IgG in the sample employs a beadbased immunoassay. Examples include the LUMINEX bead immunoassay formats that employs magnetic beads and secondary antibodies having a fluorescent label (https: / / www.thermofisher.com / bloq / behindthebench / luminex-bead-based- immunoassays-drive-immunoassays-towards-hiqher-content-biomarker-discovery / ) and the QUANTERIX platform that employs paramagnetic beads and secondary antibodies having an enzyme label (https: / / www.quanterix.com / bead-based-

[0041] In any embodiment, the bead based immunoassay comprises at least one bead coated with MPXV-A27L antigen.

[0042] In any embodiment, the bead based immunoassay comprises: at least one first bead coated with MPXV-B6R antigen; at least one second bead coated with VACV-B5R antigen; and at least one third bead coated with MPXV-A27L antigen;

[0043] Also described is a method of determining whether a subject shows a serological response to Mpox infection (as opposed to vaccination), comprising assaying a biological sample from the subject for MPXV-B6R IgG titre, and normalising the MPXV-B6R IgG titre against a MPXV-B6R IgG titre from a positive control (e.g. and average of one or more positive infection controls and / or one or more positive vaccination controls) to provide a MPXV-B6R IgGnu test value. In any embodiment, a test value of at least 0.075 IgGnu correlates with the subject exhibiting a serological response to Mpox infection.

[0044] Also described is a multiwell assay plate comprising a plurality of wells and a plurality of Orthopoxvirus antigens including: at least one well coated with MPXV-A27L antigen; optionally, at least one well coated with VACV-B5R antigen; and optionally, at least one well coated with MPXV-B6R antigen..

[0045] In any embodiment, the plurality of Orthopoxvirus antigens consists of MPXV-B6R, VACV-B5R, and MPXV-A27L.

[0046] In any embodiment, each well that is coated with antigen comprises 1 to 10 picomoles, 3-7 picomoles, or about 5 picomoles, of antigen.

[0047] In any embodiment, the multiwell assay plate comprises: at least 3, 5, 10, 20 or 30 wells coated with MPXV-A27L antigen; optionally, at least 3, 5, 10, 20 or 30 wells coated with VACV-B5 antigen; and optionally, at least 3, 5, 10, 20 or 30 wells coated with MPXV-B6R antigen.

[0048] Also described is an immunoassay kit comprising: a multiwell assay plate according to the disclosure; labelled anti-human IgG secondary antibody; and optionally, an electrochemiluminescence read buffer.

[0049] In any embodiment, the immunoassay kit comprises biological sample diluent, wash buffer and optionally serological controls.

[0050] Also described is an antibody or antibody fragment having binding specificity to MPXV-A27L. The antibody may be an IgG antibody or antibody fragment. The antibody may be an anti-human IgG antibody or antibody fragment. The antibody or antibody fragment may be labelled. The label may be a fluorescent or luminescent label, or a radio ligand.

[0051] Other aspects and preferred embodiments of the disclosure are defined and described in the other claims set out below.

[0052] Brief description of the Figures

[0053] Fig. 1 depicts the IgG Titre of MPXV-A27L across different groups.

[0054] Fig. 2 depicts the IgG Titre of MPXV-B6R across different groups.

[0055] Fig 3 depicts the IgG Titre of MPXV-B5R across different groups.

[0056] Fig 4 depicts the ratio of the BPXV-B6R to VACV-B5R in vaccinated and infected groups.

[0057] Fig 5 depicts the Receiver Operating Characteristic (ROC) curve for differentiation of infection and vaccination induced immunity.

[0058] Detailed Description of the Invention All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0059] Definitions and general preferences

[0060] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0061] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0062] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method / process steps.

[0063] “MPXV” refers to Monkeypox virus, a member of the Orthopoxvirus (OPXV) genus that is the causative agent of human Mpox infection. Other OPXVs include Variola virus, the causative agent of smallpox and Vaccinia virus (VACV) used in smallpox vaccines. OPXVs are large dsDNA viruses with extensive antigenic cross-reactivity. Within MPXV, there are two clades with 99.57% homology between clade I and clade lib responsible for the 2022 outbreak. “Mpox” refers to Monkeypox infection. “MPXV-B6R” refers to Monkeypox BR6 antigen (from Monkeypox virus Zaire-96-l- 16). This is an extracellular enveloped virions (EEV) envelope glycoprotein required for efficient cell spread, complement control protein-like. The amino acid sequence of MPXV-B6R from Monkeypox virus Zaire-96-1-16 is provided in SEQUENCE ID NO: 1 : MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLD PNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRC EEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVI GVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSP SSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEAT YHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLP

[0064] Accession Number: AAL40625.1

[0065] “MPXV-A27L” refers to Monkeypox A27L antigen (from Monkeypox virus Zaire-96-l- 16). The MPXV-A27L antigen is distinct from both the VACV-A27L antigen (Accession number NP_063708.1 (3), and the MPXV-A29L antigen (Accession Number AGR38698.1 .(2). MPXV-A27L is not a homolog of the VACV-A27L antigen (Accession number NP_063708.1 (3) as there is no significant sequence homology between the two sequences as determined by the software BLASTp accessed through the https: / / blast.ncbi.nlm.nih.qov / Blast.cqi?PAGE=Proteins. The amino acid sequence of MPXV-A27L from Monkeypox virus Zaire-96-1-16 is provided in SEQUENCE ID NO: 2:

[0066] MEVTNLIKKCTKHSKDFATEVEKLWNDELSSESGLTRKTRNVIRNILRDITKSLTTD KKSKCFRILERSTINGEQIKDVYKTIFNNGVDVESRINTTGKYVLFTVMTYAAELHLI KSDEIFALLSRFFNMICDIHRKYGCGNMFVGIPAALIVLLEIDHINKLFSVFSTRYDAK AYLYTEYFLFLNINHYLLSGSDLFINVAYGAVSFSSPISVPDYIMEALTFKACDHIMK SGDLKYTYAFTKKVKDLFNTKSDSVYQYVRLHEMSYDGVSEDTDDDDEVFAILNL SIDSSVDRYRNRVLLLTPEVASLRKEYSETEPDYKYLMDEEVPAYDKHLSKPITNT GIEEPHATGGDKEDQPIKVVHPPPNNDKDDAIKPYNPLEDPNYVPTNTRTVIGIAD YQLVINKLIEWLDKCEEECGNDGEFKTDLEEAKRKLTELNEELSDKLSKIRTLERDS VYKTERIDRLTTEIKELRDMQQNGTDDGSDSSEIDKKTIRELRESLDMEREMRSEL EKELDTIRDGKVDGSCQRELELSRMWLKQRDDDLRAEIDKRRNVEWELSRLRMD IKECDKYKEDLDKDKTTISTYVSRISTLESEIAKYQQDRDTLSVVHRELEEERRHVR DLESRLDECTRNQEDTQEVDALRSRIRELENKLTDCIESGGGNLTEISRLQSRISDL ERQLSECRGNATEITI

[0067] Accession Number: NP_536564.1

[0068] “VACV-B5R” refers to the B5R antigen of Vaccinia virus Copenhagen NCBI:txid10249. The amino acid sequence of VACV-B5R from Vaccinia virus Copenhagen NCBI:txid10249. is provided in SEQUENCE ID NO: 3: MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNNNQKVTFTCDQGYHSS DPNAVCETDKWKYENPCKKMCTVSDYISELYNKPLYEVNSTMTLSCNGETKYFR CEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYITINCDVGYEVI GASYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFILTGSPS STCIDGKWNPILPTCVRSNEKFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATY HIIIVALTIMGVIFLISVIVLVCSCDKNNDQY

[0069] Accession Number: QTC35539

[0070] The “biological sample” refers primarily to blood and blood derivatives (serum, plasma, etc) and urine, but may also include other biological fluids such as saliva, sweat, cerebrospinal fluid, semen, and lymph. In one embodiment, the liquid biological sample employed in the methods of the invention is undiluted (especially when the sample is urine). This provides an advantage insofar as the user is not required to dilute the sample, and makes the test suitable as a home-use test. In one embodiment, the sample is diluted at least x100, x200, x300, x400, x500.

[0071] “IgG” refers to Immunoglobulin G which is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. “MPXV-B6R IgG” refers to IgG present in a biological sample that specifically binds to the MPXV-B6R protein. “MPXV-A27L IgG” refers to IgG present in a biological sample that specifically binds to the MPXV-A27L protein. “VACV-B5R IgG” refers to IgG present in a biological sample that specifically binds to the VACV-B5R protein.

[0072] “IgGnu” refers to normalised units of IgG in a sample. IgGnu for an antigen in a plasma sample is calculated by dividing the antigen IgG titre in the test sample with the antigen IgG titre in a control. Typically, the control is a sample from a positive control (e.g. a sample from a patient that has been infected with Mpox). In one embodiment, the control antigen IgG titre is an average antigen IgG titre from a plurality of control samples, for example, a plurality of positive (infection) controls, or a plurality of positive (vaccination) controls, or a plurality of both positive (infection) and positive (vaccination) controls.

[0073] “MVA- vaccine” refers to Modified Vaccinia Ankara (MVA) vaccine, an attenuated vaccinia virus (VACV), that is authorised for Mpox prevention.

[0074] Exemplification

[0075] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0076] Electrochemiluminesce Assay

[0077] Each antigen was reconstituted in sterile water to a stock solution and stored at - 80°C. 96 well high-bind plates (Meso Scale Discovery (MSD), Rockville, MD) were washed 3 times with phosphate buffered saline and 0.05% tween (PBS tween) (Bio Sciences Ltd., Ireland). Each plate was coated separately with an antigen and the concentration of the coating antigen was determined using the maximum coating capacity of the high bind plates (5 picomoles of antigen per well) and incubated overnight at 4°C. Plates were washed 3 times with PBS tween and then blocked using 150ul of 1% blocker A (MSD) and incubated for 120 minutes at room temperature (RT). Thawed, ethylenediamine tetraacetic acid (EDTA)-derived plasma, diluted to 1 :500 in MSD D100 were added (25ul of diluted plasma in duplicate per well). Seven biological controls were included on each plate; three plasma samples from participants with Mpox infection, three plasma samples from participants with previous MVA vaccination, and one negative plasma sample. Plates were then incubated for 90 minutes at RT, washed, following which MSD SULFO-TAG-labelled goat anti-human IgG secondary antibody, diluted with MSD D100 was added at a concentration of 1 pg / ml and the plate was further incubated for one hour at RT. Plates were washed 3 times with PBS tween and 150ul of MSD GOLD read buffer A containing ECL substrate was added. Plates were placed in a MESO QuickPlex SQ 120 instrument (MSD). Analysis was performed using MSD Discovery Workbench Software Version 4.0. IgG titres were normalised and expressed as a ratio to the biological plate controls as IgG normalised units (IgGnu).

[0078] Statistical Analysis

[0079] To compare characteristics within groups, continuous variables such as age and days were summarised using median and interquartile range (IQR), while categorical variables were summarised with frequencies and percentages. We compared IgG titres and the difference in MPXV / VACV antigen ratio between groups, using the Kruskal-Wallis test, with post-hoc Dunn’s test or Mann-Whitney U test, respectively. To determine sensitivity and specificity of each antigen in quantifying immunity, receiver operating curves (ROC), area under the curve and Youden Index were constructed using the pROC package in R. In our ROC analyses, we compared known infection and vaccination cases to negative controls, and infection versus vaccination cases. To determine the change in antibody responses over time to vaccination and infection, scatter plots, with superimposed curves fitted using generalised additive mixed models (GAMMs), were used to depict the non-linear relationship between time since vaccination or infection and quantitative antibody levels. These models used a Gaussian function and time since vaccination or infection fitted as a spline. GAMMs were fitted using the mgcv package in R, incorporating individual participants as a random effect and an autocorrelation error structure. To compare antibody responses to infection and vaccination, Welch Two Sample t-test was used to compare the aggregated predicted means using these models. Statistical analysis was performed using R software (Version 4.4.1 ).

[0080] Study population

[0081] A total of 295 participants provided 383 samples for analysis across the four groups; Mpox group 54 samples from 28 participants, MVA vaccine group 229 samples from 167 participants, Control group 78 samples from 78 participants, and Childhood Vaccine group 22 samples from 22 participants. All participants were assigned male sex at birth, and the age (median [IQR]) of participants was similar across the groups: Mpox (32 [29-39]), MVA Vaccine (35 [30-41 ]), and Control (32 [29-38]) with the exception of the older Childhood Vaccine group (72 (56-78) . Of the total cohort of 295, 60 (20%) were living with HIV, with the prevalence of HIV similar across groups. The Mpox samples were taken at a median (IQR) of 277 (11 - 449 ) days from onset of symptoms. In the MVA Vaccine group, all participants were sampled at a median (IQR) of 238 (138-327) days post completion of vaccination course .

[0082] Using the assay to differentiate between infection and vaccination induced immune responses

[0083] ROC curves were generated comparing Mpox samples (n=54) to MVA Vaccine samples (n=229) for the three antigens IgG titre ratios. The MPXV-B6R I VACV-B5 ratio performed best in differentiating post-infectious from post-vaccination immune responses (AUC 0.850).

[0084] Combining the MPXV-A27L IgGnu with the MPXV-B6R / VACV-B5 ratio [sum of (MPXV-B6R IgGnu / VACV-B5 IgGnu) + MPXV-A27L IgGnu] resulted in the highest performing discriminator of infection from vaccination, with an AUC of 0.895. Using the Youden Index thresholds to identify the optimum threshold a [(B6R / VACVB5) + A27L IgGnu] value of 0.92 provided the best combined sensitivity (89% [80-96%]) and specificity (80% [74-84%]) to differentiate post infection from post vaccination immune responses. Within the Mpox group, 8 / 54 (15%) samples were from participants with previous infection who had also received the MVA Vaccine; 7 / 8 (88%) of these samples were identified as infection using this threshold, highlighting its utility in differentiating infection even in those with a history of both infection and vaccination. IgG responses to the MPXV protein A27 were able to distinguish post-MPXV infection from negative and post-vaccination samples with a sensitivity of 88% and specificity of 97%. Equivalents

[0085] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:1 . A method comprising: providing a biological sample from a subject; and determining an IgG titre value for each of a plurality of target Orthopoxvirus antigens in the biological sample, wherein the plurality of target Orthopoxvirus antigens comprise MPXV-B6R, VACV- B5R, and MPXV-A27L.

2. A method according to Claim 1 , in which the plurality of target Orthopoxvirus antigens consist of MPXV-B6R, VACV-B5R, and MPXV-A27L.

3. A method according to Claim 1 or 2, comprising a step of calculating [((MPXV- B6R) IgG titre value / VACV-B5R IgG titre value) + (MPXV-A27L) IgG titre value] to provide a test score for the subject.

4. A method according to Claim 1 or 2, in which the IgG titre value is selected from an IgG normalised value and an IgG concentration.

5. A method according to Claim 4, in which the IgG titre value is an IgG normalised value obtained by dividing the IgG titre by a control titre to provide an IgG normalised unit (IgGnu) value, wherein the control titre is an average of titre values obtained from a plurality of subjects that are positive for Mpox infection and I or vaccination.

6. A method according to Claim 4 or 5, which is a method of determining likelihood of a serological response in the subject being a post Mpox infection immune response or post Mpox vaccination immune response, wherein the method comprises correlating the IgG titre levels for the three antigens with likelihood of theserological response in the subject being a post Mpox infection immune response or post Mpox vaccination immune response.

7. A method according to Claim 5 wherein the correlating comprises: calculating [((MPXV-B6R) IgG titre value / VACV-B5R IgG titre value) + (MPXV-A27L) IgG titre value] to provide a test score for the subject, comparing the test score with a reference (threshold) value; and determining whether the serological response in the subject is a post Mpox infection or post Mpox vaccination immune response based on the threshold test score value.

8. A method according to Claim 6 or 7, wherein the subject has a history of both infection and vaccination.

9. A method according to Claim 7 or 8, wherein: the IgG titre values are IgG normalised units (nu); the reference (threshold) test score is 0.92; and a test score lower than 0.92 is indicative of the serological response in the subject being a post Mpox vaccination immune response and a test score higher than 0.92 is indicative of the serological response in the subject being a post Mpox infection immune response.

10. A method according to any preceding Claim, in which the step of quantifying IgG in the sample employs an immunoassay .1 1 . A method according to any preceding Claim, in which the step of quantifying IgG in the sample employs a bead-based immunoassay or an electrochemiluminescence immunoassay.

12. A method according to any preceding Claim, in which the method comprises an immunoassay and employs a multiwell assay plate comprising: at least one well coated with MPXV-B6R antigen; at least one well coated with VACV-B5R antigen; and at least one well coated with MPXV-A27L antigen.

13. A method according to Claim 12, in which each coated well comprises about 3 to about 7 picomoles of antigen.

14. A method according to Claim 12 or 13, in which the multiwell assay plate comprises: at least 10 wells coated with MPXV-B6R antigen; at least 10 wells coated with VACV-B5R antigen; and at least 10 wells coated with MPXV-A27L antigen.

15. A method according to any preceding Claim, in which the biological sample is diluted plasma, and in which the method comprises an immunoassay comprising the steps of: adding the diluted plasma to wells of a multiwell plate; incubating the diluted plasma in the wells for a first incubation period of time;21 washing the wells; adding labelled anti-human IgG secondary antibody to each well; incubating the labelled anti-human IgG secondary antibody in the wells for a second incubation period of time; and analysing the wells with a plate reader to quantify the labelled anti-human IgG secondary antibody in each well.

16. A multiwell assay plate comprising a plurality of wells and a plurality of Orthopoxvirus antigens and comprising: at least one well coated with MPXV-B6R antigen; at least one well coated with VACV-B5R antigen; and at least one well coated with MPXV-A27L antigen.

17. A multiwell assay plate according to Claim 16, in which the plurality of Orthopoxvirus antigens consist of MPXV-B6R, VACV-B5R, and MPXV-A27L.

18. A multiwell assay plate according to Claim 16 or 17, in which each well that is coated with antigen comprises about 3 to about 7 picomoles of antigen.

19. A multiwell assay plate according to any of Claims 16 to 18, in which the multiwell assay plate comprises: at least 10 wells coated with MPXV-B6R antigen; at least 10 wells coated with VACV-B5R antigen; andat least 10 wells coated with MPXV-A27L antigen.

20. An immunoassay kit comprising: a multiwell assay plate according to any of Claims 16 to 19; labelled anti-human IgG secondary antibody; and an electrochemiluminescence read buffer.21 . An immunoassay kit according to Claim 20 additionally comprising biological sample diluent, wash buffer and optionally serological controls.

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