Development and validation of a quantitative Orthopoxvirus immunoassay to evaluate and differentiate serological responses to Mpox infection and vaccination

Abstract

Background: The Mpox outbreak, caused by Monkeypox virus (MPXV), underscores the need for a serological assay to assess Mpox immunity. Modified Vaccinia Ankara (MVA) vaccine, an attenuated vaccinia virus (VACV), is authorised for Mpox prevention. We aimed to develop a quantitative immunoassay to differentiate infection- and vaccination-induced immunity and explore serological responses to Mpox infection and vaccination.

Methods: We evaluated an electrochemiluminescence assay targeting IgG to 10 MPXV and 3 VACV antigens in plasma from adults in a cohort study with previous Mpox, MVA-vaccination, or historical controls. Sensitivity and specificity to distinguish i) seropositive versus naive and ii) infection- versus vaccination-induced seropositivity were determined using ROC curves. Antibody kinetics were analysed with generalised additive models.

Findings: Eight of the thirteen IgG antibodies showed significant titre differences across groups identifying three key antigens: MPXVB6R, MPXVA27L, and VACVB5. A VACVB5 IgG titre of 0.082 IgG normalised units (nu) offered 74% (95% CI: 59-82%) sensitivity and 81% (73-96%) specificity for previous antigen exposure (infection or vaccine). For infection alone, an MPXVB6R IgG titre of 0.075 IgGnu provided 89% (82-98%) sensitivity and 94% (86-100%) specificity. To differentiate infection from vaccination-induced seropositivity, the sum of MPXVA27L IgG and the B6R/VACVB5 ratio provided 89% (80-96%) sensitivity and 80% (74-84%) specificity. VACVB5 IgG titres declined over time, with higher titres post-Mpox than post-vaccination (p < 0.0001).

Interpretation: This assay demonstrates high sensitivity and specificity in quantifying and differentiating between antibody responses to Mpox infection and vaccination. Post-Mpox antibody responses were higher than post-vaccination, though both waned over time.

Funding: Health Research Board (MONKEYVAX-2022-1), University College Dublin School of Medicine.

Keywords: Immunoassay; MVA-Vaccine; Monkeypox virus; Mpox; Orthopoxvirus.

Fig. 1

Significantly different IgG titres across groups. Each graph represents the IgG titres for that antigen for each group. Mpox (n = 54), MVA Vaccine (n = 229), Control (n = 78), Childhood Vaccine (n = 22). The error bars represent the geometric mean titre (GMT) and the 95% confidence interval of the GMT for each group. Each antigen in this figure had significantly different titres across the four groups by Kruskal–Wallis Test (all p < 0.05). Comparisons shown represent p values as per post-hoc Dunn's test to assess differences in titres between Mpox, MVA Vaccine, Control and Childhood Vaccine group with ∗p = 0.0001–0.05, ∗∗p < 0.0001, and ns = not significant.

Fig. 2

Receiver operating characteristic (ROC) curves. A: ROC curve for all antigens generated by comparing positive samples (Mpox and MVA Vaccine group, n = 283) to negative samples (Control group, n = 78). B: ROC curve for VACVB5 generated by comparing positive samples (Mpox and MVA Vaccine group, n = 283) to negative samples (Control group, n = 78). C: ROC curve for all antigens generated by comparing positive samples restricted to infection only (Mpox group, n = 54) to negative samples (Control group, n = 78). D: ROC for MPXV B6R generated by comparing positive samples restricted to infection only (Mpox group, n = 54) to negative samples (Control group, n = 78).

Fig. 3

Differentiation of infection and vaccination induced immunity. A: ROC curve for all antigens and MPXV/VACV homologue ratios generated by comparing positive samples (Mpox, n = 54) to negative samples (MVA Vaccine, n = 229). B: ROC curve for B6R/VACVB5 ratio + A27L IgGnu generated by comparing positive samples (Mpox, n = 54) to negative samples (MVA Vaccine, n = 229).

Fig. 4

Antibody responses over time. A: VACV BS lgG Responses over time post MVA-Vaccination and Mpox Infection. A: Change in VACVB5 IgGnu titres over time since onset of symptoms of Mpox or dose 2 of MVA vaccination. We modelled change in VACVB5 IgGnu over time using scatter plots with superimposed curves fitted using generalised additive mixed models (GAMM), with a Gaussian link function and time since symptom onset or vaccination fitted as a spline. B: VACV BS IgG Responses over time post MVA-Vaccination. B: VACVB5 IgGnu titres over time before and after MVA vaccination. The centre of the error bar represents the geometric mean titre with the error bars representing the 95% confidence interval of the GMT. Seropositive is defined as the threshold identified by the Youden Index with the optimum sensitivity and specificity (0.082 IgGnu) from the ROC curve analysis for VACVB5 IgG.