Development of a novel serological assay for the detection of mpox infection in vaccinated populations

Abstract

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Keywords: antibody; immunoassay; mpox; orthopoxvirus; serology.

Figure 1:. Evaluation of Non-Vaccinia Peptides for the Detection of Mpox Infection

Serum or plasma specimens from 341 presumed mpox-negative donors and 40 mpox confirmed patients were analyzed for antibody reactivity to peptide antigens derived from mpox and variola virus (VAR) by a microsphere immunoassay. (A) Median fluorescence intensity (MFI) of IgG reactivity to individual 31-mer peptides was used to generate ROC curves representing for mpox-derived B21R-A and -B (magenta) or VAR-derived B22R-A and -B (blue). (B) The log₁₀ MFI of IgG reactivity to mpox B21R-A and -B was plotted for mpox-negative, -positive, and recent vaccinee donors. The mpox-negative cohort was divided by birth year post- and pre-1972 to estimate smallpox childhood vaccination status. (C) Log₁₀ MFI of IgG reactivity to mpox B21R-A and -B was plotted for mpox-negative donors post-1972 (>50) divided by “healthy” and “HIV”. Each dot represents an individual donor. Statistical significance was determined by the non-parametric Kruskal-Wallis test where *p < 0.05 **p < 0.001 ***p < 0.0001, and ****p < 0.00001 adjusted for multiple comparisons by Dunn’s test.

Figure 2:. IgG Antibody Reactivity to Orthopoxvirus Antigens Among Mpox Negative and Positive Cohorts

Serum or plasma specimens from 341 presumed mpox-negative donors and 40 mpox confirmed patients were analyzed for antibody reactivity to recombinant protein antigens derived from mpox or vaccinia virus (VAC) by microsphere immunoassay. (A) Median fluorescence intensity (MFI) of IgG reactivity to recombinant antigens from mpox or VAC (mpox A35R, solid magenta; VAC A33R, dashed magenta; mpox H3L solid light blue; VAC B5R dashed green; VAC L1R dashed orange; mpox A29L solid dark blue, VAC A27L dashed dark blue) were used to generate ROC curves used to represent the sensitivity (%) and specificity (%) of each antigen to detect mpox infection. (B) Log₁₀ MFI of IgG reactivity to OPV antigens (A33/35, B5R, L1R, and H3L) plotted for mpox-negative, -positive, and recent vaccinee donors. Each dot represents an individual donor. Statistical significance was determined by the non-parametric Kruskal-Wallis test where *p < 0.05 **p < 0.001 ***p < 0.0001, and ****p < 0.00001 adjusted for multiple comparisons by Dunn’s test.

Figure 3:. Pattern of OPV Antigen and Mpox Peptide IgG Reactivity Among Mpox-negative and -postive Cohorts

Heat maps indicate the relative index value (MFI/cutoff value) of each antigen (Mpox A35R, VAC B5R, VAC L1R, Mpox H3L, Mpox B21R-A, and Mpox B21R-B; vertical columns) for each serum/plasma donor (horizontal rows). Yellow corresponds to a negative index value (< 1.0) below the clinical cutoff. The level of red saturation corresponds to higher index values (> 1.0) above the clinical cutoff.

Figure 4:. OPV Antigen Count and Algorithm for the Specific Serologic Detection of Mpox Infection

(A) The number of OPV antigens (A33/35, B5R, L1R, and H3L) counted as reactive (R). Reactivity was defined as an MFI value at or above the calculated clinical cutoff value. The antigen count distribution was displayed as a violin plot for mpox-confirmed and mpox-negative cohorts. The mpox-negative cohort divided by birth year post- (presumed naïve) and pre-1972 (Childhood VAC) to estimate smallpox childhood vaccination status. (B) Serum or plasma specimens tested for mpox infection using a multiplexed microsphere immunoassay will be subjected to a 2-tier algorithm using a combination of mpox-specific peptides and OPV antigens. First, specimens will be considered for IgG reactivity to a set of cross-reactive OPV antigens (VAC A33R, mpox A35R, VAC B5R, VAC L1R, and mpox H3L). Samples with reactivity to 2 or fewer antigens will be considered non-reactive (NR; MFI < clinical cutoff) overall for mpox exposure while samples that test reactive (R; MFI > clinical cutoff) to 3 or more OPV antigens will move to the second tier of the algorithm. Next, samples will be considered for their reactivity to mpox peptides B21R-A and -B. Samples with no peptide reactivity will be considered NR for mpox infection. Samples with reactivity to one or more mpox peptides will be considered R for mpox infection. (C) Serum specimens from mpox-confirmed donors (n=40) and recent vaccinees (n=15) were analzyed for IgG reactivity to viral homologues VAC A33R and mpox A35R. Mpox confirmed donors were separated by birth year to simulate vaccination status before infection. Green circles indicate recent vaccinees. Grey circles indicate a birth year prior to 1972 (childhood vaccine), and blue circles indicate a birth year after 1972 (unvaccinated). Red circles in both groups indicate individuals infected in the 2003 mpox outbreak. The dashed line in each plot indicates the reactivity cutoff for the indicated antigen as determined by ROC curve. (D) Serum IgG reactivity ratio representing the mpox A35R MFI divided by the VAC A33R MFI. Green circles indicate recent vaccinees. Grey circles indicate a birth year prior to 1972 (childhood vaccine), and blue circles indicate a birth year after 1972 (unvaccinated). Red circles in both groups indicate individuals infected in the 2003 mpox outbreak. Statistical significance was determined by the non-parametric Kruskal-Wallis test where *p < 0.05 **p < 0.001 ***p < 0.0001, and ****p < 0.00001 adjusted for multiple comparisons by Dunn’s test.