A-270 Evaluation of a multiplex panel to determine antibody profile to monkeypox virus in vaccinated, exposed, and naïve individuals
Authors: Venkateswaran N , Krishnan V , Sarwar J , Nelson W M , Venkateswaran K S 
Published: October 2024

Abstract

Background
Monkeypox Virus (MPXV) is a double-stranded DNA virus of the Orthopoxvirus genus that causes Mpox disease with symptoms such as swollen lymph nodes, chills, exhaustion, and blisters that appear on the face, inside the mouth, hands, feet etc. The genus orthopoxvirus (OPXV) has four species, namely, MPXV, smallpox-causing variola major virus (VARV), variola minor virus, and cowpox virus (CPXV), that cause disease in humans. In 1980, the World Health Assembly declared that smallpox had been eradicated due to a successful global vaccination program. This vaccine also provided cross-protection against other OPXV. Historically, the pox disease was rarely seen outside West and Central Africa. The first case of Mpox was identified in 1970 in a 9-year-old boy from the Democratic Republic of Congo (DRC), two years after the smallpox vaccination was discontinued. Since then, it has been reported in 11 African countries. In 2003, the first Mpox outbreak outside of Africa was in the USA and was linked to contact with infected pet prairie dogs. The recent 2022-2023 Mpox outbreak has generated renewed interest in the scientific community to understand the level of OPXV-specific immunity in the various geographic regions, among different age groups, and especially in the risk groups and its impact on future outbreaks by using integrated serosurveillance. To meet the need of the hour, we developed a novel multiplex assay for the detection of human IgG antibodies using Luminex® xMAP® technology for the detection of the human IgG profiles.

Methods
We have evaluated 11 different recombinant antigens from VARV, Vaccinia virus (VACV), and MPXV in a multiplex assay for detecting human IgG antibodies to OPXV using Luminex® xMAP® technology. We have covalently coupled magnetic fluorescent microspheres to three recombinant proteins from VARV, three from VACV, and five from MPXV to interrogate IgG responses. This 15-plex assay also includes four internal controls that assure the assay and reagent performance. In this preliminary evaluation, we have tested 84 de-identified serum and plasma from healthy subjects. We evaluated 24 serum samples of MPXV-exposed individuals procured commercially. We performed principal component analysis to determine which of these 11 antigens contributed the maximum differential and a result analysis algorithm was developed to predict these individuals' positive and negative antibody outcomes.

Results
The healthy subjects that were assumed to have been vaccinated showed positive response to A35R of MPXV; A36R of VARV, A33R of VACV, the homologous proteins in the three species and were distinct from unvaccinated younger individuals. There was some reactivity with B6R of MPXV protein. In MPXV-exposed subjects, we observed strong positive antibody responses to A35R of MPXV, A36R of VARV, A33R of VACV, and B6R of MPXV proteins in 19 out of 24 individuals. Fifteen individuals showed a moderate positive response to the A27L protein of VACV, and 8 out of 24 showed a weak positive response to the A29 protein of MPXV.

Conclusions
This panel is a useful tool to understand the level of OPXV-specific immunity by measuring IgG profiles for successful serosurveillance programs for public health.