Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination

IntroductionMonkeypox (mpox), an endemic zoonotic viral disease in Central and Western Africa, gained international attention in 2022 when clade IIb of the Monkeypox virus (MPXV) spread outside Africa, prompting the World Health Organization (WHO) to declare it a Public Health Emergency of Internati...

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Main Authors: Serena Marchi, Giulia Piccini, Edmond J. Remarque, Giulia Roscia, Bianca Semplici, Paolo Cantaloni, Noemi Guerrini, Roberta Zannella, Rosa Coluccio, Linda Benincasa, Niccolò Solfanelli, Claudia Maria Trombetta, Emanuele Montomoli, Alessandro Manenti
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Language:English
Published: Frontiers Media S.A. 2025-06-01
Series:Frontiers in Immunology
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Online Access:https://www.frontiersin.org/articles/10.3389/fimmu.2025.1585284/full
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author Serena Marchi
Giulia Piccini
Edmond J. Remarque
Giulia Roscia
Bianca Semplici
Paolo Cantaloni
Noemi Guerrini
Roberta Zannella
Rosa Coluccio
Linda Benincasa
Niccolò Solfanelli
Claudia Maria Trombetta
Claudia Maria Trombetta
Emanuele Montomoli
Emanuele Montomoli
Emanuele Montomoli
Emanuele Montomoli
Alessandro Manenti
author_facet Serena Marchi
Giulia Piccini
Edmond J. Remarque
Giulia Roscia
Bianca Semplici
Paolo Cantaloni
Noemi Guerrini
Roberta Zannella
Rosa Coluccio
Linda Benincasa
Niccolò Solfanelli
Claudia Maria Trombetta
Claudia Maria Trombetta
Emanuele Montomoli
Emanuele Montomoli
Emanuele Montomoli
Emanuele Montomoli
Alessandro Manenti
author_sort Serena Marchi
collection DOAJ
description IntroductionMonkeypox (mpox), an endemic zoonotic viral disease in Central and Western Africa, gained international attention in 2022 when clade IIb of the Monkeypox virus (MPXV) spread outside Africa, prompting the World Health Organization (WHO) to declare it a Public Health Emergency of International Concern (PHEIC). Although the PHEIC was lifted in 2023 due to declining global cases, a resurgence caused by clade Ib has reinstated the emergency status. Current mpox vaccines, based on live-attenuated or modified vaccinia virus (VACV), have historical use in smallpox prevention. Understanding the humoral immune response triggered by mpox vaccination and infection, as well as identifying correlates of protection, remain however critical.MethodsIn a previous study, we evaluated the neutralizing antibody response of 1,000 individuals, half born before the cessation of smallpox vaccination in Italy (pre-1975) and half after (post-1979). Higher neutralizing antibody titers against MPXV and VACV were observed in subjects vaccinated against smallpox, indicating a cross-reactive immunity to MPXV. This study further investigated these findings by analyzing the IgG response to five MPXV and five VACV antigens in a subset of the previously tested cohort, using a multiplex immunoassay. Serum samples from 370 individuals were grouped by neutralization profile (negative for both MPXV and VACV, positive for both viruses, negative for MPXV but positive for VACV, and vice versa) and age (born before 1975 and after 1979).ResultsOur data revealed stronger immune responses to specific antigens, particularly A35R/A33R and B6R/B5R, with MPXV-specific binding antibodies showing greater cross-reactivity compared to VACV ones. Furthermore, individuals born before 1975, vaccinated against smallpox, exhibited stronger binding and neutralizing antibody responses, as opposed to people born after 1979 in whom neutralization titers were lower. This suggests that prior VACV-vaccination and subsequent boosting from potential other OPXV encounters in the older population may have resulted in a more VACV-specific immune response over time.DiscussionThis study provides insights into the antigenic determinants of MPXV and VACV antibody cross-reactivity and highlights differences in immune profiles across age and exposure groups. Results obtained suggest that VACV-vaccine imprinting shapes immunity, which could guide the development of more effective vaccine strategies for preventing mpox.
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publisher Frontiers Media S.A.
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spelling doaj-art-4215d2e0ce754099a2df579673ea5fef2025-08-20T02:36:53ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-06-011610.3389/fimmu.2025.15852841585284Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccinationSerena Marchi0Giulia Piccini1Edmond J. Remarque2Giulia Roscia3Bianca Semplici4Paolo Cantaloni5Noemi Guerrini6Roberta Zannella7Rosa Coluccio8Linda Benincasa9Niccolò Solfanelli10Claudia Maria Trombetta11Claudia Maria Trombetta12Emanuele Montomoli13Emanuele Montomoli14Emanuele Montomoli15Emanuele Montomoli16Alessandro Manenti17Department of Molecular and Developmental Medicine, University of Siena, Siena, ItalyVisMederi Srl, Siena, ItalyDepartment of Virology, Biomedical Primate Research Centre, Rijswijk, NetherlandsVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyVisMederi Srl, Siena, ItalyDepartment of Molecular and Developmental Medicine, University of Siena, Siena, ItalyVaepiX, Joint Research Laboratory, University of Siena, Siena, ItalyDepartment of Molecular and Developmental Medicine, University of Siena, Siena, ItalyVisMederi Srl, Siena, ItalyVaepiX, Joint Research Laboratory, University of Siena, Siena, ItalyVisMederi Research Srl, Siena, ItalyVisMederi Srl, Siena, ItalyIntroductionMonkeypox (mpox), an endemic zoonotic viral disease in Central and Western Africa, gained international attention in 2022 when clade IIb of the Monkeypox virus (MPXV) spread outside Africa, prompting the World Health Organization (WHO) to declare it a Public Health Emergency of International Concern (PHEIC). Although the PHEIC was lifted in 2023 due to declining global cases, a resurgence caused by clade Ib has reinstated the emergency status. Current mpox vaccines, based on live-attenuated or modified vaccinia virus (VACV), have historical use in smallpox prevention. Understanding the humoral immune response triggered by mpox vaccination and infection, as well as identifying correlates of protection, remain however critical.MethodsIn a previous study, we evaluated the neutralizing antibody response of 1,000 individuals, half born before the cessation of smallpox vaccination in Italy (pre-1975) and half after (post-1979). Higher neutralizing antibody titers against MPXV and VACV were observed in subjects vaccinated against smallpox, indicating a cross-reactive immunity to MPXV. This study further investigated these findings by analyzing the IgG response to five MPXV and five VACV antigens in a subset of the previously tested cohort, using a multiplex immunoassay. Serum samples from 370 individuals were grouped by neutralization profile (negative for both MPXV and VACV, positive for both viruses, negative for MPXV but positive for VACV, and vice versa) and age (born before 1975 and after 1979).ResultsOur data revealed stronger immune responses to specific antigens, particularly A35R/A33R and B6R/B5R, with MPXV-specific binding antibodies showing greater cross-reactivity compared to VACV ones. Furthermore, individuals born before 1975, vaccinated against smallpox, exhibited stronger binding and neutralizing antibody responses, as opposed to people born after 1979 in whom neutralization titers were lower. This suggests that prior VACV-vaccination and subsequent boosting from potential other OPXV encounters in the older population may have resulted in a more VACV-specific immune response over time.DiscussionThis study provides insights into the antigenic determinants of MPXV and VACV antibody cross-reactivity and highlights differences in immune profiles across age and exposure groups. Results obtained suggest that VACV-vaccine imprinting shapes immunity, which could guide the development of more effective vaccine strategies for preventing mpox.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1585284/fullmonkeypox virusvaccinia virusbinding and neutralizing antibodiescrossreactive antibody responsehumoral immunity
spellingShingle Serena Marchi
Giulia Piccini
Edmond J. Remarque
Giulia Roscia
Bianca Semplici
Paolo Cantaloni
Noemi Guerrini
Roberta Zannella
Rosa Coluccio
Linda Benincasa
Niccolò Solfanelli
Claudia Maria Trombetta
Claudia Maria Trombetta
Emanuele Montomoli
Emanuele Montomoli
Emanuele Montomoli
Emanuele Montomoli
Alessandro Manenti
Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
Frontiers in Immunology
monkeypox virus
vaccinia virus
binding and neutralizing antibodies
crossreactive antibody response
humoral immunity
title Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
title_full Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
title_fullStr Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
title_full_unstemmed Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
title_short Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
title_sort correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination
topic monkeypox virus
vaccinia virus
binding and neutralizing antibodies
crossreactive antibody response
humoral immunity
url https://www.frontiersin.org/articles/10.3389/fimmu.2025.1585284/full
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