Table 1_Correlation between microneutralization test and a multiplexed immunoassay for evaluation of monkeypox and vaccinia virus antibodies before and after smallpox vaccination.docx

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.

引言
猴痘（Monkeypox, mpox）是中非与西非地区的地方性人畜共患病毒性疾病。2022年，猴痘病毒（Monkeypox virus, MPXV）IIb分支扩散至非洲以外地区，引发国际广泛关注，世界卫生组织（World Health Organization, WHO）将其列为国际关注的突发公共卫生事件（Public Health Emergency of International Concern, PHEIC）。尽管2023年因全球病例数下降解除了该紧急状态，但由Ib分支引发的疫情反弹重新恢复了紧急级别。当前基于减毒活痘苗病毒或修饰型痘苗病毒（modified vaccinia virus, VACV）开发的猴痘疫苗，曾广泛用于天花预防。然而，阐明猴痘疫苗接种与感染所触发的体液免疫反应（humoral immune response），明确保护相关关联因素（correlates of protection），仍是亟待解决的关键问题。

方法
在既往一项研究中，我们评估了1000名个体的中和抗体（neutralizing antibody）反应，其中半数出生于意大利天花疫苗接种停止前（1975年以前），另一半则出生于1979年之后。接种过天花疫苗的个体针对MPXV与VACV的中和抗体滴度（neutralization titers）更高，提示其对MPXV存在交叉反应性免疫（cross-reactive immunity）。本研究针对上述发现展开进一步探究：我们对既往受试队列中的一个亚组，采用多重免疫分析法（multiplex immunoassay）检测了针对5种MPXV抗原与5种VACV抗原的IgG反应。本研究纳入370名个体的血清样本（serum samples），按照中和特征（MPXV与VACV均为阴性、两种病毒均为阳性、MPXV阴性但VACV阳性，反之亦然）以及年龄（1975年以前出生与1979年以后出生）进行分组。

结果
本研究数据显示，针对特定抗原的免疫反应更为强烈，尤其是A35R/A33R与B6R/B5R抗原；相较于VACV特异性结合抗体（binding antibody），MPXV特异性结合抗体展现出更强的交叉反应性。此外，1975年以前出生、曾接种天花疫苗的个体表现出更强的结合抗体与中和抗体反应，而1979年以后出生的个体中和抗体滴度更低。这表明，老年群体既往的VACV疫苗接种，以及后续可能因接触其他正痘病毒（Orthopoxvirus, OPXV）而获得的加强免疫，可能随时间推移形成了更具VACV特异性的免疫反应。

讨论
本研究揭示了MPXV与VACV抗体交叉反应的抗原决定簇（antigenic determinants），并凸显了不同年龄与暴露群体间的免疫谱差异。研究结果提示，VACV疫苗的免疫印迹（vaccine imprinting）塑造了机体免疫状态，这可为开发更高效的猴痘预防疫苗策略提供指导。