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. 2023 Jun 1:1–13. Online ahead of print. doi: 10.1007/s11427-023-2378-x

Multi-valent mRNA vaccines against monkeypox enveloped or mature viron surface antigens demonstrate robust immune response and neutralizing activity

Niubing Zhang 1,6,7,#, Xiang Cheng 1,8,#, Yilong Zhu 2,5,#, Ouyang Mo 3,8,#, Huiqing Yu 3, Liqi Zhu 3,8, Juan Zhang 3,8, Linlin Kuang 3, Ying Gao 3,8, Ruiyuan Cao 4, Xiaozhen Liang 3,8, Haikun Wang 3,8, Honglin Li 6,7, Song Li 4, Wu Zhong 4,✉, Xuan Li 1,8,✉, Xiao Li 2,✉, Pei Hao 3,8,✉
PMCID: PMC10257374  PMID: 37300753

Abstract

Monkeypox was declared a global health emergency by the World Health Organization, and as of March 2023, 86,000 confirmed cases and 111 deaths across 110 countries have been reported. Its causal agent, monkeypox virus (MPV) belongs to a large family of double-stranded DNA viruses, Orthopoxviridae, that also includes vaccinia virus (VACV) and others. MPV produces two distinct forms of viral particles during its replication cycles: the enveloped viron (EV) that is released via exocytosis, and the mature viron (MV) that is discharged through lysis of host cells. This study was designed to develop multi-valent mRNA vaccines against monkeypox EV and MV surface proteins, and examine their efficacy and mechanism of action. Four mRNA vaccines were produced with different combinations of surface proteins from EV (A35R and B6R), MV (A29L, E8L, H3L and M1R), or EV and MV, and were administered in Balb/c mice to assess their immunogenicity potentials. A dynamic immune response was observed as soon as seven days after initial immunization, while a strong IgG response to all immunogens was detected with ELISA after two vaccinations. The higher number of immunogens contributed to a more robust total IgG response and correlating neutralizing activity against VACV, indicating the additive potential of each immunogen in generating immune response and nullifying VACV infection. Further, the mRNA vaccines elicited an antigen-specific CD4+ T cell response that is biased towards Th1. The mRNA vaccines with different combinations of EV and MV surface antigens protected a mouse model from a lethal dose VACV challenge, with the EV and MV antigens-combined vaccine offering the strongest protection. These findings provide insight into the protective mechanism of multi-valent mRNA vaccines against MPV, and also the foundation for further development of effective and safe mRNA vaccines for enhanced protection against monkeypox virus outbreak.

Supporting Information

The supporting information is available online at 10.1007/s11427-023-2378-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Keywords: monkeypox virus, enveloped and mature viron, multi-valent mRNA vaccines, immune response, neutralizing antibody

Electronic supplementary material

11427_2023_2378_MOESM1_ESM.docx (19KB, docx)

Table S1. Epitope counts of monkeypox virus surface proteins

11427_2023_2378_MOESM2_ESM.pdf (212.7KB, pdf)

Supplementary material, approximately 212 KB.

11427_2023_2378_MOESM3_ESM.xlsx (48.5KB, xlsx)

Supplementary material, approximately 48.4 KB.

11427_2023_2378_MOESM4_ESM.pdf (44.5KB, pdf)

table S3 NT50 titers against VACV (strain Tian Tan) in sera of vaccinated BALB/c mice on day57.

11427_2023_2378_MOESM5_ESM.xlsx (10.8KB, xlsx)

Table S3. PRNT50 titers against VACV (strain Tian Tan) for sera of vaccinated BALB/c mice on day 57 post 1st immunization.

11427_2023_2378_MOESM6_ESM.pdf (672.1KB, pdf)

Figure S1. Construction of expression vectors for MPV-antigens mRNA production.

11427_2023_2378_MOESM7_ESM.pdf (545.9KB, pdf)

Figure S2. Analysis of in vitro transcribed mRNA by electrophoresis.

11427_2023_2378_MOESM8_ESM.pdf (575.1KB, pdf)

Figure S3. The number (A) and proportion (B) of lymphocytes, neutrophils, monocytes, and other myeloid cells within white blood cells (WBC) in mouse blood samples obtained one day before and six days after the immunization with MPV-EM6 (total 45 μg mRNA, Materials and methods). Different cell populations were treated by lysercell WDF buffer (Sysmex, Japan) and detected using the XN-1000 automated hematology analyzer according to the manufacturer’s protocol (Sysmex, Japan). Data are shown as mean ± SEM (n=3). Comparisons were performed by Student’s t-test (*p <0.05, **p <0.01, ***p < 0.001, ****p < 0.0001).

Acknowledgements

This work was supported by the National Science and Technology Major Projects (2021YFC2300704), the National Key Research and Development Program of China (2021YFA1301402, 2018YFA0903700), the Strategic Priority Research Program of Chinese Academy of Sciences (XDA24010400), Shanghai Municipal Science and Technology Major Project (ZD2021CY001), and the National Natural Science Foundation of China (32270695, 31972881). We hope to acknowledge support from Lingang Laboratory (Shanghai, China). We thank Dr. Jianqing Xu (Shanghai Public Health Clinical Center) for providing us with VACV strain, Dr. Ziyu Li (Shanghai Pengzan Biotech Corp) for help with LNP production, Chao Shi (Laboratory of animal center, Institut Pasteur of Shanghai) for assistance with mouse immunization, and Miaolian Ma (Protein expression and purification platform, CAS Center for Excellence in Molecular Plant Sciences) for support with cell culture. We also thank Jie Gong, Jie Deng and Xintian Xu for their help with figure preparation and cell experiments.

Compliance and ethics The author(s) declare that they have no conflict of interest.

Footnotes

Contributed equally to this work

Contributor Information

Wu Zhong, Email: zhongwu@bmi.ac.cn.

Xuan Li, Email: lixuan@sippe.ac.cn.

Xiao Li, Email: skylee6226@163.com.

Pei Hao, Email: phao@ips.ac.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

11427_2023_2378_MOESM1_ESM.docx (19KB, docx)

Table S1. Epitope counts of monkeypox virus surface proteins

11427_2023_2378_MOESM2_ESM.pdf (212.7KB, pdf)

Supplementary material, approximately 212 KB.

11427_2023_2378_MOESM3_ESM.xlsx (48.5KB, xlsx)

Supplementary material, approximately 48.4 KB.

11427_2023_2378_MOESM4_ESM.pdf (44.5KB, pdf)

table S3 NT50 titers against VACV (strain Tian Tan) in sera of vaccinated BALB/c mice on day57.

11427_2023_2378_MOESM5_ESM.xlsx (10.8KB, xlsx)

Table S3. PRNT50 titers against VACV (strain Tian Tan) for sera of vaccinated BALB/c mice on day 57 post 1st immunization.

11427_2023_2378_MOESM6_ESM.pdf (672.1KB, pdf)

Figure S1. Construction of expression vectors for MPV-antigens mRNA production.

11427_2023_2378_MOESM7_ESM.pdf (545.9KB, pdf)

Figure S2. Analysis of in vitro transcribed mRNA by electrophoresis.

11427_2023_2378_MOESM8_ESM.pdf (575.1KB, pdf)

Figure S3. The number (A) and proportion (B) of lymphocytes, neutrophils, monocytes, and other myeloid cells within white blood cells (WBC) in mouse blood samples obtained one day before and six days after the immunization with MPV-EM6 (total 45 μg mRNA, Materials and methods). Different cell populations were treated by lysercell WDF buffer (Sysmex, Japan) and detected using the XN-1000 automated hematology analyzer according to the manufacturer’s protocol (Sysmex, Japan). Data are shown as mean ± SEM (n=3). Comparisons were performed by Student’s t-test (*p <0.05, **p <0.01, ***p < 0.001, ****p < 0.0001).

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