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.
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Bunge, E.M., Hoet, B., Chen, L., Lienert, F., Weidenthaler, H., Baer, L.R., and Steffen, R. (2022). The changing epidemiology of human monkeypox-a potential threat? A systematic review. PLoS Negl Trop Dis 16, e0010141.
Chen, P., Chen, M., Chen, Y., Jing, X., Zhang, N., Zhou, X., Li, X., Long, G., and Hao, P. (2022). Targeted inhibition of Zika virus infection in human cells by CRISPR-Cas13b. Virus Res 312, 198707.
Costa, S.M., Paes, M.V., Barreto, D.F., Pinhão, A.T., Barth, O.M., Queiroz, J.L.S., Armôa, G.R.G., Freire, M.S., and Alves, A.M.B. (2006). Protection against dengue type 2 virus induced in mice immunized with a DNA plasmid encoding the non-structural 1 (NS1) gene fused to the tissue plasminogen activator signal sequence. Vaccine 24, 195–205.
Davies, D.H., Molina, D.M., Wrammert, J., Miller, J., Hirst, S., Mu, Y., Pablo, J., Unal, B., Nakajima-Sasaki, R., Liang, X., et al. (2007). Proteome-wide analysis of the serological response to vaccinia and smallpox. Proteomics 7, 1678–1686.
Fang, E., Liu, X., Li, M., Zhang, Z., Song, L., Zhu, B., Wu, X., Liu, J., Zhao, D., and Li, Y. (2022). Advances in COVID-19 mRNA vaccine development. Sig Transduct Target Ther 7, 94.
Fang, Z., Monteiro, V.S., Renauer, P.A., Shang, X., Suzuki, K., Ling, X., Bai, M., Xiang, Y., Levchenko, A., Booth, C.J., et al. (2023). Polyvalent mRNA vaccination elicited potent immune response to monkeypox virus surface antigens. Cell Res 33, 407–410.
Fang, Q., Yang, L., Zhu, W., Liu, L., Wang, H., Yu, W., Xiao, G., Tien, P., Zhang, L., and Chen, Z. (2005). Host range, growth property, and virulence of the smallpox vaccine: vaccinia virus Tian Tan strain. Virology 335, 242–251.
Fine, P.E.M., Jezek, Z., Grab, B., and Dixon, H. (1988). The transmission potential of monkeypox virus in human populations. Int J Epidemiol 17, 643–650.
Fogg, C., Lustig, S., Whitbeck, J.C., Eisenberg, R.J., Cohen, G.H., and Moss, B. (2004). Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions. J Virol 78, 10230–10237.
Frey, S.E., Winokur, P.L., Hill, H., Goll, J.B., Chaplin, P., and Belshe, R.B. (2014). Phase II randomized, double-blinded comparison of a single high dose (5×108 TCID50) of modified vaccinia Ankara compared to a standard dose (1×108 TCID50) in healthy vaccinia-naïve individuals. Vaccine 32, 2732–2739.
Golden, J.W., Josleyn, M.D., and Hooper, J.W. (2008). Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies. Vaccine 26, 3507–3515.
Gonzalez-Galarza, F.F., McCabe, A., Santos, E.J.M., Jones, J., Takeshita, L., Ortega-Rivera, N.D., Cid-Pavon, G.M.D., Ramsbottom, K., Ghattaoraya, G., Alfirevic, A., et al. (2020). Allele frequency net database (AFND) 2020 update: gold-standard data classification, open access genotype data and new query tools. Nucleic Acids Res 48, D783–D788.
Hatch, G.J., Graham, V.A., Bewley, K.R., Tree, J.A., Dennis, M., Taylor, I., Funnell, S.G.P., Bate, S.R., Steeds, K., Tipton, T., et al. (2013). Assessment of the protective effect of imvamune and Acam2000 vaccines against aerosolized monkeypox virus in cynomolgus macaques. J Virol 87, 7805–7815.
Hazra, A., Rusie, L., Hedberg, T., and Schneider, J.A. (2022). Human monkeypox virus infection in the immediate period after receiving modified vaccinia Ankara vaccine. JAMA 328, 2064–2067.
Hooper, J.W., Custer, D.M., and Thompson, E. (2003). Four-gene-combination DNA vaccine protects mice against a lethal vaccinia virus challenge and elicits appropriate antibody responses in nonhuman primates. Virology 306, 181–195.
Hooper, J.W., Thompson, E., Wilhelmsen, C., Zimmerman, M., Ichou, M. A., Steffen, S.E., Schmaljohn, C.S., Schmaljohn, A.L., and Jahrling, P. B. (2004). Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J Virol 78, 4433–4443.
Huhn, G.D., Bauer, A.M., Yorita, K., Graham, M.B., Sejvar, J., Likos, A., Damon, I.K., Reynolds, M.G., and Kuehnert, M.J. (2005). Clinical characteristics of human monkeypox, and risk factors for severe disease. Clin Infect Dis 41, 1742–1751.
Hurme, A., Jalkanen, P., Heroum, J., Liedes, O., Vara, S., Melin, M., Teräsjärvi, J., He, Q., Pöysti, S., Hänninen, A., et al. (2022). Long-lasting T cell responses in BNT162b2 COVID-19 mRNA vaccinees and COVID-19 convalescent patients. Front Immunol 13, 869990.
Isidro, J., Borges, V., Pinto, M., Sobral, D., Santos, J.D., Nunes, A., Mixão, V., Ferreira, R., Santos, D., Duarte, S., et al. (2022). Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med 28, 1569–1572.
Josefson, D. (2003). Smallpox vaccination confers long lasting immunity. BMJ 326, 1164.
Kennedy, R.B., Ovsyannikova, I.G., Haralambieva, I.H., Grill, D.E., and Poland, G.A. (2022). Proteomic assessment of humoral immune responses in smallpox vaccine recipients. Vaccine 40, 789–797.
Kimball, S. (2022). WHO declares rapidly spreading monkeypox outbreak a global health emergency. Available from URL: https://www.cnbc.com/2022/07/23/who-declares-spreading-monkeypox-outbreak-a-global-health-emergency.html
Li, T., Qian, C., Gu, Y., Zhang, J., Li, S., and Xia, N. (2023). Current progress in the development of prophylactic and therapeutic vaccines. Sci China Life Sci 66, 679–710.
Likos, A.M., Sammons, S.A., Olson, V.A., Frace, A.M., Li, Y., Olsen-Rasmussen, M., Davidson, W., Galloway, R., Khristova, M.L., Reynolds, M.G., et al. (2005). A tale of two clades: monkeypox viruses. J Gen Virol 86, 2661–2672.
Lum, F.M., Torres-Ruesta, A., Tay, M.Z., Lin, R.T.P., Lye, D.C., Rénia, L., and Ng, L.F.P. (2022). Monkeypox: disease epidemiology, host immunity and clinical interventions. Nat Rev Immunol 22, 597–613.
Luna, N., Muñoz, M., Bonilla-Aldana, D.K., Patiño, L.H., Kasminskaya, Y., Paniz-Mondolfi, A., and Ramírez, J.D. (2023). Monkeypox virus (MPXV) genomics: A mutational and phylogenomic analyses of B.1 lineages. Travel Med Infect Dis 52, 102551.
Magnus, P., Andersen, E.K., Petersen, K.B., and Birch-Andersen, A. (1959). A pox-like disease in cynomolgus monkeys. Acta Pathol Microbiol Scand 46, 156–176.
Mauldin, M.R., McCollum, A.M., Nakazawa, Y.J., Mandra, A., Whitehouse, E.R., Davidson, W., Zhao, H., Gao, J., Li, Y., Doty, J., et al. (2022). Exportation of monkeypox virus from the African continent. J Infect Dis 225, 1367–1376.
McFadden, G. (2005). Poxvirus tropism. Nat Rev Microbiol 3, 201–213.
Meseda, C.A., Garcia, A.D., Kumar, A., Mayer, A.E., Manischewitz, J., King, L.R., Golding, H., Merchlinsky, M., and Weir, J.P. (2005). Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model. Virology 339, 164–175.
Nalca, A., and Zumbrun, E.E. (2010). ACAM2000: the new smallpox vaccine for United States Strategic National Stockpile. Drug Des Devel Ther 4, 71–79.
Nolen, L.D., Osadebe, L., Katomba, J., Likofata, J., Mukadi, D., Monroe, B., Doty, J., Hughes, C.M., Kabamba, J., Malekani, J., et al. (2016). Extended human-to-human transmission during a monkeypox outbreak in the Democratic Republic of the Congo. Emerg Infect Dis 22, 1014–1021.
Paran, N., Lustig, S., Zvi, A., Erez, N., Israely, T., Melamed, S., Politi, B., Ben-Nathan, D., Schneider, P., Lachmi, B., et al. (2013). Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response. Virol J 10, 229.
Pardi, N., Hogan, M.J., Porter, F.W., and Weissman, D. (2018). mRNA vaccines—a new era in vaccinology. Nat Rev Drug Discov 17, 261–279.
Pegu, A., O’Connell, S.E., Schmidt, S.D., O’Dell, S., Talana, C.A., Lai, L., Albert, J., Anderson, E., Bennett, H., Corbett, K.S., et al. (2021). Durability of mRNA-1273 vaccine-induced antibodies against SARS-CoV-2 variants. Science 373, 1372–1377.
Petersen, B.W., Harms, T.J., Reynolds, M.G., and Harrison, L.H. (2016). Use of vaccinia virus smallpox vaccine in laboratory and health care personnel at risk for occupational exposure to orthopoxviruses—recommendations of the Advisory Committee on Immunization Practices (ACIP), 2015. MMWR Morb Mortal Wkly Rep 65, 257–262.
Ramachandran, S., Satapathy, S.R., and Dutta, T. (2022). Delivery strategies for mRNA vaccines. Pharm Med 36, 11–20.
Rao, A.K., Petersen, B.W., Whitehill, F., Razeq, J.H., Isaacs, S.N., Merchlinsky, M.J., Campos-Outcalt, D., Morgan, R.L., Damon, I., Sánchez, P.J., et al. (2022). Use of JYNNEOS (smallpox and monkeypox vaccine, live, nonreplicating) for preexposure vaccination of persons at risk for occupational exposure to orthopoxviruses: recommendations of the advisory committee on immunization practices—United States, 2022. MMWR Morb Mortal Wkly Rep 71, 734–742.
Reed, L.J., and Muench, H. (1938). A simple method of estimating fifty per cent endpoints12. Am J Epidemiol 27, 493–497.
Reynisson, B., Alvarez, B., Paul, S., Peters, B., and Nielsen, M. (2020). NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res 48, W449–W454.
Saha, S., and Raghava, G.P.S. (2006). Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65, 40–48.
Sakhatskyy, P., Wang, S., Chou, T.W., and Lu, S. (2006). Immunogenicity and protection efficacy of monovalent and polyvalent poxvirus vaccines that include the D8 antigen. Virology 355, 164–174.
Tan, S., Zhang, S., Wu, B., Zhao, Y., Zhang, W., Han, M., Wu, Y., Shi, G., Liu, Y., Yan, J., et al. (2017). Hemagglutinin-specific CD4+ T-cell responses following 2009-pH1N1 inactivated split-vaccine inoculation in humans. Vaccine 35, 5644–5652.
Taub, D.D., Ershler, W.B., Janowski, M., Artz, A., Key, M.L., McKelvey, J., Muller, D., Moss, B., Ferrucci, L., Duffey, P.L., et al. (2008). Immunity from smallpox vaccine persists for decades: a longitudinal study. Am J Med 121, 1058–1064.
Thornhill, J.P., Barkati, S., Walmsley, S., Rockstroh, J., Antinori, A., Harrison, L.B., Palich, R., Nori, A., Reeves, I., Habibi, M.S., et al. (2022). Monkeypox virus infection in humans across 16 countries—April–June 2022. N Engl J Med 387, 679–691.
Viner, K.M., and Isaacs, S.N. (2005). Activity of vaccinia virus-neutralizing antibody in the sera of smallpox vaccinees. Microbes Infect 7, 579–583.
Weaver, J.R., and Isaacs, S.N. (2008). Monkeypox virus and insights into its immunomodulatory proteins. Immunol Rev 225, 96–113.
Wherry, E.J., and Barouch, D.H. (2022). T cell immunity to COVID-19 vaccines. Science 377, 821–822.
World Health Organization. (2023). 2022–23 Mpox Outbreak: Global Trends. 2022–23 Mpox (Monkeypox) Outbreak: Global Trends. Geneva: World Health Organization.
Wyatt, L.S., Earl, P.L., Eller, L.A., and Moss, B. (2004). Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge. Proc Natl Acad Sci USA 101, 4590–4595.
Xiao, Y., Aldaz-Carroll, L., Ortiz, A.M., Whitbeck, J.C., Alexander, E., Lou, H., Davis, H.L., Braciale, T.J., Eisenberg, R.J., Cohen, G.H., et al. (2007). A protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost. Vaccine 25, 1214–1224.
Xiao, Y., Zeng, Y., Schante, C., Joshi, S.B., Buchman, G.W., Volkin, D.B., Middaugh, C.R., and Isaacs, S.N. (2020). Short-term and longer-term protective immune responses generated by subunit vaccination with smallpox A33, B5, L1 or A27 proteins adjuvanted with aluminum hydroxide and CpG in mice challenged with vaccinia virus. Vaccine 38, 6007–6018.
Xu, K., An, Y., Li, Q., Huang, W., Han, Y., Zheng, T., Fang, F., Liu, H., Liu, C., Gao, P., et al. (2021). Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat spike protein RBD protects mice against COVID-19. Emerg Microbes Infect 10, 1574–1588.
Xu, X., Chen, P., Wang, J., Feng, J., Zhou, H., Li, X., Zhong, W., and Hao, P. (2020). Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci China Life Sci 63, 457–460.
Zaeck, L.M., Lamers, M.M., Verstrepen, B.E., Bestebroer, T.M., van Royen, M.E., Götz, H., Shamier, M.C., van Leeuwen, L.P.M., Schmitz, K.S., Alblas, K., et al. (2023). Low levels of monkeypox virus-neutralizing antibodies after MVA-BN vaccination in healthy individuals. Nat Med 29, 270–278.
Zhang, N., Jing, X., Liu, Y., Chen, M., Zhu, X., Jiang, J., Wang, H., Li, X., and Hao, P. (2020). Interfering with retrotransposition by two types of CRISPR effectors: Cas12a and Cas13a. Cell Discov 6, 30.
Zhang, R.R., Wang, Z.J., Zhu, Y.L., Tang, W., Zhou, C., Zhao, S.Q., Wu, M., Ming, T., Deng, Y.Q., Chen, Q., et al. (2023). Rational development of multicomponent mRNA vaccine candidates against mpox. Emerg Microbes Infect 12, 2192815.
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.
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Table S1
. Epitope counts of monkeypox virus surface proteins
11427_2023_2378_MOESM5_ESM.xlsx
Table S3. PRNT50 titers against VACV (strain Tian Tan) for sera of vaccinated BALB/c mice on day 57 post 1st immunization.
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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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Zhang, N., Cheng, X., Zhu, Y. et al. Multi-valent mRNA vaccines against monkeypox enveloped or mature viron surface antigens demonstrate robust immune response and neutralizing activity. Sci. China Life Sci. 66, 2329–2341 (2023). https://doi.org/10.1007/s11427-023-2378-x
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DOI: https://doi.org/10.1007/s11427-023-2378-x