Abstract
The global outbreak of the mpox virus (MPXV) in 2022 highlights the urgent need for safer and more accessible new-generation vaccines. Here, we used a structure-guided multi-antigen fusion strategy to design a ‘two-in-one’ immunogen based on the single-chain dimeric MPXV extracellular enveloped virus antigen A35 bivalently fused with the intracellular mature virus antigen M1, called DAM. DAM preserved the natural epitope configuration of both components and showed stronger A35-specific and M1-specific antibody responses and in vivo protective efficacy against vaccinia virus (VACV) compared to co-immunization strategies. The MPXV-specific neutralizing antibodies elicited by DAM were 28 times higher than those induced by live VACV vaccine. Aluminum-adjuvanted DAM vaccines protected mice from a lethal VACV challenge with a safety profile, and pilot-scale production confirmed the high yield and purity of DAM. Thus, our study provides innovative insights and an immunogen candidate for the development of alternative vaccines against MPXV and other orthopoxviruses.
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Change history
15 January 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41590-024-01748-6
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Acknowledgements
We thank K. Xu (Beijing Institutes of Life Science, CAS), Z. Liu (Shanxi Academy of Advanced Research and Innovation) and Y. Zhang and L. Dai (Institute of Microbiology, CAS) for their assistance in data analysis and comments. We thank Y. Chen (Institute of Biophysics, CAS) and Z. Fan (Institute of Microbiology, CAS) for their technical assistance with SPR experiments. We thank Y. Liu (Clinical Laboratory Diagnostic Center at Veterinary Teaching Hospital, China Agricultural University) for work of biochemical testing. We thank H. Liu, P. Gao and X. Wang (Institute of Microbiology, CAS) for their assistance in the performance of neutralization assays and SPR experiments. We thank Y. Deng (Chinese Center for Disease Control and Prevention) and G. Gu (Institute of Microbiology, CAS) for their assistance in virus culture. We thank Q. Wang (Institute of Microbiology, CAS) for assistance with analytical ultracentrifugation. We thank J. Li (Institute of Microbiology, CAS), C. Wong (Peking University) and X. Sun (Peking University) for their assistance with mass spectrometry. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from National Institutes of Health award P41-GM103311. This project is supported by the National Key Research and Development Program of China (grant 2021YFC2300701, to H.W.), the National Natural Science Foundation of China (grant 32270157, to H.W.) and The Fundamental Research Funds for the Central Universities, Peking University (grant 7100604396, to H.W.).
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G.F.G., H.W., Z.Z. and Q.W. designed the experiments. H.W., P.Y., T.Z., L.Q., S.L., P.H., X.Q., J.W., H.D., J.H.W., T.K., Z.G., K.D., X.Y. and S.H. performed the investigations and assays. H.W., G.F.G., Q.W., J.J.X, J.Q., M.F., X.Z., W.T. and X.C. analyzed the data. G.F.G., H.W., P.Y., T.Z. and J.W. wrote the manuscript.
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Extended data
Extended Data Fig. 1
Sequence alignment of MPXV A35 and its orthologs. The sequence alignment of MPXV A35, VACV A33, and VARV A36 was shown. The residues in the epitopes of the A2C7 and A27D7 neutralizing antibodies on VACV A33 were shown as blue and green circles, respectively.
Extended Data Fig. 2 Characteristics of the MPXV A35 ectodomain proteins.
(a–g) Analytical ultracentrifugation analysis (A35Ecto (a), A35ΔDB (b) and A35scDimer (c), A35Ecto-ΔC62 (d), A35Ecto-ΔC62A (e)) and analytical gel filtration profiles (A35Ecto-ΔC62 (f), A35Ecto-ΔC62A (g)) of the MPXV A35 ectodomain proteins were shown. Sedimentation coefficient distributions c(s) calculated from the sedimentation velocity profile with the calculated molecular weight (Mf) shown. The SDS-PAGE analyses were repeated three times. (h) The specific IgG titers elicited by the MPXV A35 ectodomain proteins (n = 5). Data indicate mean ± s.d. P values were determined by one-way ANOVA with multiple comparison tests. ns, P > 0.05.
Extended Data Fig. 3 Identification of glycosylation in the A35ΔDB by mass spectrometry.
The glycosylation sites identified by mass spectrometry in the A35ΔDB protein (p1-p8) are shown in blue (a). The glycosylation modifications of the smaller band (b) or the larger band (c) in A35ΔDB gel electrophoresis were identified by mass spectrometry. The left column shows the detectable glycosylation site, the middle column indicates the mass introduced by each glycosylation modification, and the right column presents the expectation value (the data were deemed reliable with an expectation value of less than 0.01).
Extended Data Fig. 4 Identification of glycosylation in the A35Ecto by mass spectrometry.
The glycosylation sites identified by mass spectrometry in the A35Ecto protein (p1-p19) are shown in blue (a). The glycosylation modifications of the smaller band (b) or the larger band (c) in A35Ecto gel electrophoresis were identified by mass spectrometry. The left column shows the detectable glycosylation site, the middle column indicates the mass introduced by each glycosylation modification, and the right column presents the expectation value (the data were deemed reliable with an expectation value of less than 0.01).
Extended Data Fig. 5
Sequence alignment of MPXV-M1 and its orthologs. Sequence alignment of MPXV M1 and its orthologs. The sequence alignment of MPXV M1, VACV L1 and VARV M1 was shown. The residues in the epitope of neutralizing antibody 7D11 on VACV L1 were shown as blue circles.
Extended Data Fig. 6 Cryo-EM data processing of DAM/A27D7-Fab/7D11-scFv complex (CS1).
(a) Flow chart of cryo-EM data processing. (b) Euler angle distribution of the final reconstruction. (c) The FSC curve for the reconstruction, the FSC 0.143 cutoff value is indicated by blue line.
Extended Data Fig. 7 Cryo-EM data processing of DAM/A27D7-Fab/7D11-scFv complex (CS2).
(a) Flow chart of cryo-EM data processing. (b) Euler angle distribution of the final reconstruction. (c) The FSC curve for the reconstruction, the FSC 0.143 cutoff value is indicated by blue line.
Extended Data Fig. 8 Evaluation of the safety and the T cell immune response of alum-adjuvanted DAM.
(a) PRNT assay determined the neutralizing activities against MPXV in BALB/c mice immunized with DAM at day 12 and 6 months post dose 3 (n = 5). The values indicated the mean ± s.d. P values in a and b were determined by two-sided t-test. (b)ELISA of the M1- and A35 -specific IgG antibodies after dose 1, dose 2 and dose 3 (day 19, 40 and 54 after initial immunization) in BALB/c mice (n = 5). The values indicate the mean ± s.d. In (c)-(j), the mice in DAM/Alu/10 μg group were boost immunized at 182 days after first vaccination. (c)The body weight records of mice for eight consecutive days after the boost vaccination (n = 5). Data are shown as mean ± s.e.m. (d–h)Heart, liver and kidney function were determined by blood biochemical parameters, the serum were selected at 8 days after a boost vaccination (n = 5). UREA and CREA represent kidney function (d, g). ALT, ALP and TP represent liver function (e-f). CK, and LDH represent heart function (h). Data are shown as mean ± s.d. (i) Representative histopathology (H&E) of different tissues, liver, heart, spleen, lung, kidney and intestine from PBS or VACV-VTT or DAM after a boost immunization. The H&E stained sections shown in the data are representative results from five test mice 8 days after inoculation. Scale bar = 100 μm for liver and heart, scale bar = 200 μm for other tissues. (j) Cellular immune responses for A35 and M1 at 8 days after the boost vaccination. Cellular immune responses tested by IFN-γ (n = 5), IL-2 (n = 5) and IL-4 (n = 4) ELISPOT. The values indicate the mean ± s.d. P values in (a, b and j) were analyzed with t-tests. ns, P > 0.05. P values in (e-f) were determined by one-way ANOVA with multiple comparison tests. ns, P > 0.05.
Extended Data Fig. 9 DAM expressed by the industry-standard CHO cell.
(a) SEC-HPLC profiles of DAM. (b) Reduced and non-reduced SDS-PAGE migration profiles of DAM.The SDS-PAGE analyses in b were repeated three times.
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Wang, H., Yin, P., Zheng, T. et al. Rational design of a ‘two-in-one’ immunogen DAM drives potent immune response against mpox virus. Nat Immunol 25, 307–315 (2024). https://doi.org/10.1038/s41590-023-01715-7
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DOI: https://doi.org/10.1038/s41590-023-01715-7
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