Generation and characterization of neutralizing antibodies against M1R and B6R proteins of monkeypox virus

Abstract

The global outbreak of monkeypox virus (MPXV), combined with the termination of smallpox vaccination and the lack of specific antiviral treatments, raises increasing concerns. The surface proteins M1R and B6R of MPXV are crucial for virus transmission and serve as key targets for vaccine development. In this study, a panel of human antibodies targeting M1R and B6R is isolated from a human antibody library using phage display technology. Among these antibodies, A138 against M1R and B026 against B6R show the most potent broad-spectrum neutralizing activities against MPXV and Vaccinia virus (VACV). When used in combination, A138 and B026 exhibit complementary neutralizing activity against both viruses in vitro. X-ray crystallography reveales that A138 binds to the loop regions of M1R, similar to the vulnerable epitope of 7D11 on VACV L1R. By contrast, A129 targets a more cryptic epitope, primarily comprising the β-strands of M1R. Moreover, prophylactic and therapeutic administration of A138 or B026 alone provides partial protection, while combining these two antibodies results in enhanced protection against VACV in male C57BL/6 mice. This study demonstrates of a dual-targeting strategy using two different components of the virion for the prevention and treatment of MPXV infection.

Fig. 1. Isolation and characterization of the M1R and B6R antibodies.

A Diagram of the EV and MV forms of MPXV. M1R and B6R are located on the surface of the mature and enveloped virions, respectively. B and C Frequency distribution of human IGVH and IGVL in M1R (B) and B6R antibodies (C). D The length of the CDR3 at IGVH and IGVL in M1R and B6R antibodies. (E) Binding activity and neutralization potency of the M1R and B6R antibodies. The binding assay was based on ELISA while the neutralizing activity was measured using replication-deficient MPXV. The number in the box indicates the half-maximal effective concentration (EC₅₀) or half-maximal inhibitory concentration (IC₅₀) values. Blank or excluded values were represent by an X in the corresponding table cells. No detective (N.D.) indicates that no response was observed at the highest concentration, while > 1000 or > 66.7 denotes that the EC50 or IC50 value of the antibody is higher than the maximum detectable concentration. Binding and neutralization curves are shown in Figure S2. Source data are provided as a Source Data file.

Fig. 2. Cross-neutralizing potency of M1R and B6R antibodies against VACV and replication-deficient MPXV.

A, B Neutralizing activity of individual antibodies (A129, A138, B026, and B019) or antibody cocktail targeting the same antigens against MV and EV forms of replication-deficient MPXV (A) and VACV (B). MVs of MPXV were utilized to evaluate the neutralization potency of M1R antibodies, while EVs were employed to assess the neutralizing capacity of B6R antibodies. C, D Neutralization activity of individual antibodies (A129, A138, B026, and B019) or antibody cocktail targeting the two different antigens against a mixture of MV and EV forms of replication-deficient MPXV (C) or VACV (D). E Schematic of bispecific antibodies (bsAbs). The parental mAbs contributing to the bsAbs are color-coded: red A138 and green B026. F Binding affinity of bsAbs for M1R and B6R. M1R and B6R were immobilized on the surface of biosensors and individual antibodies were tested at various concentrations. The association and dissociation of antibodies is indicated by the dashed line, which represents the fitted curve. The apparent dissociation constants (K_D, app) are shown above each plot. G Cross-neutralizing activity of bsAbs against VACV and replication-deficient MPXV. Data from a representative neutralization experiment are shown for each antibody. The experiment was replicated twice with similar results. The data represent means ± SEM of triplicates indicated by error bars. The dashed line indicates a 50% reduction of viral infectivity. Source data are provided as a Source Data file.

Fig. 3. Complex structures of A138 Fab and A129 Fab bound to M1R.

A Overall structure of antibodies in complex with M1R. MPXV M1R or VACV L1R is colored in cornflower blue; Fab of A138, M12B9, 7D11 and A129 is shown in green, brown, pink and golden, respectively. The heavy chain is shown in a darker color, and all the complex structures are positioned with M1R/L1R in the same orientation. B A129 mainly uses its heavy chain to bind the β-sheet of M1R. The buried surface area (BSA) between the A129 heavy chain and M1R is 431 Ų while that of the light chain is 192 Ų. The β-sheet of M1R is shown in red and the loops connecting β-strands with helices are shown in green. The epitope of A129 on M1R is labeled by a golden line. C A138 mainly uses its heavy chain to bind to the loops connecting β-strands and helices of M1R. The buried surface area (BSA) between A129 heavy chain and M1R is 540 Ų while that of the light chain is 258 Ų. The β-sheet of M1R is shown in red and the loops connecting β-strands with helices are shown in green. The epitope of A129 on M1R is labeled by a green line. D Sequence and secondary structures of M1R. Contacting residues in M1R for A129 and A138 are indicated by golden and green dots, respectively.

Fig. 4. The binding interface between M1R and A129/A138 Fab.

Detailed interactions of the A129 Fab (A) and A138 Fab (B) with M1R. Hydrogen bonds and salt bridges are indicated by dashed lines. M1R is shown in cornflower blue. The heavy and light chains of A129 are shown in dark goldenrod and yellow, respectively. The heavy and light chains of A138 are shown in spring green and green yellow, respectively.

Fig. 5. Epitopes analysis of B026 and B019.

A Modeled structure of the B6R protein predicted using AlphaFold2. The four domains of B6R are shown in red (domain 1), blue (domain 2), yellow (domain 3), and green (domain 4). The stalk region is shown in brown. B Schematic diagrams representing the structures of a series of truncated B6R proteins variants. All these truncated B6R constructs contain a 6×His tag at the C-terminus, which was used for protein purification. C, D Binding of B026 (C) and B019 (D) to different fragments of B6R. Different fragments of B6R were loaded onto the surface of biosensors, and individual antibodies were tested at three concentrations (75 nM, 150 nM, 300 nM). The association and dissociation curves are shown. The dashed lines represent the fitted curves based on the experimental data. Source data are provided as a Source Data file.

Fig. 6. Prophylactic and therapeutic efficacy of B026 and A138 in a high-dose VACV-challenge mouse model.

A Schematic diagram of the prophylactic and therapeutic models. C57BL/6 mice were randomly divided into 4 groups (n = 6 per group). For the prophylactic model, mice were injected intraperitoneally (i.p.) with PBS, individual antibodies (A138 or B026) or antibody cocktail (A138 and B026) at 24 hrs before challenged with a high-dose (10⁹ PFU) of VACV. For the therapeutic model, mice were injected i.p. 24 hrs after challenge with 10⁹ PFU VACV. Body weight (B, D) and survival curves (C, E) were recorded. B, C show data for prophylactic model while (D) and (E) for the therapeutic model. F, G Viral titers in the lungs and spleen of mice in the prophylactic (F) and therapeutic groups (G). The data represent means ± SEM, indicated by error bars. The significance of differences between groups was assessed using one-way ANOVAs followed by Dunnett’s multiple comparisons test for multiple comparisons. Source data are provided as a Source Data file.

Fig. 7. Prophylactic and therapeutic efficacy of B026 and A138 in a MPXV-challenged mouse model.

A Viral titers in the lungs, liver and spleen of mice in the prophylactic model. B Viral titers in the lungs, liver and spleen of mice in the therapeutic model. BALB/C mice were randomly divided into 6 groups (with n = 4). For the prophylactic model, mice were injected intraperitoneally (i.p.) with PBS, individual antibodies (A138, B026, ScFvA138B026, or 7D11) or antibody cocktail (A138 and B026) at 24 hrs before challenged with 10⁶ PFU of MPXV. For the therapeutic model, mice were injected i.p. 24 hrs after challenged with 10⁶ PFU of MPXV. The data represent means ± SEM, indicated by error bars. The significance of differences between groups was assessed using one-way ANOVAs followed by Dunnett’s multiple comparisons test for multiple comparisons. Source data are provided as a Source Data file.