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Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39233480
2401931
10.1080/22221751.2024.2401931
Version of Record
Research Article
Research Article
Identification of mpox M1R and B6R monoclonal and bispecific antibodies that efficiently neutralize authentic mpox virus
Emerging Microbes & Infections
Z. Ren et al.
Ren Zuning ab†
Li Mengjun b†
Chen Jiayin b†
Gong Xiaohua c†
Song Shuo c†
Li Delin d†
Yang Minghui e†
Yu Jianhai b
Asghar Sadia f
Cui Yanxin c
Niu Shiyu c
Liao Zhonghui c
Jiang Yushan b
Liu Jiahui b
Li Yuqing b
Zhang Bao b
Zhao Wei b
Peng Jie a
Yang Yang c
https://orcid.org/0000-0001-8607-3750
Shen Chenguang bg
a State Key Laboratory of Organ Failure Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China
b BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health; Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, People’s Republic of China
c Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Shenzhen Third People’s Hospital, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen, People’s Republic of China
d Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, People’s Republic of China
e School of Life Science, Advanced Research Institute of Multidisciplinary Science, Key Laboratory of Molecular Medicine and Biotherapy, Beijing Institute of Technology, Beijing, People’s Republic of China
f Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan
g Key Laboratory of Infectious Diseases Research in South China, Southern Medical University, Ministry of Education, People’s Republic of China
CONTACT Wei Zhao zhaowei@smu.edu.cn BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health; Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, No.1023, South Shatai Road, Baiyun District, Guangzhou, Guangdong, People’s Republic of China
Jie Peng pjie138@163.com State Key Laboratory of Organ Failure Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China
Yang Yang yyszth2018@163.com Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Shenzhen Third People’s Hospital, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen, People’s Republic of China
Chenguang Shen a124965468@smu.edu.cn BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health; Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, No.1023, South Shatai Road, Baiyun District, Guangzhou, Guangdong, People’s Republic of China
† Contributed equally.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2401931.

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https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

In 2022, the monkeypox virus (mpox virus, MPXV) exhibited global dissemination across six continents, representing a notable challenge owing to the scarcity of targeted antiviral interventions. Passive immunotherapy, such as the use of monoclonal antibodies (mAbs) and bispecific antibodies (bsAbs), has emerged as a promising option for antiviral regimens. Here, we generated several mAbs against M1R and B6R of MPXV, and subsequently characterized the antiviral activity of these antibodies both in vitro and in vivo. Two neutralizing mAbs, M1H11 and M3B2, targeting M1R, and one B6R-specific mAb, B7C9, were identified. They exhibited varying antiviral efficacy against vaccinia virus (VACV) in vitro and in vivo. A cocktail comprising M1H11 and M3B2 demonstrated a superior protective effect in vivo. A bsAb, Bis-M1M3, was engineered by conjugating the fragment crystallizable (Fc) region of the human-mouse chimeric engineered M1H11 with the single-chain fragment variable (scFv) of M3B2. In mice challenged with MPXV, Bis-M1M3 showed a notable protective effects. Analysis of neutralization mechanism showed that these mAbs and Bis-M1M3 exerted virus-neutralizing effects before the virus infects cells. In vivo pharmacokinetic experiments showed that Bis-M1M3 has a long half-life in rhesus macaques. This study provides crucial insights for further research on broad-spectrum antiviral drugs against MPXV and other orthopoxviruses.

KEYWORDS

Mpox virus
Vaccinia virus
Monoclonal antibody
Bispecific antibody
Antiviral mechanism
National Natural Science Foundation of China 10.13039/501100001809 32170939 Guangdong Basic and Applied Basic Research Foundation 10.13039/501100021171 Guangdong Science and Technology Program 10.13039/501100012245 This work was supported by the National Natural Science Foundation of China [No.32170939; No. 82371846 and No.32411540019]; Guangdong Basic and Applied Basic Research Foundation [No.2022B1515020075]; Guangdong Science and Technology Program key projects [No. 2021B1212030014]; and The Special Funds for Strategic Emerging Industry of Shenzhen [Grant No. F-2022-Z99-502266].
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pmcIntroduction

Monkeypox (mpox) is a viral zoonosis caused by the monkeypox virus (mpox virus, MPXV), which belongs to the Orthopoxvirus genus in the family Poxviridae [1]. An outbreak of mpox clade IIb has been ongoing since May 2022, and as of March 5, 2024, over 94 thousand cases of mpox had been reported from 118 countries across 6 continents, including 178 fatalities [2, 3]. However, few antiviral drugs are currently available for the treatment of mpox, especially for immunocompromised individuals. Passive immunotherapy, which refers to the use of monoclonal antibody (mAb)-based drugs that can be utilized to address a spectrum of viral infections, has emerged as a promising option in difficult-to-treat cases [4–6] Thus, the generation of neutralizing mAbs is crucial for the development of highly effective antiviral drugs against orthopoxviruses.

MPXV and its close relatives, such as variola virus (VARV) and vaccinia virus (VACV), share a high level of sequence similarity. Their surface antigens display low polymorphism [7, 8], which allows indirect protection against MPXV through cross-reactive antibodies. A VACV vaccine was used to eradicate smallpox and has been demonstrated to confer approximately 85% protection against MPXV [9]. This indicates that neutralization targets for VACV may serve as a reference for assessing the neutralizing epitopes of MPXV. For orthopoxviruses, viral replication results in the generation of various infectious virion forms. Infectious mature virions (MVs), remain inside the cell until cell lysis, while enveloped virions (EVs) are generated when mature viruses encapsulate themselves with late endosomal membranes. MVs play a crucial role in viral transmission among hosts, while EVs contribute to facilitating infection between cells [10]. VACV neutralization targets have been well characterized, frequently including A27 [11], L1R [12], H3L [13], and D8 [14] on MVs, while the effective targets on EVs are relatively fewer, typically confined to A33R and B5R [15–17]

Combining two or more mAbs may enhance the protective efficacy against VACV infection compared with that seen with the individual mAbs [18, 19]. Indeed, many bispecific antibodies (bsAbs), which can simultaneously target the recognition sites of two mAbs, have been developed for diverse viruses, including SARS-CoV-2 [20, 21], ebolaviruses [22, 23], and dengue virus [24].

L1R is a membrane protein found in VACV MVs that plays a key role in cell entry and membrane fusion [25], while B5R is a highly conserved antigen on VACV EVs of orthopoxviruses, and plays a crucial role in virus packaging [26]. In this study, we investigated the antiviral efficacy of mAbs induced by MPXV M1R and B6R, which exhibit approximately 98% and 95%-96% sequence similarity, respectively, to the L1R and B6R proteins of VACV, as well as their combination in the form of bsAb (Figure S1).

Materials and methods

Mice, cell and viruses

All in vivo experiments were approved by the Ethics Committee of the Laboratory Animal Center of Southern Medical University, and were performed according to the guidelines of the Institutional Animal Care and Use Committee, following strict guidelines for humane endpoints. Six-week-old female BALB/c mice were purchased from Guangdong Medical Laboratory Animal Center and were used for all experiments.

Sp2/0 cells were maintained in Roswell Park Memorial Institute 1640 Medium (RPMI-1640; Gibco) supplemented with 10% foetal bovine serum (FBS; Gibco). BHK-21 cell lines were purchased from IMMOCELL (Xiamen, Fujian, China) and maintained in Dulbecco Modiﬁed Eagle’s Medium (DMEM) supplemented with 10% calf serum. Vero E6 cells were cultured in Dulbecco Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% FBS. Expi293F™ cells were maintained in SMM 293-TII expression medium (Sino Biological).

VACV Tian Tan was propagated in BHK-21 cells in RPMI-1640 Medium supplemented with 10% FBS using previously described standard virus culturing techniques [27]. MPXV was propagated in Vero E6 cells in DMEM medium supplemented with 10% FBS. MVs and EVs were prepared as previously described [27]. The virus used for binding assays, animal experiments, and neutralization assays was obtained by rapidly freeze−thawing infected cells along with their culture medium three times, followed by high-speed centrifugation to collect the supernatants. The MV form of the virus was stored at −80°C, while the remainder were either used immediately or stored at 4°C for 2−3 weeks.

The expression and purification of recombinant MPXV proteins M1R and B6R

The synthesis of MPXV proteins was based on the complete genome sequence of the MPXV responsible for the current outbreak in the USA (MPXV_USA_2022_MA001, GenBank: ON563414.3). The extracellular portions of B6R (amino acids 20–275) and M1R (amino acids 1–185), which were solubilized by excluding the transmembrane domains, were engineered and ligated into the pET-25b(+) expression vector for cytosolic expression as previously described [28, 29]. BL21 receptor cells (Tsingke) were transformed with the recombinant pET-25a (+) vector. After growing to an OD600 ∼ 0.4, the cells were induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG; Solarbio) for 16 h at 20℃ with shaking (160 rpm). The cells were resuspended in lysis buffer containing 10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl and were lysed using a sonicator. The lysate was centrifuged at high speed and the resulting supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatographic column (Yeasen) and eluted with 0.2 M imidazole, 50 mM NaH2PO4, and 300 mM NaCl. After overnight dialysis against phosphate buffer saline (PBS), the proteins were stored at −80°C until use.

Mouse immunization and monoclonal antibody production

Mice were subcutaneously immunized four times with a 2-week between immunizations. Each immunization involved the injection of 50 µg of recombinant MPXV B6R or M1R protein per mouse. Three days after the final immunization, the mice were euthanized, and spleen cells were harvested and fuzed with Sp2/0 cells to create hybridomas. The subsequent procedures for hybridoma cloning and screening and antibody production and purification were executed as previously described [30].

Sequencing analysis of the mAbs

The extraction of total RNA from hybridoma cells used FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme), and the reverse transcription and identification of antibody variable region DNA sequences, were performed as previously described [30]. The sequences of antibody complementarity-determining regions (CDRs) were verified using IMGT/V-QUEST [31].

Binding analysis by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)

Assays for the binding of antibodies to recombinant proteins were undertaken by ELISA and SPR. Total IgG (EMD Millipore Corporation) was assayed by indirect ELISA in plates coated with 1μg/mL protein or 5 μg/mL inactivated whole-virus lysate of VACV. The latter was prepared by boiling a VACV (Tian Tan) suspension at 100℃ for 10 min. Subsequent steps were performed as previously described [4].

The SPR analysis was performed using the Biacore 8 K system (GE Healthcare) with the CM5 sensor chips covalently coated with M1R or B6R protein. The specific operating procedures were conducted as previously described [32].

Neutralization assays

The neutralizing activities of antibodies under test against VACV (Tian Tan) and MPXV were determined through a microneutralization assay, as previously detailed [30]. Briefly, BHK-21 cells or Vero E6 cells were washed twice with PBS and incubated in culture medium in a 96-well plate. The antibodies were serially diluted and then mixed with the virus along with fresh guinea pig serum for murine antibodies or human complement (Quidel) for mouse-human chimeric engineered antibodies. After incubation at 37°C for 2 h, a 35 μL volume of a mixture, containing 100 TCID50 (50% tissue culture infectious dose) of the virus, was added to the cells. After 1 h of incubation, the supernatant was replaced with culture medium supplemented with 2% FBS, followed by culture at 37°C with 5% CO2 for 4 days. The cytopathic effect was examined for 4 days post-infection.

Molecular docking

AlphaFold2 was used to predict the 3D structures of M1R and B6R with standard parameters and the models with the highest pLDDT score were chosen for subsequent docking [33]. Structural models of the fragment variable (Fv) domains of M1H11, M3B2, and B7C9 were generated using ABodyBuilder2 from the SAbPred antibody prediction toolbox [34]. Protein–protein docking was carried out using the online tools ZDOCK and ClusPro 2.0 [35–39] The top 10 complexes predicted by ZDOCK and the top 10 complexes predicted by ClusPro 2.0 in balanced, electrostatic-favoured, hydrophobic-favoured or VdW + Elec mode were further investigated manually and the most plausible binding models were chosen for presentation. Figures were prepared in PyMOL [40].

Therapeutic efficacy studies in mice

In vivo challenge experiments were conducted using VACV (Tian Tan) and MPXV to assess the therapeutic efficacy of the antibodies.

BALB/c female mice aged 6 weeks were used for virus challenge, with antibody treatment being administered 12–24 h post-challenge. Several mice randomly selected from each group were euthanized 2 days post-challenge and the lungs were sampled. Half of the lung samples were homogenized, and assessed for viral titre or submitted to qPCR for viral load detection. The body weights of the remaining mice were recorded daily for 10–14 days after infection. Animals that lost more than 25% of their initial body weight were euthanized according to animal ethics protocols.

Tissue staining

Mice in each group were euthanized 2 days after virus challenge. The lungs of the mice were removed, fixed in 4% paraformaldehyde, sectioned, paraffin-embedded, and stained with haematoxylin and eosin (H&E). The stained samples were analyzed using an Olympus microscope (DP73) with 10× and 40× objectives.

Nucleic acid extraction and TaqMan quantitative polymerase chain reaction (qPCR)

Lung homogenates were centrifugated at high speed and the sediment was discarded. DNA in the supernatant was extracted and stored at −80°C until use.

The viral load of the lung homogenates was measured using a TaqMan Real-Time System (ABI QuantStudio6). Briefly, standard plasmids were diluted 10-fold (from 1 × 108–1 × 102 copies/μL) and used as amplification templates. A pair of primers and probes at a concentration of 10 μM were added to each reaction system. The reaction procedure involved 5 min of pre-denaturation at 95°C, followed by 45 cycles at 95 °C for 10 s (denaturation) and 60 °C for 40 s (annealing and elongation). A TaqMan probe (5′- FAM-GAGACTCCGGAACCAAT-TAMRA-3′) was used to simultaneously detect the hemagglutinin (HA) gene of VACV. The sequences of the primers used for qPCR were forward: 5′-ACATCTGGAGAATCCACAACA-3′ and reverse: 5′-CATCATCGGTGGTTGATTTA-3′.

The engineering and production of mouse-human chimeric mAbs and bsAb

The mouse-human chimeric versions of M1H11 (cMH11), M3B2 (cM13B2) and B7C9 (cB7C9) were constructed as previously described [30]. The bsAb Bis-M1M3 was constructed based on the cM1H11 recombinant antibody structure, with an M3B2 single-chain fragment variable (scFv) linked to its Fc region using a (G4S)3 linker. Bis-M1M3 was expressed using the 293F eukaryotic expression system. The antibodies were purified from culture supernatants using protein A columns.

Immunofluorescence

Immunofluorescence assays were conducted based on the neutralization assays against VACV (Tian Tan). At 12 h post-infection, cells were fixed in 4% paraformaldehyde for 30 min at room temperature (RT), and permeabilized with PBS containing 0.5% Triton X-100 for 30 min at RT in a light-protected environment. After washing, the cells were blocked with 10% normal goat serum (Beyotime) for 30 min at RT, washed, and incubated with the primary antibody B7C9 (2 μg/mL; binds to the virus) at 4℃ for 16 h overnight, followed by incubation with fluorescein (FITC)-conjugated affinipure goat anti-mouse IgG (H + L) antibody (Proteintech) for 1 h at RT protected from light. After washing, the cells were counterstained with Hoechst 33342 (Beyotime). Images were captured using a laser confocal microscope (FV3000).

Half-life quantitation

Eight male rhesus macaques were housed and cared for in a biosafety level 2 facility. Four macaques were administered low-endotoxin Bis-M1M3 intravenously at 10 mg/kg and serum was collected before and at various time points after injection. Antibody concentrations in serum were measured by a quantitative ELISA. M1R-coated microtiter plates were used to capture Bis-M1M3 followed by detection using a HRP-conjugated anti-human IgG antibody. The other four macaques were injected with an equivalent dose of an influenza antibody as a control. Pharmacokinetic parameters were calculated in WinNonlin Software using the non-compartment model.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (v.9.5.0). For continuous variables, the data are presented as mean ± standard error of the mean (SEM). The half-maximal inhibitory concentrations (IC50) of the mAbs were calculated by non-linear regression analysis [log(inhibitor) vs. response − variable slope (four parameters)]. The half-maximal effective concentrations (EC50) of the mAbs were calculated by non-linear regression analysis [log(agonist) vs. response − variable slope (four parameters)]. One-way or Two-way ANOVA was used for comparisons among groups. All tests were two-tailed. p-values <0.05 were considered significant.

Results

The mAbs specific for M1R or B6R proteins exhibited varying degrees of binding and neutralization activities in vitro

We immunized mice with recombinant M1R and B6R proteins separately and then obtained several mAbs following the screening of hybridoma cells. These included the M1R-specific mAbs M1H11, M3B2, M4B6, and M13H, and the B6R-specific mAbs B7C9, B7E2, and B10D3. In the ELISA-based binding analysis using plates coated with inactivated VACV (Tian Tan) lysate, M3B2, and B7C9 exhibited the strongest binding activity (Figure 1A). M1H11, M3B2 and M4B6 all demonstrated robust reactivity with the M1R protein (Figure 1B); however, B7C9, which exhibited strong binding activity with viral lysates, did not react strongly with the B6R protein (Figure 1C). Figure 1. The binding, neutralizing activities, and sequences of the complementarity determining regions (CDRs) of the monoclonal antibodies (mAbs). (A–C) ELISAs for the binding activities of four M1R-targeting mAbs—M1H11, M3B2, M4B6, and M13H—and three B6R-specific mAbs—B7C9, B8A12, and B10D3—to (A) a vaccinia virus (VACV) (Tian Tan) lysate and their respective binding activities to (B) the M1R protein and (C) the B6R protein. Neutralization assays for the mAbs against the mature virion (MV) (D) or enveloped virion (EV) (E) form of VACV (Tian Tan). The error bars represent means ± SEM. (F) The binding affinities of the mAbs for the target antigens obtained through surface plasmon resonance (SPR) analysis. (G) The DNA sequences of the CDRs of three mAbs.

Among the mAbs targeting the MV surface protein M1R, M1H11 and M3B2 showed the most promising MV neutralizing activity (Figure 1D). Meanwhile, among the mAbs targeting the EV surface protein B6R, only B7C9 exhibited EV neutralizing activity (Figure 1E).

We further characterized the three mAbs—M1H11 and M3B2, which specifically target M1R, and B7C9, which is specific for B6R—owing to their relatively superior neutralization activities against the MV and EV forms of orthopoxviruses, respectively. All three mAbs were characterized as IgG antibodies featuring κ-type light chains. Specifically, M1H11 was classified within the IgG2a subclass, whereas both M3B2 and B7C9 were categorized within the IgG1 subclass. The results of the SPR analysis indicated that the affinities of M1H11 and M3B2 for M1R were higher than that of B7C9 for B6R, which was consistent with the ELISA results (Figure 1F). The DNA sequences of the variable regions of these three mAbs, and their CDR sequences are shown in Figure 1G.

Three mAbs may recognize distinct epitopes on MPXV

Subsequently, we conducted a molecular docking assay to understand the interactions between M1R or B6R and the three monoclonal antibodies to predict their epitopes (Figure 2). The structure of M1R was predicted by AlphaFold2. The architecture of M1R was found to be similar to poxvirus L1 protein (PDB 1YPY) and consists of an α-helical bundle packed against a pair of β-sheets. Three pairs of disulfide bonds are formed between Cys34 and Cys57, Cys49 and Cys136, and Cys116 and Cys158 [28]. Molecular docking analysis showed that α1, α2 and α4 may be involved in the binding to M1H11 (Figure 2A, B), while β1, β3, α5 and the α4-β3 linker might participate in the binding to M3B2 (Figure 2D, E). CDR1, and CDR3 from the heavy chain and CDR1 and CDR2 from the light chain of M1H11 contribute polar interactions to the three α helices (Figure C). Except for CDR2 from the light chain of M3B2, all the CDRs are involved in the binding to M1R through hydrogen bonds and electrostatic interactions (Figure 2F). Mpox virus B6R was predicted to consist of 4 consecutive complement control protein (CCP) domains and a transmembrane helix at the C-terminal, resembling the smallpox inhibitor of complement enzymes (SPICE) [41]. Eight pairs of disulfide bonds are formed within the 4 CCP domains (Figure 2G, H). Molecular docking analysis indicated that CCP2 and CCP3 may be involved in the binding to B7C9 (Figure 2I). CDR1 and CDR3 from the heavy chain and CDR1 from the light chain seem to contribute to hydrogen bonds and electrostatic interactions with polar and charged residues on CCP2 and CCP3 of B7C9. Figure 2. Model of the binding of M1H11 and M3B2 to M1R, and the binding of B7C9 to B6R. The structures of the MPXV proteins M1R and B6R were predicted by AlphaFold2 and the Fv domain of the monoclonal antibodies (mAbs) was predicted by SAbPred. The docked complexes were created by ZDOCK, ClusPro, and AlphaFold2 and were then further investigated manually. (A) The most plausible result for the overall architecture of M1R bound to M1H11. The structure of M1R is shown in magenta. The cysteines that form disulfide bonds are shown as sticks. The heavy and light chains of M1H11 are shown in blue and cyan, respectively. (B) The M1R-M1H11 interface. M1R and the Fv domain of M1H11 are shown in cartoon and surface representation, respectively. The residues on the heavy and light chains involved in M1R binding are shown as sticks and coloured in blue and cyan. (C) The detailed interactions between M1R and the complementarity-determining regions (CDRs) on M1H11. Residues on M1R are shown in stick representation and coloured in magenta. Residues on the heavy and light chains of M1H11 are shown in stick representation and coloured in blue and cyan. (D) The most plausible result for M1R-M3B2 binding. The structure of M1R is shown as a magenta cartoon. The cysteines that form disulfide bonds are shown as sticks. The heavy and light chains of M3B2 are coloured blue and cyan, respectively. (E) The M1R-M3B2 interface. M1R and the Fv domain of M3B2 are shown in cartoon and surface representation, respectively. The residues on the heavy and light chains involved in M1R binding are shown as sticks and coloured in blue and cyan. (F) The detailed interactions between M1R and the CDRs on M3B2. Residues on M1R are shown in stick representation and coloured in magenta. Residues on the heavy chain and light chain of M3B2 are shown in stick representation and coloured in blue and cyan. (G) The most plausible result for the overall architecture of B6R-B7C9. The structure of B6R is shown as a magenta cartoon. The cysteines that form disulfide bonds are shown as sticks. The heavy and light chains of B7C9 are coloured blue and cyan, respectively. (H) The B6R-B7C9 interface. B6R and the Fv domain of B7C9 are shown in cartoon and surface representation, respectively. The residues on the heavy and light chains involved in B6R binding are shown as sticks and coloured in blue and cyan. (I) The detailed interactions between B6R and the CDRs on B7C9. Residues on B6R are shown in stick representation and coloured in magenta. Residues on the heavy and light chains of B7C9 are shown in stick representation and coloured in blue and cyan.

The therapeutic administration of mAbs and their cocktails conferred different degrees of protection against VACV infection in mice

To delve deeper into the protective effects of M1H11, M3B2, and B7C9 against orthopoxvirus in vivo, we examined each antibody for its therapeutic efficacy in a murine VACV (Tian Tan) challenge model. We established cocktail groups with either M1H11 & M3B2, M1H11 & B7C9 or M3B2 & B7C9 to evaluate the potential additive protective effects of the mAbs in vivo. All the mice survived throughout the observation period. Meanwhile, the average body weights of mice in all the mAb-treatment groups were notably higher than those in the control group (Figure 3A, B). Moreover, the post-challenge viral loads in the lungs of mice treated with any of the mAbs were significantly lower than those in the control group (Figure 3C). The protective effect of the M1H11 & M3B2 cocktail was markedly pronounced, with mice in this group displaying minimal post-infection weight loss among the groups including the the mAbs along, or the cocktail comprising M1H11& B7C9 or M3B2 & B7C9. H & E staining of mouse lungs indicated that alveolar structure was almost lost in mice of the control group accompanied by a significant infiltration of inflammatory cells. In contrast, alveolar damage and inflammation were alleviated to varying degrees in the mAb-treated groups, with the most pronounced effects observed in the cocktail groups (Figure 3D). Figure 3. The therapeutic effects of monoclonal antibodies (mAbs) in mice infected with the vaccinia virus (VACV) (Tian Tan). Mice (n = 8 per group) were infected with 2.5 × 105 TCID50 doses of VACV (Tian Tan) and then treated with the indicated antibodies (10 mg/kg) via intraperitoneal administration 12 h later. The lungs of mice (n = 3 per group) were sampled 2 days post-VACV (Tian Tan) challenge. (A) The body weight changes (n = 5 per group) within 10 days post-challenge. Two-way ANOVA was used for comparisons of post-challenge weight changes among the groups. “#” indicates p < 0.05 in comparison between the M1H11 & M3B2 cocktail group and other groups; “$” indicates p < 0.05 in comparison between the IgG control group and other groups. (B) Survival curves monitored within 10 days post-challenge. (C) Lung viral loads 2 days post-challenge. Error bars represent means ± SEM. One-way ANOVA was used for comparisons between the indicated antibody treatment groups and the IgG control group. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. (D) Haematoxylin and eosin (H&E) staining of the lungs of mice 2 days post-challenge. The yellow arrows indicate alveolar wall granulocyte infiltration, the red arrows indicate perivascular and peribronchiolar lymphocyte infiltration, and the blue arrows indicate bronchiolar epithelial cell necrosis.

A bsAb, designed based on the two M1R-specific mAbs, exhibited strong neutralizing activity in vitro

Based on the in vivo experiments, the M1H11 & M3B2 cocktail exhibited remarkably robust protective effects. Consequently, leveraging IgG1 as a framework, we designed and expressed mouse-human chimeric engineered M1H11 (cM1H11) and M3B2 (cM3B2). Subsequently, we engineered and generated a bsAb based on the cM1H11, with the scFv of M3B2 linked to its Fc region. This bsAb was designated Bis-M1M3 (Figure 4A, B). Figure 4. In vitro assessment of the functionality of the bispecific antibody (bsAb) Bis-M1M3. (A) A simple model of Bis-M1M3. (B) SDS–PAGE (with DTT) of Bis-M1M3, cM1H11, and cM3B2. ELISA-based analysis of the binding activities of mouse-human chimeric engineered mAbs and bsAb against (C) M1R and (D) inactivated vaccinia virus (VACV) (Tian Tan) lysate. (E) The binding affinity of Bis-M1M3 for the M1R protein obtained through SPR analysis. (F) The neutralization activities of Bis-M1M3, cM1H11, cM3B2, and the cM1H11 & cM3B2 cocktail against MPXV. The error bars represent means ± SEM.

ELISA results showed that the binding activities of Bis-M1M3 to M1R protein (Figure 4C) and inactivated VACV (Tian Tan) lysate (Figure 4D) were similar to that of cM1H11. The KD value of Bis-M1M3, measured via SPR, was 1.47 × 10−10 M, indicative of a high affinity (Figure 4E).

We also assessed the in vitro neutralization activity of Bis-M1M3 against MPXV. The IC50 of Bis-M1M3 was lower than that of cM1H11 alone or the cocktail comprising cM1H11 & cM3B2 (Figure 4F).

BsAb conferred robust resistance against MPXV infection in mice

To investigate the in vivo protective effects of Bis-M1M3, we subjected mice to MPXV challenge. Mice treated with Bis-M1M3 after MPXV challenge showed significantly reduced mortality (Figure 5B), less weight loss (Figure 5A), and reduced viral load in the lungs (Figure 5C). Additionally, Bis-M1M3 outperformed the cM1H11 & cM3B2 cocktail in mitigating virus-induced weight loss and lowering lung viral titres. Figure 5. The therapeutic effects of the bispecific antibody (bsAb) Bis-M1M3 in mice infected with MPXV. Mice (n = 8 per group) were infected with 5 × 105 TCID50 doses of MPXV and then treated with the indicated antibodies (5 mg/kg) via intraperitoneal administration 24 h later. The lungs of mice (n = 3 per group) were sampled 2 days post-MPXV challenge. (A) The body weight changes (n = 3 per group) within 14 days post-challenge. Two-way ANOVA was used for post-challenge comparisons of weight changes among groups. “#” indicates p < 0.05 in comparison between the Bis-M1M3 group and other groups. “&” indicates p < 0.05 in comparison between the cM1H11 & cM3B2 cocktail group and other groups. “$” indicates p < 0.05 in comparison between the IgG control group and other groups. (B) Survival curves monitored within 14 days post-challenge. (C) Lung viral loads sampled 2 days post-challenge. The error bars represent means ± SEM. One-way ANOVA was used for comparisons among the groups. **** p < 0.0001; “$” indicates p < 0.05 in comparison between the IgG control group and other groups. (D) Haematoxylin and eosin (H&E) staining of the lungs of mice 2 days post-challenge. The yellow arrows indicate alveolar wall granulocyte infiltration, the red arrows indicate perivascular and peribronchiolar lymphocyte infiltration, and the blue arrows indicate bronchiolar epithelial cell necrosis.

The neutralizing activities of both the mAbs and the bsAb Bis-M1M3 are primarily achieved before viral entry into the cells

To understand the mechanism underlying mAb- and Bis-M1M3-mediated virus neutralization, we conducted an indirect immunofluorescence analysis using confocal microscopy based on VACV (Tian Tan) neutralization assays. Two experimental groups were established based on the timing of antibody–virus interaction; one group (pre-incubation group) involved incubating antibodies and viruses for 1 h before infecting the cells with varying antibody concentrations (25.0 μg/mL, 8.3 μg/mL or 2.8 μg/mL); the other group (post-infection group), the cells were first infected with the virus for 1 h, followed by the addition of antibodies and 2 h of incubation.

As depicted in Figure 6, a decreasing neutralization effect was observed under consecutive dilutions of antibody concentrations in the pre-incubation group. Notably, the neutralizing activity was significantly higher in the Bis-M1M3 group than in the other groups at all concentrations tested. In the post-infection group, the viral titre of indicated antibodies was markedly high, broadly in line with the IgG control. This result suggested that both the mAbs and Bis-M1M3 can impede cell infection by the virus. However, once the virus has entered the cells, the neutralizing effects of the antibodies diminish. Figure 6. The inhibitory effects of the mouse-human chimeric engineered monoclonal antibodies (mAbs) and bispecific antibody (bsAb) against the vaccinia virus (VACV) (Tian Tan) as determined by indirect immunofluorescence analysis using confocal microscopy. Pre-incubation group: VACV (Tian Tan) and antibodies were incubated together for 1 h before cell infection. Post-infection group: Cells were first infected with VACV (Tian Tan) and then the antibodies were added. The green fluorescence indicates the presence of the VACV (Tian Tan). Nuclei were counterstained with Hoechst 33342 (with DAPI, blue). The image shows the merged FITC and DAPI signals. Magnification = ×40.

Bis-M1M3 has a long half-life in rhesus macaques

To expand the clinical application potential of Bis-M1M3 against mpox, we determined the half-life of Bis-M1M3 in rhesus macaques. Subsequently, the antibodies were infused into the macaques, and their serum levels of the antibodies were analyzed over 14 days (Figure 7A). The in vivo half-life of Bis-M1M3 was found to be 5.142 days based on serum concentrations over the 14 days, which was comparable to that of the control mAb (Figure 7B). Figure 7. Serum antibody concentrations in rhesus macaques infused with Bis-M1M3. (A) Schematic diagram showing the antibody pharmacokinetics in rhesus monkeys. The rhesus macaques (n = 4 per group) were injected with a 10 mg/kg dose of antibody on Day 0, and serum was collected on Days 1, 3, 5, 7, 9, 11, and 14; the antibody concentration was determined by ELISA. (B) The half-life of Bis-M1M3 and control mAb 12G6 (an influenza antibody) in rhesus macaques was measured in serum over 14 days after the intravenous administration of a single 10 mg/kg dose of each antibody.

Discussion

The currently available therapeutic options for MPXV infection are limited. Additionally, there is a risk of development of drug resistance particularly in immunocompromised individuals [42, 43]. Passive immunotherapy, such as the use of neutralizing mAbs and bsAbs, has emerged as a promising approach for overcoming these drawbacks. M1R and B6R proteins are both essential MPXV neutralization targets and exhibit high homology with the L1R and B5R proteins of VACV, respectively. Several neutralizing mAbs specifically targeting the L1R [12, 44] and B5R [18, 45] proteins of VACV have been previously developed. In this study, we identified three MPXV-specific mAbs – M1H11 and M3B2, which target the M1R protein, and B7C9, which shows specificity for the B6R protein. They were found to recognize different epitopes on MPXV and to exhibit varying degrees of antiviral efficacy against orthopoxviruses both in vitro and in vivo; meanwhile, a cocktail of M1H11 and M3B2 exhibited pronounced markedly superior protective effects compared with either mAb alone. Subsequently, we designed and generated a bsAb based on the variable regions of M1H11 and M3B2, and this bsAb showed potent in vivo antiviral protection against MPXV infection. The bispecific form of the antibody showed a long in vivo half-life in rhesus macaques, indicating that its immunogenicity was not strong in vivo, thereby showing promise for potential future applications.

In this study, the neutralization activity of mAbs targeting the B6R protein was significantly lower than that of M1R-specific mAbs. It has been suggested that the relatively low density of target antigens on EVs of VACV allows them to escape direct neutralization due to antibody binding [46, 47], this explain why many studies have reported that EV-specific mAbs exhibit poor neutralization activity [48, 49]. Furthermore, we observed that B7C9 demonstrated robust binding affinity towards viral lysates and displayed relatively enhanced neutralization efficacy against EVs, yet its reactivity toward the B6R protein was relatively weak. This disparity may arise from variations between the configuration of the viral protein in viral particles and that of its soluble form. Alternatively, post-translational modifications, such as glycosylation or phosphorylation within the viral protein in cellular contexts, may potentially alter antibody recognition specificity and affinity.

We employed two virus challenge models in our study, namely, VACV(Tian Tan) and MPXV challenge model. VACV(Tian Tan) exhibits low virulence and is mostly non-lethal in mice, similar to the low mortality rate seen in real-world mpox infections. Although differences in survival rates were not observed, the efficacy of the in vivo protective abilities of the antibodies could be inferred from their reduced effects on body weight. In contrast, MPXV is highly virulent and lethal, which allowed us to determine that mAb or bsAb administration post-virus infection could prevent or reduce mouse mortality. Nevertheless, to advance therapeutic antibodies for use in the clinic, it is necessary to conduct in vivo experiments in non-human primates, an approach that we intend to pursue in the future studies.

In addition, in this study, the molecular interactions and epitope predictions were simulated using molecular docking programmes, but without experimental validation. We sought to divide the full-length M1R protein into several short peptides and use western blotting to detect their binding to mAbs. The results indicated that M1H11 and M3B2 did not bind to any of the generated short peptides, suggesting that these antibodies likely recognize discontinuous conformational epitopes. Subsequently, based on molecular docking results, we selected five predicted epitopes for alanine scanning and assessed antibody binging to the mutated proteins by ELISA. The results showed that D66 is the key epitope on M1R recognized by M1H11 (Figure S2). However, mutations in the five predicted epitopes did not affect the binding of M3B2, indicating the need for caution in interpreting molecular docking results. Studies have identified the neutralizing epitope targeted by L1R mAbs through the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography [12]. We plan to further refine these analyses in our future work. Additionally, it would be preferable to generate actual structures of the mAbs and bsAbs by cryo-electron microscopy., an approach we also hope to undertake in future in-depth studies. The precise identification of antigenic epitopes and the unravelling of antiviral mechanisms can contribute to elucidating why the M1H11 & M3B2 cocktail has better antiviral effects than M1H11 or M3B2 alone, whereas the combination of M1H11 and B7C9 does not.

Authors’ contributions

Experimental design: CS, JP, YY, WZ

Proteins expression and vaccination: ZR, JC, JL, YL, JY, SN

Virus isolation, titration and animal experiments: ZR, ML

In vitro neutralization experiments of MPXV and the binding assays by SPR: SS, YC, ZL

Molecular docking assay: DL, BZ

Manuscript preparation and statistical analyses: CS, ZR, SA

Supplementary Material

Supplemental Material

Supplemental Material

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.
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