Article 4

Global Impact and Epidemiology of Monkeypox (Mpox)

• Definition and Classification: Monkeypox (mpox) is a viral zoonosis caused by the monkeypox virus (MPXV), which is a member of the Orthopoxvirus genus within the Poxviridae family.
• 2022 Outbreak Statistics: Since May 2022, a global outbreak of mpox clade IIb has spread across six continents.     * Case Count: As of March 5, 2024, more than $94,000$ cases have been reported.     * Geographic Reach: Cases identified in $118$ countries.     * Mortality: $178$ fatalities recorded globally.
• Treatment Challenges: There is a notable scarcity of targeted antiviral interventions, particularly for immunocompromised individuals. Passive immunotherapy using monoclonal antibodies (mAbs) or bispecific antibodies (bsAbs) is a promising therapeutic alternative.

Orthopoxvirus Structure and Antigenic Cross-Reactivity

• Sequence Similarity: MPXV shares high sequence similarity with other orthopoxviruses, including the variola virus (VARV/smallpox) and vaccinia virus (VACV).
• Protective Cross-Reactivity: The VACV vaccine, used to eradicate smallpox, provides approximately $85\%$ protection against MPXV due to low polymorphism in surface antigens.
• Infectious Virion Forms:     * Mature Virions (MVs): These remain inside the host cell until cell lysis occurs and are primary drivers of host-to-host transmission.     * Enveloped Virions (EVs): Created when mature viruses are encapsulated by late endosomal membranes; these facilitate cell-to-cell infection within a host.
• Neutralization Targets:     * MV Targets: Key proteins include A27, L1R, H3L, and D8.     * EV Targets: Primarily A33R and B5R.
• Specific Study Homologs:     * M1R: An MPXV protein with approximately $98\%$ sequence similarity to the VACV L1R protein (involved in cell entry and membrane fusion).     * B6R: An MPXV protein with approximately $95\%-96\%$ sequence similarity to the VACV B5R protein (involved in virus packaging).

Methodology: Protein Expression, Immunization, and Sequencing

• Protein Synthesis: Based on the MPXV genome sequence responsible for the USA outbreak (MPXV_USA_2022_MA001, GenBank: ON563414.3).     * B6R Protein: Extracellular portion (amino acids $20\text{--}275$).     * M1R Protein: Extracellular portion (amino acids $1\text{--}185$).
• Expression System: Solubilized proteins (excluding transmembrane domains) ligated into pET-25b(+) vectors and expressed in BL21 receptor cells. Induction was achieved using $0.5\,mM$ isopropyl-\beta-D-thiogalactoside (IPTG) for $16\,h$ at $20\,^\circ\text{C}$.
• Mice and Immunization:     * Subject: Six-week-old female BALB/c mice.     * Protocol: Subcutaneous immunization four times with $2$-week intervals.     * Dosage: $50\,\mu g$ of recombinant M1R or B6R protein per mouse.
• Antibody Production: Spleen cells were fused with Sp2/0 cells to create hybridomas three days after the final immunization.
• Sequencing and Characterization: Total RNA extracted using the FastPure Cell/Tissue Total RNA Isolation Kit. Complementarity-determining regions (CDRs) were verified using IMGT/V-QUEST.

Identification and In Vitro Activity of Monoclonal Antibodies

• Identified Antibodies:     * M1R-specific: M1H11, M3B2, M4B6, and M13H.     * B6R-specific: B7C9, B7E2, and B10D3.
• Binding Efficacy (ELISA): M3B2 and B7C9 showed the strongest binding to inactivated VACV (Tian Tan) lysate. M1H11, M3B2, and M4B6 reacted robustly with the M1R protein.
• Neutralization Potential:     * MV Neutralization: M1H11 and M3B2 were most effective against the MV form.     * EV Neutralization: Only B7C9 exhibited effective neutralizing activity against the EV form.
• Antibody Isotypes:     * M1H11: IgG2a subclass, \kappa-type light chain.     * M3B2: IgG1 subclass, \kappa-type light chain.     * B7C9: IgG1 subclass, \kappa-type light chain.
• Surface Plasmon Resonance (SPR) Affinities: M1H11 and M3B2 demonstrated higher affinities for M1R compared to B7C9’s affinity for B6R.

Structural Modeling and Epitope Prediction

• Molecular Docking (AlphaFold2/ZDOCK/ClusPro 2.0):     * M1R Structure: Consists of an \alpha-helical bundle packed against a pair of \beta-sheets. Disulfide bonds: Cys34-Cys57, Cys49-Cys136, and Cys116-Cys158.     * M1H11 Interaction: Binds to \alpha1, \alpha2, and \alpha4 helices of M1R through polar interactions from CDR1 and CDR3 (heavy chain) and CDR1 and CDR2 (light chain).     * M3B2 Interaction: Involves \beta1, \beta3, \alpha5, and the \alpha4-\beta3 linker. All CDRs except light chain CDR2 participate via hydrogen bonds and electrostatic interactions.     * B6R Structure: Consists of 4 consecutive complement control protein (CCP) domains and a C-terminal transmembrane helix. Includes 8 pairs of disulfide bonds.     * B7C9 Interaction: Binds to CCP2 and CCP3 domains of B6R through heavy chain CDR1/3 and light chain CDR1 interactions.
• Alanine Scanning Validation: Identified $D66$ as the critical epitope on M1R for M1H11 binding.

Therapeutic Efficacy in Mouse Challenge Models

• VACV (Tian Tan) Challenge:     * Dosage: $2.5 \times 10^5\,TCID_{50}$.     * Treatment: $10\,mg/kg$ administered $12\,h$ post-challenge.     * Observations: All treated mice survived. The M1H11 & M3B2 cocktail produced the most significant reduction in lung viral load and weight loss compared to individual mAbs or other cocktail combinations.     * Lung Pathology: Control mice showed lost alveolar structure and granulocyte infiltration. Cocktail-treated groups showed significantly alleviated inflammation.
• MPXV Challenge:     * Dosage: $5 \times 10^5\,TCID_{50}$.     * Treatment: $5\,mg/kg$ administered $24\,h$ post-challenge.     * Observations: Bis-M1M3 (the bispecific antibody) significantly reduced mortality, viral load, and weight loss, outperforming the cM1H11 & cM3B2 cocktail.

Engineering and Performance of Bis-M1M3 Bispecific Antibody

• Design: Human-mouse chimeric M1H11 (cM1H11) served as the framework. The single-chain fragment variable (scFv) of M3B2 was linked to the cM1H11 Fc region using a $(G_4S)_3$ linker.
• Binding Affinity: Bis-M1M3 yielded a $K_D$ value of $1.47 \times 10^{-10}\,M$ via SPR, indicating high affinity.
• Neutralization Efficacy: The IC50 of Bis-M1M3 and MPXV was lower than that of cM1H11 alone or the cM1H11 & cM3B2 cocktail, suggesting superior in vitro neutralization potency.

Neutralization Mechanisms and In Vivo Pharmacokinetics

• Timing of Neutralization:     * Pre-incubation Group: Strong neutralizing activity observed when antibodies and viruses were mixed $1\,h$ before infection.     * Post-infection Group: Neutralizing effects were significantly diminished when antibodies were added after the virus had already entered the cells.     * Conclusion: The mAbs and Bis-M1M3 primarily work by impeding viral entry into the host cell.
• Pharmacokinetics (Rhesus Macaque Model):     * Subjects: $8$ male rhesus macaques ($n=4$ per antibody group).     * Administration: Single Intravenous infusion at $10\,mg/kg$.     * Half-life: Bis-M1M3 recorded an in vivo half-life of $5.142$ days, which is comparable to standard control mAbs (e.g., influenza antibody 12G6).

Discussion of Clinical Potential and Future Research

• EV Resistance: The study noted that B6R-specific mAbs (targeting EVs) show lower neutralization than M1R-specific mAbs (targeting MVs). This is likely because low antigen density on EVs allows them to escape direct antibody binding.
• Conformational Epitopes: Hybridoma testing with short peptides failed, suggesting M1H11 and M3B2 recognize discontinuous conformational epitopes rather than linear sequences.
• Future Directions: The researchers plan to use cryo-electron microscopy to generate precise structures of these antibodies and conduct further efficacy studies in non-human primates to advance clinical translation potential.