The genus Orthopoxvirus contains many viruses that can cause severe pox diseases in humans and animals, including the monkeypox virus.（MPXV）, Vaccinia Virus（CPXV）And the most famous smallpox virus（VARV）Although smallpox was officially eradicated worldwide in 1980 following a historic global vaccination campaign, orthopoxvirus infections remain a significant public health threat. To this day, multi-country mpox outbreaks continue. As a DNA virus, MPXV theoretically does not mutate as frequently as RNA viruses. However, adaptive mutations have recently been discovered in the genome of the ongoing mpox outbreak. Therefore, against the backdrop of the global mpox epidemic, the possibility of MPXV undergoing further adaptive mutations and causing larger-scale outbreaks cannot be ruled out. There is an urgent need to develop effective therapeutic strategies to combat orthopoxviruses.
A variety of treatment methods have been developed so far, including antiviral inhibitors and human vaccinia immunoglobulin.（VIG）Orthopoxvirus-specific monoclonal antibodies. Increasing evidence highlights the importance of orthopoxvirus antibodies in viral control and recovery from primary and secondary infections. The development of therapeutic monoclonal antibodies represents a promising strategy for the prevention or treatment of orthopoxvirus infections. Currently, monoclonal antibodies targeting VACV A33, VACV B5, MPXV M1, and VACV A27 have demonstrated effectiveness against several orthopoxviruses in research.(Including VACV, CPXV, MPXV, and VARV)Broad cross-neutralizing activity.
March 27, 2024Deng Yongqiang and Qin Chengfeng's research team from the Academy of Military Sciences collaborates with Abogen BiosciencesInNatureSub-JournalSignal Transduction and Targeted TherapyPublished an article titled“Rapid development of double-hit mRNA antibody cocktail against orthopoxviruses”research paper. This study is the first to report an mRNA antibody therapy targeting orthopoxviruses with in vivo protective effects, delivered through lipid nanoparticles.（LNP）The encapsulated mRNA platform has constructed a set of mRNAs encoding broadly neutralizing antibodies against orthopoxviruses. A single intravenous injection of each mRNA antibody can rapidly produce effective neutralizing antibodies in mice. More importantly, the dual-target attack on EEV-type and IMV-type viruses.（double-hit）mRNA Antibody Combination（Cocktail）Mix2a shows excellent protective efficacy against lethal VACV attacks in mice.
Construction and Characterization of mRNA Encoding Orthopoxvirus AntibodiesThe research team first selected four antibodies, mAb22, mAb283, mAb26, and mAb301, which have been verified to possess broad cross-neutralizing activity, targeting the VACV-A33, VACV-B5, MPXV-M1, and VACV-A27 proteins, respectively. SubsequentlyOptimize the heavy chain of each candidate monoclonal antibody（HC）and light chain（LC）Coding sequence, and insert separatelyPlasmid ABOP-028In China.The prepared mRNA contains a 5' cap, 5' UTR, signal peptide, codon-optimized coding sequence for the candidate mAb, 3' UTR, and poly(A) tail (Fig. 1a).Figure 1. Construction and Characterization of mRNA Encoding Orthopoxvirus AntibodiesIn Vivo Characterization of LNP-mRNAThe mRNAs encoding four antibodies were combined and encapsulated into LNP formulations, named mRNA-mab22-LNP, mRNA-mab283-LNP, mRNA-mab26-LNP, and mRNA-mab301-LNP, respectively. Dynamic light scattering analysis showed uniform particle sizes ranging from 73.12 to 75.39 nm, with a polydispersity index.（PDI）Below 0.1. The concentration of human IgG in mouse serum was evaluated using ELISA, with a range of 269-2598 ng/mL.(Figure 2)。Figure 2. In Vivo Characterization of LNP-mRNATo further determine the efficacy of these mRNA antibodies, plaque reduction neutralization assays were performed using VACV EEV and IMV in BS-C-1 cells.（PRNT）。High levels of anti-EEV neutralizing antibodies were measured in the serum of mice injected with mRNA-mab22-LNP and mRNA-mab283-LNP, while injection with mRNA-mab26-LNP and mRNA-mab301-LNP contributed to the neutralization of the IMV form of VACV.Among them, the neutralization titer of the EEV candidate drug is much higher than that of the IMV candidate drug, thus it is speculated that the EEV candidate drug is crucial for superior protective efficacy.Protection Efficiency against VACV Infection
BySince lung tissue is the primary infected organ in mice infected with VACV, the infectivity of viral particles in the lungs of infected mice was further measured using a standard plaque assay in BS-C-1 cells.Treatment with mRNA-mab22-LNP completely controlled VACV replication in the lungs, with no detectable infectious viral particles.In addition, using H&E staining and immunohistochemistry（IHC）Analysis of pathological lung lesions was conducted. The results showed that VACV infection led to mild to significant diffuse degeneration and necrosis of the epithelial lining in the control group of mice.(Black Arrow), accompanied by bleeding and edema(Red Arrow)And fibrin exudation into the surrounding alveoli(Blue Arrow), and antibody therapy encoded by mRNA(mRNA-mab283-LNP excluded)Significantly blocked VACV-induced lung injury(Figure 3)Moreover, IHC analysis using anti-VACV D8 monoclonal antibodies showed D8L protein-positive cells in the lungs of control group mice, while no virus protein-positive cells were detected in the lung tissues of the mRNA antibody treatment group.Figure 3. Study on the Protective Efficacy Against VACV InfectionDual-Strike mRNA Combination Contributes to Achieving Superior ProtectionTo determine the optimal dual-hit mRNA antibody mixture for neutralizing IMV-type and EEV-type viruses, two candidate drugs were prepared accordingly, referred to as Mix2a.(mRNA-mab22-LNP plus mRNA-mab26-LNP)And Mix2b(mRNA-mab283-LNP and mRNA-mab26-LNP)
Experiments show that Mix2a and Mix2b contribute to the functional neutralization of EEV and IMV forms of VACV, with Mix2a generating higher levels of functional antibodies than Mix2b.Both can protect mice from weight loss after infection with a lethal dose of VACV, completely block VACV-induced pathological lung lesions, and no virus protein-positive cells were detected by IHC analysis.These results indicate that,Optimal protection against orthopoxvirus infection requires the cooperation of EEV and IMV targeted neutralizing antibodies., and emphasized that Mix2a is the most effective dual-target mRNA antibody combination candidate in current research.Figure 4. Dual-Target mRNA Antibody Combination Enhances Protective EfficiencyOverall, a single injection of the mRNA antibody mixture Mix2a prevented infection with orthopoxvirus in mice. Human antibodies using customized mRNACombinationRepresents a promising antiviral strategy that can rapidly respond to combat current and future outbreaks of human poxviruses.It should be noted that in the formulation approach of this study, it is necessary to encapsulate two mRNAs encoding the light chain and heavy chain into the LNP formulation. The correct pairing of homologous light and heavy chains is crucial for antibody function, making multi-component mRNA antibodies more complex than multi-component mRNA vaccines. Additionally, research has found that simultaneous administration of different mRNAs expressing antibodies may lead to low production efficiency of functional antibodies. The levels of functional antibodies in the Mix2a and Mix2b groups were slightly lower than those obtained from administering a single antibody, indicating some degree of antibody interference. Therefore, further research is needed to determine the interactions between different mRNA antibodies and the specific mechanisms affecting their functionality.Reference: Chi, H., Zhao, SQ., Chen, RY.et al. Rapid development of double-hit mRNA antibody cocktail against orthopoxviruses. Sig Transduct Target Ther 9, 69 (2024). https://doi.org/10.1038/s41392-024-01766-8
Source:RNAScript
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