Introduction

Within the Orthopoxvirus genus, MPXV is a double-stranded DNA virus.1 Of the twelve viral families that were found to be among the fifty viruses with the greatest potential for spillover, two of these top-ranked viruses were found in the family Poxviridae.2 In particular, the MPXV and cowpox viruses, in terms of spillover risk, were ranked 24th and 28th, respectively. In terms of its viral structure, clinical symptoms, and medication treatment, it shows similarities to the smallpox virus. The viral structure of MPXV is critically important for drug development. During the prodromal phase, the virus is not infectious. This stage is primarily characterized by non-specific immune response symptoms such as fever, lymphadenopathy, and myalgia. The rash typically emerges on the face within 1 to 3 days following the onset of fever and lymphadenopathy, subsequently spreading rapidly in a centrifugal pattern to involve the entire body. Fever symptoms generally subside on the day the rash appears or within three days thereafter.3,4 MPXV was initially identified in the 1950s during the investigation of cynomolgus monkeys in Copenhagen5 and was detected in a nine-month-old boy in the Congo region in 1970, which was the first verified case of human infection caused by MPXV.6 Since then, mpox has mostly spread from Congo to other countries. While the virus was originally identified in Africa, the epidemiological pattern of mpox cases among travelers from Nigeria has evolved, resulting in its dissemination to novel geographic areas lacking any direct links to West or Central Africa.7,8

Despite a substantial increase in publications related to mpox in recent years, the existing literature exhibits significant fragmentation. Specifically, clinical evidence for antiviral drugs, preclinical data, and computational prediction results are often indistinctly categorized, lacking systematic integration based on levels of evidence. Moreover, the clinical translation pathways for emerging strategies remain unclear. This article presents a comprehensive review of extensive mpox-related literature, summarizing the virus’s developmental history, scientific discoveries, and reliable data concerning viral structure, binding targets, therapeutic regimens, and intervention approaches, to address these gaps. The review covers publications from the initial emergence of mpox in 1950 through 2025, encompassing databases including PubMed, CNKI, Web of Science, ClinicalTrials.gov, and the WHO database. Search terms combined MeSH headings and free-text keywords, focusing on core concepts such as “Mpox/Monkeypox/MPXV,” “Antiviral medicine,” “Clinical trial,” and “Virus structure,” with search strategies tailored to the characteristics of each database. Also, it differentiates between approved therapies, preclinical drug candidates, and computationally predicted compounds, emphasizing the evaluation of existing antiviral agents’ clinical efficacy and resistance risks, progress in innovative approaches like host-targeted therapies, and barriers to drug accessibility in low- and middle-income countries. Although several reviews have summarised the epidemiology, virology and targeted prevention and control measures for MPXV, comprehensive reviews that take a holistic approach—considering vaccines, antibody therapies, antiviral drugs and natural products—from the perspective of drug development remain relatively limited. The principal contribution of this review lies in providing a systematic analysis of current therapeutic strategies, their molecular mechanisms, clinical progress and research and development challenges, with the aim of highlighting new opportunities in the field of next-generation MPXV prevention and treatment.

Epidemiology and Evolution

In order to attain a more comprehensive understanding of the prevention and control strategies for mpox, it is essential to examine its epidemiological history and evolutionary development. Back in 1958, an outbreak of a non-lethal pox-like illness was reported among cynomolgus monkeys in the Copenhagen region.4 Although the virus was originally discovered in monkeys, the name is somewhat inaccurate—non-human primates are not the natural reservoir, as subsequent observations have documented its presence in a diverse range of mammalian species, such as squirrels, as well as rodents including rats, mice, and prairie dogs. These animals serve as natural hosts of MPXV, carrying the virus without developing symptoms and maintaining viral circulation. The role of small mammals in forests is widely acknowledged as serving a pivotal function in the propagation of zoonotic diseases.9,10 Non-human primates, prairie dogs, and genetically defective mice have been used as susceptible animals for mpox research, but none serve as natural reservoirs. The initial human case of mpox was identified in a nine-month-old infant in the Congo region in 1970.5 According to official reports from the Centers for Disease Control and Prevention (CDC), since the emergence of mpox up to the year 2003, more than 40 cases have been identified in the Midwestern United States. Among these, 47 cases were attributed to direct or indirect contact with prairie dogs infected with mpox.11 The most severe outbreak transpired in Nigeria, approximately four decades following the previous documented case.12 The United Kingdom is where the current MPXV Clade IIb 2022 outbreak first appeared, with patients similarly having recently traveled to Nigeria in May in the same year. It is characterized by an unprecedented rise in cases, setting it apart from all prior outbreaks. Not long after, the WHO proclaimed mpox to be a Public Health Emergency of International Concern (PHEIC). As reported as of September 22, 2022, a worldwide cumulative total of 64,290 cases had been documented (63,711 of which originated from regions with no prior history of mpox), with 20 confirmed deaths.13 Since then, mpox has attracted global attention. Although the pandemic gradually diminished in 2022, the virus continued to evolve within the African continent. Subsequently, the outbreak of MPXV clade Ib in Congo in 2023 and the increase in mpox cases in Africa in 2024 have forced the WHO to once again pay attention to mpox. On August 14, 2024, WHO declared that mpox once more represents PHEIC. This declaration constitutes the second instance within a span of nearly two years in which a public health emergency related to mpox has been declared. MPXV is characterized by two genetically distinct clades: the Central African (Congo Basin) clade and the West African clade. These classifications are supported by genetic divergence as well as clinically observed differences.14 Clade I is designated as the Central African clade, whereas the West African clade is identified as clade II. Notably, the Central African clade contains the mpox inhibitor of complement enzymes, which is absent in the West African clade. Consequently, the virulence of the West African clade is considered lower than that of the Congo Basin clade, with its case fatality rate estimated between 1% and 3%. In contrast, the case fatality rate for clade I is notably higher, approximating 10%.15,16 Among seven American sample sequences collected in May 2022, five formed a monophyletic group together with European MPXV sequences collected in the same year. Within this clade, most genomes exhibited zero to two nucleotide variations in non-repetitive regions. This clade will be designated as the predominant variant responsible for the 2022 mpox outbreak, clade IIb.1. The poxvirus caused by MPXV clade IIb is spreading internationally, and the first case was reported in the United States on May 17, 2022. As of May 1, 2024, according to CDC reports, more than 99,000 cases of MPXV Clade IIb have been documented across a minimum of 118 countries, with the United States reporting more than 33,000 cases and 60 deaths.17 After 2022, clade IIb infection will gradually decrease in regions such as the United States and Europe, but the incidence of MPXV infection in Africa is expected to progressively rise and experience a novel evolutionary trajectory. From September 2023 to January 2024, the Democratic Republic of Congo saw the most extensive clade I outbreak that has been reported to date, with more than 20,000 suspected cases and hundreds of deaths reported.18 This new strain is different from the previously prevalent strain and belongs to the evolved new Clade Ib. This strain exhibits considerable genetic divergence from former virus sequences obtained during prior outbreaks and has accumulated mutations linked to the activity of the APOBEC3. This suggests a divergence from previous animal-to-human transmission patterns, suggesting that direct human-to-human transmission is emerging as the primary mode of mpox spread in large outbreaks.19–21

The majority of mpox outbreaks have been concentrated in Africa. Although they have spread to non-endemic areas in recent years, given that most countries can respond to emergencies, the risk of large-scale mpox epidemics outside of Africa is relatively low. Amid the context of the global pandemic, the epidemic prevention and control landscape in China demonstrates unique and distinguishing features. To this day, mpox outbreaks in China remain at a low level. In 2022, there were mostly scattered cases. Earlier than the mainland, Taiwan, China, reported a case of mpox imported from Germany, which was also the first instance of mpox imported into China. Then, China, Hong Kong, in June 2022, and Chongqing, China, in September 2022, reported imported mpox cases from abroad.22,23 All patients reported a history of residence abroad. Subsequently, the National Health Commission promulgated technical guidelines for the prevention and control of mpox, guaranteeing that areas designated as high-risk implement health declaration requirements for incoming populations to strengthen entry quarantine measures and bolster the capacity of prevention and control. Until June 2023, all mpox cases in China were imported. However, data collected during the latter half of 2023 reveals a substantial change in the status of the pandemic; the initial detection of mpox cases without a history of international travel in Beijing, Guangzhou, and other places marked the emergence of local transmission chains.24,25 The number of cases will gradually increase in the second half of 2023, but it will still maintain a low-level epidemic status. Clade IIb is the epidemic strain, according to Gene sequencing, with low pathogenicity.26 Subsequently, the number of mpox cases progressively rose in comparison to earlier years; however, it stayed mostly sporadic. In January 2025, a cluster outbreak of imported Clade Ib occurred in Zhejiang, Guangdong, and other places in China. The mortality rate of this branch decreased, but the transmission increased. In April 2025, a foreigner who entered the country from Congo was diagnosed with MPXV Clade Ib infection after testing. This represents the second documented occurrence of Clade Ib in China. This branch has high pathogenicity and transmission, which has garnered significant attention from public health departments. With the introduction of the Clade Ib, this marks a new Phase In China’s mpox prevention and control efforts. Despite the fact that the spread is restricted, its high pathogenicity poses a severe challenge to the public health system.

Virus Structure

Comprehending the epidemiological features of MPXV necessitates a foundational understanding of its virological properties. MPXV is categorized under the genus Orthopoxvirus of the family Poxviridae, a group that also encompasses VARV and VACV, among other related viruses. MPXV possesses a complicated and well-defined structure, with its structural features forming the basis of its infectivity and immunogenicity. MPXV, recognized as one of the biggest animal viruses, displays a characteristic brick-shaped or elliptical morphology, with mean dimensions measuring approximately 313×267 × 236 nanometers. In comparison to the more rectangularly shaped VACV mature viruses (MVs), the dimensions of which are 347×260×240 nanometers. The major long axis of these MPXV MVs is noticeably shorter, while the intermediate axis is markedly elongated.27,28 MPXV’s genome is roughly 197,205 base pairs, characterized by variable regions located at both termini and a highly conserved central region. Gene encoding critical enzymes are found in the central region, including DNA polymerase and a capping enzyme, in addition to structural proteins such as M1R, E8L, H3L, A29L, A35R, and B6R.29 MPXV infection as well as replication takes place within the cytoplasm, involving the mechanisms of adsorption, entry, and uncoating, DNA and protein production, viral assembly, maturation, as well as release.30 Infectious viral particles of MPXV exist in two forms: namely, extracellular enveloped virus (EEV) and intracellular mature virus (IMV). They are produced at various phases of the life cycle and possess distinct structures, surface proteins, and functions.

IMVs accumulate within infected cells and can be shed upon cell death. Early micro-electron microscopy and cryo-electron tomography experiments imaged and reconstructed MPXV IMVs, and their overall structure consists of a protein-coated lipid envelope, two side bodies, as well as a double-cavity viral core that encircles genomic DNA.28,31 The DNA genome and numerous viral enzymes are found in the biconcave viral core. The viral genome encompasses the complete set of genetic information, which is crucial for the assembly and replication of viruses, situated at the central core of the virus. Following the fusion of the IMV envelope with the host cell membrane, the side bodies dissociate from the central viral core and then release effector proteins into the cytoplasm of the host cell. Subsequently, the central viral core is liberated and functions as an early transcription factory, using its own enzymatic reactions to transcribe and synthesize early viral genes, thereby protecting the viral genome.28,32 This structurally stable form serves as the primary vehicle for viral transmission in the environment or between cells. The lipid envelope consists of viral-encoded proteins and lipids. Specialized tubular or lipoprotein structures decorate the surface, with proteins such as M1 and A29 embedded within this membrane layer. These serve as the major targets for antibody neutralization of IMV.

Besides IMV, MPXV also exhibits an alternative form of viral particle characterized by unique properties. EEVs are secreted from the cell membrane via exocytosis to increase their diffusion throughout the host. Essentially, they are IMVs with an extra layer of membrane, possessing a more complex structure than IMVs. They possess both a viral membrane and a host cell membrane. The viral envelope originates from the Golgi apparatus or endoplasmic reticulum of infected cells and is rich in virus-specific proteins. Proteins such as B6 and A35 are embedded in this membrane layer, serving as primary targets for antibodies that neutralize EEVs and prevent intercellular transmission. The host cell membrane is present only in EEVs, located at the virus’s outermost layer. This structure is unstable: the outer membrane is easily disrupted, sensitive to environmental conditions, and prone to losing infectivity.33

The genome meticulously encodes these intricate viral structures. For example, the MPXV genome encodes a variety of proteins that serve specific functions in viral replication, pathogenesis, and evasion of host immune responses. Among these, the MPXV EEV contains three important structural proteins: B6R, C19L, and A35R. The key proteins of the IMV include A29L, H3L, M1R, E8L, I5L, and A43R, which are crucial for viral particle formation and stability. Additionally, several non-structural proteins are present but do not constitute integral components of the viral particle. However, these proteins play vital roles in immune evasion, modulation of host cellular processes, viral replication, and virion assembly.34 Proteins that are conserved across the Orthopoxvirus genus represent ideal drug targets, providing a structural foundation for the development of broad-spectrum anti-Orthopoxvirus therapeutics.

Prevention and Treatment Biological Agent Vaccination

In order to prevent epidemics, vaccination has always been a vital and efficient tactic. According to a review of the literature, during the mpox outbreak, people who were vaccinated had a lower risk of serious problems and long-term consequences than those who were not vaccinated.35 Historical evidence suggests that conventional vaccination methods have elicited safety concerns, thereby motivating the advancement of next-generation vaccine technologies.

With the outbreak of mpox in 2022, the prevention and treatment of mpox have garnered worldwide attention. Given the continuous evolution of mpox resulting in increased person-to-person transmission, the development of targeted vaccines for mpox remains an ongoing process. The types and characteristics of the vaccines used in this study are listed in Table 1.

Table 1 Summary of Vaccination

At present, most of the marketed vaccines for mpox (JYNNEOS in the United States, IMVANEX within the European Union, and IMAMUNE in Canada) are the Ankara strain of the basic modified smallpox virus, which is a specific vaccine developed for smallpox. As a result of the significant structural resemblance between the smallpox virus and MPXV, studies indicate that smallpox vaccination can induce neutralizing antibodies with cross-protective effects, thereby shielding people from MPXV infection.36,37 Numerous studies have demonstrated that individuals immunized against VACV exhibit cross-reactive immune responses to specific MPXV surface proteins.33 An enhanced attenuated virus vaccine, the Ankara vaccine, is difficult to cause systemic infections because it cannot replicate effectively in mammalian cells. So the MVA-based smallpox vaccine JYNNEOS was extensively utilized in the 2022 MPXV outbreak. Nonetheless, the vaccine did not provide protection in primates exhibiting significantly damaged T-cell functionality.38 So far, JYNNEOS and the ACAM2000 are effective against MPXV in macaque models.39 The preclinical study Results showed that a cohort of 40 animals administered two doses of the JYNNEOS vaccine combined with a single dose of the ACAM2000 vaccine exhibited total protection against severe clinical manifestations and mortality related to the illness.40 On the other hand, administration of just one dose of the JYNNEOS vaccine proved inadequate in mitigating disease severity and the risk of death. The JYNNEOS vaccine received regulatory approval in Europe in 2013 and subsequently received authorization from the US Food and Drug Administration in 2019. The product was introduced to the commercial market, but the CDC does not recommend continuing vaccination after the onset of mpox symptoms.41–43 Besides JYNNEOS, ACAM2000 represents another authorized vaccine that employs a different technological methodology. ACAM2000 was approved for marketing in 2007; however, the virus can undergo replication in cells, which can lead to various adverse events, including autoinoculation of the ocular region, disseminated vaccinia, eczema vaccinatum, progressive vaccinia, myocarditis, and mortality.44 Compared to ACAM2000, JYNNEOS is a defective vaccine with a lower risk of adverse events, making it the preferred choice for mpox vaccines. However, due to its high price, JYNNEOS has poor popularity in low- and middle-income households. In September 2024, according to the WHO, the MVA-BN vaccine has emerged as the first MPXV vaccine to be included in its prequalification list. It is anticipated that this approval for prequalification will enhance access to the vaccine for communities in need, reduce viral transmission, and contribute to outbreak control.45 However, as live virus vaccines, they are not yet fully understood, and the effects of their gene products on the immune system, as well as the possibility of unfavorable outcomes, remain inadequately characterized. Hence, it is essential to develop vaccines specifically designed to target MPXV in order to attain comprehensive protective efficacy.46 To overcome the limitations associated with the previously mentioned vaccines, researchers have developed a candidate vaccine belonging to the third generation. LC16m8 is a third-generation vaccine for smallpox that is safer and has fewer side effects relative to the first- and second-generation smallpox vaccines available during the same time. It has also been researched as a vaccine against MPXV. Developed from the Lister strain of vaccinia virus, LC16m8 is an attenuated, replicating smallpox vaccine with current marketing authorization in Japan.47 The strong safety record of LC16m8 is demonstrated by its minimal neurovirulence in animal models and the absence of significant adverse events—including complications or fatalities—among a large group of vaccinated individuals in Japan. Beyond its ability to generate protective immunity against MPXV, LC16m8 may also confer cross-protection against other orthopoxviruses.48 The key advantage of this vaccine lies in its ability to achieve an optimal balance between robust immunogenicity and a favorable safety profile. While it demonstrates significantly greater safety compared to ACAM2000, its safety is theoretically marginally inferior to that of the fully non-replicating JYNNEOS vaccine. Nonetheless, extensive clinical data have substantiated its overall safety as excellent.

Recent advancements in messenger RNA (mRNA) technology have significantly expanded the potential pathways for vaccine development. Given the outbreak of novel coronavirus pneumonia, the mRNA vaccine has received unprecedented attention. In comparison to live virus and DNA vaccines, mRNA vaccines demonstrate superior immunogenicity and an enhanced safety profile.49,50 Recent experimental investigations have demonstrated that multivalent vaccines using mRNA technology platforms have been shown to elicit strong immune responses in preclinical models, including both rodent and non-human primate studies, thereby conferring protection against poxviruses, including VACV and MPXV. The advancement of the BNT166 vaccine has specifically demonstrated the viability of the mRNA platform. BNT166 is an efficient mRNA MPXV vaccine candidate that provides broad protection against various orthopoxviruses, particularly VACV and MPXV. The vaccine has demonstrated strong potential in animal experiments, while BNT166 is currently undergoing a Phase I/II clinical trial (NCT05988203) to explore its safety profile and immunogenic potential.51 As of July 2026, the data have not been released, and the Phase III trial has not yet commenced. In addition to mRNA vaccines, the VGPox series, which utilizes a fusion protein design, presents an alternative technical strategy. The VGPox vaccine series targets homologous M1R and A35R regions of MPXV and VACV, employing innovative fusion protein design to enhance immunogenicity. Following two immunizations, VGPox 1, 2, and 3 conferred complete protection against lethal VACV challenge in murine models, preventing weight loss and mortality while achieving complete viral clearance from the lungs. The VGPox vaccine series generates rapid and durable protective immunity. Using VACV as a challenge model, it demonstrates that the vaccine-induced immune response confers cross-protection against orthopoxviruses, which is critical for addressing different branches of MPXV. Its performance outperforms that of traditional attenuated live virus vaccines, establishing it as a promising alternative to traditional whole-virus vaccines, which could help alleviate safety concerns associated with orthopoxvirus immunization.52

Additionally, multiple studies have employed mRNA-lipid nanoparticle (LNP) technology platforms to achieve intercellular transmembrane transport, thereby enhancing initiation of immune responses within both deep muscle and superficial regions. The multivalent mRNA vaccine confers a wider range of protection, inducing heightened neutralizing antibody responses targeting both MPXV and VACV, demonstrating cross-neutralizing activity against both live VACV and MPXV, and blocking MPXV-induced cytopathic impact. There is no inflammatory response or abnormal skin manifestations at the injection site with the mRNA-LNP vaccine, outperforming traditional vaccines like ACAM2000 in terms of local adverse reactions.53,54 Experimental investigations have revealed several benefits of mRNA vaccines compared to conventional vaccine platforms. However, considerable obstacles persist in the process of applying laboratory research findings to clinical settings. Existing research mainly utilizes murine models, lacks data derived from non-human primates, and has not incorporated challenge evaluation using circulating strains of MPXV. Consequently, determining whether mRNA vaccines can serve as substitutes for attenuated live virus vaccines necessitates additional clinical studies to assess their safety profile and long-term effectiveness.

Amid the international competition to develop vaccines, China has recently achieved considerable advancements. Currently, no MPXV-specific antiviral medicines are available worldwide. Following the global mpox outbreak, it is widely accepted that conventional smallpox vaccines offer effective protection against MPXV infection. Therefore, newer vaccines with fewer side effects, based on the original smallpox vaccine, have been approved and are widely used in some countries. However, as of now, no MPXV vaccine products have been launched in China. With the new wave of mpox outbreaks in Africa, particularly the widespread circulation of clade Ib, mpox has once again drawn global public health attention. On September 9, 2024, the MVA strain MPXV attenuated live vaccine, independently developed by the Shanghai Institute for Biological Products of China National Pharmaceutical Group (Sinopharm), received a clinical trial notification from the National Medical Products Administration (NMPA), becoming China’s first MPXV vaccine approved for clinical trials. This attenuated MPXV vaccine is based on the replication-deficient VACV strain MVA, whose safety and efficacy have been thoroughly validated by clinical data. This significant accomplishment not only addresses the existing gap in the development of MPXV vaccines in China but also offers a Chinese contribution to the global efforts in pandemic prevention and control. This approval marks the entry of China’s domestically developed MPXV vaccine into the clinical trial phase, bringing new hope for global epidemic prevention and control efforts.

Monoclonal Antibodies

Monoclonal antibodies are highly promising therapeutic agents with great potential for clinical applications and are instrumental for the effective clinical management of herpesvirus infections, aiding in both diagnosis and treatment.55 Nevertheless, owing to the complicated proteomic composition of orthopoxviruses and the heterogeneity of their viral particles, the development of therapeutic antibodies presents considerable difficulties and challenges. To date, although numerous orthopoxvirus-targeting drugs have been developed with an emphasis on VACV structural components, only a limited number have been specifically designed for MPXV.56–58 Consequently, researchers have directed their focus toward studies concerning smallpox viruses, given their structural resemblance to MPXV. Prior research has identified key neutralizing antibody targets on both MV surface proteins (H3L, E8L, A29L, M1R, A28L, A17L) and EV surface proteins (B6R, A35R), underscoring the complementary roles of these viral forms in immune recognition.33 The specificity of these target structures provides a strong theoretical foundation for antibody development. See the details in the section, Table 2 provides an overview of different antibodies.

Table 2 Summary of Antibodies

Within this theoretical framework, the proteins A29L and A35R have been prominently highlighted as key representative targets. A29L, the IMV surface envelope protein of MPXV, is an ortholog of the A27 strain of VACV, while the A35R protein, which is the EEV envelope component of MPXV, shares the same homology with the A33 protein of VACV. Based on these findings, ELISA screening against MPXV A35 and A29L identified seven A35-specific mAbs and six A29L-specific mAbs, with most A35R-targeting antibodies showing no neutralizing activity. Among the three A29L-specific mAbs (9F8, 3A1, and 2D1), broad-spectrum binding and neutralizing activity against orthopoxviruses were confirmed, with 9F8 demonstrating the highest neutralizing potency. In in vivo antiviral prevention and treatment trials, 9F8 alone achieved complete protective activity, while 3A1 and 2D1 alone showed limited protective activity. When these were used in combination, they had a strong synergistic antiviral activity.56,57

Subsequent investigations have identified antibodies targeting A29 that exhibit increased specificity. Monoclonal antibody mAb 69–126-3-7 selectively recognizes the heparin-binding region of the MPXV A29 protein. The MPXV A29 protein’s structure is similar to that of VACV A27, although mAb 69–126-3-7 displays excellent specificity, binding only to MPXV A29 and showing no cross-reactivity with A27 homologous proteins from other orthopoxviruses. At the same time, A27 protein performs multiple key functions during the replication cycle of viruses, including facilitating attachment to cell surface heparan sulfate and controlling membrane fusion; in addition, it makes it easier for IMV to assemble and produce EEV.59,60 MPXV A29 exhibits binding affinity similar to VACV A27 with heparin. While this antibody does not neutralize the virus, its high specificity makes it a strong candidate for developing mpox-specific diagnostic tools.61

The emergence of recovered patients has provided researchers with the opportunity to directly identify highly potent antibodies derived from human immune responses. In recent years, researchers have endeavored to isolate memory B cells from antigen-antibody mixtures in peripheral blood mononuclear cells of recovered patients to identify specific antibodies. By employing the MPXV A35 and H3L proteins as dual antigens, they sorted CD19+CD27+ memory B cells that simultaneously bound both targets using flow cytometry. This methodology facilitated the identification of neutralizing antibodies that exclusively recognized the A35 protein, which were subsequently classified as MV127, MV128, and MV129. Among these, MV129 emerged as the most promising neutralizing antibody, demonstrating high affinity and potent neutralizing efficacy. While the A35-targeted antibody was successfully generated, the study also identified limitations inherent in the existing screening methodology. The study was limited to the identification of antibodies against A35, failing to isolate neutralizing antibodies targeting H3L, and primarily focused on in vitro validation without further experimental confirmation.62 Subsequent research successfully generated human monoclonal antibodies exhibiting improved protective efficacy through the refinement of the screening methodology. Researchers have identified three high-affinity human monoclonal antibodies—EV35-2, EV35-6, and EV35-7—from a convalescent individual infected with Clade IIb, which specifically target the A35 protein of MPXV. Multidimensional functional validation confirmed that their epitopes are highly conserved and bind to A35/A33 homologous proteins across multiple orthopoxviruses. EV35-2 largely depends on Fc function; upon Fc segment mutation-induced inactivation, its protective capacity dramatically declined. EV35-6 and EV35-7 exhibited potent direct neutralization activity. In prophylactic protection studies, mice treated with EV35-6 or EV35-7 were 100% protected against infection, while one mouse in the EV35-2 group died on day 11, resulting in a 75% survival rate. This study first characterized the structure, potential functional activity, and potent in vivo protective effects of human A35 mAbs against MPXV, suggesting that A35 could serve as a novel target for next-generation vaccines or offer new therapeutic options for severe MPXV infections.63

Concurrently, nanobodies have gained prominence as a significant area of research owing to their distinctive small-molecule properties. In addition to human monoclonal antibodies, researchers also screened and identified three nanobody mAbs (VHH-1, VHH-2, VHH-3) using phage display technology. All three nanobodies specifically bind to A35R, with VHH-1 showing the best performance and great potential for diagnostic and therapeutic development.57

Concurrent efforts are being made in the research and development of antibodies aimed at various structural proteins of the virus. M1R and B6R are important neutralization targets in MPXV, with antibodies against them targeting the MV and EV viral particle forms, respectively. At the monoclonal antibody level, M1R-targeting antibodies (M1H11, M3B2, M4B6, M13H) and B6R-targeting antibodies (B7C9, B7E2, B10D3) were obtained by immunizing mice. Among these, M1H11 and M3B2 demonstrate potent neutralizing effects against the MV of VACV and MPXV, while B7C9 exhibits inhibitory effects against the EV of the virus, albeit slightly weaker than M1R-targeting antibodies.58 Beyond the utilization of mouse-derived antibodies, the direct screening of fully human antibodies from human antibody libraries constitutes a significant alternative approach. Antibodies were extracted from a human antibody library (ST-ST-HuNAL) using phage display technology, yielding 37 M1R-targeting antibodies and 40 B6R-targeting antibodies. Among these, A138 targeting the M1R loop region and B026 targeting B6R exhibited the strongest broad-spectrum neutralizing activity. The combination of targets A138 and B026 demonstrated the most potent neutralizing efficacy against the MV+EV hybrid virus.64 Expanding on the achievements of monoclonal antibody combinations, researchers have advanced the development of bispecific antibodies to simultaneously engage multiple targets within a single molecular entity. At the level of bispecific antibodies, Bis-M1M3 was assembled with cM1H11 as the scaffold. The scFv of M3B2 was fused to the Fc region of cM1H11 via a (G4S)3 linker, yielding neutralizing activity against MPXV superior to that of the parental antibody combination.58 Substantial advancements have been achieved in the development of bispecific antibodies derived from A138 and B026. Based on A138 and B026, seven distinct bispecific antibodies were designed. ScFvA138B026 demonstrated superiority among these seven antibodies, exhibiting stronger neutralizing activity than monoclonal antibodies but slightly lower than the A138+B026 bispecific antibody combination.64

In studies targeting B6, an additional class of non-competitive antibodies has been found, presenting a potential therapeutic option. hMB621 and hMB668 are non-competitive humanized neutralizing antibodies targeting the MPXV B6 protein, exhibiting broad-spectrum antiviral potential against orthopoxviruses, particularly suitable for immunocompromised individuals. They represent potential therapeutic candidates against MPXV and other orthopoxvirus infections. Furthermore, the SCR3-4 region recognized by hMB621 reveals a unique neutralizing epitope on the B6 protein, providing a structural foundation for subsequent antibody drug design.65

Researchers designed a bispecific antibody targeting both IMV protein A29 and EEV protein B6 (bsAb A9F8-B7C9) or the A29 bivalent epitope (bsAb A9F8-A3A1), which exhibited potent neutralizing activity against both VACV and MPXV in vitro and provided complete protection against VACV and MPXV infection in mouse models. Among these, bsAb A9F8-B7C9 demonstrated high affinity for both A29 and B6, whereas A9F8-A3A1 exhibited strong binding solely to A29.66 Researchers isolated A35A3 and A35A9 antibodies targeting A35, as well as B6H1 and B6G1 antibodies targeting B6, from peripheral blood mononuclear cells derived from donors vaccinated with the recombinant vaccinia virus (rTV), using flow cytometry-based sorting to obtain A35- and B6-specific memory B cells. All four antibodies exhibited cross-binding activity against both VACV and MPXV; furthermore, the dual combination of A35A3 and B6H1 demonstrated superior efficacy compared to either antibody alone.67

In studies targeting M1R, there are also numerous studies available. From peripheral blood mononuclear cells of volunteers who had received VARV vaccination for over 40 years, the antibodies ME5, MB11, MD3, MD7, MB7, MD6, MD11, and MB6 targeting M1R were identified, with MB7 exhibiting the highest affinity. This suggests that MB7 may represent a protective antibody against orthopoxviruses and requires further validation through animal studies.68 Meanwhile, researchers screened and prepared VHH antibodies targeting the MPXV surface antigen M1R by taking advantage of the preparation technology for single-domain (VHH) antibodies. After bivalentization, the mutants bi-M1A8 and bi-M1C2 exhibited affinity improvements ranging from hundreds to tens of thousands of-fold. These antibodies demonstrated cross-neutralizing activity against both MPXV and vaccinia virus in vitro, and significantly reduced viral load and pathological damage in the lungs of mice infected with MPXV.69

Beyond the previously mentioned targets, the E8 protein, recognized as a critical molecule involved in viral entry, has emerged as a novel focal point for the development of antibodies. Three fully human monoclonal antibodies (C5, C9, F8) targeting the MPXV E8 protein were identified by screening and isolating E8-specific memory B cells from recombinant vaccinia virus vaccine recipients utilizing the MPXV E8 protein as a probing agent via flow cytometry. Among these antibodies, C9 is the only E8 antibody capable of neutralizing MPXV, demonstrating cross-neutralizing activity and both in vitro and in vivo biological protection. This indicates that the E8 protein serves as a critical conserved target for broad-spectrum prevention and treatment against orthopoxviruses. Combining it with antibodies targeting other viral proteins could boost therapeutic efficacy against MPXV.70

Theoretically, antibodies directed against viral surface proteins have the potential to facilitate viral infection via Fc receptor-mediated enhancement. Nonetheless, to date, no definitive evidence of antibody-dependent enhancement has been documented in studies involving poxviruses. The majority of MPXV-specific mAbs remain in preclinical or early clinical development stages, with no significant toxicity observed in animal model investigations. Drawing upon the previously discussed research findings concerning antibodies directed against various viral proteins, mAbs exhibit several distinct advantages as a therapeutic strategy for MPXV. MAbs hold significant potential in countering the threat of MPXV due to their engineering flexibility, scalable production platforms, and potent preclinical efficacy. By targeting key viral structural proteins and combining rational antibody engineering with animal model validation, mAbs are poised to become a core component of next-generation antiviral therapeutics and preventive tools. Overall, current MPXV antibody research is advancing toward greater efficiency and precision. Researchers are leveraging technologies such as bispecific design, humanization modifications, and upgrades from monovalent to multivalent formats to develop therapeutic antibody drugs with enhanced efficacy and broader coverage.

Chemicals

Most patients without severe immunodeficiency recover with basic therapeutic measures and pain management plans. However, serious patients and those with high-risk diseases who face serious mpox require mpox-specific treatment. To date, no antiviral therapy has been shown effective against mpox. However, certain compounds have demonstrated potential as antiviral agents against orthopoxviruses, have received emergency use authorization in some countries, and are currently being tested in clinical trials. In this chapter, we have listed numerous compounds with potential therapeutic efficacy against mpox, which will be presented in detail in Table 3.

Table 3 Summary of Chemicals

Among these agents, tecovirimat represents the first approved targeted therapeutic, functioning by inhibiting the viral p37 protein. This protein is essential for orthopoxvirus replication and is critically involved in the assembly of the viral envelope. Thus, tecovirimat can inhibit viral release from cells.71,72 On July 13, 2018, based on tecovirimat’s efficacy in animal models infected with orthopoxviruses, the FDA approved it for treating VACV infections. This remained the primary indication until the 2022 mpox outbreak. The onset of the pandemic in 2022 expedited the accessibility of this medication for the treatment of mpox. On August 9, 2022, the FDA granted an Emergency Use Authorization permitting oral administration of tecovirimat capsules to adults and pediatric patients weighing a minimum of 13 kilograms. Subsequently, an intravenous formulation was approved, offering an alternative for patients with swallowing issues. During the ongoing clade II outbreak, over 7,100 mpox patients received tecovirimat through EA-IND protocols, with only mild adverse reactions reported.73 Although initial data appeared promising, a randomized controlled trial PALM007, conducted in 2024 demonstrated a more intricate clinical scenario. A 2024 randomized controlled trial of oral tecovirimat revealed that the persistence of rash in MPXV clade I infections was not diminished as expected.74 The STOMP trial for clade II demonstrated no significant difference in outcomes between the tecovirimat group and the placebo group.75 The results of the UNITY trial were first presented at the IAS conference in July 2025, confirming that tecovirimat is ineffective against MPXV clade II. To date, these trial results have not been formally published in a peer-reviewed journal, but they have been cited by regulatory agencies such as the European Medicines Agency (EMA), along with the PALM007 and STOMP trials, as evidence supporting the restriction of tecovirimat’s use for the treatment of mpox.76 Tecovirimat is primarily used in severely ill patients, and drug resistance has garnered significant attention. CDC researchers have confirmed that resistance in mpox patients correlates with mutations in the viral F13L gene. Although the overall incidence remains low, patients with impaired immune function still experience a higher incidence rate during long-term treatment.77 Although tecovirimat resistance occurs infrequently in the general treatment population, it has become a real and serious challenge in treating immunocompromised mpox patients.

Beyond agents directed at viral envelope proteins, nucleoside analogues that disrupt viral DNA replication have likewise exhibited significant therapeutic efficacy. Cidofovir and brincidofovir act as nucleoside analogs by blocking the activity of viral DNA polymerase, thus suppressing the replication of the virus.78 They exhibit antiviral activity against multiple viruses. In 1996, the FDA authorized the use of cidofovir for management of cytomegalovirus retinitis in individuals with AIDS. Although cidofovir has exhibited antiviral activity in both in vitro experiments and animal models targeting orthopoxviruses, there were no clinical trials against mpox, and cidofovir exhibits severe nephrotoxicity.34 To overcome the safety limitations associated with cidofovir, researchers have developed an enhanced prodrug formulation. Brincidofovir is a prodrug precursor to cidofovir. Unlike intravenous cidofovir, brincidofovir is an orally administered antiviral agent that is transformed to cidofovir in the body. The primary adverse reaction observed with brincidofovir is upregulated transaminases; however, its nephrotoxicity is greatly decreased compared to cidofovir.79 In animal studies, brincidofovir showed better antiviral activity against orthopoxviruses.80,81 Consequently, the FDA approved brincidofovir for smallpox in 2021 and later extended it for mpox therapy during the outbreak. In vitro studies suggest brincidofovir has enhanced antiviral activity against VARV compared to cidofovir. Additionally, both drugs exhibit a lower likelihood of resistance.81

There are no FDA-approved treatments for MPXV infections, so researchers conducted numerous studies based on drugs previously used to treat mpox. To overcome the safety limitations associated with cidofovir, researchers have developed an enhanced prodrug formulation. The E8L is one of the key proteins involved in the MPXV invasion of host cells. Researchers attempted to use computational studies to screen compounds with potent effects against the MPXV E8L protein, with diosmin and flavin adenine dinucleotide (FAD) demonstrating the most promising outcomes.82 Meanwhile, tecovirimat analogs ABX-1431, alflutinib, avacopan, casopitant, and darapalib can bind to the E8L protein, blocking viral interaction with host cell surface glycosaminoglycans, with superior docking efficacy compared to tecovirimat.83 DrugBank database searches reveal that brinzolamide, dorzolamide, and methazolamide also bind to E8L, although further experimental validation is still required.84 Given the homology between VACV and MPXV protein structures, molecular docking and computational ADMET predictions suggest that natural drug monomers such as curcumin, quercetin, and riboflavin exhibit favorable binding to the D8L protein. Meanwhile, silibinin, oleanolic acid, and ursolic acid show binding to the A26 and H3 proteins with relatively low toxicity, exhibiting clinical advantages.85,86

According to research, medications like fludarabine, nilotinib, conivaptan, and ponatinib may function as MPXV A6R inhibitors and have the potential to be anti-smallpox virus agents. Among these, fludarabine exhibits higher binding stability with A6R than cidofovir.87 Additional essential enzymes implicated in viral DNA replication have likewise been identified as targets for pharmaceutical development. Studies on screening have shown that the MPXV I4L protein has been targeted to inhibit DNA synthesis by using anticancer medications such as gemcitabine, hydroxyurea, and fludarabine. Zidovudine exhibits a low binding affinity but considerable hepatotoxicity, while being chosen based on MPXV L2R binding.84

Research on the possibility of switching to tetracycline antibiotics has found tigecycline, eravacycline, omadacycline, and minocycline as promising candidate drugs against MPXV infection.88 Investigations into the translocation of the E12 subunit of the virus have also led to the identification of several promising candidate compounds. FDA-approved drugs (rosuvastatin, 5-hydroxy-L-tryptophan, deferasirox) and bioactive chemicals (rutin, quercitrin, epigallocatechin) exhibit low binding fractions but strong binding affinity with the MPXV E12 subunit, indicating high potential drug value.89 Researchers have resolved the crystal structure of the broad-spectrum methyltransferase inhibitor Sinefungin complex, revealing its binding to MPXV 2′-O-MTase VP39, though the stability of the binding complex was moderate.90 In contrast, folic acid and 1,2,4,6561 tetragalloylglucose demonstrated superior performance in virtual screening according to the crystal structure of VP39-sinefungin, exhibiting significantly higher stability than sinefungin.91

Additional critical enzymes implicated in viral DNA metabolism have likewise garnered considerable scholarly interest. MPXV DNA topoisomerase I (TOP1) plays a key role in the early phase of viral replication. Quercitrin, myricitrin, and rosmarinic acid were identified through virtual screening of natural substances as compounds with moderate affinity for MPXV TOP1. Viral DNA polymerase plays an important role in the replication of the viral genome. By preventing its activity, viral DNA synthesis can be successfully inhibited, making it a crucial target in antiviral medication development. Small-molecule inhibitors screened from Moringa oleifera phytochemicals exhibited higher affinity than Cidofovir and brincidofovir.92 The viral core cysteine protease I7L participates in IMV maturation. Using structure-based virtual screening and drug design, researchers identified compounds such as SC75741, ammonium glycyrrhizinate, CHEMBL32926, CHEMBL4861364, gallicynoic acid F, and H2-erythro-neopterin, which demonstrated high binding affinity validated by molecular dynamics simulations and MM/GBSA binding free energy analyses. Natural product screening further revealed that shikonin and myricetin form covalent interactions with the C328 residue of the I7L protein, indicating significant bioactivity.93–95

Advancing from computational predictions to empirical verification, the cell infection model screening substantiated the antiviral efficacy of multiple commercially available drugs. Three approved drugs (Atovaquone, Mefloquine, and Molnupiravir) exhibit antiviral activity against MPXV through distinct mechanisms, and the combination of Atovaquone and Tecovirimat demonstrates synergistic effects.96 Nonetheless, to overcome the challenges of toxicity and drug resistance linked to current nucleoside analogues, the development of structurally optimized novel prodrugs has become a primary focus in the field. Given the limited efficacy, susceptibility to drug resistance, and hepatotoxicity associated with current treatments like tecovirimat and cidofovir, researchers designed and synthesized three novel prodrug nucleoside analogs. Among these, the most promising activity was shown by ODE-(S)-HPMPA formate across multiple experiments.97 In vitro experiments showed that ODE-(S)-HPMPA formate significantly outperformed brincidofovir in inhibiting MPXV, but showed significantly reduced hepatotoxicity. In addition to directly targeting viral proteins, analyses grounded in network pharmacology of host-virus interactions have identified novel strategies for therapeutic intervention. By analyzing the interaction network between MPXV and human proteins, three licensed pharmaceuticals have been identified by researchers, which may have therapeutic potential—fostamatinib, trilostane, and raloxifene—possessing the capability to simultaneously target both viral and human proteins. Fostamatinib is indicated for treating chronic immune thrombocytopenia in adults with insufficient response to previous therapy; Trilostane is a low-molecular-weight pharmaceutical agent used in the treatment of Cushing’s syndrome; raloxifene is a selective estrogen receptor modulator.98 Concurrently, large-scale high-throughput screening methodologies have effectively facilitated the identification of novel inhibitors targeting host factors. High-throughput screening identified three low-toxicity, potent host-targeted anti-MPXV compounds: IRAK4-IN-6, SM-7368, and KRAS inhibitor-10. These compounds showed strong antiviral activity both in vitro and in vivo, positioning them as potential candidate drugs for future clinical research and development, particularly for topical treatment of mpox skin lesions.99

The capsid protein D13L, a component of viral structural proteins, constitutes a significant category of potential drug targets, which is the main capsid protein of poxviruses, forming trimeric complexes that are critical for viral development. Previous investigations on VACV demonstrated that rifampin interacts with the D13L gene within VACV; However, the later emergence of drug resistance has limited its clinical application. Experimental studies have identified simeprevir as a potent inhibitor of D13L with greater binding stability compared to rifampin.100–102 In a separate screening utilizing a cellular infection model, researchers identified compounds exhibiting broad spectrum antiviral activity against acne-associated viruses. Scientists screened chemical libraries by using MPXV-infected cell models and identified gemcitabine, trifluridine, and mycophenolic acid as potent inhibitors of MPXV cell proliferation with broad-spectrum activity against orthopoxviruses.103 Aurintricarboxylic acid, mitoxantrone, tetrapyroles, as well as distamycin showed promising potential in in vitro anti-apoxvirus assays but have not undergone clinical assessment.104 In vitro studies have shown that lipoxygenase pathway inhibitors, including ETYA and BW755c, specifically block orthopoxvirus replication.105 Immunomodulatory molecules originating from the host also demonstrate indirect antiviral activities. Prostaglandins display encouraging antiviral activity against orthopoxviruses, but further experimental data are required. Consequently, the current clinical value of prostaglandins against orthopoxvirus infection remains limited.106,107 Human β-defensin, produced by keratinocytes and induced to release prostaglandins following skin damage, has demonstrated potent in vitro efficacy against VACV.108

Traditional Chinese Medicine

The drugs currently under development are largely chemically synthesized substances or natural drug monomers. Concurrently, the investigation of therapeutic approaches derived from traditional medical systems is increasingly recognized as a prominent area of research. As treasures of TCM, Chinese medicines have held a significant position throughout thousands of years of history. Since the beginning of disease, people have tried to treat illnesses and alleviate symptoms with herbal medicines. Chinese herbal medicine has played a long-standing role in preventing and treating epidemics and poxvirus infections throughout Chinese history, possessing a rich theoretical and practical foundation. The earliest documented accounts of infectious diseases resembling pox can be found in the ancient Chinese medical treatise Huangdi Neijing, which dates back more than two millennia. This text provides the earliest documented nomenclature and pathogenesis of infectious pox-like viral diseases, attributing urticaria to yin energy obstructing the cutaneous channels. These classical theories laid the foundation for developing therapeutic strategies against contagious poxviral infections.109 Currently, researchers are ancient classical literature, screening TCM databases, and using techniques such as network pharmacology to find herbal remedies that may target MPXV. Numerous experimental methods have been used to verify their clinical usefulness; the chemicals and principles derived from TCM are gaining attention as potential lead structures for the design of specific and effective medications targeting a range of medical conditions.

Specifically, the rich experience of traditional Chinese medicine in the prevention and treatment of smallpox provides important insights for contemporary research. MPXV shares this taxonomic classification with VARV as an orthopoxvirus. With declining smallpox vaccination rates, the susceptibility to poxvirus infections like MPXV has increased. Researchers are urgently seeking effective components for mpox therapy, including components from traditional Chinese herbs, while accumulated experience with formulas targeting VARV can inform modern combo treatments. A total of 2,344 formulas were compiled from the LTM-TCM database as well as 12 classical Chinese medical texts. The data analysis identifies several promising herbal medicines and bioactive compounds that may target infection. Based on relative citation frequency (RFC ≥ 0.10), researchers identified 19 herbs frequently employed in the management of poxvirus infections, along with 64 related biological functional modules. At the same time, Researchers selected compounds that met the ADME criteria (oral bioavailability > 30%, pharmacophore score > 0.18) and appeared frequently in at least five herbal medicines as the selection criteria. 29 lead compounds exhibiting anti-inflammatory, antibacterial, as well as antiviral activities were screened and identified. Both the plants and these lead compounds show promising potential for the treatment of poxvirus infections. Although the use of TCM is mostly empirical and unvalidated, researchers think that most formulas for treating poxviruses share a typical biological process. Docking analysis indicates that these Chinese herbs and their active ingredients collectively target CD4+ T cell regulatory pathways to enhance immune responses, thereby obstructing key phases of the viral replication process, such as DNA synthesis, RNA capping, and the assembly of mature virions. Among the 19 commonly used herbs, GanCao was the most frequently employed, while RenShen, DangGui, FangFeng, JieGeng, NiuBangZi, and HuangQin were also frequently used to treat ulcers. The 29 high-frequency active substances primarily exhibited multifaceted activities in anti-inflammatory, anticancer, antibacterial, immunomodulatory, and antioxidant effects.110

Given the multi-target characteristics of the aforementioned active ingredients, researchers further explored traditional Chinese medicine treatment protocols targeting specific symptoms of mpox. Up to now, treatment for mpox has large