It is suggested that some important components of the cellular membrane might be involved in the initial binding of the poxvirus membrane to the host cells, such as glycosaminoglycans (including heparan sulfate and chondroitin), and laminin (Moss, 2012). On the other hand, there is evidence of important vaccinia virus proteins, encompassing D8 (Matho et al., 2018), A27 (Chung et al., 1998), A34 (Blasco et al., 1993), A26 (Chiu et al., 2007), and H3 (Lin et al., 2000) that entail binding to the host cell membrane. Other transmembrane proteins, including L1 and A27, participate in hemifusion (Duncan and Smith, 1992).

The viral core, released into the cytoplasm, contains enzymes responsible for initiating a series of events that lead to transcription (Rosales et al., 1994), DNA replication, and protein synthesis. Infectious poxvirus particles contain a transcriptional system including early, intermediate, and late genes (Knipe and Howley, 2007).

Following the entry into the cytoplasm, the DNA-dependent RNA polymerase, present in the viral core, activates the transcription of early mRNA that is translated into a variety of proteins, including factors necessary for the synthesis of viral DNA and the transcription of intermediate genes. With the disruption of the viral core, the expression of early genes ceases, whereas DNA replication progresses to form concatemeric molecules. The progeny of DNA activates the transcription of intermediate genes in intermediate mRNA, which are translated into enzymes and factors for the late gene expression. The final products of late genes include virion structural proteins, enzymes and early transcription factors. The formation of membrane structures and viral genome, that derived from the segregation of concatemeric DNA intermediate, are assembled and packaged in immature virions (IV) and then into the intracellular mature virions (MV), the first infectious form.

In the cytoplasm, MVs may behave in different ways: i) they remain free in cytoplasm; ii) they move to the cell surface for exocytosis through microvilli; iii) they acquire a second envelope from the trans-Golgi network to form wrapped virions (WVs), which bud from the host cell as extracellular enveloped virions (EVs) (Roberts and Smith, 2008).

MVs are more abundant than EVs and are thought to be responsible for the transmission between hosts, whereas EVs facilitate virus dissemination within an infected host (Schmidt et al., 2012).

The outer envelope of EVs presents specific transmembrane surface proteins, that include A33 (Roper et al., 1996), A34, A36 (Parkinson and Smith, 1994), A56 (Shida, 1986), F13 (Moss, 2011) and B5 (Yu et al., 2021), which are important for infectivity and cell-to-cell spread. In particular, A33, A36, B5, and A34 contribute to the formation of actin tails and microvilli, which are essential for viral dissemination among cells.

Once infected, the innate immune system activates different signaling pathways: (TLR)-dependent and (TLR)-independent toll-like receptors, various host pattern recognition receptors (PRRs), including DNA and RNA sensors, and inflammasome components to detect the virus. They are triggered by the expression of antiviral responses (Yu et al., 2021). Poxviruses have several immune evasion mechanisms, such as the synthesis of a variety of immunomodulatory proteins, that interfere with the antiviral defenses, triggered by the host pattern recognition receptors (PRRs).

Infections by poxviruses manifest a broad spectrum of pathogenicity. The infection can be either localized, self-limited in the skin, or systemic, and characterized by a generalized rash. The rash progresses through different stages such as macules, papules, vesicles, pustules and scabs (Obermeier et al., 2024).

The type of infection is dependent on the species of poxviruses, the route of entry, the genus/species of the susceptible animal and its immune status (Knipe and Howley, 2007). Although it is difficult to understand the natural pathogenesis due to the lack of data on the disease pathogenesis in its natural host, there is clinical evidence from animal models, such as ectromelia infection in mice and MPOX infection in non-human primates, that depicts the specifics of human pathogenesis (Knipe and Howley, 2007). Pathogenesis is divided into two phases: primary viremia and prodromal stage (Kumar et al., 2022). Primary viremia starts with the accumulation and replication of the virus in the primary inoculation site, including the nasopharyngeal, oropharyngeal, and the intradermal region. Then, the virus disseminates to all the lymph nodes and the secondary organs, where it subsequently replicates. When the virus ultimately reaches the skin and the tertiary organs, the prodromal stage (or secondary viremia) starts (Majie et al., 2023).

Poxviruses’ transmission may occur through i) skin lesions (cuts, abrasions, or bites) of infected animals; ii) body fluids or respiratory droplets of infected individuals; iii) ingestion of contaminated food or water; iv) contaminated objects; or iv) cannibalism (Buller and Palumbo, 1991; Grant et al., 2020; Vivancos et al., 2022).

Among human poxvirus infections, only smallpox is not a zoonotic one. Zoonotic infections may spread as a primary transmission (from animal to human) or as a secondary transmission (from human to human). Transmission by infected animals to humans occurs through animal body secretions or animal bites. Transmission from human to human occurs mainly through the inhalation of large airborne respiratory droplets from infected persons, especially during prolonged face-to-face exposure or close contact. Transmission via contaminated objects (fomites) in household and healthcare settings or contact with infectious lesions from the rash or scab may also occur (Vivancos et al., 2022).

In recent MPOX outbreaks, it was observed that most confirmed cases were linked to unprotected intercourses and anogenital lesions. Although MPOX is not classified as a sexually transmitted infection, evidence suggests that direct sexual contact is a primary route of acquisition.

Multiple studies demonstrated that up to 95% of reported cases are middle-aged males, particularly among men who have sex with men (MSM) (Bhat et al., 2023; Kumar et al., 2025). A disproportionate gender distribution of patients was also observed. However, it remains unclear whether the virus may infect sperm cells or reproduce in the genital canal (Kumar et al., 2025).

Although there are very limited data on MPOX infection during pregnancy, a few studies described fetal infection with MPXV in one out of four infected pregnant women had fetal death, of which two delivered miscarriages in their first trimester, and one delivered healthy baby (Mbala et al., 2017), thus, demonstrating the existence of vertical transmission of MPXV (Fahrni and Choudhary, 2022).

The most frequent human-to-human transmission of MPXV occurred in 99% of cases through the close contact among adult men, 94% of reported cases being due to male-to-male sexual contact or intimate contact 3 weeks before the symptoms of infection (Parums, 2024). These data suggested that sexual contact may be a novel route of transmission, even though MPOX was not considered a sexually transmitted disease before (Sadeghpour et al., 2021).

Although the MPXV genome has a low mutation rate (Isidro et al., 2022), phylogenetic analysis indicates that the 2022 strain depicts an ongoing viral evolution from the 2017–2018 Nigeria outbreak. A means of 50 single-nucleotide polymorphisms (SNPs) has been identified, some of which may have been triggered by apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) enzymes. APOBEC3 belongs to the family of cytidine deaminases and it is an essential component of the innate immune system. These enzymes can introduce mutations into viral DNA through deaminase and deaminase-independent mechanisms. In some circumstances, APOBEC3 might not completely inactivate the viruses, and mutated variants can acquire mechanisms that escape from the immune system. The precise role of APOBEC3 in the introduction of these mutations and its contribution to the evolution of virus remains to be fully elucidated.

The roots of smallpox vaccination were found in the variolation practices from the 10th to the 18th centuries to control disease, including intranasally insufflation or cutaneous inoculation of smallpox material scabs.

This precursor methodology led to the term of “vaccination” and to the development of the first smallpox vaccine, pioneered by Edward Jenner in 1798, that introduced a safer and more reliable alternative to variolation using cowpox material. This represented the initial use of poxviruses as vaccines.

In the subsequent years of the Jennerian vaccine, smallpox vaccines did not use CPXV, but other OPXVs, which for a long time have been known as “vaccine virus.” The origins of VACV are unknown. VACV might have been the hybrid result between CPXV and VARV.

The first-generation smallpox vaccines were based on live, non-attenuated VACV strains and production systems. The most common VACV strains used worldwide include the New York City Board of Health (NYCBH) (North America and West Africa), Lister (UK), EM-63 (Russia and India) and TianTan (also known as Temple of Heaven, China), usually administered percutaneously, by scarification with a bifurcated needle, or with a jet injector (Moss, 2011).

There was a steady improvement in the methods of preparing vaccines. Early smallpox vaccine manufacturing introduced the “vaccine farms,” that were rapidly adopted around the world and were considered a safe method at the time (Esparza et al., 2020). It consisted in the propagation and harvesting of VACV strains from the skin of live animals (e.g., calf, sheep, water buffalo). Based on WHO requirements, other production methods were applied such as lyophilization and dried frozen vaccine batches to provide a better efficacy and safety profile (Kmiec and Kirchhoff, 2022). This contributed significantly to the success of smallpox eradication.

Although the first-generation vaccines were used for the eradication of the smallpox, significant adverse events were reported, particularly in individuals with immune deficiency, such as eczema vaccinatum, progressive vaccinia, and post-vaccinal encephalitis (Fulginiti et al., 2003).

To reduce adverse events, a second-generation of live vaccines were designed, that were produced in tissue cultures such as rabbit kidney (RK) cells, chick embryo fibroblasts (CEF), human MRC-5 lung cells, and African green monkey kidney epithelial cells (Vero). There are two VACV strains used in the second-generation vaccines, such as Lister (used for the production of RIVM, Lister, and Elstree-Bavarian-Nordic vaccines) and NYCBH (used for the production of ACAM2000 and CJ-5030022 vaccines) (Monath et al., 2004).

In 2007, the Food and Drug Administration (FDA) approved the use of the ACAM2000 vaccine for over 18-year-old healthy people that were at high-risk for smallpox infection (Food and Drug Administration US, 2007). ACAM2000 is under surveillance of Expanded Access Investigational New Drug (EN-IND) application for MPOX prevention (Sehulster and Chinn, 2003).

Although they have a better safety profile compared to the first-generation vaccines, they still carry the risk of serious adverse effects. Given that ACAM-2000 is a live VACV-based vaccine, it is contraindicated in individuals with eczema, human immunodeficiency virus (HIV) infection, cardiovascular diseases, pregnant women, and other specific conditions (Frey et al., 2009). Clinical data have observed that ACAM2000 can cause myocarditis and/or pericarditis, with an average of 5.7 cases per 1,000 primary vaccinees. Attenuated vaccines were thus developed, sustaining immunogenicity and protective effectiveness (Jacobs et al., 2009).

In contrast with live VACV-based vaccines used in the first and second generation, the third-generation vaccines were developed through classical attenuation after multiple passages of VACV strains in specific tissue cultures. During this process, gene deletions and mutations were introduced into the virus genome, that resulted in the attenuation of the virus pathogenicity, which could not replicate in human cells anymore (Perdiguero et al., 2023). There are four VACV strains used in the third-generation vaccines, such as Ankara, Copenhagen, Lister, and NYCBH. Among these, two of them represent the most important vaccines, that include MVA-BN (Modified Vaccinia Ankara - Bavarian Nordic, that used the Ankara strain) and the LC16m8 (that used the Lister strain) (Paran and Sutter, 2009) both approved for smallpox and MPOX in high-risk groups.

According to clinical data, the third-generation vaccines, and in particular the MVA vaccine, are safer and suitable for a broader range of individuals, including immune-compromised subjects (Volz and Sutter, 2017).

Because the LC16m8 vaccine is partially replicating, it is contraindicated for immune-suppressed individuals or during pregnancy (Gubser et al., 2004).

The history of the generation of the different poxvirus-based vaccines is illustrated in Figure 3.

As WHO has recently declared MPOX a Public Health Emergency, several organisations, such as the U.S. Center for Disease Control and Prevention (CDC), Food and Drug Administration (FDA), European Medicines Agency (EMA), and Japanese KM Biologics, have adopted several mitigation approaches to control and prevent the current and future MPOX outbreak.

CDC recommends two doses of the JYNNEOS vaccine as the “best protection” against MPXV (Prevention US C.D.C, 2024). Meanwhile, Food and Drug Administration US (2024) expanded the approval of the ACAM2000 vaccine to individuals at high risk of MPOX infection. European Medicines Agency (2024) has recently recommended the vaccination of adolescents aged from 12 to 17 with IMVANEX. Although LC16, produced by Japanese KM Biologics is not yet internationally commercialized, a single dose of the vaccine has been approved domestically in Japan and in the DRC since June 2024 (Organization, 2024e) ⁵ .

From September 2023 to November 2024, 1,137,000 doses of the MVA-BN vaccine were allocated to nine African countries by the Access and Allocation Mechanism, involving African CDC, Coalition for Epidemic Preparedness Innovations, Gavi, Vaccine Alliance, UNICEF and WHO (MPOX report, 2024) ^{6, 7} .

High vaccination rates lead to successful vaccination campaigns and effective control of infection. Factors that lead to successful vaccination campaigns are not only related to the development of safe and effective vaccines, but they had to ensure logistical issues and an equitable distribution (Gates, 2022).

A meta-analysis, including 61 studies with 263,857 participants from 87 countries, evaluated the prevalence of MPOX vaccine acceptance and uptake (Sulaiman et al., 2024). The overall global rate of intention to vaccinate against MPOX was 60.9%, and there was a substantial variation observed across the six WHO regions. The highest rate of intention to vaccinate was in the Western Pacific Region at 73.5% (95% CI, 63.0%–82.9%), whereas the African Region had the lowest rate at 41.9% (95% CI, 36.6%–47.4%).

Obstacles preventing the global distribution of MPOX vaccines include their high cost, low availability, and poor accessibility. Logistical obstacles impede the global use of the MPOX vaccine since, in nonendemic regions, there is a limited availability and accessibility, whereas in higher-income countries stockpiles of millions of doses are available. Although in 2024 African countries accounted for more than 95% of all cases and deaths due to MPOX, few vaccines were available for prevention. Vaccine equity is critical to the fight against MPOX, and it necessitates collaboration among the different stakeholders (Shafaati et al., 2025).

Vaccines against MPOX may be administered to individuals at high risk as primary preventive vaccination before exposure or as post-exposure vaccination after contacts with an MPOX case (European Center D.P.C, 2025) ⁸ . A meta-analysis, including 61 studies with 263,857 participants from 87 countries reported a prevalence of MPOX vaccine uptake among people living with HIV (35.7%) compared to the general public (20.2%) and among the LGBTQI+ community (39.8%) (Sulaiman et al., 2024). The higher rates of acceptance and uptake observed among the LGBTQI+ community (73.6% and 39.8%) may indicate the group’s higher risk perception, which plays a high role in vaccine acceptance. Among the healthcare workers, the prevalence of intention to be vaccinated against MPOX (51.9%) is comparable to the acceptance rate of the COVID-19 vaccine (55.9%–65.7%) (Sulaiman et al., 2024).

The effective control of the MPOX outbreak requires widespread acceptance and uptake of the vaccine, especially among high-risk groups such as the LGBTQI+ community and frontline healthcare workers. The outbreak of MPOX occurred at a time when global vaccine hesitancy was at an all-time high levels period (Bergen et al., 2023). Vaccine hesitancy, defined by WHO as “a delay in the acceptance or refusal of vaccination despite the availability of vaccination services”, jeopardizes the success of vaccination programs (MacDonald and SAGE Working Group on Vaccine Hesitancy, 2015). Several studies have attempted to identify key determinants of intention and hesitancy to vaccinate against MPOX.

High-risk groups and people with poor vaccination histories should be targeted. People living with HIV, men who have sex with men (MSM), and people with pre-exposure prophylaxis may need a priority status for the MPOX vaccine. In a survey conducted in France, among MSM persons living with HIV or with pre-exposure prophylaxis, 52 out of 155 (33.6%) of the participants declared to be hesitant about MPOX vaccination (Zucman et al., 2022). Factors associated with the acceptance behavior were subjects with an increased number of sexual partners during the previous months, the fear about MPXV infection or the endorsement of mandatory COVID-19 vaccination in high-risk groups (Zucman et al., 2022). Participants declaring that the smallpox vaccine should be compulsory for people at risk were significantly associated with higher acceptance of MPXV vaccination (p < 0.001) (Zucman et al., 2022).

Another study conducted in Africa revealed that individuals who have never been vaccinated against other diseases were at a higher risk of MPOX vaccination hesitancy for themselves and for their children, despite the vaccine being available (Du et al., 2025).

A meta-analysis aiming at evaluating the rate of MPOX vaccine acceptance and uptake globally found that age, sex, level of education, and level of income are among the most reported sociodemographic determinants of MPOX vaccine acceptance (Sulaiman et al., 2024).

Therefore, public health intervention programs should consider these sociodemographic characteristics to design tailored strategies to address vaccine hesitancy and optimize the uptake rates. Modifiable behavioral factors associated with MPOX vaccine acceptance, such as concerns about the disease, the perception of risk, and knowledge about MPOX, should be considered to increase knowledge and awareness, especially among high-risk groups such as the LGBTQI+ community.

The source of information is another important factor to be considered when implementing strategies to combat misinformation. Studies have found that “infodemic”, defined by WHO as “too much information, including false or misleading information in digital and physical environments during a disease outbreak” was strongly linked to vaccine hesitancy (Pierri et al., 2022). Interventions should aim at increasing the public’s positive perception of MPOX vaccines and encourage them to get information from reliable sources like health institutions.

Engaging health professionals and institutions in media campaigns has been recommended as a potent method to face the problem (Shasha et al., 2022). It is important to address vaccine hesitancy through health education programs, especially in risk populations, to promote MPOX vaccine uptake. A meta-analysis involving 16 studies with 9,066 participants reported poor knowledge about MPOX vaccines in 53.4% of them (Tanashat et al., 2024).

Tailored long-term public messages targeting specific groups should be structured by health authorities. Healthcare professionals should increase their knowledge and awareness about MPOX vaccines. Trust in the safety and effectiveness of vaccines highly influences the decision to get vaccinated.

Clinical development of ACAM 2000, MVA-BN, and LC16m8 was initiated and progressed as a measure against the potential use of smallpox as a biological weapon (Nalca and Zumbrun, 2010; Grabenstein and Hacker, 2024). At that time, MPOX was rare and unpredictable, making it difficult to conduct clinical trials.

In the last two decades, multiple studies have evaluated the safety of these vaccines in human volunteers through clinical trials. These studies were in line with the current clinical guidelines, with sample sizes ranging from several dozen to several hundred participants (Volz and Sutter, 2013; von Krempelhuber et al., 2010; Pittman et al., 2019; Morino et al., 2024).

A multicenter randomized controlled study testing ACAM2000 has concluded that this vaccine is effective and suitable as a booster dose for individuals previously vaccinated against smallpox (Food and Drug Administration US, 2022). LC16m8 has demonstrated potential in animal models, but further research is necessary to determine its long-lasting protection (Saijo et al., 2006; Hirani et al., 2023). In a study by Okumura et al. (2024) 1,006 adults were vaccinated with LC16m8, including those living with HIV (346 individuals), with an acceptance rate of 90.3% in the HIV group and 94.6% in the uninfected group. None of the participants developed MPOX, and vaccine’s efficacy could not be measured. There is also an ongoing clinical trial conducted with LC16m8 in Colombia, but the results are not yet available (Tomotsugu et al., 2025). A total of twenty-two clinical trials established the strong efficacy and safety of the MVA-BN vaccine, as it ensured protection of individuals of various age groups from MPOX infection (Stittelaar et al., 2005; von Krempelhuber et al., 2010).

An analysis of adverse events, reported from the surveillance systems of America, Australia, Canada, and the Netherlands, revealed no unexpected adverse events following immunization related to MVA-BN vaccine (Muller et al., 2024; Duffy et al., 2022; van der Boom and van Hunsel, 2023).

JYNNEOS' safety was evaluated in more than 7,800 individuals (Organization, 2023) ⁹ . A case-control study estimated the vaccine’s effectiveness as 36% after a single dose and 66% after two doses (Deputy et al., 2023). According to another study in the German population, a single dose of the MVA-BN vaccine’s effectiveness was 84.1% in people without HIV compared to 34.9% in people living with HIV (PLHIV) (Hillus et al., 2025).

In a cohort of 849 healthcare workers from the DRC, who were vaccinated with two doses of MVA-BN, most of Congolese participants remained seropositive (Priyamvada et al., 2022) 2 years after the vaccination. A study by Berry et al. (2024) estimated that the vaccine’s effectiveness remains >59% for up to 10 years, even after a single dose. MVA-BN is well tolerated in children, as none of the 87 English children (median age, 5 years old), who received one MVA-BN dose in a study conducted from 1 June to 30 November 2022, experienced serious adverse events nor developed MPOX disease after vaccination. Among survey respondents, 36% reported no symptoms, 40% reported only injection-site reactions, and 24% experienced systemic symptoms with or without local reactions (Ladhani et al., 2023). Furthermore, in October 2024, Bavarian Nordic announced a clinical trial to expand the vaccine’s approval to children aged from 2 to 11 (Bavarian Nordic, 2024).

In a pivotal Phase 3 randomized clinical trial, MVA-BN showed a more favorable safety profile compared to ACAM 2000 (Pittman et al., 2019). Nevertheless, a single dose of MVA-BN was shown to induce low titers of VACV-specific and MPVX-specific neutralizing antibody titers compared to a single dose of ACAM 2000 (Mason et al., 2024). However, the vaccine’s efficacy against MPOX infections for one dose of MVA-BN suggests high effectiveness.

A recent study suggests that smallpox vaccination does not provide full protection against MPOX during the ongoing outbreak (Clinical Trials, 2024), and ACAM2000 has been linked to significant adverse events, underscoring the need for the development of MPOX-specific vaccines (EPAR, 2024). The clinical studies related to MPOX vaccines are summarized and illustrated in Table 3.