Exploring computational approaches to design mRNA Vaccine against vaccinia and Mpox viruses.

BACKGROUND: Messenger RNA (mRNA) vaccines emerged as a powerful tool in the fight against infections. Unlike traditional vaccines, this unique type of vaccine elicits robust and persistent innate and humoral immune response with a unique host cell-mediated pathogen gene expression and antigen presentation. METHODS: This offers a novel approach to combat poxviridae infections. From the genome of vaccinia and Mpox viruses, three key genes (E8L, E7R, and H3L) responsible for virus attachment and virulence were selected and employed for designing the candidate mRNA vaccine against vaccinia and Mpox viral infection. Various bioinformatics tools were employed to generate (B cell, CTL, and HTL) epitopes, of which 28 antigenic and immunogenic epitopes were selected and are linked to form the mRNA vaccine construct. Additional components, including a 5' cap, 5' UTR, adjuvant, 3' UTR, and poly(A) tail, were incorporated to enhance stability and effectiveness. Safety measures such as testing for human homology and in silico immune simulations were implemented to avoid autoimmunity and to mimics the immune response of human host to the designed mRNA vaccine, respectively. The mRNA vaccine's binding affinity was evaluated by docking it with TLR-2, TLR-3, TLR-4, and TLR-9 receptors which are subsequently followed by molecular dynamics simulations for the highest binding one to predict the stability of the binding complex. RESULTS: With a 73% population coverage, the mRNA vaccine looks promising, boasting a molecular weight of 198 kDa and a molecular formula of C8901H13609N2431O2611S48 and it is said to be antigenic, nontoxic and nonallergic, making it safe and effective in preventing infections with Mpox and vaccinia viruses, in comparison with other insilico-designed vaccine for vaccinia and Mpox viruses. CONCLUSIONS: However, further validation through in vivo and in vitro techniques is underway to fully assess its potential.

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Description of Sheep Pox Outbreak in Spain in 2022-2023: Challenges Found and Lessons Learnt in Relation with Control and Eradication of This Disease.

Sheep pox and goat pox are infectious viral diseases that affect ovine and caprine animals and are caused by two viruses of the family Poxviridae, genus Capripoxvirus. Sheep pox has been traditionally endemic in Africa, the Middle East, and several Southeast Asian countries, but it is considered a transboundary disease capable of affecting previously free countries epidemically. It is a disease of compulsory immediate notification to the World Organization for Animal Health (WOAH) and the European Union (EU). On 19 September 2022, the disease reemerged in Spain, which had been free of it since 1968, causing a total of 30 outbreaks until 17 May 2023, when the last outbreak of the disease was reported. The control and eradication measures implemented were those laid down in EU legislation, based on the total stamping out of positive herds, zoning and restriction of movement, and strengthening of biosecurity and passive surveillance. This manuscript describes the outbreak, as well as assesses the challenges and lessons learned in relation to its management, with the aim of helping in the effective management of future outbreaks of this disease.

Formulation of next-generation polyvalent vaccine candidates against three important poxviruses by targeting DNA-dependent RNA polymerase using an integrated immunoinformatics and molecular modeling approach.

BACKGROUND: Poxviruses comprise a group of large double-stranded DNA viruses and are known to cause diseases in humans, livestock animals, and other animal species. The Mpox virus (MPXV; formerly Monkeypox), variola virus (VARV), and volepox virus (VPXV) are among the prevalent poxviruses of the Orthopoxviridae genera. The ongoing Mpox infectious disease pandemic caused by the Mpox virus has had a major impact on public health across the globe. To date, only limited repurposed antivirals and vaccines are available for the effective treatment of Mpox and other poxviruses that cause contagious diseases. METHODS: The present study was conducted with the primary goal of formulating multi-epitope vaccines against three evolutionary closed poxviruses i.e., MPXV, VARV, and VPXV using an integrated immunoinformatics and molecular modeling approach. DNA-dependent RNA polymerase (DdRp), a potential vaccine target of poxviruses, has been used to determine immunodominant B and T-cell epitopes followed by interactions analysis with Toll-like receptor 2 at the atomic level. RESULTS: Three multi-epitope vaccine constructs, namely DdRp_MPXV (V1), DdRp_VARV (V2), and DdRp_VPXV (V3) were designed. These vaccine constructs were found to be antigenic, non-allergenic, non-toxic, and soluble with desired physicochemical properties. Protein-protein docking and interaction profiling analysis depicts a strong binding pattern between the targeted immune receptor TLR2 and the structural models of the designed vaccine constructs, and manifested a number of biochemical bonds (hydrogen bonds, salt bridges, and non-bonded contacts). State-of-the-art all-atoms molecular dynamics simulations revealed highly stable interactions of vaccine constructs with TLR2 at the atomic level throughout the simulations on 300 nanoseconds. Additionally, the outcome of the immune simulation analysis suggested that designed vaccines have the potential to induce protective immunity against targeted poxviruses. CONCLUSIONS: Taken together, formulated next-generation polyvalent vaccines were found to have good efficacy against closely related poxviruses (MPXV, VARV, and VPXV) as demonstrated by our extensive immunoinformatics and molecular modeling evaluations; however, further experimental investigations are still needed.

Novel strategy for Poxviridae prevention: Thermostable combined subunit vaccine patch with intense immune response.

Poxviruses gained international attention due to the sharp rise in monkeypox cases in recent years, highlighting the urgent need for the development of a secure and reliable vaccine. This study involved the development of an innovative combined subunit vaccine (CSV) targeting poxviruses, with lumpy skin disease virus (LSDV) serving as the model virus. To this end, the potential sites for poxvirus vaccines were fully evaluated to develop and purify four recombinant proteins. These proteins were then successfully delivered to the dermis in a mouse model by utilizing dissolvable microneedle patches (DMPs). This approach simplified the vaccination procedure and significantly mitigated the associated risk. CSV-loaded DMPs contained four recombinant proteins and a novel adjuvant, CpG, which allowed DMPs to elicit the same intensity of humoral and cellular immunity as subcutaneous injection. Following immunization with SC and DMP, the mice exhibited notable levels of neutralizing antibodies, albeit at a low concentration. It is noteworthy that the CSV loaded into DMPs remained stable for at least 4 months at room temperature, effectively addressing the storage and transportation challenges. Based on the study findings, CSV-loaded DMPs are expected to be utilized worldwide as an innovative technique for poxvirus inoculation, especially in underdeveloped regions. This novel strategy is crucial for the development of future poxvirus vaccines.

Molecular Virology of Orthopoxviruses with Special Reference to Monkeypox Virus.

Poxviruses are large (200-450 nm) and enveloped viruses carrying double-stranded DNA genome with an epidermal cell-specific adaptation. The genus Orthopoxvirus within Poxviridae family constitutes several medically and veterinary important viruses including variola (smallpox), vaccinia, monkeypox virus (MPXV), and cowpox. The monkeypox disease (mpox) has recently emerged as a public health emergency caused by MPXV. An increasing number of human cases of MPXV have been documented in non-endemic nations without any known history of contact with animals brought in from endemic and enzootic regions, nor have they involved travel to an area where the virus was typically prevalent. Here, we review the MPXV replication, virus pathobiology, mechanism of viral infection transmission, virus evasion the host innate immunity and antiviral therapies against Mpox. Moreover, preventive measures including vaccination were discussed and concluded that cross-protection against MPXV may be possible using antibodies that are directed against an Orthopoxvirus. Despite the lack of a specialised antiviral medication, several compounds such as Cidofovir and Ribavirin warrant consideration against mpox.

Poxvirus Vaccines: Past, Present, and Future.

Smallpox was a significant cause of mortality for over three thousand years, amounting to 10% of deaths yearly. Edward Jenner discovered smallpox vaccination in 1796, which rapidly became a smallpox infection preventive practice throughout the world and eradicated smallpox infection by 1980. After smallpox eradication, monkeypox vaccines have been used primarily in research and in outbreaks in Africa, where the disease is endemic. In the present, the vaccines are being used for people who work with animals or in high-risk areas, as well as for healthcare workers treating patients with monkeypox. Among all orthopoxviruses (OPXV), monkeypox viral (MPXV) infection occurs mainly in cynomolgus monkeys, natural reservoirs, and occasionally causes severe multi-organ infection in humans, who were the incidental hosts. The first case of the present epidemic of MXPV was identified on May 7, 2022, and rapidly increased the number of cases. In this regard, the WHO declared the outbreak, an international public health emergency on July 23, 2022. The first monkeypox vaccine was developed in the 1960s by the US Army and was based on the vaccinia virus, which is also used in smallpox vaccines. In recent years, newer monkeypox vaccines have been developed based on other viruses such as Modified Vaccinia Ankara (MVA). These newer vaccines are safer and can provide longer-lasting immunity with fewer side effects. For the future, there is ongoing research to improve the current vaccines and to develop new ones. One notable advance has been the development of a recombinant vaccine that uses a genetically modified vaccinia virus to express monkeypox antigens. This vaccine has shown promising results in pre-clinical trials and is currently undergoing further testing in clinical trials. Another recent development has been the use of a DNA vaccine, which delivers genetic material encoding monkeypox antigens directly into cells. This type of vaccine has shown effectiveness in animal studies and is also undergoing clinical testing in humans. Overall, these recent advances in monkeypox vaccine development hold promise for protecting individuals against this potentially serious disease.

Poxviruses as Agents of Biological Warfare: The Importance of Ensuring Ethical Standards for Research with Viruses.

Historically, biological agents have been used to target various populations. One of the earliest examples could be the catastrophic effect of smallpox in Australia in the eighteenth century (as alleged by some historians). Modern biological techniques can be used to both create or provide protection against various agents of biological warfare. Any microorganism (viruses, bacteria, and fungi) or its toxins can be used as biological agents. Minnesota Department of Health has listed Smallpox (variola major) as a category A bioterrorism agent, even though it has been eradicated in 1980 through an extensive vaccination campaign. Category A agents are considered the highest risk to public health. Laboratory-associated outbreaks of poxviruses could cause unprecedented occupational hazards. Only two WHO-approved BSL-4 facilities in the United States and Russia are allowed to perform research on the variola virus. So, poxviruses present themselves as a classical case of a dual-use dilemma, since research with them can be used for both beneficial and harmful purposes. Although the importance of ethics in scientific research requires no further elaboration, ethical norms assume greater significance during experimentation with poxviruses. In this chapter, we will update the readers on the sensitive nature of conducting research with poxviruses, and how these viruses can be a source of potential biological weapons. Finally, specified ethical guidelines are explored to ensure safe research practices in virology.

Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections.

The recent outbreak of mpox epidemic, caused by monkeypox virus (MPXV), poses a new threat to global public health. Here, we initially assessed the preexisting antibody level to the MPXV B6 protein in vaccinia vaccinees born before the end of the immunization program and then identified two monoclonal antibodies (MAbs), hMB621 and hMB668, targeting distinct epitopes on B6, from one vaccinee. Binding assays demonstrate that both MAbs exhibit broad binding abilities to B6 and its orthologs in vaccinia (VACV), variola (VARV) and cowpox viruses (CPXV). Neutralizing assays reveal that the two MAbs showed potent neutralization against VACV. Animal experiments using a BALB/c female mouse model indicate that the two MAbs showed effective protection against VACV via intraperitoneal injection. Additionally, we determined the complex structure of B6 and hMB668, revealing the structural feature of B6 and the epitope of hMB668. Collectively, our study provides two promising antibody candidates for the treatment of orthopoxvirus infections, including mpox.

Safety and Efficacy of the Modified Vaccinia Ankara-Bavaria Nordic Vaccine Against Mpox in the Real World: Systematic Review and Meta-Analysis.

In May 2022, mpox began to spread worldwide, posing a serious threat to human public health. Modified Vaccinia Ankara-Bavaria Nordic (MVA-BN) is a live attenuated orthopoxvirus vaccine that has been authorized by the U.S. Food and Drug Administration as the vaccine of choice for the prevention of mpox. In this study, we conducted a meta-analysis of all currently published literature on the efficacy and safety of the MVA-BN vaccine in the real world, showing that the MVA-BN vaccine is effective and safe, with efficacy of up to 75% with a single dose and up to 80% with a two-dose vaccine. Meanwhile, we found that subcutaneous injection has lower local and systemic adverse events than intradermal injection, regardless of single- or two-dose vaccination, and subcutaneous injection is better tolerated in children, the elderly, or people with underlying medical conditions. These results have important reference value for clinical practice.

Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection.

The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses.

Orthopox viruses: is the threat growing?

BACKGROUND: Smallpox was a major cause of human mortality until its eradication, but the threat of orthopox viruses has not disappeared. Since the eradication of smallpox and the cessation of the related vaccination campaigns, the threat has been growing, as evidenced by the currently ongoing worldwide Mpox outbreak. In addition to threats of an evolving Mpox, we must also be aware of a myriad of other threats that remain. Many countries still lack biosecurity regulations reflecting the recent technological advances, and the threat of bioterrorism remains ever present. Reconstruction of smallpox is a distinct possibility, as are other scenarios whereby other orthopox viruses may be made more fit for transmission in humans. OBJECTIVES: To outline and discuss potential biosafety and biosecurity threats posed by orthopox viruses. SOURCES: Published scientific literature, news articles, and international agreements. CONTENT AND IMPLICATIONS: It would be wise to take steps to mitigate these threats now. Vaccination campaigns should be considered in areas with frequent orthopox outbreaks, and more efforts must be made to put a final end to the Mpox outbreak. In many countries, national biosafety and biosecurity regulations may need to be revised and strengthened to better reflect the threats posed by new technologies, including controls on synthesis of smallpox sequences. Furthermore, more international cooperation and aid is needed. The present global Mpox outbreak could likely have been prevented had areas where Mpox is endemic not been neglected. Future outbreaks could be much worse.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Percepción del riesgo en el proceso de producción de vacunas aviares / Risk perception in the avian vaccine production process

La importancia que tienen para la avicultura cubana el virus de la enfermedad infecciosa de la bolsa (Gumboro) y el virus de la viruela aviar, así como la producción de vacunas que permitan controlar las enfermedades producidas por estos agentes biológicos, justifican la necesidad del establecimiento de una buena gestión de la bioseguridad, ya que el desconocimiento de los peligros y riesgos del personal que labora en estas vacunas puede provocar accidentes de consecuencias indeseables para el producto, escapes de estos microorganismos durante sus procesos productivos y la consecuente contaminación del medio ambiente. El objetivo de la presente investigación fue realizar un análisis de la percepción de riesgo existente en el personal responsable del proceso de producción de dos vacunas aviares. Para ello se utilizó el software RISKPERCEP en una instalación de producción de vacunas aviares; su aplicación mostró variables que demostraron subestimación del riesgo por el personal expuesto y variables con tendencia a la sobrestimación, asociadas fundamentalmente al incorrecto diseño de la instalación. Se concluye que existe la necesidad de una buena capacitación y que se impartan cursos de actualización de bioseguridad donde se tengan en cuenta todos los aspectos del diseño del laboratorio que puedan solucionarse(AU)

The importance of infectious bursal disease virus and fowl pox virus for Cuban poultry farming, as well as the production of vaccines to control the diseases caused by these biological agents, justifies the need for establishment of a good Biosafety management; since the ignorance of the dangers and risks on the part of the personnel that works in them can cause accidents with undesirable consequences for the product, escapes of these microorganisms during their production processes and the consequent contamination of the environment. The objective of the research was to carry out an analysis of the perception of risk in the personnel responsible for the production process of two avian vaccines. The RISKPERCEP software was used in an avian vaccine production facility; its application showed variables that demonstrated underestimation of the risk by the exposed personnel and variables with a tendency to overestimate; fundamentally associated with the incorrect design of the facility. Finally, it is proposed that biosafety update courses be given and that all aspects of the laboratory design that can be solved are taken into account(AU)

Varíola, erradicaçäo e doenças infecciosas; editorial / Smallpox, eradication and infectious disease; editorial

O artigo descreve o estado atual dos conhecimentos epidemiológicos sobre a varíola e sua erradicaçäo. Ressalta três aspectos fundamentais: em 1§ lugar, o significado ecológico da varíola simiana e de outras eventuais infecçöes por ortopoxvírus no homem. Em 2§ lugar, a utilizaçäo do vírus da vacínia e suas conseqüências. Finalmente, a crença na erradicaçäo de outras doenças infecciosas, encarada sob o ponto de vista da teoria evolucionista em ecologia.