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Monkeypox (MPXV) is one of the infectious viruses which caused morbidity and mortality problems in these years. Despite its danger to public health, there is no approved drug to stand and handle MPXV. On the other hand, drug repurposing is a promising screening method for the low-cost introduction of approved drugs for emerging diseases and viruses which utilizes computational methods. Therefore, drug repurposing is a promising approach to suggesting approved drugs for the MPXV. This paper proposes a computational framework for MPXV antiviral prediction. To do this, we have generated a new virus-antiviral dataset. Moreover, we applied several machine learning and one deep learning method for virus-antiviral prediction. The suggested drugs by the learning methods have been investigated using docking studies. The target protein structure is modeled using homology modeling and, then, refined and validated. To the best of our knowledge, this work is the first work to study deep learning methods for the prediction of MPXV antivirals. The screening results confirm that Tilorone, Valacyclovir, Ribavirin, Favipiravir, and Baloxavir marboxil are effective drugs for MPXV treatment.

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Monkeypox (Mpox), a zoonotic illness triggered by the monkeypox virus (MPXV), poses a significant threat since it may be transmitted and has no cure. This work introduces a computational method to predict Protein-Protein Interactions (PPIs) during MPXV infection. The objective is to discover prospective drug targets and repurpose current potential Food and Drug Administration (FDA) drugs for therapeutic purposes. In this work, ensemble features, comprising 2-5 node graphlet attributes and protein composition-based features are utilized for Deep Learning (DL) models to predict PPIs. The technique that is used here demonstrated an excellent prediction performance for PPI on both the Human Integrated Protein-Protein Interaction Reference (HIPPIE) and MPXV-Human PPI datasets. In addition, the human protein targets for MPXV have been identified accurately along with the detection of possible therapeutic targets. Furthermore, the validation process included conducting docking research studies on potential FDA drugs like Nicotinamide Adenine Dinucleotide and Hydrogen (NADH), Fostamatinib, Glutamic acid, Cannabidiol, Copper, and Zinc in DrugBank identified via research on drug repurposing and the Drug Consensus Score (DCS) for MPXV. This has been achieved by employing the primary crystal structures of MPXV, which are now accessible. The docking study is also supported by Molecular Dynamics (MD) simulation. The results of our study emphasize the effectiveness of using ensemble feature-based PPI prediction to understand the molecular processes involved in viral infection and to aid in the development of repurposed drugs for emerging infectious diseases such as, but not limited to, Mpox. The source code and link to data used in this work is available at: https://github.com/CMATERJU-BIOINFO/In-Silico-Drug-Repurposing-Methodology-To-Suggest-Therapies-For-Emerging-Threats-like-Mpox .

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Monkeypox is an infectious disease caused by the monkeypox virus (MPXV), a member of the Orthopoxvirus genus closely related to smallpox. The structure of the A42R profilin-like protein is the first and only available structure among MPXV proteins. Biochemical studies of A42R were conducted in the 1990s and later work also analyzed the protein's function in viral replication in cells. This study aims to screen tripeptides for their potential inhibition of the A42R profilin-like protein using computational methods, with implications for MPXV therapy. A total of 8000 tripeptides underwent molecular docking simulations, resulting in the identification of 20 compounds exhibiting strong binding affinity to A42R. To validate the docking results, molecular dynamics simulations and free energy perturbation calculations were performed. These analyses revealed two tripeptides with sequences TRP-THR-TRP and TRP-TRP-TRP, which displayed robust binding affinity to A42R. Markedly, electrostatic interactions predominated over van der Waals interactions in the binding process between tripeptides and A42R. Three A42R residues, namely Glu9, Ser12, and Arg38, appear to be pivotal in mediating the interaction between A42R and the tripeptide ligands. Notably, tripeptides containing two or three tryptophan residues demonstrate a pronounced binding affinity, with the tripeptide comprising three tryptophan amino acids showing the highest level of affinity. These findings offer valuable insights for the selection of compounds sharing a similar structure and possessing a high affinity for A42R, potentially capable of inhibiting its enzyme activity. The study highlights a structural advantage and paves the way for the development of targeted therapies against MPXV infections.

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BACKGROUND: Monkeypox is a viral zoonotic disease commonly reported in humans in parts of Central and West Africa. This protocol is for an Expanded Access Programme (EAP) to be implemented in the Central African Republic, where Clade I monkeypox virus diseases is primarily responsible for most monkeypox infections. The objective of the programme is to provide patients with confirmed monkeypox with access to tecovirimat, a novel antiviral targeting orthopoxviruses, and collect data on clinical and virological outcomes of patients to inform future research. METHODS: The study will be conducted at participating hospitals in the Central African Republic. All patients who provide informed consent to enrol in the programme will receive tecovirimat. Patients will remain in hospital for the duration of treatment. Data on clinical signs and symptoms will be collected every day while the patient is hospitalised. Blood, throat and lesion samples will be collected at baseline and then on days 4, 8, 14 and 28. Patient outcomes will be assessed on Day 14 -end of treatment-and at Day 28. Adverse event and serious adverse event data will be collected from the point of consent until Day 28. DISCUSSION: This EAP is the first protocolised treatment programme in Clade I MPXV. The data generated under this protocol aims to describe the use of tecovirimat for Clade I disease in a monkeypox endemic region of Central Africa. It is hoped that this data can inform the definition of outcome measures used in future research and contribute to the academic literature around the use of tecovirimat for the treatment of monkeypox. The EAP also aims to bolster research capacity in the region in order for robust randomised controlled trials to take place for monkeypox and other diseases. TRIAL REGISTRATION: {2a & 2b}: ISRCTN43307947.

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Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

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BACKGROUND: While the world is still facing the global pandemic COVID-19, another zoonosis monkeypox (Mpox) has emerged posing a great threat to society. Insight into the pathogenesis, symptoms, and management strategies will aid in the development of potent therapeutics for the treatment of monkeypox virus infection. OBJECTIVES: To get insight into the current treatment and prevention strategies will aid in effectively coping with the disease. METHODS: For obtaining information regarding the ongoing treatment and prevention strategies and the drugs under pipeline, we referred to Google Scholar, Pub Med, Pub Chem, and WHO official site. RESULTS: There are a few drugs that came out to be effective for the treatment of Mpox. Tecovirimat acts by inhibiting viral replication and viral wrapping. Another drug is cidofovir, which hinders the activity of viral DNA polymerase but has the drawback of nephrotoxicity. To overcome this, a conjugate of cidofovir is being used-known as brincidofovir-which has a similar mechanism as cidofovir but lesser toxicity. Ribavirin acts via inhibiting inosine monophosphate dehydrogenase (IMPDPH) thus disrupting viral translation. It also interferes with helicase activity. Tiazofurin, Adenosine N1 oxide, and HPMPA have shown efficacy in in-vitro studies by inhibiting IMPDH, DNA polymerase, and viral mRNA translation respectively. In-silico studies have proven the effect of nilotinib, simeprevir, and dihydroergotamine for Mpox treatment. They have shown binding affinity for proteins required for the growth and release of MPXV. Vaccines have also been employed for the prevention of Mpox, which includes JYNNEOS, ACAM2000, and VIGIV. CONCLUSION: This review highlights the pathogenesis of the virus, disease manifestations, drugs, and vaccines that are being used and those under pipeline for the treatment and prevention of Mpox.

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Mpox virus (formerly monkeypox virus [MPXV]) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.026 to 0.89 µM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the postentry step to reduce the intracellular production of virions. Knockdown of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. This evidence shows that IMPDH is a potential target for the development of anti-MPXV agents. IMPORTANCE Mpox is a zoonotic disease caused by infection with the mpox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against mpox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against mpox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-mpox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited mpox virus propagation and exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested IMP dehydrogenase as a potential target for the development of anti-mpox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-mpox virus activity than mycophenolic acid.

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BACKGROUND: Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. METHODS: We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. RESULTS: Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 µM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. CONCLUSIONS: These data suggest that atovaquone would be a potential candidate for treating mpox.

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The monkeypox outbreak has become a global public health emergency. The lack of valid and safe medicine is a crucial obstacle hindering the extermination of orthopoxvirus infections. The identification of potential inhibitors from natural products, including Traditional Chinese Medicine (TCM), by molecular modeling could expand the arsenal of antiviral chemotherapeutic agents. Monkeypox DNA topoisomerase I (TOP1) is a highly conserved viral DNA repair enzyme with a small size and low homology to human proteins. The protein model of viral DNA TOP1 was obtained by homology modeling. The reliability of the TOP1 model was validated by analyzing its Ramachandran plot and by determining the compatibility of the 3D model with its sequence using the Verify 3D and PROCHECK services. In order to identify potential inhibitors of TOP1, an integrated library of 4103 natural products was screened via Glide docking. Surface Plasmon Resonance (SPR) was further implemented to assay the complex binding affinity. Molecular dynamics simulations (100 ns) were combined with molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) computations to reveal the binding mechanisms of the complex. As a result, three natural compounds were highlighted as potential inhibitors via docking-based virtual screening. Rosmarinic acid, myricitrin, quercitrin, and ofloxacin can bind TOP1 with KD values of 2.16 µM, 3.54 µM, 4.77 µM, and 5.46 µM, respectively, indicating a good inhibitory effect against MPXV. The MM/PBSA calculations revealed that rosmarinic acid had the lowest binding free energy at -16.18 kcal/mol. Myricitrin had a binding free energy of -13.87 kcal/mol, quercitrin had a binding free energy of -9.40 kcal/mol, and ofloxacin had a binding free energy of -9.64 kcal/mol. The outputs (RMSD/RMSF/Rg/SASA) also indicated that the systems were well-behaved towards the complex. The selected compounds formed several key hydrogen bonds with TOP1 residues (TYR274, LYS167, GLY132, LYS133, etc.) via the binding mode analysis. TYR274 was predicted to be a pivotal residue for compound interactions in the binding pocket of TOP1. The results of the enrichment analyses illustrated the potential pharmacological networks of rosmarinic acid. The molecular modeling approach may be acceptable for the identification and design of novel poxvirus inhibitors; however, further studies are warranted to evaluate their therapeutic potential.

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Monkeypox virus (MPXV) is a member of the Orthopoxvirus genus and the Poxviridae family, which instigated a rising epidemic called monkeypox disease. Proteinases are majorly engaged in viral propagation by catalyzing the cleavage of precursor polyproteins. Therefore, proteinase is essential for monkeypox and a critical drug target. In this study, high-throughput virtual screening (HTVS) and molecular dynamics simulation were applied to detect the potential natural compounds against the proteinase of the monkeypox virus. Here, 32,552 natural products were screened, and the top five compounds were selected after implementing the HTVS and molecular docking protocols in series. Gallicynoic Acid F showed the minimum binding score of -10.56 kcal/mole in the extra precision scoring method, which reflected the highest binding with the protein. The top five compounds showed binding scores ≤-8.98 kcal/mole. These compound complexes were tested under 100 ns molecular dynamics simulation, and Vaccinol M showed the most stable and consistent RMSD trend in the range of 2 Å to 3 Å. Later, MM/GBSA binding free energy and principal component analysis were performed on the top five compounds to validate the stability of selected compound complexes. Moreover, the ligands Gallicynoic Acid F and H2-Erythro-Neopterin showed the lowest binding free energies of -61.42 kcal/mol and -61.09 kcal/mol, respectively. Compared to the native ligand TTP-6171 (ΔGBind = -53.86 kcal/mol), these two compounds showed preferable binding free energy, suggesting inhibitory application against MPXV proteinase. This study proposed natural molecules as a therapeutic solution to control monkeypox disease.

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Mpox virus is an emergent human pathogen. While it is less lethal than smallpox, it can still cause significant morbidity and mortality. In this review, we explore 3 antiviral agents with activity against mpox and other orthopoxviruses: cidofovir, brincidofovir, and tecovirimat. Cidofovir, and its prodrug brincidofovir, are inhibitors of DNA replication with a broad spectrum of activity against multiple families of double-stranded DNA viruses. Tecovirimat has more specific activity against orthopoxviruses and inhibits the formation of the extracellular enveloped virus necessary for cell-to-cell transmission. For each agent, we review basic pharmacology, data from animal models, and reported experience in human patients.

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A TECNOLOGIA: descrição da tecnologia: A vacina smallpox e monkeypoxa , conhecida pelos nomes comerciais Imvanex®, Jynneos®, Imvamune® ou MVA (Modified Vaccinia Ankara) é composta pelo vírus vaccinia Ankara vivo, atenuado e modificado1 . Como o nome já diz, a vacina é indicada para a profilaxia, em adultos, de infecções provocadas pelos vírus Smallpox (causador da varíola humana) e Monkeypox, ambos do gênero ortopoxvírus. Essa vacina é produzida pela empresa Bavarian Nordic, sendo sua apresentação farmacêutica em frasco com suspensão injetável na concentração 0,5 mlb por dose. É administrada por via subcutânea na posologia de duas doses de 0,5 ml em um intervalo de 28 dias. A vacina deve ser armazenada em temperatura de -20°C a +/-5°C, tendo validade de 36 meses. Pode ser conservada entre -60°C a -40ºC por 60 meses a partir da data de fabricação e, após descongelada, poderá ser mantida a 2°C a 8°C por 12 horas. Condição clínica: O vírus que causa a doença monkeypox é um ortopoxvírus, membro da família Poxviridae. Os sintomas são semelhantes aqueles da varíola humana (smallpox), mas menos graves. A família Poxviridae é composta por vírus epiteliotróficos, ou seja, que são capazes de afetar pele e mucosa em vários sítios corporais. São capazes de infectar uma variedade de animais, incluindo insetos, pássaros, répteis, marsupiais e mamíferos. O mais conhecido de todos os ortopoxvírus é o Smallpox, agente causador da varíola humana (variola major ou smallpox disease); outros membros da família são os vírus Monkeypox, Cowpox, Orf e vaccínia. INFORMAÇÕES REGULATÓRIAS: Informações sobre registro: A Agência Nacional de Vigilância Sanitária (Anvisa) aprovou no dia 25 de agosto de 2022 a dispensa de registro para que o Ministério da Saúde importe e utilize no Brasil a vacina (nomes comerciais Jynneos® ou Imvanex®) para imunização contra a monkeypox. A dispensa temporária e excepcional se aplica somente ao Ministério da Saúde e terá validade de seis meses, desde que não seja expressamente revogada pela Anvisa2 . A indicação está condicionada à prevenção de smallpox e monkeypox em adultos com idade a partir de 18 anos e alto risco de infecção por essas doenças. A administração deve ser realizada em posologia de duas doses (0,5 ml) com um intervalo de 28 dias. PANORAMA DE DESENVOLVIMENTO: Estratégia de busca: Os ensaios clínicos com o uso da vacina smallpox e monkeypox (Imvanex®, Jynneos® ou Imvamune) para a prevenção da monkeypox foram identificados, inicialmente, na base de pesquisa clínica clinicaltrials.gov em 04 de agosto de 2022, com atualização em 26 de agosto de 2022. Foram incluídos ensaios clínicos em qualquer fase em andamento e/ou finalizados, em até cinco anos com o uso de tecnologias para a indicação de monkeypox. Além disso, foram consultadas as bases eletrônicas MEDLINE (via PubMed), EMBASE (via Periódicos Capes), Cochrane Library e o Cortellis da Clarivate Analytics1 em 26 de agosto de 2022. As estratégias de busca foram elaboradas com os termos relacionado à doença e à tecnologia, assim como seus sinônimos e códigos de pesquisa, sem filtro para a fase de desenvolvimento. CONSIDERAÇÕES FINAIS: A monkeypox tem sido considerada uma doença tropical negligenciada da África Ocidental e Central há alguns anos. Entretanto, desde o início de maio de 2022, um surto da doença envolveu a maioria dos países europeus, bem como as Américas do Norte e do Sul, fazendo com que as autoridades de saúde trabalhassem rapidamente para controlar sua disseminação. Uma das estratégias avaliadas é a vacinação da população para a prevenção da doença. O vírus que causa a monkeypox é um ortopoxvírus, membro da família Poxviridae, mesma família da smallpox (varíola humana). A monkeypox possui sintomas semelhantes aos observados no passado em pacientes com a varíola humana, embora seja clinicamente menos grave. Com a erradicação da varíola humana, em 1980, e o subsequente encerramento da vacinação em todo o mundo, a monkeypox emergiu como o ortopoxvírus mais importante para a saúde pública. Embora a vacinação contra a varíola humana tenha sido protetora no passado, atualmente, pessoas com menos de 40 a 50 anos de idade (dependendo do país) podem ser mais suscetíveis à monkeypox devido à cessação das campanhas de vacinação contra a smallpox em todo o mundo após a erradicação da doença. Nesse sentido, a vacinação que vem ocorrendo em alguns países com a vacina para a prevenção da monkeypox na população mais susceptível, principalmente profissionais de saúde, tem grande importância para a saúde pública. Vale ressaltar que a vacina, cujo nome comercial é Imvanex® no Reino Unido e na Europa, Jynneos® nos EUA e Imvamune® no Canadá, são o mesmo produto e contém a vacina de vírus Ankara modificado (vivo atenuado de replicação deficiente), na concentração com título não inferior a 5 x 10 Inf. U (Inf. U = infectious units ou unidades de infecção) por dose de 0,5 ml, todos fabricados pela mesma empresa Bavarian Nordic. Em 25 de agosto de 2022, a Anvisa concedeu a dispensa de registro temporário dessa vacina para o Ministério da Saúde, considerando a emergência em saúde do momento e o atendimento aos interesses do Sistema Único de Saúde, para garantir a celeridade no acesso à vacina para a população em risco. Os dados que embasaram os registros nas agências sanitárias internacionais, bem como a decisão da Anvisa, foram obtidos de estudos em animais, os quais demostraram proteção contra o vírus monkeypox em primatas não humanos vacinados com Imvanex® e de estudos para a proteção contra smallpox em humanos. Assim, a empresa Bavarian Nordic tem patrocinado estudos observacionais que estão sendo conduzidos durante o surto de monkeypox na Europa para confirmar os benefícios da vacina contra a doença.

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Decades after the eradication of smallpox and the discontinuation of routine smallpox vaccination, over half of the world's population is immunologically naïve to variola virus and other orthopoxviruses (OPXVs). Even in those previously vaccinated against smallpox, protective immunity wanes over time. As such, there is a concomitant increase in the incidence of human OPXV infections worldwide. To identify novel antiviral compounds with potent anti-OPXV potential, we characterized the inhibitory activity of PAV-866 and other methylene blue derivatives against the prototypic poxvirus, vaccinia virus (VACV). These compounds inactivated virions prior to infection and consequently inhibited viral binding, fusion and entry. The compounds exhibited strong virucidal activity at non-cytotoxic concentrations, and inhibited VACV infection when added before, during or after viral adsorption. The compounds were effective against other OPXVs including monkeypox virus, cowpox virus and the newly identified Akhmeta virus. Altogether, these findings reveal a novel mode of inhibition that has not previously been demonstrated for small molecule compounds against VACV. Additional studies are in progress to determine the in vivo efficacy of these compounds against OPXVs and further characterize the anti-viral effects of these derivatives.

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Smallpox, caused by Variola virus (VARV), was eradicated in 1980; however, VARV bioterrorist threats still exist, necessitating readily available therapeutics. Current preparedness activities recognize the importance of oral antivirals and recommend therapeutics with different mechanisms of action. Monkeypox virus (MPXV) is closely related to VARV, causing a highly similar clinical human disease, and can be used as a surrogate for smallpox antiviral testing. The prairie dog MPXV model has been characterized and used to study the efficacy of antipoxvirus therapeutics, including recently approved TPOXX (tecovirimat). Brincidofovir (BCV; CMX001) has shown antiviral activity against double-stranded DNA viruses, including poxviruses. To determine the exposure of BCV following oral administration to prairie dogs, a pharmacokinetics (PK) study was performed. Analysis of BCV plasma concentrations indicated variability, conceivably due to the outbred nature of the animals. To determine BCV efficacy in the MPXV prairie dog model, groups of animals were intranasally challenged with 9 × 105 plaque-forming units (PFU; 90% lethal dose [LD90]) of MPXV on inoculation day 0 (ID0). Animals were divided into groups based on the first day of BCV treatment relative to inoculation day (ID-1, ID0, or ID1). A trend in efficacy was noted dependent upon treatment initiation (57% on ID-1, 43% on ID0, and 29% on ID1) but was lower than demonstrated in other animal models. Analysis of the PK data indicated that BCV plasma exposure (maximum concentration [Cmax]) and the time of the last quantifiable concentration (AUClast) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival.IMPORTANCE Preparedness activities against highly transmissible viruses with high mortality rates have been highlighted during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Smallpox, caused by variola virus (VARV) infection, is highly transmissible, with an estimated 30% mortality. Through an intensive vaccination campaign, smallpox was declared eradicated in 1980, and routine smallpox vaccination of individuals ceased. Today's current population has little/no immunity against VARV. If smallpox were to reemerge, the worldwide results would be devastating. Recent FDA approval of one smallpox antiviral (tecovirimat) was a successful step in biothreat preparedness; however, orthopoxviruses can become resistant to treatment, suggesting the need for multiple therapeutics. Our paper details the efficacy of the investigational smallpox drug brincidofovir in a monkeypox virus (MPXV) animal model. Since brincidofovir has not been tested in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

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Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX®, SIGA Technologies' smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak. TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis. It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity. Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.

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The lack of antiviral drugs for the treatment of orthopoxvirus disease represents an unmet medical need, particularly due to the threat of variola virus (the causative agent of smallpox) as an agent of biowarfare or bioterrorism (Henderson, 283:1279-1282, 1999). In addition to variola, monkeypox, cowpox, and vaccinia viruses are orthopoxviruses of concern to human health (Lewis-Jones, 17:81-89, 2004). Smallpox vaccination, using the closely related vaccinia virus, is no longer provided to the general public leading to a worldwide population increasingly susceptible not only to variola but to monkeypox, cowpox, and vaccinia viruses as well. Orthopoxviruses share similar life cycles (Fenner et al., WHO, Geneva, 1988), and significant nucleotide and protein homology, and are immunologically cross-protective against other species within the genus, which was the basis of the highly successful vaccinia virus vaccine. These similarities also serve as the basis for screening for antivirals for dangerous pathogens such as variola and monkeypox virus using generally safer viruses such as cowpox and vaccinia. Methods for preliminary screening and initial characterization of potential orthopoxvirus antivirals in vitro, using vaccinia virus as a relatively safe surrogate for more pathogenic orthopoxviruses, are described herein. They include candidate identification in a viral cytopathic effect (CPE) assay as well as evaluation of the antiviral activity in inhibition assays to determine mean effective (or inhibitory) concentrations (EC50 or IC50). These assays were utilized in the identification and early characterization of tecovirimat (ST-246) (Yang et al., 79:13,139-13,149, 2005). These initial steps in identifying and characterizing the antiviral activity should be followed up with additional in vitro studies including specificity testing (for other orthopoxviruses and against other viruses), single-cycle growth curves, time of addition assays, cytotoxicity testing, and identification of the drug target.

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