Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections.

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

Variola Virus and Clade I Mpox Virus Differentially Modulate Cellular Responses Longitudinally in Monocytes During Infection.

Variola virus (VARV), the etiological agent of smallpox, had enormous impacts on global health prior to its eradication. In the absence of global vaccination programs, mpox virus (MPXV) has become a growing public health threat that includes endemic and nonendemic regions across the globe. While human mpox resembles smallpox in clinical presentation, there are considerable knowledge gaps regarding conserved molecular pathogenesis between these 2 orthopoxviruses. Thus, we sought to compare MPXV and VARV infections in human monocytes through kinome analysis. We performed a longitudinal analysis of host cellular responses to VARV infection in human monocytes as well as a comparative analysis to clade I MPXV-mediated responses. While both viruses elicited strong activation of cell responses early during infection as compared to later time points, several key differences in cell signaling events were identified and validated. These observations will help in the design and development of panorthopoxvirus therapeutics.

Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus.

Smallpox, caused by the solely human pathogen Variola virus (VARV), was declared eradicated in 1980. While known VARV stocks are secure, smallpox remains a bioterrorist threat agent. Recent U.S. Food and Drug Administration approval of the first smallpox anti-viral (tecovirimat) therapeutic was a successful step forward in smallpox preparedness; however, orthopoxviruses can become resistant to treatment, suggesting a multi-therapeutic approach is necessary. Animal models are required for testing medical countermeasures (MCMs) and ideally MCMs are tested directly against the pathogen of interest. Since VARV only infects humans, a representative animal model for testing therapeutics directly against VARV remains a challenge. Here we show that three different humanized mice strains are highly susceptible to VARV infection, establishing the first small animal model using VARV. In comparison, the non-humanized, immunosuppressed background mouse was not susceptible to systemic VARV infection. Following an intranasal VARV challenge that mimics the natural route for human smallpox transmission, the virus spread systemically within the humanized mouse before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. Our identification of a permissive/representative VARV animal model can facilitate testing of MCMs in a manner consistent with their intended use.

Exportation of Monkeypox Virus From the African Continent.

BACKGROUND: The largest West African monkeypox outbreak began September 2017, in Nigeria. Four individuals traveling from Nigeria to the United Kingdom (n = 2), Israel (n = 1), and Singapore (n = 1) became the first human monkeypox cases exported from Africa, and a related nosocomial transmission event in the United Kingdom became the first confirmed human-to-human monkeypox transmission event outside of Africa. METHODS: Epidemiological and molecular data for exported and Nigerian cases were analyzed jointly to better understand the exportations in the temporal and geographic context of the outbreak. RESULTS: Isolates from all travelers and a Bayelsa case shared a most recent common ancestor and traveled to Bayelsa, Delta, or Rivers states. Genetic variation for this cluster was lower than would be expected from a random sampling of genomes from this outbreak, but data did not support direct links between travelers. CONCLUSIONS: Monophyly of exportation cases and the Bayelsa sample, along with the intermediate levels of genetic variation, suggest a small pool of related isolates is the likely source for the exported infections. This may be the result of the level of genetic variation present in monkeypox isolates circulating within the contiguous region of Bayelsa, Delta, and Rivers states, or another more restricted, yet unidentified source pool.

Identification of Small Molecules with Improved Potency against Orthopoxviruses from Vaccinia to Smallpox.

The genus Orthopoxvirus contains several human pathogens, including vaccinia, monkeypox, cowpox, and variola virus, the causative agent of smallpox. Although there are a few effective vaccines, widespread prophylactic vaccination has ceased and is unlikely to resume, making therapeutics increasingly important to treat poxvirus disease. Here, we described efforts to improve the potency of the anti-poxvirus small molecule CMLDBU6128. This class of small molecules, referred to as pyridopyrimidinones (PDPMs), showed a wide range of biological activities. Through the synthesis and testing of several exploratory chemical libraries based on this molecule, we identified several compounds that had increased potency from the micromolar into the nanomolar range. Two compounds, designated (12) and (16), showed inhibitory concentrations of 326 nM and 101 nM, respectively, which was more than a 10-fold increase in potency to CMLDBU6128 with an inhibitory concentration of around 6 µM. We also expanded our investigation of the breadth of action of these molecules and showed that they can inhibit the replication of variola virus, a related orthopoxvirus. Together, these findings highlighted the promise of this new class of antipoxviral agents as broad-spectrum small molecules with significant potential to be developed as antiviral therapy. This would add a small molecule option for therapy of spreading diseases, including monkeypox and cowpox viruses, that would also be expected to have efficacy against smallpox.

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.

Rapid and sensitive point-of-care detection of Orthopoxviruses by ABICAP immunofiltration.

BACKGROUND: The rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. Commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce. RESULTS: In this work we present a point-of-care test for the detection of Orthopoxviruses (OPV). The OPV ABICAP assay detects down to 1 × 104 plaque forming units/mL of OPV particles within 45 min. It can be applied to clinical material like skin crusts and detects all zoonotic OPV infecting humans, including Vaccinia, Cowpox, Monkeypox, and most importantly Variola virus. CONCLUSIONS: Given the high sensitivity and the ease of handling, the novel assay could be highly useful for on-site diagnostics of suspected Monkeypox virus infections in areas lacking proper laboratory infrastructure as well as rapid on-site testing of suspected bioterrorism samples.

Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity.

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

In vitro efficacy of brincidofovir against variola virus.

Brincidofovir (CMX001), a lipid conjugate of the acyclic nucleotide phosphonate cidofovir, is under development for smallpox treatment using "the Animal Rule," established by the FDA in 2002. Brincidofovir reduces mortality caused by orthopoxvirus infection in animal models. Compared to cidofovir, brincidofovir has increased potency, is administered orally, and shows no evidence of nephrotoxicity. Here we report that the brincidofovir half-maximal effective concentration (EC50) against five variola virus strains in vitro averaged 0.11 µM and that brincidofovir was therefore nearly 100-fold more potent than cidofovir.

Phylogenetic and ecologic perspectives of a monkeypox outbreak, southern Sudan, 2005.

Identification of human monkeypox cases during 2005 in southern Sudan (now South Sudan) raised several questions about the natural history of monkeypox virus (MPXV) in Africa. The outbreak area, characterized by seasonally dry riverine grasslands, is not identified as environmentally suitable for MPXV transmission. We examined possible origins of this outbreak by performing phylogenetic analysis of genome sequences of MPXV isolates from the outbreak in Sudan and from differing localities. We also compared the environmental suitability of study localities for monkeypox transmission. Phylogenetically, the viruses isolated from Sudan outbreak specimens belong to a clade identified in the Congo Basin. This finding, added to the political instability of the area during the time of the outbreak, supports the hypothesis of importation by infected animals or humans entering Sudan from the Congo Basin, and person-to-person transmission of virus, rather than transmission of indigenous virus from infected animals to humans.

Efficacy of tecovirimat (ST-246) in nonhuman primates infected with variola virus (Smallpox).

Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfare/bioterrorist weapon (F. Fleck, Bull. World Health Organ. 81:917-918, 2003). While effective, the smallpox vaccine is currently not recommended for routine use in the general public due to safety concerns (http://www.bt.cdc.gov/agent/smallpox/vaccination). Safe and effective countermeasures, particularly those effective after exposure to smallpox, are needed. Currently, SIGA Technologies is developing the small-molecule oral drug, tecovirimat (previously known as ST-246), as a postexposure therapeutic treatment of orthopoxvirus disease, including smallpox. Tecovirimat has been shown to be efficacious in preventing lethal orthopoxviral disease in numerous animal models (G. Yang, D. C. Pevear, M. H. Davies, M. S. Collett, T. Bailey, et al., J. Virol. 79:13139-13149, 2005; D. C. Quenelle, R. M. Buller, S. Parker, K. A. Keith, D. E. Hruby, et al., Antimicrob. Agents Chemother., 51:689-695, 2007; E. Sbrana, R. Jordan, D. E. Hruby, R. I. Mateo, S. Y. Xiao, et al., Am. J. Trop. Med. Hyg. 76:768-773, 2007). Furthermore, in clinical trials thus far, the drug appears to be safe, with a good pharmacokinetic profile. In this study, the efficacy of tecovirimat was evaluated in both a prelesional and postlesional setting in nonhuman primates challenged intravenously with 1 × 10(8) PFU of Variola virus (VARV; the causative agent of smallpox), a model for smallpox disease in humans. Following challenge, 50% of placebo-treated controls succumbed to infection, while all tecovirimat-treated animals survived regardless of whether treatment was started at 2 or 4 days postinfection. In addition, tecovirimat treatment resulted in dramatic reductions in dermal lesion counts, oropharyngeal virus shedding, and viral DNA circulating in the blood. Although clinical disease was evident in tecovirimat-treated animals, it was generally very mild and appeared to resolve earlier than in placebo-treated controls that survived infection. Tecovirimat appears to be an effective smallpox therapeutic in nonhuman primates, suggesting that it is reasonably likely to provide therapeutic benefit in smallpox-infected humans.

Variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and SRC family tyrosine kinases.

Vaccinia virus (VacV) enters mammalian cells, replicates extranuclearly, and produces virions that move to the cell surface along microtubules, fuse with the plasma membrane, and move from infected cells toward apposing cells on actin-filled membranous protrusions or actin tails. To form actin tails, cell-associated enveloped virions (CEV) require Abl and Src family tyrosine kinases. Furthermore, release of CEV from the cell requires Abl but not Src family tyrosine kinases and is blocked by imatinib mesylate (STI-571; Gleevec), an Abl family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Here we demonstrate that the Poxviridae family members monkeypox virus (MPX) and variola virus (VarV) use conserved mechanisms for actin motility and extracellular enveloped virion (EEV) release. Furthermore, we show that imatinib mesylate is effective in a mouse model of infection with VacV, whether delivered prophylactically or postinfection, and restricts spread of virions from the site of inoculation. While inhibitors of both Src and Abl family kinases, such as dasatinib (BMS-354825; Sprycel), are effective in limiting dissemination of VacV, VarV, and MPX in vitro, members of this class of drugs appear to have immunosuppressive effects in vivo that preclude their use as anti-infectives. Together, these data suggest a possible utility for imatinib mesylate in treating smallpox or MPX infections or complications associated with vaccination.

Vaccinia virus infections in martial arts gym, Maryland, USA, 2008.

Vaccinia virus is an orthopoxvirus used in the live vaccine against smallpox. Vaccinia virus infections can be transmissible and can cause severe complications in those with weakened immune systems. We report on a cluster of 4 cases of vaccinia virus infection in Maryland, USA, likely acquired at a martial arts gym.

Poxvirus complement control proteins are expressed on the cell surface through an intermolecular disulfide bridge with the viral A56 protein.

The vaccinia virus (VACV) complement control protein (VCP) is an immunomodulatory protein that is both secreted from and expressed on the surface of infected cells. Surface expression of VCP occurs though an interaction with the viral transmembrane protein A56 and is dependent on a free N-terminal cysteine of VCP. Although A56 and VCP have been shown to interact in infected cells, the mechanism remains unclear. To investigate if A56 is sufficient for surface expression, we transiently expressed VCP and A56 in eukaryotic cell lines and found that they interact on the cell surface in the absence of other viral proteins. Since A56 contains three extracellular cysteines, we hypothesized that one of the cysteines may be unpaired and could therefore form a disulfide bridge with VCP. To test this, we generated a series of A56 mutants in which each cysteine was mutated to a serine, and we found that mutation of cysteine 162 abrogated VCP cell surface expression. We also tested the ability of other poxvirus complement control proteins to bind to VACV A56. While the smallpox homolog of VCP is able to bind VACV A56, the ectromelia virus (ECTV) VCP homolog is only able to bind the ECTV homolog of A56, indicating that these proteins may have coevolved. Surface expression of poxvirus complement control proteins may have important implications in viral pathogenesis, as a virus that does not express cell surface VCP is attenuated in vivo. This suggests that surface expression of VCP may contribute to poxvirus pathogenesis.

Detection of North American orthopoxviruses by real time-PCR.

The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad.

Pharmacokinetics and Efficacy of a Potential Smallpox Therapeutic, Brincidofovir, in a Lethal Monkeypox Virus Animal Model.

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.

Discovery of Retro-1 Analogs Exhibiting Enhanced Anti-vaccinia Virus Activity.

Orthopoxviruses (OPXVs) are an increasing threat to human health due to the growing population of OPXV-naive individuals after the discontinuation of routine smallpox vaccination. Antiviral drugs that are effective as postexposure treatments against variola virus (the causative agent of smallpox) or other OPXVs are critical in the event of an OPXV outbreak or exposure. The only US Food and Drug Administration-approved drug to treat smallpox, Tecovirimat (ST-246), exerts its antiviral effect by inhibiting extracellular virus (EV) formation, thereby preventing cell-cell and long-distance spread. We and others have previously demonstrated that host Golgi-associated retrograde proteins play an important role in monkeypox virus (MPXV) and vaccinia virus (VACV) EV formation. Inhibition of the retrograde pathway by small molecules such as Retro-2 has been shown to decrease VACV infection in vitro and to a lesser extent in vivo. To identify more potent inhibitors of the retrograde pathway, we screened a large panel of compounds containing a benzodiazepine scaffold like that of Retro-1, against VACV infection. We found that a subset of these compounds displayed better anti-VACV activity, causing a reduction in EV particle formation and viral spread compared to Retro-1. PA104 emerged as the most potent analog, inhibiting 90% viral spread at 1.3 µM with a high selectivity index. In addition, PA104 strongly inhibited two distinct ST-246-resistant viruses, demonstrating its potential benefit for use in combination therapy with ST-246. These data and further characterizations of the specific protein targets and in vivo efficacy of PA104 may have important implications for the design of effective antivirals against OPXV.

Conserved Oligomeric Golgi (COG) Complex Proteins Facilitate Orthopoxvirus Entry, Fusion and Spread.

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1-COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.

Antiviral Ranpirnase TMR-001 Inhibits Rabies Virus Release and Cell-to-Cell Infection In Vitro.

Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity. TMR-001, a proprietary bulk drug substance solution of ranpirnase, was evaluated against rabies virus in three cell types: mouse neuroblastoma, BSR (baby hamster kidney cells), and bat primary fibroblast cells. When TMR-001 was added to cell monolayers 24 h preinfection, rabies virus release was inhibited for all cell types at three time points postinfection. TMR-001 treatment simultaneous with infection and 24 h postinfection effectively inhibited rabies virus release in the supernatant and cell-to-cell spread with 50% inhibitory concentrations of 0.2-2 nM and 20-600 nM, respectively. TMR-001 was administered at 0.1 mg/kg via intraperitoneal, intramuscular, or intravenous routes to Syrian hamsters beginning 24 h before a lethal rabies virus challenge and continuing once per day for up to 10 days. TMR-001 at this dose, formulation, and route of delivery did not prevent rabies virus transit from the periphery to the central nervous system in this model (n = 32). Further aspects of local controlled delivery of other active formulations or dose concentrations of TMR-001 or ribonuclease analogues should be investigated for this class of drugs as a rabies antiviral therapeutic.