Monkeypox: features of spread after cancellation of mandatory pox immunization

Features of spread of monkeypox after eradication of smallpox and cancellation of mandatory pox immunization are examined. In the condition of cancellation of mandatory pox immunization, a decrease of population immunity to pox in the population, a lack of vigilance in most of the medical specialists to diseases caused by other pathogenic for human orthopoxviruses was noted. This situation complicates the prognosis of the development of possible outbreaks of infection of humans by monkeypox. In such situation only constant vigilance with respect to this zooanthroponosis, use of express diagnostics methods, as well as means of effective protection will allow to stop outbreaks of monkeypox at the early stages of the development.     

Subunit recombinant vaccine protects against monkeypox

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20-155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.     

Immunomodulatory proteins of orthopoxviruses

The review considers recent data on the structural-functional organization of the genome of orthopoxviruses pathogenic for humans, including the variola, monkeypox, cowpox, and vaccinia viruses. Emphasis was placed on the structure of molecular virulence factors that suppress the inflammatory reactions, immune response, and interferon effects induced by virus infection.     

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.     

Substrate specificity of homogeneous monkeypox virus uracil-DNA glycosylase

Weak or nonexistent smallpox immunity in today's human population raises concerns about the possibility of natural or provoked genetic modifications leading to re-emergence of variola virus and other poxviruses. Thus, the development of new antiviral strategies aimed at poxvirus infections in humans is a high priority. The DNA repair protein uracil-DNA glycosylase (UNG) is one of the viral enzymes important for poxvirus pathogenesis. Consequently, the inhibition of UNG is a rational therapeutic strategy for infections with poxviruses. Monkeypox virus, which occurs naturally in Africa, can cause a smallpox-like disease in humans. Here, the monkeypox virus UNG (mpUNG) is characterized and compared to vaccinia virus UNG (vUNG) and human UNG (hUNG). The mpUNG protein excises uracil preferentially from single-stranded DNA. Furthermore, mpUNG prefers the U.G pair over the U.A pair and does not excise oxidized bases. Both mpUNG and vUNG viral proteins are strongly inhibited by physiological concentrations of NaCl and MgCl2. Although the two viral DNA repair enzymes have similar substrate specificities, the kcat/KM values of mpUNG are higher than those of vUNG. The mpUNG protein was strongly inhibited by 5-azauracil and to a lesser extent by 4(6)-aminouracil and 5-halogenated uracil analogues, whereas uracil had no effect. To develop antiviral drugs toward mpUNG, we also validated a repair assay using the molecular beacons containing multiple uracil residues. Potential targets and strategies for combating pathogenic orthopoxviruses, including smallpox, are discussed.     

Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus

The use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naive and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and immunocompromised animals, and its efficacy against smallpox is currently being addressed. Here we directly compare the efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus and is therefore an important candidate for the protection of humans against smallpox.     

Immunomodulatory Proteins of Orthopoxviruses

The review considers recent data on the structural–functional organization of the genome of orthopoxviruses pathogenic for humans, including the variola, monkeypox, cowpox, and vaccinia viruses. Emphasis was placed on the structure of molecular virulence factors that suppress the inflammatory reactions, immune response, and interferon effects induced by virus infection.     

Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.     

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.     

Variola virus immune evasion proteins

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.     

Assessment of the Protective Effect of Imvamune and Acam2000 Vaccines against Aerosolized Monkeypox Virus in Cynomolgus Macaques

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.     

Monkeypox virus: a re-emergent threat to humans

Human monkeypox (MPX) is a rare zoonotic infection characterized by smallpox-like signs and symptoms. It is caused by monkeypox virus (MPXV), a double stranded DNA virus belonging to the genus Orthopoxvirus. MPX was first identified in 1970 and mostly prevailed in the rural rainforests of Central and West Africa in the past. Outside Africa, MPX was reported in the United Kingdom, the USA, Israel, and Singapore. In 2022, the resurgence of MPX in Europe and elsewhere posed a potential threat to humans. MPXV was transmitted by the animals-human or human-human pathway, and the symptoms of MPXV infection are similar to that of smallpox, but in a milder form and with lower mortality (1%–10%). Although the smallpox vaccination has been shown to provide 85% protection against MPXV infection, and two anti-smallpox virus drugs have been approved to treat MPXV, there are still no specific vaccines and drugs against MPXV infection. Therefore it is urgent to take active measures including the adoption of novel anti-MPXV strategies to control the spread of MPXV and prevent MPX epidemic. In this review, we summarize the biological features, epidemiology, pathogenicity, laboratory diagnosis, and prevention and treatment strategies on MPXV. This review provides the basic knowledge for prevention and control of future outbreaks of this emerging infection.     

Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus

Monkeypox virus (MPXV) infection in humans results in clinical symptoms very similar to ordinary smallpox. Aerosol is a route of secondary transmission for monkeypox, and a primary route of smallpox transmission in humans. Therefore, an animal model for aerosol exposure to MPXV is needed to test medical countermeasures. To characterize the pathogenesis in cynomolgus macaques (Macaca fascicularis), groups of macaques were exposed to four different doses of aerosolized MPXV. Blood was collected the day before, and every other day after exposure and assessed for complete blood count (CBC), clinical chemistry analysis, and quantitative PCR. Macaques showed mild anorexia, depression, and fever on day 6 post-exposure. Lymphadenopathy, which differentiates monkeypox from smallpox, was observed in exposed macaques around day 6 post-exposure. CBC and clinical chemistries showed abnormalities similar to human monkeypox cases. Whole blood and throat swab viral loads peaked around day 10, and in survivors, gradually decreased until day 28 post-exposure. Survival was not dose dependent. As such, doses of 4×10⁴ PFU, 1×10⁵ PFU, or 1×10⁶ PFU resulted in lethality for 70% of the animals, whereas a dose of 4×10⁵ PFU resulted in 85% lethality. Overall, cynomolgus macaques exposed to aerosolized MPXV develop a clinical disease that resembles that of human monkeypox. These findings provide a strong foundation for the use of aerosolized MPXV exposure of cynomolgus macaques as an animal model to test medical countermeasures against orthopoxviruses.     

Orthopoxvirus genes for Kelch-like proteins. I. Analysis of species specific differences by gene structure and organization

Genes and proteins of the kelch superfamily were structurally analyzed in the smallpox (SPV), monkeypox (MPV), cowpox (CPV), and vaccinia (VV) viruses. Genes potentially coding for the kelch-like proteins were found only in the variable terminal regions of the orthopoxvirus genome. The set and sizes of their protein products varied with species. All genes of the superfamily proved to be disrupted by mutations in SPV, which is highly pathogenic for its only host, man. The largest set of kelch-like proteins was observed for CPV, which is low-pathogenic for humans and has the broadest animal host range. The kelch-like proteins of one virus showed low homology to each other, whereas isologs of different viruses were highly homologous. The results testified to the earlier assumption that CPV is the most ancient and an ancestor of the other orthopoxviruses pathogenic for humans.     

Smallpox inhibitor of complement enzymes (SPICE): regulation of complement activation on cells and mechanism of its cellular attachment

Despite eradication of smallpox three decades ago, public health concerns remain due to its potential use as a bioterrorist weapon. Smallpox and other orthopoxviruses express virulence factors that inhibit the host's complement system. In this study, our goals were to characterize the ability of the smallpox inhibitor of complement enzymes, SPICE, to regulate human complement on the cell surface. We demonstrate that SPICE binds to a variety of cell types and that the heparan sulfate and chondroitin sulfate glycosaminoglycans serve as attachment sites. A transmembrane-engineered version as well as soluble recombinant SPICE inhibited complement activation at the C3 convertase step with equal or greater efficiency than that of the related host regulators. Moreover, SPICE attached to glycosaminoglycans was more efficient than transmembrane SPICE. We also demonstrate that this virulence activity of SPICE on cells could be blocked by a mAb to SPICE. These results provide insights related to the complement inhibitory activities of poxviral inhibitors of complement and describe a mAb with therapeutic potential.     

Correlates of protective immunity in poxvirus infection: where does antibody stand?

Even though smallpox has been eradicated, the threat of accidental or intentional release has highlighted the fact there is little consensus about correlates of protective immunity or immunity against re-infection with the causative poxvirus, variola virus (VARV). As the existing vaccine for smallpox has unacceptable rates of side effects and complications, new vaccines are urgently needed. Surrogate animal models of VARV infection in humans, including vaccinia virus (VACV) and ectromelia virus (ECTV) infection in mice, monkeypox virus (MPXV) infection in macaques have been used as tools to dissect the immune response to poxviruses. Mousepox, caused by ECTV, a natural mouse pathogen, is arguably the best surrogate small-animal model, as it shares many aspects of virus biology, pathology and clinical features with smallpox in humans. The requirements for recovery from a primary ECTV infection have been well characterized and include type I and II interferons, natural killer cells, CD4T cells, CD8T cell effector function and antibody. From a vaccine standpoint, it is imperative that the requirements for recovery from secondary infection are also identified. We have investigated host immune parameters in response to a secondary ECTV infection, and have identified that interferon and CD8T cell effector functions are not essential; however, T- and B-cell interaction and antibody are absolutely critical for recovery from a secondary challenge. The central role of antibody has been also been identified in the secondary response to other poxviruses. These findings have important clinical implications and would greatly assist the design of therapeutic interventions and new vaccines for smallpox.     

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.