Toxicity and pathophysiology of palytoxin congeners after intraperitoneal and aerosol administration in rats

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Toxicon 150 (2018): 235-250, doi:10.1016/j.toxicon.2018.06.067.Preparations of palytoxin (PLTX, derived from Japanese Palythoa tuberculosa) and the congeners 42-OH-PLTX (from Hawaiian P. toxica) and ovatoxin-a (isolated from a Japanese strain of Ostreopsis ovata), as well as a 50:50 mixture of PLTX and 42-OH-PLTX derived from Hawaiian P. tuberculosa were characterized as to their concentration, composition, in-vitro potency and interaction with an anti-PLTX monoclonal antibody (mAb), after which they were evaluated for lethality and pathophysiological effects by intraperitoneal (IP) and aerosol administration to rats. Once each preparation was characterized as to its toxin composition by LC-HRMS and normalized to a total PLTX/OVTX concentration using HPLC-UV, all four preparations showed similar potency towards mouse erythrocytes in the erythrocyte hemolysis assay and interactions with the anti-PLTX mAb. The IP LD50 values derived from these experiments (1-3 μg/kg for all) were consistent with published values, although some differences from the published literature were seen. The aerosol LD50 values (.03-.06 μg/kg) confirmed the exquisite potency of PLTX suggested by the literature. The pathophysiological effects of the different toxin preparations by IP and aerosol administration were similar, albeit with some differences. Most commonly affected tissues were the lungs, liver, heart, kidneys, salivary glands, and adrenal glands. Despite some differences, these results suggest commonalities in potency and mechanism of action among these PLTX congeners.This work was supported by the Defense Threat Reduction Agency, through the Joint Program Executive Office for Chemical and Biological Defense, Contract number CB10396. Additional support to DMA and DLK was provided by National Science Foundation (Grant OCE-1314642) and National Institutes of Health (NIEHS-1P50-ES021923-01) through the Woods Hole Center for Oceans and Human Health

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

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

Proteomic Basis of the Antibody Response to Monkeypox Virus Infection Examined in Cynomolgus Macaques and a Comparison to Human Smallpox Vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92–95% (166–192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome

ACAM2000™: The new smallpox vaccine for United States Strategic National Stockpile

Aysegul Nalca, Elizabeth E ZumbrunCenter for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD, USAAbstract: Smallpox was eradicated more than 30 years ago, but heightened concerns over bioterrorism have brought smallpox and smallpox vaccination back to the forefront. The previously licensed smallpox vaccine in the United States, Dryvax® (Wyeth Laboratories, Inc.), was highly effective, but the supply was insufficient to vaccinate the entire current US population. Additionally, Dryvax® had a questionable safety profile since it consisted of a pool of vaccinia virus strains with varying degrees of virulence, and was grown on the skin of calves, an outdated technique that poses an unnecessary risk of contamination. The US government has therefore recently supported development of an improved live vaccinia virus smallpox vaccine. This initiative has resulted in the development of ACAM2000™ (Acambis, Inc.™), a single plaque-purified vaccinia virus derivative of Dryvax®, aseptically propagated in cell culture. Preclinical and clinical trials reported in 2008 demonstrated that ACAM2000™ has comparable immunogenicity to that of Dryvax®, and causes a similar frequency of adverse events. Furthermore, like Dryvax®, ACAM2000™ vaccination has been shown by careful cardiac screening to result in an unexpectedly high rate of myocarditis and pericarditis. ACAM2000™ received US Food and Drug Administration (FDA) approval in August 2007, and replaced Dryvax® for all smallpox vaccinations in February 2008. Currently, over 200 million doses of ACAM2000™ have been produced for the US Strategic National Stockpile. This review of ACAM2000™ addresses the production, characterization, clinical trials, and adverse events associated with this new smallpox vaccine.Keywords: smallpox, vaccinia, variola, vaccine, efficacy, safet

Comparison of experimental respiratory tularemia in three nonhuman primate species

Tularemia is a zoonotic disease caused by Francisella tularensis, which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10–50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than \u3e30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of theanimals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemiadisease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis

Serum chemistries in macaques exposed to aerosolized MPXV.

<p>The dotted lines indicate the normal reference range; n: number of animals. Graphs show average <b>A</b>) total protein, <b>B</b>) albumin, <b>C</b>) lactate dehydrogenase (LDH), <b>D</b>) C-reactive protein, <b>E</b>) aspartate transaminase (AST), <b>F</b>) and alanine transaminase (ALT), <b>G</b>) urea nitrogen.</p