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

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

Quorum Sensing Inhibition Selects for Virulence and Cooperation in Pseudomonas aeruginosa

With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed

Two Component Systems: Physiological Effect of a Third Component

Signal transduction systems mediate the response and adaptation of organisms to environmental changes. In prokaryotes, this signal transduction is often done through Two Component Systems (TCS). These TCS are phosphotransfer protein cascades, and in their prototypical form they are composed by a kinase that senses the environmental signals (SK) and by a response regulator (RR) that regulates the cellular response. This basic motif can be modified by the addition of a third protein that interacts either with the SK or the RR in a way that could change the dynamic response of the TCS module. In this work we aim at understanding the effect of such an additional protein (which we call “third component”) on the functional properties of a prototypical TCS. To do so we build mathematical models of TCS with alternative designs for their interaction with that third component. These mathematical models are analyzed in order to identify the differences in dynamic behavior inherent to each design, with respect to functionally relevant properties such as sensitivity to changes in either the parameter values or the molecular concentrations, temporal responsiveness, possibility of multiple steady states, or stochastic fluctuations in the system. The differences are then correlated to the physiological requirements that impinge on the functioning of the TCS. This analysis sheds light on both, the dynamic behavior of synthetically designed TCS, and the conditions under which natural selection might favor each of the designs. We find that a third component that modulates SK activity increases the parameter space where a bistable response of the TCS module to signals is possible, if SK is monofunctional, but decreases it when the SK is bifunctional. The presence of a third component that modulates RR activity decreases the parameter space where a bistable response of the TCS module to signals is possible

Comparison of the Safety and Pharmacokinetics of ST-246® after IV Infusion or Oral Administration in Mice, Rabbits and Monkeys

ST-246® is an antiviral, orally bioavailable small molecule in clinical development for treatment of orthopoxvirus infections. An intravenous (IV) formulation may be required for some hospitalized patients who are unable to take oral medication. An IV formulation has been evaluated in three species previously used in evaluation of both efficacy and toxicology of the oral formulation. plasma concentrations. These effects were eliminated using slower IV infusions. associated toxicity. Shorter infusions at higher doses in NHP resulted in decreased clearance, suggesting saturated distribution or elimination. Elimination half-lives in all species were similar between oral and IV administration. The administration of ST-246 was well tolerated as a slow IV infusion

Melatonin prevents inflammation and oxidative stress caused by abdominopelvic and total body irradiation of rat small intestine

We investigated the day-night differences in intestinal oxidative-injury and the inflammatory response following total body (TB) or abdominopelvic (AP) irradiation, and the influence of melatonin administration on tissue injury induced by radiation. Rats (male Wistar, weighing 220-280 g) in the irradiated groups were exposed to a dose of 8 Gy to the TB or AP region in the morning (resting period - 1 h after light onset) or evening (activity span - 13 h after light onset). Vehicle or melatonin was administered immediately before, immediately after and 24 h after irradiation (10, 2.0 and 10 mg/kg, ip, respectively) to the irradiated rats. AP (P < 0.05) and TB (P < 0.05) irradiation applied in the morning caused a significant increase in thiobarbituric acid reactive substance (TBARS) levels. Melatonin treatment in the morning (P < 0.05) or evening (P < 0.05) decreased TBARS levels after TB irradiation. After AP irradiation, melatonin treatment only in the morning caused a significant decrease in TBARS levels (P < 0.05). Although we have confirmed the development of inflammation after radiotherapy by histological findings, neither AP nor TB irradiation caused any marked changes in myeloperoxidase activity in the morning or evening. Our results indicate that oxidative damage is more prominent in rats receiving TB and AP irradiation in the morning and melatonin appears to have beneficial effects on oxidative damage irrespective of the time of administration. Increased neutrophil accumulation indicates that melatonin administration exerts a protective effect on AP irradiation-induced tissue oxidative injury, especially in the morning