The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signalregulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.Anthrax is caused by Bacillus anthracis, a large, rod-shaped, spore-forming, gram-positive bacterium (27). Stable B. anthracis spores form the basis of potential biological or bioterrorism weapons. The virulence of B. anthracis is dependent on the genes carried by two plasmids, pXO1 and pXO2. The genes for the synthesis of an antiphagocytic poly-␥-D-glutamic acid capsule are encoded by pXO2. Plasmid pXO1 contains three genes, pag, lef, and cya, which encode protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively (26). These three proteins form two toxins, edema toxin (EdTx; PA plus EF) and lethal toxin (LeTx; PA plus LF). PA is the receptor-binding component of the anthrax toxins and mediates their entry into host cells. Once PA binds to the receptor, it is cleaved at the N-terminal region by a host cell surface protease (3). The resulting 63-kDa protein heptamerizes and forms a ring structure with competitive binding sites for three molecules of LF and/or EF (28). The toxin complex is then taken up via receptor-mediated endocytosis (5).The cellular receptors for PA are expressed on a wide variety of cell lines and tissues, including peripheral blood leukocytes, at moderate to low levels (3, 43). EF is a calmodulin-dependent adenylate cyclase that forms cyclic AMP (cAMP) from ATP (23), and LF is a zinc metalloprotease with mitogenactivated...

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.Transmission of malaria occurs after a female mosquito ingests infected blood, thereby initiating subsequent parasite sexual and sporogonic development. Within minutes in response to midgut environmental cues, gametocytes fertilize to form zygotes that transform into motile ookinetes over the following 15-25 h. Mature midgut lumen ookinetes penetrate and traverse the peritrophic matrix and midgut epithelium and then develop into oocysts on the luminal side of the epithelial basement membrane. Strategies to block the transmission of parasites from vertebrate host to mosquitoes seek to interrupt parasite development at some point in the continuum from gametocytes to ookinete penetration of the mosquito midgut epithelium.The Plasmodium ookinete contains a single type of specialized secretory organelle, the microneme, which is thought to be involved in host-cell recognition, binding, and motility via secretion of soluble and cell surface molecules involved in interaction with different compartments within the mosquito midgut. Ookinetes differ from other Plasmodium invasive stages, such as sporozoites and merozoites, in that they lack rhoptry and dense granule organelles. Plasmodium ookinetes appear to secrete micronemal contents constitutively, and it has not been determined whether ookinete apic...

Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machinelearning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2-and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dosedependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was ϳ2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD.

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

Recent outbreaks of the Ebola virus (EBOV) have focused attention on the dire need for antivirals to treat these patients. We identified pyronaridine tetraphosphate as a potential candidate as it is an approved drug in the European Union which is currently used in combination with artesunate as a treatment for malaria (EC50 between 420 nM—1.14 μM against EBOV in HeLa cells). Range-finding studies in mice directed us to a single 75 mg/kg i.p. dose 1 hr after infection which resulted in 100% survival and statistically significantly reduced viremia at study day 3 from a lethal challenge with mouse-adapted EBOV (maEBOV). Further, an EBOV window study suggested we could dose pyronaridine 2 or 24 hrs post-exposure to result in similar efficacy. Analysis of cytokine and chemokine panels suggests that pyronaridine may act as an immunomodulator during an EBOV infection. Our studies with pyronaridine clearly demonstrate potential utility for its repurposing as an antiviral against EBOV and merits further study in larger animal models with the added benefit of already being used as a treatment against malaria.