The mucosal surfaces of all vertebrates have been exposed to similar evolutionary pressures for millions of years. In terrestrial vertebrates such as birds and mammals, the nasopharynx-associated lymphoid tissue (NALT) represents a first line of immune defence. Here we propose that NALT is an ancient arm of the mucosal immune system not restricted to terrestrial vertebrates. We find that NALT is present in rainbow trout and that it resembles other teleost mucosa-associated lymphoid tissues. Trout NALT consists of diffuse lymphoid cells and lacks tonsils and adenoids. The predominant B-cell subset found in trout NALT are IgT + B cells, similar to skin and gut. The trout olfactory organ is colonized by abundant symbiotic bacteria, which are coated by trout secretory immunoglobulin. Trout NALT is capable of mounting strong anti-viral immune responses following nasal delivery of a live attenuated viral vaccine. Our results open up a new tool for the control of aquatic infectious diseases via nasal vaccination.
Influenza viruses are respiratory pathogens that are responsible for both seasonal influenza epidemics and occasional influenza pandemics. The narrow therapeutic window of oseltamivir, coupled with the emergence of drug resistance, calls for the next-generation of antivirals. With our continuous interest in developing AM2-S31N inhibitors as oral influenza antivirals, we report here the progress of optimizing the in vitro pharmacokinetic (PK) properties of AM2-S31N inhibitors. Several AM2-S31N inhibitors, including compound 10b, were discovered to have potent channel blockage, single to submicromolar antiviral activity, and favorable in vitro PK properties. The antiviral efficacy of compound 10b was also synergistic with oseltamivir carboxylate. Interestingly, binding kinetic studies (K, K, and K) revealed several AM2-S31N inhibitors that have similar K values but significantly different K and K values. Overall, this study identified a potent lead compound (10b) with improved in vitro PK properties that is suitable for the in vivo mouse model studies.
Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection. IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevir—an FDA-approved drug used to treat hepatitis C virus (HCV) infections—as a potent antiviral with a novel mechanism of action toward 2Apro. 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68.
Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as the acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68, we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and releasing of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, western blot, RT-qPCR, viral heat inactivation, serial viral passage and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.
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