Zhenyu Tian

Generation of influenza A viruses as live but replication-incompetent virus vaccines

Genetic code expansion and orthogonal translation machinery are used to generate live, attenuated viral vaccines. Protecting by changing the code Live attenuated vaccines can be very potent, but their potential to revert to their pathogenic form limits their use. In an attempt to get around this, Si et al. expanded the genetic code of influenza A viruses. They propagated viruses that were mutated to encode premature termination codons (PTCs) in a cell line engineered to be able to express these flu proteins. Despite not being able to replicate in conventional cells, PTC-containing viruses were highly immunogenic and protected mice, guinea pigs, and ferrets against influenza challenge. Science, this issue p. 1170 The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)–harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell–mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus.

Design, synthesis and biological activity evaluation of novel conjugated sialic acid and pentacyclic triterpene derivatives as anti-influenza entry inhibitors

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Recently, plant-derived pentacyclic triterpenes have been shown to act as highly potent anti-viral agents by efficiently preventing the attachment of the virion to the host cells. In this report, we conjugated sialic acid with oleanolic acid (OA), a natural product with broad antiviral entry activity, as well as three other analogs echinocystic acid (EA), ursolic acid (UA) and betulinic acid (BA). A total of 24 conjugated sialic acid and pentacyclic triterpene derivatives with different linkers were synthesized and evaluated for antiviral activity against influenza A/WSN/33 (H1N1) virus in MDCK cell culture. The most potent compound had an IC50 of 41.2 μM. Time-of-addition, hemagglutination inhibition (HI), surface plasmon resonance (SPR) and molecular docking assays demonstrated that compound 20a acted as an influenza virus entry inhibitor by preventing the binding of influenza virus hemagglutinin (HA) protein to host cells.

Recent progress in the antiviral activity and mechanism study of pentacyclic triterpenoids and their derivatives

Viral infections cause many serious human diseases with high mortality rates. New drug‐resistant strains are continually emerging due to the high viral mutation rate, which makes it necessary to develop new antiviral agents. Compounds of plant origin are particularly interesting. The pentacyclic triterpenoids (PTs) are a diverse class of natural products from plants composed of three terpene units. They exhibit antitumor, anti‐inflammatory, and antiviral activities. Oleanolic, betulinic, and ursolic acids are representative PTs widely present in nature with a broad antiviral spectrum. This review focuses on the recent literatures in the antiviral efficacy of this class of phytochemicals and their derivatives. In addition, their modes of action are also summarized.

Precision Fluorescent Labeling of an Adeno-Associated Virus Vector to Monitor the Viral Infection Pathway

Adeno-associated virus 2 (AAV2) is a common vehicle for the delivery of a variety of therapeutic genes. A better understanding of the process of infection of AAV2 will advance our knowledge of AAV2 biology and allow for the optimization of AAV2 capsids with favorable transduction profiles. However, the precise fluorescent labeling of an AAV2 vector for probing virus tracking without affecting the nature of the virus remains a challenge. In this study, a lab-synthesized azide-moieties on the viral capsid at modifiable sites is precisely displayed. Upon bioorthogonal copper-less click reaction, fluorophores are subsequently conjugated to AAV2 vectors for visualization of particles. Using this principle, the authors demonstrate that it can be used for visibly studying the cell entry, and intracellular trafficking of AAV2 particles, enabling the monitoring of the intracellular dynamics of AAV2 infection. This study provides new insights into the precision labeling of AAV2 particles with important implications for a better understanding of the molecular mechanism of therapeutic gene delivery.

Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity

Adeno-associated virus (AAV) is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs) through click chemistry. Using this approach, only the chosen site(s) was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

Synthesis and In Vitro Anti-Influenza Virus Evaluation of Novel Sialic Acid (C-5 and C-9)-Pentacyclic Triterpene Derivatives

The emergence of drug resistant variants of the influenza virus has led to a great need to identify novel and effective antiviral agents. In our previous study, a series of sialic acid (C-2 and C-4)-pentacyclic triterpene conjugates have been synthesized, and a five-fold more potent antiviral activity was observed when sialic acid was conjugated with pentacyclic triterpene via C-4 than C-2. It was here that we further reported the synthesis and anti-influenza activity of novel sialic acid (C-5 and C-9)-pentacyclic triterpene conjugates. Their structures were confirmed by ESI-HRMS, 1H-NMR, and 13C-NMR spectroscopic analyses. Two conjugates (26 and 42) showed strong cytotoxicity to MDCK cells in the CellTiter-Glo assay at a concentration of 100 μM. However, they showed no significant cytotoxicity to HL-60, Hela, and A549 cell lines in MTT assay under the concentration of 10 μM (except compound 42 showed weak cytotoxicity to HL-60 cell line (10 μM, ~53%)). Compounds 20, 28, 36, and 44 displayed weak potency to influenza A/WSN/33 (H1N1) virus (100 μM, ~20–30%), and no significant anti-influenza activity was found for the other conjugates. The data suggested that both the C-5 acetylamide and C-9 hydroxy of sialic acid were important for its binding with hemagglutinin during viral entry into host cells, while C-4 and C-2 hydroxy were not critical for the binding process and could be replaced with hydrophobic moieties. The research presented herein had significant implications for the design of novel antiviral inhibitors based on a sialic acid scaffold.

Recent progresses on pentacyclic triterpene-based antiviral agents

Pentacyclic triterpenes are a class of secondary plant metabolites widely distributed throughout the plant kingdom with promising pharmacological activities including antitumor, anti-infection, immunomodulatory and so on. This review will focus on the anti-viral aspect of pentacyclic triterpene together with their derivatives and analogs on HIV, HCV and influenza viruses. This will be valuable for the development of novel pentacyclic triterpene-based antiviral therapeutic agents.