Chufang Li

Visualizing influenza virus infection in living mice

Preventing and treating influenza virus infection remain a challenge because of incomplete understanding of the host–pathogen interactions, limited therapeutics and lack of a universal vaccine. So far, methods for monitoring the course of infection with influenza virus in real time in living animals are lacking. Here we report the visualization of influenza viral infection in living mice using an engineered replication-competent influenza A virus carrying luciferase reporter gene. After intranasal inoculation, bioluminescence can be detected in the chest and nasopharyngeal passage of living mice. The intensity of bioluminescence in the chest correlates with the dosage of infection and the viral load in the lung. Bioluminescence in the chest of infected mice diminishes on antiviral treatment. This work provides a novel approach that enables real-time study of influenza virus infection and effects of antiviral therapeutics in living animals.

Identification of hits as matrix-2 protein inhibitors through the focused screening of a small primary amine library.

Although amantadine derivatives are the only M2 drugs for influenza virus A, their use is limited in the U.S. because of drug resistance. Here we report the identification of multiple M2 inhibitors that were rapidly generated through focused screening of a small primary amine library that was designed using a scaffold-hopping strategy based on amantadine. These compounds are as active as amantadine and might be hits for further lead generation processes.

The highly potent and selective dipeptidyl peptidase IV inhibitors bearing a thienopyrimidine scaffold effectively treat type 2 diabetes

New dipeptidyl peptidase IV inhibitors were designed based on Alogliptin using a scaffold-hopping strategy. All of the compounds constructed on a thienopyrimidine scaffold demonstrated good inhibition and selectivity for DPP-IV. Compound 10d exhibited subnanomolar (IC(50)=0.33nM) DPP-IV inhibitory activity, good in vivo efficacy and an acceptable pharmacokinetic profile. A pharmacokinetic-driven optimization of 10d may lead to a new class of clinical candidate DPP-IV inhibitors.

All-normal dispersion, figure-eight, tunable passively mode-locked fiber laser with an invisible and changeable intracavity bandpass filter

We report dissipative solitons generation in the figure-8 all-normal dispersion fiber ring laser. The steep edge of the output optical spectrum results from the filtering effect of NOLM together with polarization controllers (PCs). No any physical bandpass filters are used in the cavity. Moreover, the output wavelength can be tuned in a wide range of about 20 nm. In the long-wavelength region, the spectral filtering effect of NOLM becomes weaker and output spectrum shows only one steep side. Filtering effect can perform periodically with increasing the pump power. The highly chirped pulse has good potential for application to the chirped pulse amplification (CPA) system.

Discovery of highly potent agents against influenza A virus.

We previously reported several new M2 inhibitors as active as amantadine against influenza A virus and validated by three types of in vitro assays. Herein, we further modified one of the most potent hits in a viral inhibition assay and conducted structure-activity relationship studies on this scaffold. As a result, compound 8e was identified to be the most potent inhibitor against wild-type influenza A virus, being nearly 240-fold more active than amantadine.

Potent neutralizing monoclonal antibodies against Ebola virus infection

Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection.

Regulation of SIV antigen-specific CD4+ T cellular immunity via autophagosome-mediated MHC II molecule-targeting antigen presentation in mice.

CD4+ T cell-mediated immunity has increasingly received attention due to its contribution in the control of HIV viral replication; therefore, it is of great significance to improve CD4+ T cell responses to enhance the efficacy of HIV vaccines. Recent studies have suggested that macroautophagy plays a crucial role in modulating adaptive immune responses toward CD4+ T cells or CD8+ T cells. In the present study, a new strategy based on a macroautophagy degradation mechanism is investigated to enhance CD4+ T cell responses against the HIV/SIV gag antigen. Our results showed that when fused to the autophagosome-associated LC3b protein, SIVgag protein can be functionally targeted to autophagosomes, processed by autophagy-mediated degradation in autolysosomes/lysosomes, presented to MHC II compartments and elicit effective potential CD4 T cell responses in vitro. Importantly, compared with the SIVgag protein alone, SIVgag-LC3b fusion antigen can induce a stronger antigen-specific CD4+ T cell response in mice, which is characterized by an enhanced magnitude and polyfunctionality. This study provides insight for the immunological modulation between viral and mammalian cells via autophagy, and it also presents an alternative strategy for the design of new antigens in the development of effective HIV vaccines.

Proton channel activity of influenza A virus matrix protein 2 contributes to autophagy arrest

ABSTRACT Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Background The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal Findings The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/Significance Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.

Improvement of influenza vaccine strain A/Vietnam/1194/2004 (H5N1) growth with the neuraminidase packaging sequence from A/Puerto Rico/8/34.

H5N1 influenza candidate vaccine viruses were developed using the “6+2” approach. The hemagglutinin (HA) and neuraminidase (NA) genes were derived from the popular H5N1 virus and the remaining six internal segments were derived from the A/Puerto Rico/8/34 strain (H1N1, PR8). However, some of these candidate strains have been reported to produce relatively low yields in vaccine manufacture. In this study, we found that the NA vRNA of the A/Vietnam/1194/2004 strain (H5N1, VN1194) was poorly packaged into recombinant viruses with a backbone of PR8 genes, which resulted in the formation of defective virions that did not include the NA vRNA in the genome. Using recombinant DNA techniques, we constructed a chimeric NA gene with the coding region of VN1194 NA flanked by the packaging signal sequence of the PR8 NA gene (41 bp form the 3′ end of the vRNA and 67 bp from the 5′ end). The packaging of the NA vRNA was restored to normal levels in the recombinant viruses containing the chimeric NA gene. Recombinant viruses containing the chimeric NA replicated much better in chicken embryonated eggs than viruses with the wild-type NA from VN1194. These findings suggest a novel strategy to improve in ovo growth of vaccine strains and to increase the number of vaccine doses available to save people if a pandemic were to occur.