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Abstract: 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. [Copyright &y& Elsevier]

Ethnopharmacological relevance Isatis indigotica is a traditional Chinese medicine. Its dried roots named “ban lan gen” in Chinese, are used for clinical treatment of virus infection, tumor, inflammation with a long history. However, its anti-influenza active ingredient and the underlying mechanism remain unclear. In this study, the anti-influenza and anti-inflammatory effects of a lignan glycoside: lariciresinol-4-O-β- D -glucopyranoside isolated from the root of I. indigotica on human alveolar epithelial cell line A549 infected with influenza A virus were investigated. Materials and methods Chemical and spectroscopic methods were employed to identify the structure of the lignan glycoside. Cytotoxicity of the lignan glycoside was analyzed using methylthiazolyltetrazolium (MTT) assay. The inhibitory activity against influenza virus of the lignan was determined by CPE inhibition assay. HEK-293 cells stably co-transfected with NF-κB responsive firefly luciferase and constitutively expressing GFP were employed for monitoring the effect of the lignan on NF-κB signal pathway activation. Nuclear export of viral ribonucleoprotein (RNP) complexes was monitored by indirect immunofluorescence. Quantitative real-time PCR was used to quantify the expression profiling of cytokines and chemokines after infection with influenza virus. Results We showed that the lignan glycoside treatment was effective against the influenza A virus-induced cytopathic effect (CPE) in MDCK cells. Further study demonstrated the lignan glycoside attenuated virus-induced NF-κB activation, but did not affect export of viral ribonucleoprotein (RNP) complexes from the nucleus in late stages of infection. We revealed that the lignan glycoside suppressed influenza A virus (H1N1)-induced expression of the pro-inflammatory molecules IL-6, TNF-α, IL-8, MCP-1, IP-10 and IFN-α. Moreover, the cytokines and chemokines profiles induced by H9N2 virus resembled those of influenza virus H1N1, but the lignan glycoside reduced the expression of IP-10 and TNF-α. Conclusions Our results suggest that the lignan glycoside is a bioactive component of I. indigotica which may contribute an adjunct to pharmacotherapy for influenza virus infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Proteasome-mediated proteolysis is a primary protein degradation pathway in cells. The present study demonstrates that c-Abl and Arg (abl-related gene) tyrosine kinases associate with and phosphorylate the proteasome PSMA7 (α4) subunit at Tyr-153. Consequently, proteasome-dependent proteolysis is compromised. Notably, cells expressing a phosphorylation mutant of PSMA7(Y153F) display impaired G1/S transition and S/G2 progression, highlighting the biological significance of tyrosine phosphorylation of a proteasome subunit as an important cellular regulatory control. [Copyright &y& Elsevier]

Zika virus (ZIKV) infection can cause neonatal microcephaly and neurological disorders. Currently, there is no designated drug for treating ZIKV infection and preventing neonatal microcephaly. In this study, we evaluated the effect of chloroquine, an anti-malaria drug, in ZIKV infected cells and mouse models. Chloroquine significantly inhibited ZIKV infection in multiple mammalian cell lines. Chloroquine treatment significantly improved the survival of ZIKV-infected 1-day old suckling SCID Beige mice and reduced viremia in adult SCID Beige mice. Importantly, chloroquine protected the fetus from maternal infection by reducing placenta to fetus viral transmission. We found that chloroquine exerts at least two mechanisms in protecting against ZIKV infection: 1) inhibiting endosomal disassembly of the internalized virus and thus reducing the release of viral RNA to the cytoplasm for replication; 2) inhibiting ZIKV RNA replication through blocking ZIKV induced autophagy. Our study suggests that chloroquine treatment warrants to be considered as a mitigation strategy for treating ZIKV infection and preventing ZIKV-associated microcephaly in pregnant women. • Chloroquine significantly inhibits Zika virus infection in multiple mammalian cell lines. • Chloroquine protects against Zika virus infection in immunocompromised SCID Beige adult mice and neonatal mice. • Chloroquine prevents maternal to fetal Zika virus transmission in an immunocompromised SCID Beige mouse model. • Chloroquine blocks the release of Zika virus RNA from the endosome into the cytoplasm. • Chloroquine inhibits Zika virus replication through blocking ZIKV-induced autophagy. [ABSTRACT FROM AUTHOR]

Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development. [ABSTRACT FROM AUTHOR]

Abstract: Current treatments for allergic asthma primarily ameliorate symptoms rather than inhibit disease progression. Regulating the excessive T helper type 2 (Th2) responses may prevent asthma exacerbation. In this study, we investigated the protective effects of Ad5-gsgAM, an adenovirus vector carrying two mycobacterial antigens Ag85A and Mtb32, against allergic asthma. Using an ovalbumin (OVA)-induced asthmatic mouse model, we found that Ad5-gsgAM elicited much more Th1-biased CD4+T and CD8+T cells than bacillus Calmette-Guérin (BCG). After OVA challenge, Ad5-gsgAM-immunized mice showed significantly lowered airway inflammation in comparison with mice immunized with or without BCG. Total serum immunoglobulin E and pulmonary inducible-nitric-oxide-synthase were efficiently reduced. The cytokine profiles in bronchial-alveolar-lavage-fluids (BALFs) were also modulated, as evidenced by the increased level of interferon-γ (IFN-γ) and the decreased level of interleukin (IL)-4, IL-5, and IL-13. Anti-inflammatory cytokine IL-10 was sharply increased, whereas pro-inflammatory cytokine IL-33 was significantly decreased. Importantly, exogenous IL-33 abrogated the protective effects of Ad5-gsgAM, revealing that the suppression of IL-33/ST2 axis substantially contributed to protection against allergic inflammation. Moreover, regulatory T cells were essential for regulating aberrant Th2 responses as well as IL-33/ST2 axis. These results suggested that modulating the IL-33/ST2 axis via adenovirus-vectored mycobacterial antigen vaccination may provide clinical benefits in allergic inflammatory airways disease.Key messages: •Ad5-gsgAM elicits Th1 responses and suppresses Th2-mediated allergic asthma in mice.•Ad5-gsgAM inhibits IL-33/ST2 axis by reducing IL-33 secretion but not ILC2 recruiting.•Treg is essential for modulating Th2 responses and IL-33/ST2 axis by Ad5-gsgAM. [ABSTRACT FROM AUTHOR]

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a coronavirus (CoV), MERS-CoV. With the continuing spread of MERS-CoV, prophylactic and therapeutic treatments are urgently needed. In this study, we prepared purified equine F(ab’) 2 from horses immunized with MERS-CoV virus-like particles (VLPs) expressing MERS-CoV S, M and E proteins. Both IgG and F(ab’) 2 efficiently neutralized MERS-CoV replication in tissue culture. Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab’) 2 for potential use in the prophylactic or therapeutic treatment of exposed or infected patients. [ABSTRACT FROM AUTHOR]

Highlights: [•] The immunogenicity of hemagglutinin (HA) of newly emerged influenza A virus H7N9 need to be known. [•] Inactivated whole-virion containing HA and NA of H7N9 in the PR8 background was evaluated. [•] Antibody titers were assessed in different doses of HA alone, or with alum, and/or with a booster. [•] Immunization could produce neutralizing antibodies in mice and rhesus macaques. [•] Mice received sera from immunized animals could be protected from lethal challenge of H7N9 virus. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 (IC50 =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. [Copyright &y& Elsevier]

Abstract: The CCAAT/enhancer-binding protein beta (C/EBPβ) is a critical transcription factor that regulates gene expression during numerous biological processes, including differentiation, metabolism, homeostasis, proliferation, tumorigenesis, inflammation, and apoptosis. In this study, interactions between C/EBPβ and either the Abelson murine leukemia viral oncogene homolog 1 (c-Abl) or the Abl-related gene (Arg) were demonstrated in vitro and in vivo with a direct binding assay and by co-immunoprecipitation, respectively. The Y79 amino acid residue of C/EBPβ was phosphorylated by c-Abl or Arg. The phosphorylation of C/EBPβ resulted in an increased C/EBPβ stability and a potentiation of C/EBPβ transcription activation activity in cells. Expression of the C/EBPβ(Y79F) mutant in HEK293, and K562, and in other cell lines, resulted in less of a delay in the cell cycle compared to the wild type C/EBPβ; furthermore, the C/EBPβ (Y79F) mutant induced an increased apoptosis compared to the wild type C/EBPβ. These findings suggest that the c-Abl family non-receptor tyrosine kinases have a role in the regulation of the C/EBPβ transcription factor. [Copyright &y& Elsevier]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro , making its promising candidate treatment for controlling COVID-19 disease. [ABSTRACT FROM AUTHOR]

Highly pathogenic influenza H7N9 viruses that emerged in the fifth wave of H7N9 outbreak pose a risk to human health. The World Health Organization has updated the candidate vaccine viruses for H7N9 viruses recently. In this study, we evaluated the immune response to an updated H7N9 candidate vaccine virus, which derived from the highly pathogenic A/Guangdong/17SF003/2016 (GD/16) in mice and rhesus macaques. GD/16 vaccination elicited robust neutralizing, virus-specific immunoglobulin G antibodies and effective protection, but poor hemagglutination inhibition antibody titers. Furthermore, mouse and rhesus macaque serum raised against the previous H7N9 CVV A/Anhui/1/2013 (AH/13) were tested for its cross-reactivity to GD/16 virus. We found that although AH/13-immune serum has poor hemagglutination inhibition reactivity against GD/16 virus, AH/13 elicit efficient cross-neutralizing antibodies and in vivo protection against GD/16. Further studies showed that the hemagglutinin of GD/16 has strong receptor binding avidity, which might be associated with the decreased hemagglutination inhibition assay sensitivity. This study underscores the point that receptor binding avidity should be taken into account when performing quantitative interpretation of hemagglutination inhibition data. A combination of multiple serological assays is required for accurate vaccine evaluation and antigenic analysis of influenza viruses. • Systemically evaluated the immune response to H7N9 CVV A/Guangdong/17SF003/2016 (GD/16) in mice and rhesus macaques. • GD/16 elicited robust neutralizing and IgG antibodies, but poor hemagglutination inhibition antibody titers. • Receptor binding avidity should be considered in interpretation of HI data for evaluation of influenza antigenic variation. [ABSTRACT FROM AUTHOR]

Background: SARS-Cov is the etiologic agent of severe acute respiratory syndrome. An understanding of the antibody responses to the viral components is very important for diagnosis and vaccine development. Objective: The spectrum of SARS-specific antibody profiles in SARS patients was investigated from 7 to 210 days after the onset of the symptoms. Study design: Serial serum samples from 14 SARS patients were isolated from 7 to 210 days after the onset of the symptoms, and were tested for anti-viral IgG and IgM by indirect immunofluorescence tests (IFA), anti-nucleocaspid antibody by ELISA tests and viral neutralization. Results: Anti-viral (IgG) and anti-nucleocaspid antibodies were observed in 13 of 14 patients at 14 days after the onset of symptoms, and in all 14 patients at 30–210 days thereafter. Anti-viral antibody (IgM) was detected maximally at 30 days, later than that for the IgG class. IgM antibody declined and became undetectable between 60 to 180 days after the onset of the symptoms. Neutralizing viral antibodies were demonstrated in the sera from all of the patients with SARS symptoms. Conclusions: Anti-viral IgG, IgM, and anti-nucleocaspid antibodies were detected 7–30 days in patients after the onset of SARS symptoms. Anti-viral IgM antibodies disappeared earlier than IgG. Viral neutralization was demonstrated in the sera from the convalescent patients. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]