Alloferon and Zanamivir Show Effective Antiviral Activity against Influenza A Virus (H1N1) Infection In Vitro and In Vivo

The use of vaccines is the most effective and reliable method for the prevention of viral infections. However, research on evaluation of effective therapeutic agents for use in treatment after infection is necessary. Zanamivir was administered through inhalation for treatment of pandemic influenza A/H1N1 in 2009. However, the emergence of drug-resistant strains can occur rapidly. Alloferon, an immunomodulatory drug developed as an NK cell activator, exerts antiviral effects against various viruses, particularly influenza viruses. Therefore, alloferon and zanamivir were administered in combination in an effort to improve the antiviral effect of zanamivir by reducing H1N1 resistance. First, we confirmed that administration of combined treatment would result in effective inhibition of viral proliferation in MDCK and A549 cells infected with H1N1. Production of IL-6 and MIP-1α in these cells and the activity of p38 MAPK and c-Jun that are increased by H1N1 were inhibited by combined treatment. Mice were then infected intranasally with H1N1, and examination of the antiviral efficacy of the alloferon/zanamivir combination was performed. The results showed that combined treatment after infection with H1N1 prevented weight loss, increased the survival rate, and improved lung fibrosis. Combined treatment also resulted in reduced infiltration of neutrophils and macrophages into the lungs. Combined treatment effectively inhibited the activity of p38 MAPK and c-Jun in lung tissue, which was increased by infection with H1N1. Therefore, the combination of alloferon/zanamivir effectively prevents the development of H1N1-mediated inflammation in the lungs by inhibiting the production of inflammatory mediators and migration of inflammatory cells into lung tissue.     

静注甲型H1N1流感人免疫球蛋白的研制

目的研制高效价静注甲型H1N1流感人免疫球蛋白。方法用甲型H1N1流感疫苗(简称甲流)对固定供血浆者按疫苗说明书免疫后2周开始采集原料血浆,采用血凝抑制法检测原料血浆和制品的甲流抗体效价;采用柱层析法从高效价甲流免疫血浆中提取富含IgM免疫球蛋白的样品,模拟低温乙醇蛋白分离法工艺从小量混合血浆分离IgG样品,测定血浆和提取样品的甲流抗体效价,分析甲流中和抗体效价与IgG、IgM含量的对应关系,判断甲流中和抗体的免疫球蛋白类型;按本公司静脉注射人免疫球蛋白(Intravenous immunoglobulin,IVIG)生产工艺制备静注甲型H1N1流感人免疫球蛋白,并与普通IVIG及相应合并血浆的甲流抗体效价进行比较。结果筛选到甲流抗体效价≥320 HU/ml的合格血浆9 866袋,合格率达33.5%,其中抗体效价≥640 HU/ml的血浆占合格血浆的49%,血浆质量符合《中国药典》三部(2010版)"血液制品生产用人血浆规程"要求;甲流抗体效价与IgG含量呈相应比例关系,而与IgM含量无关,甲流中和抗体的免疫球蛋白类型以IgG为主;制备的甲流人免疫球蛋白制品的甲流中和抗体效价达2 560 HU/ml,为普通IVIG的197倍,其他质量指标符合《中国药典》三部(2010版)"静注人免疫球蛋白制检规程"要求。结论成功研制了静注甲型H1N1流感人免疫球蛋白,在甲流疫情得到有效控制的情况下,仍具有战略储备意义。     

人甲型流感病毒H1N1亚型NS1蛋白的原核表达及纯化

目的表达并纯化人甲型流感病毒H1N1亚型NS1蛋白。方法用RT-PCR法从人甲型流感病毒株A/PR/8/34(H1N1)中扩增NS1基因,克隆入原核表达载体pTXB1,构建重组原核表达质粒pTXB1/NS1,经酶切鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达形式和表达量。经几丁质柱亲和层析纯化表达蛋白,串联飞行时间质谱仪检测其相对分子质量。结果所构建的重组表达质粒pTXB1/NS1序列完整,插入的基因片段全长690bp。以1.0mmol/LIPTG37℃诱导4h,重组蛋白表达量最高,占菌体总蛋白的50%以上。破菌上清及沉淀中均有目的蛋白表达。纯化的NS1蛋白纯度达95%以上,相对分子质量约为26000。结论已成功表达并纯化了人甲型流感病毒H1N1亚型NS1蛋白,为其进一步的研究奠定了基础。     

国产甲型H1N1流感病毒裂解疫苗的安全性及免疫原性

目的观察国产甲型H1N1流感病毒裂解疫苗的安全性及免疫原性。方法按照随机、对照、盲法的原则,0、21d程序选择老年组、少年组和少儿组各220人,按1︰1比例随机接种15μg、30μg甲型H1N1流感疫苗;成年组330人,按1︰1︰1比例随机接种15μg、30μg甲型H1N1流感疫苗和安慰剂对照(PBS);0、28d程序只选择成年组220人,按1︰1比例随机接种15μg、30μg甲型H1N1流感疫苗。观察各组接种后的总体不良反应率、全身和局部不良反应率以及免疫后HI抗体阳转率、保护率、GMT水平和平均增长倍数。结果1210名观察对象总体不良反应发生率为21.82%,均以1级不良反应为主,未观察到3级及3级以上不良反应、其他异常反应和严重不良事件。30μg剂量组免疫后HI抗体阳转率和保护率与15μg剂量组比较,差异无统计学意义。两接种程序同一剂量组内第1针免疫后HI抗体阳转率、保护率、免疫后GMT及增长倍数各指标比较,差异均无统计学意义。结论国产甲型H1N1流感病毒裂解疫苗具有良好的安全性和免疫原性,按照0、21d程序各接种1针15μg甲型H1N1流感疫苗,即可在12～60岁人群中产生良好的免疫效果。     

Combining Molecular Docking and Molecular Dynamics to Predict the Binding Modes of Flavonoid Derivatives with the Neuraminidase of the 2009 H1N1 Influenza A Virus

Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R(2) = 0.75.     

Antiviral Gene Expression in Young and Aged Murine Lung during H1N1 and H3N2

Influenza is a respiratory virus that alone or in combination with secondary bacterial pathogens can contribute to the development of acute pneumonia in persons >65 years of age. Host innate immune antiviral signaling early in response to influenza is essential to inhibit early viral replication and guide the initiation of adaptive immune responses. Using young adult (3 months) and aged adult mice infected with mouse adapted H1N1 or H3N2, the results of our study illustrate dysregulated and/or diminished activation of key signaling pathways in aged lung contribute to increased lung inflammation and morbidity. Specifically, within the first seven days of infection, there were significant changes in genes associated with TLR and RIG-I signaling detected in aged murine lung in response to H1N1 or H3N2. Taken together, the results of our study expand our current understanding of age-associated changes in antiviral signaling in the lung.     

Synthesis and In Vitro Evaluation of C-7 and C-8 Luteolin Derivatives as Influenza Endonuclease Inhibitors

The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg²⁺ or Mn²⁺ ions located in the enzyme’s catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3′,4′-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC₅₀ values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.     

Isocyanides as Influenza A Virus Subtype H5N1 Wild‐Type M2 Channel Inhibitors

Basic bulky amines such as amantadine are well‐characterized M2 channel blockers, useful for treating influenza. Herein we report our surprising findings that charge‐neutral, bulky isocyanides exhibit activities similar to—or even higher than—that of amantadine. We also demonstrate that these isocyanides have potent growth inhibitory activity against the H5N1 virus. The ?NH₂ to ?N≡C group replacement within current anti‐influenza drugs was found to give compounds with high activities at low‐micromolar concentrations. For example, a tenfold improvement in potency was observed for 1‐isocyanoadamantane ( 27 ), with an EC₅₀ value of 0.487 μm against amantadine‐sensitive H5N1 virus as determined by both MTT and plaque‐reduction assays, without showing cytotoxicity. Furthermore, the isocyanide analogues synthesized in this study did not inhibit the V27A or S31N mutant M2 ion channels, according to electrophysiology experiments, and did not exhibit activity against amantadine‐resistant virus strains.     

Antiviral Activity of N1,N3-Disubstituted Uracil Derivatives against SARS-CoV-2 Variants of Concern

Despite the widespread use of the COVID-19 vaccines, the search for effective antiviral drugs for the treatment of patients infected with SARS-CoV-2 is still relevant. Genetic variability leads to the continued circulation of new variants of concern (VOC). There is a significant decrease in the effectiveness of antibody-based therapy, which raises concerns about the development of new antiviral drugs with a high spectrum of activity against VOCs. We synthesized new analogs of uracil derivatives where uracil was substituted at the N₁ and N₃ positions. Antiviral activity was studied in Vero E6 cells against VOC, including currently widely circulating SARS-CoV-2 Omicron. All synthesized compounds of the panel showed a wide antiviral effect. In addition, we determined that these compounds inhibit the activity of recombinant SARS-CoV-2 RdRp. Our study suggests that these non-nucleoside uracil-based analogs may be of future use as a treatment for patients infected with circulating SARS-CoV-2 variants.     

人用H5N1禽流感裂解疫苗的制备工艺

目的设计3种工艺,制备不同裂解程度的禽流感疫苗,并观察其免疫效果。方法用禽流感毒种R1203株制备3种禽流感疫苗(全病毒、裂解-1和裂解-2),并分别以不同剂量免疫大鼠和家兔,肌肉接种2针(间隔14d),初免后14d和28d静脉采血,检测动物血清中血凝抑制抗体和中和抗体。结果两种动物在疫苗接种1针后14d,血抑抗体和中和抗体滴度均较低;接种2针后14d,均显著高于1针;裂解-2疫苗接种两种动物后的抗体反应均高于其他两种疫苗,且家兔的中和抗体量-效反应明显。结论裂解-2疫苗的工艺优于其他两种疫苗,其中和抗体能正确反映疫苗的质量。     

H5N1高致病性人禽流感DNA疫苗表达质粒的构建及其免疫原性

目的构建H5N1高致病性人禽流感DNA疫苗表达质粒,并观察其免疫原性。方法分析H5N1人禽流感病毒HA基因的进化关系,人工合成人禽流感病毒HA基因(包含多数H5N1人禽流感病毒共有序列),构建含细胞毒性T淋巴细胞抗原4(CTLA4)和HA融合基因的真核表达质粒pVAX-CLHA及HA基因的真核表达质粒pVAX-HA,经酶切及测序鉴定正确后,转染293-T细胞,经RT-PCR法检测转染细胞中HA基因mRNA的转录水平,间接免疫荧光法(IFA)检测其表达;分别以质粒pVAX-CLHA及pVAX-HA免疫BALB/c小鼠,测定血清HI抗体效价。结果重组DNA疫苗表达质粒pVAX-CLHA和pVAX-HA经酶切及测序证明构建正确,转染的293-T细胞可检测到目的基因的转录及蛋白的表达;3次免疫后均能诱导BALB/c小鼠产生HI抗体,pVAX-CLHA诱导的HI抗体高于pVAX-HA。结论已成功构建了含H5N1HA及CTLA4融合基因的DNA疫苗表达质粒,其对小鼠具有良好的免疫效果,为H5N1高致病性人禽流感DNA疫苗的开发奠定了基础。     

甲型H1N1流感病毒实时荧光PCR诊断试剂盒的制备

目的制备甲型H1N1流感病毒实时荧光PCR诊断试剂盒,并进行验证。方法采用磁珠法从甲型H1N1流感疑似患者咽拭子样品中提取病毒RNA,逆转录合成cDNA。根据NCBI最新公布的大流行甲型H1N1流感病毒(2009)基因序列,设计针对编码基质蛋白M基因的引物和探针,检测甲型流感病毒;设计针对血凝素(HA)基因和神经氨酸酶(NA)基因特异性的引物和探针,检测甲型H1N1流感病毒;同时针对人的RNaseP基因设计用于内部控制的引物和探针。所有探针均为Taqman探针,5'端标记FAM,3'端标记BHQ1。对最佳荧光PCR反应条件进行优化,在此基础上组装成甲型H1N1流感病毒实时荧光PCR诊断试剂盒,对其特异性、灵敏度、精密性和稳定性进行验证。与市售试剂盒的检测结果进行对比,并对63份临床甲型H1N1流感疑似患者咽拭子样品进行检测。结果设计的PCR引物及探针能对甲型H1N1流感病毒进行准确检测,与流感病毒的其他型和亚型无交叉反应;试剂盒的灵敏度为0.004个血凝素单位;试验内变异系数小于2.5%,批间变异系数小于5%;试剂盒放置-20℃保存,稳定性良好;检测20份甲型H1N1流感疑似患者咽拭子样品的结果与市售试剂盒一致;检测63份流感疑似患者咽拭子样品,其中大流行甲型H1N1流感病毒阳性36份,普通甲型流感病毒阳性5份。结论所制备的甲型H1N1流感病毒实时荧光PCR诊断试剂盒具有较高的灵敏度、特异性、精密性和稳定性,可用于目前流行的甲型H1N1流感病毒的快速检测。     

甲型H1N1流感疫苗免疫安全性观察

目的观察国产甲型H1N1流感疫苗的免疫安全性。方法对3～65岁的人群接种国产甲型H1N1流感疫苗和接种季节性流感疫苗进行接种副反应观察。结果 78972人接种甲型H1N1流感疫苗者,有91人次局部轻微红肿,另有6人出现发热,1人出现过敏,接种反应率为0.124%。季节性流感疫苗接种24899人次,局部轻微红肿26人次,发热2人次,接种反应率为0.112%,结论国产甲型H1N1流感疫苗副反应少,免疫安全性可靠,群众可放心接种。     

H5N1亚型禽流感病毒免疫血清精制工艺的建立

目的建立马抗H5N1亚型禽流感病毒免疫血清的精制工艺。方法以硫酸铵盐析法提取免疫马血浆中的IgG抗体,以8～256μg/mgIgG胃蛋白酶(活性单位1:3000)进行消化,以阳离子交换层析柱纯化F(ab’)2抗体,并参照《中国药典》三部(2005版)要求,对试制的样品进行检定。结果经一步50%硫酸铵和多步33%硫酸铵盐析,获得了较为纯净的IgG抗体。8μg胃蛋白酶用量可完全消化1mgIgG抗体分子,阳离子交换方法分离F(ab’)2抗体纯度可达90%以上,高于常规工艺制备的抗体纯度。以此工艺试制的样品,各项质量指标均符合《中国药典》三部(2005版)质量标准。结论已初步建立了马抗H5N1亚型禽流感病毒免疫血清的精制工艺。     

表达甲型H1N1流感病毒神经氨酸酶的重组腺病毒的构建及其免疫原性

目的构建表达甲型H1N1流感病毒神经氨酸酶(Neuraminidase,NA)的重组腺病毒,并检测其免疫原性。方法从质粒pMD19T-simple-NA中扩增NA基因,克隆至穿梭质粒pShuttleCMV中,经同源重组获得重组腺病毒质粒,转染Ad-293细胞,包装出重组腺病毒Ad-NA,RT-PCR和免疫荧光法检测NA基因在Vero细胞中的转录和表达。CsCl密度梯度离心纯化重组腺病毒,免疫小鼠,ELISA法检测免疫小鼠血清中抗NA抗体滴度。结果重组腺病毒质粒经PacⅠ酶切鉴定表明带有目的基因的穿梭质粒已整合到腺病毒基因组中;NA基因在Vero细胞中成功转录和表达;重组腺病毒可刺激小鼠产生抗NA抗体,初免后4周,抗体水平达最高,为1∶100 000。结论成功构建了表达甲型H1N1流感病毒NA蛋白的重组腺病毒,其可刺激小鼠产生有效的免疫应答,为甲型H1N1流感病毒基因工程疫苗的研发奠定了基础。     

Identification of Small Molecules that Interfere with H1N1 Influenza A Viral Replication

Successful replication of the influenza A virus requires both viral proteins and host cellular factors. In this study we used a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We used an established reporter assay to assess influenza viral replication by monitoring the activity of co‐expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7 , which inhibits a novel yet elusive target. Quantitative real‐time PCR studies confirmed the dose‐dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 is effective in rescuing high‐dose influenza infection in an in vivo mouse model. As oseltamivir‐resistant influenza strains emerge, compound 7 could be further investigated as a new and potentially suitable scaffold for the development of anti‐influenza agents that act on novel targets.     

Design,Synthesis, and Biological Evaluation of Pyrazoline‐Based Hydroxamic Acid Derivatives as Aminopeptidase N (APN) Inhibitors

Aminopeptidase N (APN) has been recognized as a target for anticancer treatment due to its overexpression on diverse malignant tumor cells and association with cancer invasion, metastasis and angiogenesis. Herein we describe the synthesis, biological evaluation, and structure–activity relationship study of two new series of pyrazoline analogues as APN inhibitors. Among these compounds, 5‐(2‐(2‐(hydroxyamino)‐2‐oxoethoxy)phenyl)‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazole‐1‐carboxamide (compound 13 e ) showed the best APN inhibition with an IC₅₀ value of 0.16±0.02 μm , which is more than one order of magnitude lower than that of bestatin (IC₅₀=9.4±0.5 μm ). Moreover, compound 13 e was found to inhibit the proliferation of diverse carcinoma cells and to show potent anti‐angiogenesis activity. At the same concentration, compound 13 e presents significantly higher anti‐angiogenesis activity than bestatin in human umbilical vein endothelial cells (HUVECs) capillary tube formation assays. The putative binding mode of 13 e in the active site of APN is also discussed.     

Synthesis,Biological Evaluation,and Molecular Docking of Combretastatin and Colchicine Derivatives and their hCE1-Activated Prodrugs as Antiviral Agents

Recent studies indicate that tubulin can be a host factor for vector-borne flaviviruses like dengue (DENV) and Zika (ZIKV), and inhibitors of tubulin polymerization such as colchicine have been demonstrated to decrease virus replication. However, toxicity limits the application of these compounds. Herein we report prodrugs based on combretastatin and colchicine derivatives that contain an ester cleavage site for human carboxylesterase, a highly abundant enzyme in monocytes and hepatocytes targeted by DENV. Relative to their parent compounds, the cytotoxicity of these prodrugs was reduced by several orders of magnitude. All synthesized prodrugs containing a leucine ester were hydrolyzed by the esterase in vitro. In contrast to previous reports, the phenylglycine esters were not cleaved by human carboxylesterase. The antiviral activity of combretastatin, colchicine, and selected prodrugs against DENV and ZIKV in cell culture was observed at low micromolar and sub-micromolar concentrations. In addition, docking studies were performed to understand the binding mode of the studied compounds to tubulin.     

H7N7亚型马流感病毒单克隆抗体的制备

目的 制备抗H7N7亚型马流感病毒(Equine influenza virus,EIV)的单克隆抗体,以建立特异、灵敏、简便的H7N7亚型流感病毒金标试纸检测方法.方法 以H7N7亚型EIV为抗原免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,通过血凝抑制(HI)试验和间接ELISA筛选能稳定分泌抗H...