The neutralization effect of montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies

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清热解毒方体外抗人乳头瘤病毒感染及作用机制

目的:采用高危型人乳头瘤状病毒16(human papillomavirus 16,HPV16)体外假病毒感染模型评价清热解毒方体外抗HPV16感染活性及作用方式,并探讨清热解毒方的作用机制。方法:首先,将HPV16假病毒加入到293T/17细胞中,建立HPV假病毒体外荧光感染模型,以此模型评价清热解毒方(20~1 000 g·L~(-1))体外抗HPV16作用,探讨清热解毒方抗HPV感染的量效关系;其次,采用HPV假病毒模型,通过预处理细胞、与病毒同时作用于细胞、病毒处理后作用于细胞的不同处理方法,探讨清热解毒方(20 g·L~(-1))抗HPV的作用方式;最后,从清热解毒方作用后HPV转化的He La细胞中提取致癌蛋白E7,细胞外信号调节激酶(ERK)以及核转录因子-κB(NF-κB),用蛋白免疫印迹法(Western blot)检测E7,ERK,NF-κB蛋白表达,探讨清热解毒方(3. 33~20 g·L~(-1))在肿瘤相关蛋白水平上的作用机制。结果:与病毒组比较,清热解毒方可以明显抑制HPV16假病毒的感染(P 0. 05),且呈现明显的浓度依赖关系;且清热解毒方能够通过保护细胞、抑制病毒吸附并抑制病毒感染后过程的多种方式发挥其抗HPV16作用,其中保护细胞的效果更显著(P 0. 01),说明清热解毒方能够显著预防HPV16感染;与空白组比较,清热解毒方能够明显抑制E7致癌蛋白的表达,并抑制ERK,NF-κB蛋白的表达(P 0. 05,P 0. 01),达到抗肿瘤的作用。结论:清热解毒方具有较好的抑制HPV病毒的效果,该研究为清热解毒方清除HPV病毒和防治宫颈癌提供了创新性的理论依据。     

Detection of HPV types and neutralizing antibodies in Gansu province,China

A total of 82 samples from patients with cervical cancer (Group 1) and 50 samples from patients with other genital diseases (Group 2) were collected in Gansu, China. All 132 samples were tested for HPV DNA with a typing kit that can detect 21 types of HPV, and also tested for neutralizing antibodies against HPV‐16, ‐18, ‐58, ‐45, ‐6, and ‐11 using pseudovirus‐based neutralization assays. The results revealed that 28% (23/82) of sera in Group 1 were positive for type‐specific neutralizing antibodies with a titer range of 160–640, of which 23.2% (19/82), 2.4% (2/82), 2.4% (2/82), 1.2% (1/82), and 1.2% (1/82) were against HPV‐16, ‐58, ‐6, ‐18, and ‐45, respectively. Only one serum (2%) in Group 2 was positive for neutralizing antibodies, which were against HPV‐6 with a titer of 2,560. Overall, 85.4% (70/82) of samples in Group 1 were HPV DNA‐positive, compared with 28% (14/50) of samples in Group 2. The seven most common types detected in Group 1 were HPV‐16 (80%), HPV‐52 (7.1%), HPV‐66 and HPV‐11 (5.7% each), and HPV‐58, HPV‐18, and HPV‐33 (4.3% each), while the four most common types in Group 2 were HPV‐16 (12%), HPV‐52 and HPV‐11 (6% each), and HPV‐68 (4%). The concordance between HPV DNA and corresponding neutralizing antibodies was 32.9% (27/82) with a significant difference (P < 0.005). More specifically, the concordance was 42.7% (35/82) for HPV‐16 in Group 1. The full‐length sequences of six HPV types (HPV‐16, ‐58, ‐33, ‐59, ‐11, and ‐68) were determined and showed 99% identities with their reported genomes. J. Med. Virol. 81:693–702, 2009 © 2009 Wiley‐Liss, Inc.     

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses

Molecular characterization of the severe acute respiratory syndrome coronavirus has revealed genetic diversity among isolates. The spike (S) glycoprotein, the major target for vaccine and immune therapy, shows up to 17 substitutions in its 1,255-aa sequence; however, the biologic significance of these changes is unknown. Here, the functional effects of S mutations have been determined by analyzing their affinity for a viral receptor, human angiotensin-converting enzyme 2 (hACE-2), and their sensitivity to Ab neutralization with viral pseudotypes. Although minor differences among eight strains transmitted during human outbreaks in early 2003 were found, substantial functional changes were detected in S derived from a case in late 2003 from Guangdong province [S(GD03T0013)] and from two palm civets, S(SZ3) and S(SZ16). S(GD03T0013) depended less on the hACE-2 receptor and was markedly resistant to Ab inhibition. Unexpectedly, Abs that neutralized most human S glycoproteins enhanced entry mediated by the civet virus S glycoproteins. The mechanism of enhancement involved the interaction of Abs with conformational epitopes in the hACE-2-binding domain. Finally, improved immunogens and mAbs that minimize this complication have been defined. These data show that the entry of severe acute respiratory syndrome coronaviruses can be enhanced by Abs, and they underscore the need to address the evolving diversity of this newly emerged virus for vaccines and immune therapies.     

Previous SARS-CoV-2 Infection Increases B.1.1.7 Cross-Neutralization by Vaccinated Individuals

With the spread of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a need to assess the protection conferred by both previous infections and current vaccination. Here we tested the neutralizing activity of infected and/or vaccinated individuals against pseudoviruses expressing the spike of the original SARS-CoV-2 isolate Wuhan-Hu-1 (WH1), the D614G mutant and the B.1.1.7 variant. Our data show that parameters of natural infection (time from infection and nature of the infecting variant) determined cross-neutralization. Uninfected vaccinees showed a small reduction in neutralization against the B.1.1.7 variant compared to both the WH1 strain and the D614G mutant. Interestingly, upon vaccination, previously infected individuals developed more robust neutralizing responses against B.1.1.7, suggesting that vaccines can boost the neutralization breadth conferred by natural infection.     

新型冠状病毒假病毒的构建及其在血清中和抗体测定中的应用

目的 构建新型冠状病毒(SARS-CoV-2)假病毒,优化假病毒制备条件并应用于中和抗体活性检测.方法 优化合成SARS-CoV-2刺突蛋白(S)基因,进行假病毒包装;通过Western blot检测S基因的表达;应用定量ELI-SA检测接种新冠灭活疫苗后血清中新型冠状病毒IgG抗体的滴度,同时应用假病毒中和实验对接种...     

Safe Pseudovirus-based Assay for Neutralization Antibodies against Influenza A(H7N9) Virus

Serologic studies are urgently needed to assist in understanding an outbreak of influenza A(H7N9) virus. However, a biosafety level 3 laboratory is required for conventional serologic assays with live lethal virus. We describe a safe pseudovirus–based neutralization assay with preliminary assessment using subtype H7N9–infected samples and controls.