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Anti-HBc mirrors the activation of HBV-specific CD8+ T cell immune response and exhibits a direct effect on HBV control 抗 HBc 可反映 HBV 特异性 CD8+ T 细胞免疫反应的激活,并对 HBV 控制产生直接影响。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-31 DOI: 10.1016/j.antiviral.2024.105975
Guofu Ye , Chengcong Chen , Yongjun Zhou , Libo Tang , Jianzhong Cai , Yiyan Huang , Jiayue Yang , Yaoting Feng , Liangxing Chen , Yuhao Wang , Yanchen Ma , Guanfeng Lin , Yingsong Wu , Xiaotao Jiang , Jinlin Hou , Yongyin Li

Background

Hepatitis B core antibody (anti-HBc) is commonly present in patients with chronic hepatitis B virus (HBV) infection and serves as a marker of humoral immunity. Herein, we aim to investigate the correlation between anti-HBc and antiviral immune response and its putative role in HBV control.

Methods

Quantitative anti-HBc and levels of anti-HBc subtypes were measured in chronic hepatitis B (CHB) patients. The effects of anti-HBc on immune cells and HBV replication were evaluated using the HBV mouse models and human hepatoma cell lines.

Results

Baseline levels of IgG1 and IgG3 anti-HBc were elevated in CHB patients with favorable treatment response, and correlated with the virological response observed at week 52. Additionally, increased levels of IgM and IgG1 anti-HBc were observed exclusively in CHB patients with liver inflammation. Notably, significant correlations were identified between quantitative levels of anti-HBc and the frequencies of HBcAg-specific CD8+ T cells. Intriguingly, HBcAg efficiently activates T cells aided by B cells in vitro experiments. Moreover, anti-HBc inhibits HBV replication either by a direct effect or through complement-mediated cytotoxicity in HBV-producing cell lines.

Conclusions

Anti-HBc reflects the activation of an HBV-specific CD8+ T cell immune response and may have anti-HBV activity.

背景:乙型肝炎核心抗体(抗-HBc)常见于慢性乙型肝炎病毒(HBV)感染患者,是体液免疫的标志物。在此,我们旨在研究抗 HBc 与抗病毒免疫反应之间的相关性及其在 HBV 控制中的作用:方法:测定慢性乙型肝炎(CHB)患者的抗 HBc 定量和抗 HBc 亚型水平。使用 HBV 小鼠模型和人类肝癌细胞系评估了抗 HBc 对免疫细胞和 HBV 复制的影响:结果:治疗反应良好的慢性乙型肝炎患者的 IgG1 和 IgG3 抗 HBc 基线水平升高,并与第 52 周观察到的病毒学反应相关。此外,IgM 和 IgG1 抗 HBc 水平的升高只出现在有肝脏炎症的慢性乙型肝炎患者中。值得注意的是,抗 HBc 的定量水平与 HBcAg 特异性 CD8+ T 细胞的频率之间存在明显的相关性。耐人寻味的是,在体外实验中,HBcAg 能在 B 细胞的帮助下有效激活 T 细胞。此外,抗 HBc 可通过直接作用或补体介导的细胞毒性抑制 HBV 生产细胞系中的 HBV 复制:结论:抗 HBc 反映了 HBV 特异性 CD8+ T 细胞免疫反应的激活,可能具有抗 HBV 活性。
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引用次数: 0
β-d-N4-hydroxycytidine, a metabolite of molnupiravir, exhibits in vitro antiviral activity against rabies virus β-d-N4-羟基胞嘧啶是莫仑吡韦的一种代谢产物,在体外对狂犬病毒具有抗病毒活性。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-31 DOI: 10.1016/j.antiviral.2024.105977
Kei Konishi , Shinji Kusakabe , Nijiho Kawaguchi , Takao Shishido , Naoto Ito , Michiko Harada , Satoshi Inoue , Ken Maeda , William W. Hall , Yasuko Orba , Hirofumi Sawa , Michihito Sasaki , Akihiko Sato

Rabies is a fatal neurological disorder caused by rabies virus (RABV) infection. Approximately 60,000 patients die from rabies annually, and there are no effective treatments for this disease. Nucleoside analogs are employed as antiviral drugs based on their broad antiviral spectrum, and certain nucleoside analogs have been reported to exhibit anti-RABV activity. The nucleoside analog β-d-N4-hydroxycytidine (NHC) has antiviral effects against a range of RNA viruses. Molnupiravir (MPV), a prodrug of NHC, is clinically used as an oral antiviral drug for coronavirus infections. Despite its broad-spectrum activity, the antiviral activity of NHC against RABV remains unclear. In this study, we reveal that NHC exhibits comparable in vitro anti-RABV activity as ribavirin and favipiravir (also known as T-705) with a 90% effective concentration of 6 μM in mouse neuroblastoma cells. NHC reduced viral loads in neuronal and nonneuronal cells in a dose-dependent manner. Both laboratory and field RABVs (fixed and street strains, respectively) were susceptible to NHC. However, no increase in survival or reduction in viral titers in the brain was observed in RABV-infected mice treated prophylactically with MPV. These findings highlight the potential and challenges of NHC in the treatment of RABV infection.

狂犬病是一种由狂犬病毒(RABV)感染引起的致命神经系统疾病。每年约有 6 万名患者死于狂犬病,目前尚无有效的治疗方法。核苷类似物具有广泛的抗病毒谱,因此被用作抗病毒药物,据报道,某些核苷类似物具有抗狂犬病病毒的活性。核苷类似物 β-d-N4-hydroxycytidine (NHC) 对一系列 RNA 病毒具有抗病毒作用。莫能吡韦(MPV)是 NHC 的一种原药,临床上用作治疗冠状病毒感染的口服抗病毒药物。尽管 NHC 具有广谱抗病毒活性,但其对 RABV 的抗病毒活性仍不清楚。在这项研究中,我们发现 NHC 在小鼠神经母细胞瘤细胞中的体外抗 RABV 活性与利巴韦林和法非比拉韦(又称 T-705)相当,其 90% 的有效浓度为 6 μM。NHC 能以剂量依赖的方式降低神经细胞和非神经细胞中的病毒载量。实验室和野外 RABV(分别为固定株和街头株)都对 NHC 易感。然而,用 MPV 预防性治疗 RABV 感染的小鼠的存活率没有提高,脑内病毒滴度也没有降低。这些发现凸显了 NHC 在治疗 RABV 感染方面的潜力和挑战。
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引用次数: 0
An mpox quadrivalent mRNA vaccine protects mice from lethal vaccinia virus challenge 一种 mpox 四价 mRNA 疫苗能保护小鼠免受致命的疫苗病毒挑战。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-31 DOI: 10.1016/j.antiviral.2024.105974
Entao Li , Qizan Gong , Jiachen Zhang , Xiaoping Guo , Wenyu Xie , Da Chen , Yanqiong Shen , Dongxiang Hong , Zhihao Li , Qianqian Wang , Chao Wang , Yucai Wang , Sandra Chiu

The outbreak of 2022 monkeypox virus (MPXV) infection in nonendemic regions is a global public health concern. A highly effective and safe MPXV vaccine that is available to the general public is urgently needed to control the mpox pandemic. Here, we developed a multivalent mRNA vaccine candidate, MPXV-1103, which expresses the full-length B6, A35, A29 and M1 proteins with three flexible linkers (G4S1)3 in a single sequence. Compared with the monovalent MPXV mRNA vaccine candidates or the quadrivalent mRNA vaccine from mixtures of the four monovalent MPXV mRNA vaccines, MPXV-1103 elicits a robust humoral response and an MPXV-specific T-cell response and protects mice from lethal vaccinia virus (VACV) challenge, with no live virus detected in the nasal or lungs even at dosages as low as 1 μg. Furthermore, analysis of complete blood counts and photomicrographs of tissue from the main organs of mice vaccinated with MPXV-1103 at doses of 5 μg and 20 μg revealed that two doses of MPXV-1103 did not cause any observable pathological changes in the mice. Collectively, our results suggest that MPXV-1103, with features of high efficacy, safety and a simplified manufacturing process, is a promising vaccine candidate for defending against MPXV infection.

2022 年猴痘病毒 (MPXV) 感染在非流行地区的爆发是一个全球性的公共卫生问题。为控制猴痘病毒的大流行,迫切需要一种高效、安全且可供大众使用的猴痘病毒疫苗。在此,我们开发了一种多价 mRNA 候选疫苗 MPXV-1103,它在单一序列中表达了全长的 B6、A35、A29 和 M1 蛋白以及三个柔性连接体(G4S1)3。与单价 MPXV mRNA 候选疫苗或由四种单价 MPXV mRNA 疫苗的混合物制成的四价 mRNA 疫苗相比,MPXV-1103 能引起强大的体液反应和 MPXV 特异性 T 细胞反应,并保护小鼠免受致命性疫苗病毒 (VACV) 的挑战,即使剂量低至 1 μg,鼻腔或肺部也检测不到活病毒。此外,对接种 5 μg 和 20 μg 剂量 MPXV-1103 的小鼠进行的全血细胞计数和主要器官组织显微照片分析表明,两种剂量的 MPXV-1103 均未对小鼠造成任何可观察到的病理变化。总之,我们的研究结果表明,MPXV-1103 具有高效、安全和生产工艺简化等特点,是一种很有希望的用于预防 MPXV 感染的候选疫苗。
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引用次数: 0
Activating adaptive immunity by bispecific, T-cell engager antibodies bridging infected and immune-effector cells is a promising novel therapy for chronic hepatitis B 通过连接感染细胞和免疫效应细胞的双特异性 T 细胞吸引抗体来激活适应性免疫,是治疗慢性乙型肝炎的一种前景广阔的新疗法。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-30 DOI: 10.1016/j.antiviral.2024.105972
Zhe Xie , Ulrike Protzer

Bispecific antibodies (bsAbs) are engineered immunoglobulins that combine two different antigen-binding sites in one molecule. BsAbs can be divided into two molecular formats: IgG-like and non-IgG-like antibodies. Structural elements of each format have implications for engaging the immune system. T cell engager antibodies (TCEs) are bsAbs designed to engage T cells with target cells. TCEs can be applied not only in cancer but also in infectious disease therapy to activate T-cell responses. In this review, we focus on current literature on the design and use of bsAbs as an innovative strategy to enhance adaptive antiviral immune responses. We summarized the novel T cell-related immunotherapies with a focus on TCEs, that are developed for the treatment of chronic hepatitis B. Chronic infection with the hepatitis B virus (HBV) had a death toll of 1.1 million humans in 2022, mainly due to liver cirrhosis and hepatocellular carcinoma developing in the more than 250 million humans chronically infected. A curative treatment approach for chronic hepatitis B is lacking. Combining antiviral therapy with immune therapies activating T-cell responses is regarded as the most promising therapeutic approach to curing HBV and preventing the sequelae of chronic infection. Attracting functionally intact T cells that are not HBV-specific and, therefore, have not yet been exposed to regulatory mechanisms and activating those at the target site in the liver is a very interesting therapeutic approach that could be achieved by TCEs. Thus, TCEs redirecting T cells toward HBV-positive cells represent a promising strategy for treating chronic hepatitis B and HBV-associated hepatocellular carcinoma.

双特异性抗体(bsAbs)是在一个分子中结合了两个不同抗原结合位点的工程免疫球蛋白。双特异性抗体可分为两种分子形式:IgG 样抗体和非 IgG 样抗体。每种形式的结构元素都会影响免疫系统的参与。T 细胞吸引抗体(TCEs)是专为吸引 T 细胞与靶细胞而设计的 bsAbs。TCEs 不仅可用于癌症治疗,还可用于传染病治疗,以激活 T 细胞反应。在这篇综述中,我们重点讨论了目前有关设计和使用 bsAbs 作为增强适应性抗病毒免疫反应的创新策略的文献。2022 年,慢性乙型肝炎病毒(HBV)感染造成的死亡人数达 110 万,主要原因是超过 2.5 亿慢性感染者出现肝硬化和肝细胞癌。目前还缺乏治疗慢性乙型肝炎的方法。将抗病毒疗法与激活 T 细胞反应的免疫疗法相结合,被认为是治愈 HBV 和预防慢性感染后遗症的最有希望的治疗方法。吸引功能完好的非 HBV 特异性 T 细胞,并在肝脏靶点激活这些细胞,是一种非常有趣的治疗方法,TCEs 可以实现这一目标。因此,将 T 细胞重新定向到 HBV 阳性细胞的 TCE 是治疗慢性乙型肝炎和 HBV 相关肝细胞癌的一种很有前景的策略。
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引用次数: 0
Inhibition of human cytomegalovirus entry into mucosal epithelial cells 抑制人类巨细胞病毒进入黏膜上皮细胞
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-27 DOI: 10.1016/j.antiviral.2024.105971
Li He , Laura Hertel , Claire D. James , Iain M. Morgan , Aloysius J. Klingelhutz , Tong-Ming Fu , Lawrence M. Kauvar , Michael A. McVoy

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

人类巨细胞病毒(CMV)会导致宫内感染的新生儿在母亲经口感染后出现严重的发育障碍。因此,母体唾液中的中和抗体有可能预防母体感染,进而预防胎儿传播和疾病。根据标准的细胞培养模型,CMV 的进入介质(以及中和目标)是细胞类型依赖性的:进入成纤维细胞需要糖蛋白 B(gB)和由糖蛋白 H、L 和 O 组成的三聚体复合物(TC),而进入内皮细胞和上皮细胞则需要由糖蛋白 H 和 L 与 UL128、UL130 和 UL131A 组成的五聚体复合物(PC)。然而,由于粘膜细胞进入的介质和细胞分化的潜在影响仍不清楚,本研究利用突变病毒、中和抗体和可溶性 TC 受体来确定感染粘膜细胞系和原发性扁桃体上皮细胞所需的进入介质。进入未分化细胞主要依赖 PC,但分化可诱导 PC 依赖性进入。此外,还观察到依赖于 TC 的进入,并因细胞系和分化情况而异。一些供体的原代扁桃体细胞感染完全不依赖于 TC。相反,gB 抗体或使用肝素破坏病毒附着会阻止病毒进入所有细胞。这些研究结果表明,CMV 进入体内各种细胞可能比标准细胞培养模型所显示的更为复杂,可能受到病毒包膜糖蛋白复合物的相对丰度以及细胞类型、来源组织和分化状态的影响。
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引用次数: 0
Nirmatrelvir and molnupiravir maintain potent in vitro and in vivo antiviral activity against circulating SARS-CoV-2 omicron subvariants Nirmatrelvir 和 Molnupiravir 在体外和体内对循环的 SARS-CoV-2 Omicron 亚变体都具有很强的抗病毒活性。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-25 DOI: 10.1016/j.antiviral.2024.105970
Romel Rosales , Briana L. McGovern , M. Luis Rodriguez , Rocio Leiva-Rebollo , Randy Diaz-Tapia , Jared Benjamin , Devendra K. Rai , Rhonda D. Cardin , Annaliesa S. Anderson , Emilia Mia Sordillo , Harm van Bakel , Viviana Simon , Adolfo García-Sastre , Kris M. White

Variants of SARS-CoV-2 pose significant challenges in public health due to their increased transmissibility and ability to evade natural immunity, vaccine protection, and monoclonal antibody therapeutics. The emergence of the highly transmissible Omicron variant and subsequent subvariants, characterized by an extensive array of over 32 mutations within the spike protein, intensifies concerns regarding vaccine evasion. In response, multiple antiviral therapeutics have received FDA emergency use approval, targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro) regions, known to have relatively fewer mutations across novel variants. In this study, we evaluated the efficacy of nirmatrelvir (PF-07321332) and other clinically significant SARS-CoV-2 antivirals against a diverse panel of SARS-CoV-2 variants, encompassing the newly identified Omicron subvariants XBB1.5 and JN.1, using live-virus antiviral assays. Our findings demonstrate that while the last Omicron subvariants exhibited heightened pathogenicity in our animal model, nirmatrelvir and other clinically relevant antivirals consistently maintained their efficacy against all tested variants, including the XBB1.5 subvariant.

SARS-CoV-2 的变异体由于其传播性增强,能够逃避自然免疫、疫苗保护和单克隆抗体疗法,给公共卫生带来了重大挑战。具有高度传播性的 Omicron 变体和随后出现的亚变体,其特点是尖峰蛋白中出现了超过 32 个突变,这加剧了人们对疫苗逃避能力的担忧。作为回应,多种抗病毒疗法已获得美国食品及药物管理局的紧急使用批准,这些疗法针对的是 SARS-CoV-2 RNA 依赖性 RNA 聚合酶(RdRp)和主蛋白酶(Mpro)区域,已知这些区域在各种新型变体中的突变相对较少。在这项研究中,我们使用活病毒抗病毒试验评估了 nirmatrelvir (PF-07321332) 和其他具有临床意义的 SARS-CoV-2 抗病毒药物对多种 SARS-CoV-2 变异株的疗效,其中包括新发现的 Omicron 亚变异株 XBB1.5 和 JN.1。我们的研究结果表明,虽然最后的 Omicron 亚变异体在我们的动物模型中表现出更强的致病性,但 nirmatrelvir 和其他临床相关的抗病毒药物对包括 XBB1.5 亚变异体在内的所有测试变异体始终保持有效。
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引用次数: 0
Highly specific SARS-CoV-2 main protease (Mpro) mutations against the clinical antiviral ensitrelvir selected in a safe, VSV-based system 在基于 VSV 的安全系统中筛选出针对临床抗病毒药物 ensitrelvir 的高特异性 SARS-CoV-2 主要蛋白酶 (Mpro) 突变。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-23 DOI: 10.1016/j.antiviral.2024.105969
Stefanie Rauch , Francesco Costacurta , Helge Schöppe , Ju-Yi Peng , David Bante , Ela Emilie Erisoez , Bernhard Sprenger , Xi He , Seyed Arad Moghadasi , Laura Krismer , Anna Sauerwein , Anne Heberle , Toni Rabensteiner , Dai Wang , Andreas Naschberger , Theresia Dunzendorfer-Matt , Teresa Kaserer , Dorothee von Laer , Emmanuel Heilmann

In the SARS-CoV-2 pandemic, the so far two most effective approved antivirals are the protease inhibitors nirmatrelvir, in combination with ritonavir (Paxlovid) and ensitrelvir (Xocova). However, antivirals and indeed all antimicrobial drugs are sooner or later challenged by resistance mutations. Studying such mutations is essential for treatment decisions and pandemic preparedness. At the same time, generating resistant viruses to assess mutants is controversial, especially with pathogens of pandemic potential like SARS-CoV-2. To circumvent gain-of-function research with non-attenuated SARS-CoV-2, a previously developed safe system based on a chimeric vesicular stomatitis virus dependent on the SARS-CoV-2 main protease (VSV-Mpro) was used to select mutations against ensitrelvir. Ensitrelvir is clinically especially relevant due to its single-substance formulation, avoiding drug-drug interactions by the co-formulated CYP3A4 inhibitor ritonavir in Paxlovid. By treating VSV-Mpro with ensitrelvir, highly-specific resistant mutants against this inhibitor were selected, while being still fully or largely susceptible to nirmatrelvir. We then confirmed several ensitrelvir-specific mutants in gold standard enzymatic assays and SARS-CoV-2 replicons. These findings indicate that the two inhibitors can have distinct viral resistance profiles, which could determine treatment decisions.

在 SARS-CoV-2 大流行的情况下,迄今为止已批准的两种最有效的抗病毒药物是蛋白酶抑制剂 nirmatrelvir(与利托那韦联用)(Paxlovid)和 ensitrelvir(Xocova)。然而,抗病毒药物乃至所有抗菌药物迟早都会受到抗药性突变的挑战。研究这种突变对治疗决策和大流行病防备至关重要。与此同时,产生抗药性病毒以评估突变体是有争议的,特别是对于像 SARS-CoV-2 这样具有大流行潜力的病原体。为了避免使用非减毒 SARS-CoV-2 进行功能增益研究,我们使用了以前开发的基于依赖 SARS-CoV-2 主蛋白酶的嵌合型水泡性口炎病毒(VSV-Mpro)的安全系统来选择抗 Ensitrelvir 的突变体。Ensitrelvir 在临床上具有特别重要的意义,因为它采用单一药物制剂,避免了 Paxlovid 中共同配制的 CYP3A4 抑制剂利托那韦的药物相互作用。通过用恩西特韦处理 VSV-Mpro,我们筛选出了几种对这种抑制剂具有高度特异性耐药性的突变体,而它们对尼马特韦仍然完全或基本敏感。然后,我们在金标准酶测定和 SARS-CoV-2 复制子中证实了几种 ensitrelvir 特异性突变体。这些研究结果表明,这两种抑制剂可能具有不同的病毒耐药性特征,从而决定治疗方案。
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引用次数: 0
A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19 ACE2 的一种模拟肽能防止 SARS-CoV-2 感染,并减轻与 COVID-19 有关的肺部炎症。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-14 DOI: 10.1016/j.antiviral.2024.105968
Ernna H. Oliveira , Ana C. Monteleone-Cassiano , Lucas Tavares , Jadson C. Santos , Thais M. Lima , Giovanni F. Gomes , Pedro P. Tanaka , Cintia J. Monteiro , Matheus Munuera , Sabrina S. Batah , Alexandre T. Fabro , Vitor M. Faça , Ana P. Masson , Eduardo A. Donadi , Mariangela Dametto , Rodrigo Bonacin , Ronaldo B. Martins Jr. , Eurico Arruda Neto , Luis Lamberti P. daSilva , Thiago M. Cunha , Geraldo A. Passos

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22–46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

由于人类血管紧张素转换酶 2(ACE2)是 SARS-CoV-2 的主要受体,因此鉴定 ACE2 允许 SARS-CoV-2 进入人类细胞的区域对于设计基于肽的抗病毒阻断剂和阐明病毒的发病机制至关重要。我们发现并合成了一种25聚合物拟态肽(包含ACE2α-螺旋α1的22-46位),它与S1受体结合域(RBD)-ACE2界面有关。体外模拟(野生型,WT)ACE2 多肽能显著抑制 SARS-CoV-2 对人类肺 Calu-3 细胞的感染。根据硅学蛋白质建模预测,ACE2 α-螺旋α1的F28、K31、F32、F40和Y41残基对于SARS-CoV-2的原始株、Delta株和Omicron株建立Spike RBD-ACE2界面至关重要。用丙氨酸(A)或天冬氨酸(D)取代这些残基会削弱多肽的抗病毒保护作用,这表明这些位置对病毒进入肺细胞至关重要。分子动力学模拟显示,WT ACE2 肽与 SARS-CoV-2 S1 RBD 有明显的相互作用,而 A 或 D 突变肽则没有。通过确定 ACE2 α1α-螺旋的关键氨基酸残基,我们证明了 WT ACE2 肽能保护易感 K18-hACE2 小鼠免受体内 SARS-CoV-2 感染,并能有效治疗 COVID-19。
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引用次数: 0
High throughput profiling identified PA-L106R amino acid substitution in A(H1N1)pdm09 influenza virus that confers reduced susceptibility to baloxavir in vitro 高通量剖析确定了甲型 H1N1 pdm09 流感病毒中的 PA-L106R 氨基酸替代物,该替代物可降低体外对巴洛沙韦的敏感性。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-13 DOI: 10.1016/j.antiviral.2024.105961
Dongdong Chen , Wen Su , Ka-Tim Choy , Yan Sing Chu , Chi Ho Lin , Hui-Ling Yen

Baloxavir acid (BXA) is a pan-influenza antiviral that targets the cap-dependent endonuclease of the polymerase acidic (PA) protein required for viral mRNA synthesis. To gain a comprehensive understanding on the molecular changes associated with reduced susceptibility to BXA and their fitness profile, we performed a deep mutational scanning at the PA endonuclease domain of an A (H1N1)pdm09 virus. The recombinant virus libraries were serially passaged in vitro under increasing concentrations of BXA followed by next-generation sequencing to monitor PA amino acid substitutions with increased detection frequencies. Enriched PA amino acid changes were each introduced into a recombinant A (H1N1)pdm09 virus to validate their effect on BXA susceptibility and viral replication fitness in vitro. The I38 T/M substitutions known to confer reduced susceptibility to BXA were invariably detected from recombinant virus libraries within 5 serial passages. In addition, we identified a novel L106R substitution that emerged in the third passage and conferred greater than 10-fold reduced susceptibility to BXA. PA-L106 is highly conserved among seasonal influenza A and B viruses. Compared to the wild-type virus, the L106R substitution resulted in reduced polymerase activity and a minor reduction of the peak viral load, suggesting the amino acid change may result in moderate fitness loss. Our results support the use of deep mutational scanning as a practical tool to elucidate genotype-phenotype relationships, including mapping amino acid substitutions with reduced susceptibility to antivirals.

巴洛沙韦酸(BXA)是一种泛流感抗病毒药物,其靶标是病毒 mRNA 合成所需的聚合酶酸性蛋白(PA)的帽依赖性内切酶。为了全面了解与对 BXA 敏感性降低有关的分子变化及其适应性特征,我们对 A(H1N1)pdm09 病毒的 PA 内切酶结构域进行了深度突变扫描。在 BXA 浓度不断增加的情况下,对重组病毒文库进行体外连续传代,然后进行下一代测序,以监测检测频率增加的 PA 氨基酸置换。将富集的 PA 氨基酸变化分别引入重组 A(H1N1)pdm09 病毒,以验证它们对 BXA 敏感性和病毒体外复制适应性的影响。在重组病毒文库中,我们在 5 个连续传递周期内都检测到了已知会降低对 BXA 敏感性的 I38T/M 取代。此外,我们还发现了一个新的 L106R 替换,该替换在第三次传代中出现,使病毒对 BXA 的敏感性降低了 10 倍以上。PA-L106 在季节性甲型和乙型流感病毒中高度保守。与野生型病毒相比,L106R置换导致聚合酶活性降低,病毒载量峰值略有下降,这表明氨基酸变化可能会导致适度的适应性损失。我们的研究结果支持将深度突变扫描作为一种实用工具,用于阐明基因型与表型之间的关系,包括绘制对抗病毒药物敏感性降低的氨基酸替代图。
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引用次数: 0
Cold-adapted influenza vaccine carrying three repeats of a respiratory syncytial virus (RSV) fusion glycoprotein epitope site protects BALB/c mice and cotton rats against RSV infection 冷适应流感疫苗携带三个重复的呼吸道合胞病毒(RSV)融合糖蛋白表位位点,可保护 BALB/c 小鼠和棉鼠免受 RSV 感染。
IF 4.5 2区 医学 Q1 PHARMACOLOGY & PHARMACY Pub Date : 2024-07-08 DOI: 10.1016/j.antiviral.2024.105960

Respiratory syncytial virus is the major cause of respiratory viral infections, particularly in infants, immunocompromised populations, and the elderly (over 65 years old), the prevention of RSV infection has become a priority. In this study, we generated a chimeric influenza virus, termed LAIV/RSV/HA-3F, using reverse genetics technology which contained three repeats of the RSV fusion protein neutralizing epitope site II to the N terminal in the background of the hemagglutinin (HA) gene of cold adapted influenza vaccine A/California/7/2009 ca. LAIV/RSV/HA-3F exhibited cold-adapted (ca) and attenuated (att) phenotype. BALB/c mice immunized intranasally with LAIV/RSV/HA-3F showed robust immunogenicity, inducing viral-specific antibody responses against both influenza and RSV, eliciting RSV-specific humoral, cellular and mucosal immune responses. LAIV/RSV/HA-3F also conferred protection as indicated by reduced viral titers and improved lung histopathological alterations against live RSV virus challenge. Mechanismly, single-cell RNA sequencing (scRNA-seq) and single-cell T cell antigen receptor (TCR) sequencing were employed to characterize the immune responses triggered by chimeric RSV vaccine, displaying that LAIV/RSV/HA-3F provided protection mainly via interferon-γ (IFN-γ). Moreover, we found that LAIV/RSV/HA-3F significantly inhibited viral replication in the challenged lung and protected against subsequent RSV challenge in cotton rats without causing lung disease. Taken together, our findings demonstrated that LAIV/RSV/HA-3F has potential as a promising bivalent vaccine with dual purpose candidate for the prevention of influenza and RSV, and preclinical and clinical studies warrant further investigations.

呼吸道合胞病毒是呼吸道病毒感染的主要病因,尤其是在婴儿、免疫力低下人群和老年人(65 岁以上)中,预防 RSV 感染已成为当务之急。在这项研究中,我们利用反向遗传学技术生成了一种名为 LAIV/RSV/HA-3F 的嵌合型流感病毒,它在冷适应流感疫苗 A/California/7/2009 ca 的血凝素(HA)基因背景的 N 端含有三个重复的 RSV 融合蛋白中和表位点 II。LAIV/RSV/HA-3F表现出冷适应(ca)和减毒(att)表型。用 LAIV/RSV/HA-3F 经鼻免疫 BALB/c 小鼠显示出很强的免疫原性,可诱导针对流感和 RSV 的病毒特异性抗体反应,引起 RSV 特异性体液、细胞和粘膜免疫反应。LAIV/RSV/HA-3F还具有保护作用,这表现在病毒滴度降低和肺组织病理学改变改善,从而抵御活的RSV病毒挑战。从机制上讲,我们采用了单细胞RNA测序(scRNA-seq)和单细胞T细胞抗原受体(TCR)测序来描述嵌合型RSV疫苗引发的免疫反应,结果显示LAIV/RSV/HA-3F主要通过干扰素-γ(IFN-γ)提供保护。此外,我们还发现,LAIV/RSV/HA-3F 能显著抑制受挑战小鼠肺部的病毒复制,并能保护棉鼠免受随后的 RSV 挑战,而不会导致肺部疾病。总之,我们的研究结果表明,LAIV/RSV/HA-3F 有潜力成为一种具有双重用途的双价疫苗,用于预防流感和 RSV,临床前和临床研究值得进一步研究。
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Antiviral research
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