H2AX

▲ 详细日程见文末 ▲摘要:伪狂犬病毒（PRV）是一种高度传染性疾病，可感染大多数哺乳动物，猪是唯一的天然宿主，给世界养猪业造成了巨大的经济损失。先天性免疫是宿主抵御病原体攻击的第一道防线，对于适应性免疫的正常建立至关重要。宿主会利用先天性免疫反应来抵御PRV的入侵，但PRV会利用各种策略来抑制先天性免疫反应，从而促进病毒的复制。目前，世界范围内用于预防猪感染PRV的减毒活疫苗，如Bartha K61。然而，越来越多的数据表明，这些疫苗并不能完全抵御自2011年底以来出现的PRV新变种。本文综述了PRV与宿主天然免疫之间的相互作用以及减毒活疫苗的研究现状，旨在促进新型、高效PRV疫苗的研制。关键词：伪狂犬病毒，先天免疫，免疫逃避，减毒活疫苗，重组疫苗。1背景PRV是导致伪狂犬病（PR）的病原体，也被称为奥杰斯基病（AD，Aujeszky’s disease）。PRV属于α-疱疹病毒亚家族，宿主范围很广，虽然猪是它的天然宿主，但包括人、猫、狗、山羊和牛在内的许多哺乳动物也可能感染。猪感染PRV可引起呼吸道、生殖和神经系统症状。处于不同生长阶段的猪会出现不同的症状。受PRV感染的新生仔猪会出现神经系统症状，死亡率高达 100%，而成年猪一般只表现出轻微的呼吸道症状，但会在神经系统中形成终生潜伏感染，这种潜伏感染可被重新激活，成为病毒的动态贮存库。PRV具有大约143kb 的双链DNA基因组，紧密排列，至少70个开放阅读框，并含有两个反向重复序列。几种 PRV 蛋白发挥调节宿主先天性免疫反应的功能，主要包括干扰素(IFN)系统、细胞凋亡和自噬，以促进病毒的产生。先天免疫对病毒感染反应迅速，但病原体清除和预防再感染的关键依赖于后天免疫系统，而高质量的先天免疫反应是建立后天免疫系统的先决条件。因此，了解 PRV 的先天免疫逃避机制对于疫苗的开发具有重要意义。自PRV流行以来，以 Bartha K61为代表的减毒活疫苗在预防和控制该病方面发挥了重要作用。然而，自2011年以来，中国出现了新的变异。Bartha K61对PRV变异株感染不能提供完全的保护作用。新的疫苗的开发是一个必要条件，一些学者认为，增强先天免疫能引起病毒的衰减。病毒先天免疫逃避的破坏可以减少病毒的毒力，同时建立强有力的免疫应答。本文综述了PRV先天性免疫逃避的多种策略，主要包括干扰素系统、自噬和细胞凋亡，以及PRV减毒活疫苗的研究进展。我们希望这篇综述能够为开发针对新型PRV变异体的疫苗提供一些思路。2PRV的先天免疫调节机制病毒感染诱导先天免疫应答，并通过模式识别受体 (PRR) 识别病原体相关分子模式 (PAMPs) 激活抗病毒先天免疫应答。据报道，宿主可以通过几种PRR识别PRV感染: Toll样受体2 (TLR2)，环磷酸鸟苷-磷酸腺苷合酶 (cGAS)，RIG-I样受体 (RLRs)，DEAD (Asp-Glu-Ala-Asp) 盒多肽41 (DDX41) 和IFN调节因子的DNA依赖性激活剂。随后，特异性信号传导途径被激活以响应病原体感染而产生干扰素、促炎因子和趋化因子。我们总结了参与先天性免疫系统调控的PRV编码蛋白，重点是那些参与逃避 IFN 介导的免疫反应的蛋白（图1；表1）。图1 PRV通过编码多种蛋白质调节IFN介导的先天性免疫应答表1 PRV 因子与宿主的先天免疫系统相互作用2.1 IFN-I系统IFN-I 介导的先天性免疫应答是宿主抵抗病毒感染最有效的防御策略之一。然而，病毒在长期的进化过程中，已经发展出了各种各样的逃避宿主免疫的策略。PRV 通过几种策略调节宿主的先天性免疫反应。采用微阵列技术，用野生型 PRV 株感染细胞后，IFN-β 通常诱导的一部分基因表达下降。在病毒感染后，病毒 DNA 首先被细胞质 DNA 传感器 cGAS 识别，导致 cGAS 中的构象开关催化第二信使 cGAMP 的产生，然后 cGAMP 与位于内质网上的 STING 结合。它们转运到高尔基体，招募TANK-结合激酶1（TBK1）和IRF3/7，并使IRF3/7磷酸化，最后磷酸化的IRF3/7被转运到细胞核，激活编码IFN-I的基因转录。随后，JAK-STAT通路被IFN-I激活，启动数百个ISGs的转录，从而调节细胞因子。PRV 编码一系列蛋白质来规避 IFN-I 介导的先天免疫，其主要靶标是 cGAS-STING 信号通路和 IFN-I 信号通路（表 1）。STING是cGAS-STING 通路中的关键蛋白之一，一项可靠的研究表明，PRV UL13 招募 E3 连接酶 RING-finger 蛋白 5 (RNF5)，以促进 STING 的 K27-/K29 链接泛素化和降解。过氧化物酶1（PRDX1）通过与TBK1和IκB激酶ε（IKKε）相互作用来促进IFN诱导，Lv等人证实UL13能降解PRDX1，从而抑制IFN的产生，这取决于其激酶活性。研究表明，UL13 可通过泛素化和蛋白酶体介导的降解作用降解 IRF3。Bo等人通过筛选50种PRV蛋白发现UL13限制了ifn-β 转录。进一步的研究发现，UL13利用其激酶活性诱导IRF3磷酸化，从而减弱IRF3与其启动子的结合。gE和US3也以多种方式抑制IRF3活性。gE通过降解irf3的伴侣CREB结合蛋白 (CBP) 来抑制IFN的产生。US3抑制IRF3的活性，通过蛋白酶体途径机械降解IRF3。UL24通过降解irf7损害DNA传感途径。ifn-i介导的先天免疫反应是宿主抵抗病毒感染的最有效防御策略之一。然而，病毒已经开发了各种策略来逃避宿主免疫。在漫长的进化过程中。PRV以几种策略调节宿主先天免疫应答。当使用微阵列技术用野生型PRV株感染细胞时，通常由ifn-β 诱导的基因子集的表达降低。病毒感染后，病毒DNA首先被细胞质DNA传感器cGAS识别，导致cGAS中的构象转换以催化第二信使cGAMP的产生，然后cGAMP与位于内质网上的STING结合。JAK/STAT 信号通路在 IFN-I 介导的 ISG 产生中起重要作用。JAK/STAT 通路受 PRV 广泛调控。PRV 阻断了 JAK/STAT 信号通路重要节点 STAT1的磷酸化。几种病毒蛋白在这一过程中发挥积极作用，包括 EP0，US3，UL24，UL42和 UL50。EP0蛋白是通过抑制 IRF9的转录来拮抗天然宿主干扰素应答所必需的。Bcl-2相关转录因子1(Bclaf1)维持 STAT1和 STAT2磷酸化，并通过与 ISRE 和 STAT2直接相互作用来转录 ISG 来促进 ISGF3与 ISRE 的结合。US3通过蛋白酶体途径降解 Bclaf1抑制 ISG 的产生，促进 PRV 的免疫逃避。此外，UL42还干扰 ISGF3与 ISRE 的结合，其他一些研究发现 UL24抑制 ISG (包括 OASL，ISG20等)的转录。发现 UL50降解 IFN 受体以下调 IFN-α 免疫应答。Janus 激酶磷酸化并传导干扰素的抗病毒信号，最近发现 PRV 通过诱导蛋白酶体降解而降解 Janus 激酶。然而，其中涉及的机制仍不清楚。核因子-κB（NF-κB）在调节免疫、炎症反应、细胞增殖和存活方面起着至关重要的作用。NF-κB 的主要形式是由 p50 和 RelA 亚基组成的异源二聚体，它们以抑制活性的状态与 IκB 家族蛋白结合。当细胞受到应激、细胞因子或病原体入侵等各种刺激时，NF-κB 抑制蛋白 IκBα 被激活并与 NF-κB 分离，通过蛋白酶体途径降解，NF-κB 进一步转运到细胞核，激活各种基因的转录。细胞 NF-κB 信号通路受到 PRV 感染的严重影响。最近的研究发现，PRV编码蛋白UL13通过抑制RIG-I和MDA5的转录来调节NF-κB的活化，从而抑制RLR介导的IFN-β的产生，以逃避先天宿主免疫。此外，UL24 与 p65 相互作用，介导蛋白酶体途径中的 p65 降解，从而影响 NF-κB 的激活。总之，PRV 从多个方面调控 IFN 的功能，包括 IFN 的产生和 IFN 信号通路。病毒抑制 IFN 信号转导的能力与毒力之间存在密切的相关性。通过删除 STAT1/2 拮抗剂可以减弱麻疹病毒。一项重要研究发现，PRV 的糖蛋白 gE/gI 复合物能显著抑制浆细胞树突状细胞（pDCs）分泌 IFN。与野生型（WT）PRV 株相比，删除了 gE/gI 的 Bartha K61 减毒疫苗株通过 pDC 触发了更强的 IFN-I 反应。恢复甚至改善在野生型 PRV 感染中受损的 IFN 系统是一种很有前景的抗病毒策略。尽管 IFN 系统并不是病毒毒性的唯一决定因素，但它仍具有重要作用。PRV 的免疫逃逸因子在病毒感染中所起的关键作用为疫苗和抗病毒药物的开发提供了潜在的目标。2.1 细胞凋亡、自噬和坏死细胞凋亡是一种程序性细胞死亡，其特征是细胞膜起泡、染色质凝结和 DNA 断裂，同时保持膜的完整性，形成凋亡小泡。细胞凋亡是宿主抵抗病原体入侵的一种手段，它使受感染的细胞过早死亡，导致病毒增殖中止，并通过轻度释放抗原诱导特异性免疫。有报道称 PRV 诱导细胞凋亡。PRV 的 US3 和 gE 蛋白不仅能拮抗 IFN 系统，还能通过不同途径抑制细胞凋亡。研究表明，US3 可激活 PI3K/Akt 和 NF-κB 通路，从而抑制细胞凋亡；gE 可使细胞外信号调节激酶 1/2（ERK1/2）磷酸化，从而降解促凋亡蛋白Bim。DNA 损伤反应（DDR）对维持基因组完整性至关重要，它根据损伤程度导致 DNA 修复、细胞周期停滞和细胞凋亡。PRV 感染会造成宿主细胞的 DNA 损伤，导致细胞凋亡，最近的研究表明 PRV 与 DDR 之间存在更深入的对话。磷酸化的 H2AX 是 DDR 的标志，而 PRV 的 UL13 可与 H2AX 相互作用并磷酸化 H2AX，从而促进其复制。富亮氨酸 PPR 矩阵蛋白（LRPPRC）是一种多功能蛋白，也是第一个独立于 Caspase 蛋白之外的凋亡蛋白。最近，Xu 等人发现 PRV 的 UL16 蛋白与 LRPPRC 相互作用，减弱了 LRPPRC 对 NF-κB 的激活，而 NF-κB 是 PRV 复制的宿主限制因子。这些基因是开发新型减毒活疫苗时有望减弱 PRV 的靶标。自噬可用于修复细胞内成分，并在细胞饥饿时为细胞提供重要的能量和物质。越来越多的研究表明，自噬是免疫反应不可或缺的一部分。PRV 可诱导细胞自噬，通过经典的 Beclin-1-Atg7-Atg5 途径促进复制。Wnt/β-Catenin信号通路调节细胞增殖、分化和存活。最近的研究发现，PRV 感染会显著激活 Wnt/β-Catenin 信号通路，这可能会通过上调病毒诱导的自噬促进病毒增殖。自噬通路似乎被 PRV 利用，Sun等人的研究表明，PRV 对自噬的调控十分精妙，在感染早期诱导细胞自噬，这与病毒复制无关，随着病毒复制的进展，自噬水平下降。进一步的研究发现，US3 可激活 PI3-K/AKT 通路来抑制自噬。US3 对细胞自噬的抑制有利于细胞存活，并可能促进 PRV 的复制。Bcl2-associated athanogene 3（BAG3）参与调节细胞凋亡、发育、细胞骨架组织和自噬。BAG3 可抑制 PRV 复制，PRV 蛋白 UL56 与 BAG3 相互作用，并通过溶酶体途径降解 BAG3；但在 WT PRV感染中却观察不到这种现象。PRV的其他一些蛋白可能会拮抗UL56降解BAG3的活性，具体细节还有待进一步探讨，BAG3可能是生成稳定细胞系以生产高水平PRV减毒活疫苗的潜在靶标。坏死是一种不同于细胞凋亡的细胞死亡形式。在受体相互作用蛋白激酶 3（RIPK3）及其伴侣蛋白（RIPK1、TRIF、DAI 或其他）的帮助下，混合谱激酶结构域（MLKL）被激活，导致细胞破裂，释放出大量与损伤相关的分子，并引发炎症反应。PRV 以依赖 RIPK3 的方式导致细胞坏死，而抑制坏死会促进 PRV 复制。PRV 是否会阻断坏死尚不清楚，但其他一些疱疹病毒，如小鼠巨细胞病毒（MCMV）编码的 M45/vIRA 可阻断 RIPK3 的活性，同样，HSV-1 编码的核糖核苷酸还原酶大亚基（ICP6）可与人体细胞中的 RIPK3 相互作用，从而抑制坏死。这些信息表明，PRV 可能具有某种潜在的能力来抑制细胞坏死，从而促进其复制。细胞凋亡、自噬和坏死是细胞死亡的三种典型形式，随着时间的推移，这些形式的细胞死亡的独立性和相关性得到了越来越多的认识。当细胞受到刺激并相互转化时，可以同时诱导多种类型的细胞死亡。PRV 感染与宿主死亡率之间的关系是复杂的，PRV 与宿主之间的相互作用是研制抗 PRV 感染的新型疫苗和抗病毒药物的基础。3PRV变异体的基因组进化目前，许多欧美国家已宣布自己不再感染 PRV，中国已通过接种 Bartha K61疫苗有效地控制了 PRV 感染。自2011年以来，中国东部和北部的几个养猪场发生了 PRV 感染暴发，更令人担忧的是，这些受感染的猪已经接种了PRV疫苗。2011年以前，尽管不同的 PRV 分离株的生物学特性不同，但只有一种PRV血清型。他等人根据 PRV全长序列构建了最大似然树，结果表明目前的PRV分离株可分为基因型1和基因型2两个不同的分支。利用 gC 基因构建的最大进化枝可信度树证实了 PRV 分为进化枝1和进化枝2，同时表明这两个进化枝已经独立传播了几个世纪。最大似然树分别基于 gB 和 gD 构建，PRV 分为两个进化枝，毫无疑问，Bartha K61和2011年后的变异被分配到不同的进化枝。有趣的是，2011年以后，中国的 PRV 分离株主要是基因型2，这部分解释了为什么 Bartha K61疫苗(基因型1)不能提供对新型 PRV 变异体的完全保护。GB、 gC 和 gD 是主要的抗原基因，易发生突变，因此经常用于 PRV 系统发育分析。抗原基因的独立系统发育分析使得更容易理解疫苗和不同毒力株之间的关系。研究表明，PRV 变异体 gC 和 gD 的突变在逃避 Bartha K61诱导的免疫中起关键作用。天然突变和重组体促进 PRV 菌株多样性的变异。翟等人于2017-2019年间从中国东部的养猪场采集了 PRV 样本。根据 gC 基因，这些 PRVs 属于进化分支2。主要 PRV 糖蛋白 gB，gC，gD 和 gE 的测序分析显示分离物内氨基酸序列分歧最大，分别为4.0,0.7,0.3和1.1% 。与 Bartha K61疫苗株相比，这些分离株在几个蛋白位点有突变，特别是 S41G，R162H 和 S345L 在 gC 上的变化可以改变受体结合和/或糖基化，这可能导致变异 PRV 的出现。自然重组加速了病毒基因组的进化，生物信息学分析表明，在 PRV 基因组中经常发生包被和包被重组。Tan 等人发现了一种新的 HN2019 PRV 毒株，它来源于经典 PRV (如 Ea)和 HB-98疫苗毒株的重组。胡等人利用 Bartha K61对所有中国分离株进行重组预测，得到23个重组事件。Bo 等人发现 Bartha 菌株为重组 JSY13变体提供了部分基因。早期伪狂犬病病毒 SC 株是介于中国地方性病毒株和巴萨疫苗样病毒株之间的重组病毒株。这些报道表明，重组在中国 PRV 的演变中起着关键作用。Bartha K61是近半个世纪前引入中国的，长期免疫对 PRV 的突变和重组有很大的贡献。因此，PRV 的监测和进化分析对于 PR 疾病的预防和控制非常重要，而这些信息可以为 PRV 疫苗的开发和使用提供关键信息。4PRV活疫苗接种疫苗是预防传染病的最有效策略之一。根除 PRV 运动的成功主要归功于应用了优良的 PRV 疫苗。然而，传统的疫苗接种并不是一劳永逸的解决方案，人类与传染病的斗争仍在继续。PRV 变异体在中国的流行促使我们集中精力开发新的和更有效的疫苗来阻止 PRV 变异体大流行。减毒疫苗保留了病原体的大部分自然结构。尽管如此，它们在体内的复制能力和毒力降低，现在普遍认为减毒疫苗提供更好和更完整的免疫保护，特别是 T 细胞介导的免疫(图2)。减毒活疫苗是目前应用最为广泛和发展最为迅速的疫苗。本节总结了目前减毒活疫苗和基于病毒载体的活疫苗研制的进展和关键问题，并对新型活疫苗的研制提供了见解。图2 野生型伪狂犬病毒和减毒活疫苗可诱导体内产生后天免疫系统4.1减毒活疫苗大多数 PRV 疫苗是基于部分基因缺失的减毒活疫苗。PRV的美国区基因US3、 US和US8是主要的毒力基因，被认为是对PRV复制不重要的非核心基因。US2在大多数疱疹病毒中保守，通过调节ERK的定位间接调节MAPK的活性。另一项研究表明，缺失PRV us2基因会增加病毒滴度。US9是一种膜蛋白，在病毒传播过程中发挥着至关重要的作用疱疹病毒(包括 PRV)的顺式神经传输。UL23编码一个与PRV毒力相关的非结构蛋白 PK，几乎不具有免疫原性。目前这些基因常被用作PRV衰减剂。第一株PRV减毒活疫苗 Bartha K61株是最好的改良活疫苗之一。对巴沙 K61疫苗株进行了体外连续传代，获得了减毒特性。后来，研究人员在 Bartha K61的美国区域发现了许多缺失，包括 US8和 US9基因的完全缺失和 US7和 US2基因的部分缺失。随着 PRV 遗传学和分子生物学的进步，PRV 基因的作用还没有被恢复，更多的基因缺失的 PRV 疫苗正在通过各种技术被开发。基因工程技术，如同源重组、细菌人工染色体和 CRISPR/Cas9基因编辑系统，可以快速构建基因缺失的菌株，作为活的减毒性病毒候选菌株。这主要包括单基因缺失、双基因缺失、三基因缺失、四基因缺失和五基因缺失疫苗(表2)。表2 PRV减毒活疫苗概述尽管减毒活疫苗对PR的预防和控制具有重大意义，但一些研究人员对减毒活疫苗在易感动物中的安全性提出了质疑。减毒性病毒菌株rPRvtJ-delgE/gI对猪是无毒的，但对绵羊是致命的，这表明gE和gI的缺失可能不足以减弱。另一组基于PRVΔTK&gE-AH02株构建TK/gE双基因缺失疫苗，导致1日龄无PRV抗体仔猪死亡。基于PRVΔTK和gE-AH02的PK基因缺失导致PRV病毒进一步减弱，并且在接种没有 PRV 抗体的新生仔猪后没有观察到临床症状。最近的一项研究发现，基于gE/TK缺失菌株的 US3基因的进一步缺失在具有更高水平的中和抗体，细胞因子和组织驻留记忆T细胞(TRM)的小鼠中具有更好的免疫原性。值得注意的是 US3编码的PK蛋白，它广泛参与PRV的免疫逃逸，如第1.2节所述。因此US3基因可能成为活体减毒性病毒设计和开发的有希望的目标。越来越多的基于突变体的减毒PRV疫苗已经开发出来，但仍有一些问题需要进一步考虑。首先，减毒剂仅限于US2、 US3、 US7、 US8、 US9和UL23的单个或多个缺失，下一步需要进一步开发新的减毒相关基因; 其次，多基因缺失后的病毒生长特性很少受到关注，一些基因缺失可能会降低病毒滴度，影响减毒活疫苗的生产; 最后，该疫苗在除猪以外的动物中的安全性有待进一步研究，特别是在非人灵长类动物中。为了控制新的 PRV 变种，更新疫苗株以匹配循环株是最关键的。因此，必须继续监测现场菌株的变化，并据此构建新的候选疫苗。我们的一般概念是，疫苗的安全性与毒力相关基因缺失的数量呈正相关，但更多的基因缺失对疫苗株的免疫原性和产量有更大的影响。减弱病毒免疫逃避是构建减毒疫苗的一种策略。先前的研究显示，US3、 US8、 UL13、 UL16、 UL24、 UL42、 UL50和 EP0有助于 PRV 逃避宿主的先天免疫，因此这些基因具有构建减毒性病毒的潜力。免疫逃逸基因的引入可以降低 PRV 的毒力，提高其安全性。例如，Wang 等人构建的 PRV EP0缺失突变体对 IFN-α 介导的抗病毒免疫更敏感。另一个研究小组构建了一个 UL50缺失的 PRV 突变体，该突变体对 IFN-α 介导的病毒抑制类似敏感。尽管缺乏动物研究来验证这些基因降低毒力的能力，将 PRV 免疫逃逸基因导入目前的减毒活疫苗是一个有希望的策略来产生新的 PRV 活疫苗。4.2. 抗多种病原体的减毒重组PRV活疫苗猪至少受到40种病原体的威胁，其中包括16种病毒、15种细菌、8种寄生虫和1种原生动物。近年，猪只传染病如非洲猪瘟、口蹄疫、猪只繁殖与呼吸系统综合症、猪细小病毒和流感等已被广泛记录。针对每一次潜在的疫情爆发，采用适当的疫苗接种是最有效的方法，但现场存在许多混合感染。不同疫苗在短期培养周期内多次正确接种是很难实现的。异源抗原基因在病毒基因组中的插入和表达导致额外的免疫保护或增强的免疫效应是一个令人兴奋的研究课题。PRV 有一个很大的基因组，如前所述，一些基因的缺失使其减弱而不会显著影响其生长能力和免疫原性。几种基于伪狂犬病毒的载体疫苗已经研制成功。例如，共同表达流行性乙型脑炎病毒(JEV)的 PrM-E，经典猪瘟病毒(CSFVs)的 E2，口蹄疫病毒 p12A 和3C，PPV VP2，猪三角冠病毒(PDCoV)穗(S) ，PRRSV gP5和 M，ASFV 和SARS-CoV-2S 或 N 的 CP204L (p30) ，CP530R (pp62) ，E183L (p54) ，B646L (p72)和 EP402R (CD2v)基因。这些多价疫苗对许多疾病具有预防作用，但其在猪体内的安全性和有效性仍有待进一步研究。这些研究表明，基因缺失的减毒 PRV 病毒可以作为一种有前途的载体来开发针对两种或两种以上病原体的多价疫苗。分子佐剂通过多种机制调节免疫系统，从而提高疫苗的效力。设计合适的分子佐剂来构建基于基因缺陷PRV的重组活疫苗是一个很有前景的方向。Zheng等人构建了表达PPV VP2蛋白和猪IL-6的重组PRV，显著增加了 PPV 特异性淋巴细胞的增殖。将Fms相关酪氨酸激酶3配体（Flt3L）插入到TK和gE缺陷的PRV中，构建重组HNX-TK-/gE--Flt3L，可显著促进树突状细胞的成熟和活化(DCs)。5结论PRV仍然是猪的主要传染源之一。此外，人类感染PRV的持续发生增加了 PRV 的风险水平。PRV利用多种策略逃避宿主先天免疫。先天性免疫逃逸基因的修饰或缺失是开发新型减毒活疫苗的一个有前途的策略。减毒性病毒活菌株也是研制新型多价疫苗的良好载体，可对抗病毒、细菌和支原体等两种或多种病原体，特别是与呼吸道有关的病原体。另外，合适的分子佐剂的插入可以大大提高疫苗的保护效率。尽管已有几种 PRV 疫苗应用于临床，但加快新型疫苗的研制是当前PRV变种和其他病原体流行的迫切需要。参考资料：Nie Z, Zhu S, Wu L, Sun R, Shu J, He Y, Feng H. Progress on innate immune evasion and live attenuated vaccine of pseudorabies virus. Front Microbiol. 2023 Mar 3;14:1138016. doi: 10.3389/fmicb.2023.1138016. PMID: 36937252; PMCID: PMC10020201.为了推动兽用生物制品行业交流，共同探讨该领域的最新研发进展、产业化现状及未来发展趋势，生物制品圈联合四叶草会展、乘风济海将于2024年4月17日-18日在南京共同举办“兽用生物制品研发和产业化大会”，系中国医药全产业链新资源大会（CBC大会）中的一个分领域专业会议。诚邀全国相关领域研究者共享学术盛会。名称：兽用生物制品研发和产业化大会时间：2024年4月17日-18日（周三-周四）地点：南京国际展览中心主办单位：生物制品圈、抗体圈、四叶草会展，乘风济海媒体支持：药时空、细胞基因研究圈报名方式：扫描下方二维码或点击文章最底部“阅读原文”→ 填写表格 → 报名成功组委会获得报名信息后，根据报名信息进行初筛，并进一步与报名者沟通确认，实现精准邀请（严格审核通过）。大会日程注：大会日程以会议现场为准。中国医药全产业链新资源大会（CBC大会）是一场将“政、产、学、研、用、管、投”各方精英围绕全产业链新资源展开的合作大会，将于2024年4月16日-18日在南京国际展览中心举办。大会将主打“全产业链新资源对接”，围绕投资、立项、临床前研发、临床研究、生产、供应链国产化、销售、MAH合作、国际品种合作、公司股权合作与并购全产业链，为中国医药同仁带来全新的资源与商机。大会下设的同期会议包括：宠物药品、食品、保健品新资源大会兽用生物制品研发和产业化大会透皮技术研发生产与注册开年分享会吸入制剂研发、生产与注册新机遇新进展分享会中国改良新药与缓控释制剂全产业链合作大会陆续更新中......识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

Preliminary Results for the year ended 31 December 2022 FDA CLEARANCE A MAJOR BREAKTHROUGH FOR PARSORTIX LIQUID BIOPS Parsortix system substantially out-performs standard of care for ovarian cancer diagnosis Prostate cancer partnership signed with Solaris Health Pipeline of opportunities growing strongly GUILDFORD, SURREY / ACCESSWIRE / April 21, 2023 / ANGLE plc (OTCQX:ANPCY)(AIM:AGL), a world-leading liquid biopsy company, today announces audited preliminary results for the year ended 31 December 2022. 2022 was a breakthrough year for ANGLE, with both FDA clearance and excellent results from the ovarian cancer study. These achievements have placed ANGLE in a strong position to play a leading role in the emerging liquid biopsy market for personalised cancer care and is reflected in strong growth in unaudited revenues in Q1 2023 year-on-year. Operational Highlights Products · FDA De Novo clearance received for the Parsortix® PC1 Clinical System for its intended use with metastatic breast cancer (MBC) patients - first ever FDA product clearance to harvest intact cancer cells from a patient blood sample for subsequent user-validated analysis - multiple global distribution agreements secured to support commercial roll-out Pharma services · Increased pharma industry engagement post FDA clearance - repeat contract, worth up to $1.2 million, from large-scale pharma customer - assay development contract successfully delivers DNA Damage Repair (DDR) assay - ISO 15189 accreditation received for the United States laboratory Clinical uses · Ovarian cancer - excellent headline results from ovarian study with ROC-AUC 95.4% - results demonstrate clinical validity employing molecular analysis of cancer cells captured using the Parsortix system in a difficult to diagnose real-world setting · Prostate cancer - partnership established with Solaris Health, a major United States urology group to evaluate the Parsortix system in prostate cancer - clinical study now underway and expected to complete during 2023 Financial Highlights · Revenue £1.0 million (2021: £1.0 million) as previously communicated · Loss for the year £21.7 million reflecting planned investment (2021: loss £15.0 million) · Fundraising from institutional investors, including existing and new institutional investors, raising gross proceeds of £20.1 million (£18.9 million net of expenses) · Cash and cash equivalents at 31 December 2022 of £31.9 million (2021: £31.8 million) with R&D Tax Credits due at 31 December 2022 of £2.8 million (2021: £4.5 million) 2023 Progress and Outlook · 2023 product and services revenues both progressing well with unaudited Q1 2023 revenue strongly ahead year-on-year · Pharma services business growing well with new customers, such as Crescendo Biologics, and a growing pipeline of opportunities under discussion · Strong repeat pharma services business model being demonstrated with existing customers signing additional contracts · Prostate cancer pilot study enrolment on track for headline data around the end of 2023 · Corporate deal signed with BioView for development of a HER2 breast cancer test, to deliver revenues of c. £1.2 million in the initial phase, and with the prospect of adding other large corporate partners in due course · Encouraging initial results from third-party molecular tests on the Parsortix CTC harvests opening the potential for high value molecular tests in the future for pharma services and clinical use · Shortly after the year-end, two new Non-Executive Directors were appointed strengthening the Board for the next phase of the Company's development · Current pipeline of commercial opportunities supports management's confidence in delivering strong growth in 2023 and beyon Garth Selvey, Non-Executive Chairman of ANGLE plc, commented: "2022 was a breakthrough year for ANGLE with the world's first ever FDA product clearance for a system to harvest CTCs, intact living cancer cells, from metastatic breast cancer patient blood for subsequent analysis. This was followed by ANGLE's ovarian cancer study demonstrating the clinical validity of analysing Parsortix CTCs for real-world clinical applications. We are executing on our strategy to commercialise the Parsortix system through: a product business, with distribution partners for Parsortix instruments and consumables; and a services business, to utilise the Parsortix system in cancer drug trials and as an accelerator and demonstrator to support product sales. In both business areas, ANGLE is looking to leverage corporate partnerships to accelerate multiple commercial opportunities through clinical validation, regulatory approval and reimbursement authorities. The increasing number of published studies for a variety of cancer types combined with the FDA clearance have placed ANGLE in a strong position to play a leading role in the emerging liquid biopsy market for personalised cancer care. The CTCs harvested by the Parsortix system have wide applicability for diagnosis, treatment selection and monitoring to improve patient outcomes and reduce healthcare costs. We are pleased to see that 2023 has started strongly and look forward to continued commercial progress in the year ahead." Details of webcast A virtual meeting for analysts will be held at 10:00 am BST today. A live webcast of the analyst meeting can be accessed via ANGLE's Investor Centre page, , with Q&A participation reserved for analysts only. Please register in advance and log on to the webcast approximately 5 minutes before 10:00 am on the day of the results. A recording of the webcast will be made available on ANGLE's website following the results meeting. ANGLE plc +44 (0) 1483 343434 Andrew Newland, Chief Executive Ian Griffiths, Finance Director Andrew Holder, Head of Investor Relations Berenberg (NOMAD and Joint Broker) Toby Flaux, Ciaran Walsh, Milo Bonser +44 (0) 20 3207 7800 Jefferies (Joint Broker) Thomas Bective, Shaam Vora +44 (0) 20 7029 8000 FTI Consulting Simon Conway, Ciara Martin Matthew Ventimiglia (US) +44 (0) 203 727 1000 +1 (212) 850 5624 For Frequently Used Terms, please see the Company's website on The information contained within this announcement is deemed by the Company to constitute inside information as stipulated under the EU Market Abuse Regulation (596/2014). Upon the publication of this announcement via a regulatory information service, this information is considered to be in the public domain. These Preliminary Results may contain forward-looking statements. These statements reflect the Board's current view, are subject to a number of material risks and uncertainties and could change in the future. Factors that could cause or contribute to such changes include, but are not limited to, the general economic climate and market conditions, as well as specific factors including the success of the Group's research and development, commercialisation strategies, the uncertainties related to clinical study outcomes and regulatory clearance, obtaining reimbursement and payor coverage, acceptance into national guidelines and the acceptance of the Group's products by customers. CHAIRMAN'S STATEMENT Introduction The ground-breaking FDA product clearance for the Parsortix PC1 Clinical System in metastatic breast cancer heralds a new era for personalised cancer care. Large-scale medtech and pharma companies now have an FDA cleared platform on which to develop new diagnostic solutions for personalised cancer care and ANGLE is now moving to commercial roll-out of the system. I am pleased to welcome two new non-executive directors to the Board, who joined in January 2023. Juliet Thompson, who brings specialist knowledge in the areas of financing, strategy and corporate governance, and Dr. Joe Eid, who brings extensive experience of biomarkers in oncology and a wide pharma industry network. Overview of Financial Results Revenue of £1.0 million in the year (2021: £1.0 million) came mainly from research use sales of the Parsortix system with an initial contribution from the newly established pharma services business. ANGLE continued substantial investment in studies to develop and validate the clinical application and commercial use of the Parsortix system and to expand its commercial team ahead of anticipated customer demand, resulting in operating costs for the year of £24.8 million (2021: £18.0 million) and a loss for the year of £21.7 million (2021: loss £15.0 million). In July 2022, ANGLE moved rapidly post FDA clearance to complete a capital raise of £20.1 million (£18.9 million net of expenses) to support the Company's commercialisation plans through to mid-2024. The orderly wind down of the site in Toronto, Canada, and resultant streamlining of the Company's operations in the second half of 2022 further increases the cash runway into H2 2024, leaving ANGLE in a strong position to deliver on planned objectives and milestones. The Company is tightly controlling its cash resources and, post year end, the decision was taken not to pay cash bonuses in relation to 2022 despite strong performance against agreed objectives during the year. Instead share options and LTIP options were granted with a three year vesting period and a further two year holding period for executive directors. Share price performance conditions were set for senior management and executive directors, which must be met as a precondition if options are to be exercised. Commercial strategy ANGLE's vision is to secure widespread adoption of the Parsortix system by providing CTCs as the "best sample" for analysis in the emerging multi-US$ billion liquid biopsy oncology market. To drive commercialisation, ANGLE has established both a product business and a services business. Both business areas are supported by a growing body of scientific evidence and clinical studies from leading cancer centres in published peer-reviewed journals. Outlook 2022 was a breakthrough year for ANGLE, with both FDA clearance and the growing level of scientific evidence increasing the pipeline of opportunities for both our product and pharma services businesses. The Company is engaging with some of the largest pharma companies, medtech companies and clinical laboratories globally, with the capacity to drive Parsortix adoption through multiple clinical validation, specific regulatory approvals and acceptance by clinical service payers. ANGLE is focusing on the most immediate commercial opportunities and has the resources in place to deliver on its strategic and commercial plans. The current year has started well with several new customers and orders confirmed and revenues are up strongly in Q1 2023 year-on-year. Garth Selvey Chairman 20 April 2023 CHIEF EXECUTIVE'S STATEMENT Commercial strategy ANGLE's vision is to secure widespread adoption of the Parsortix technology by providing CTCs as the "best sample" for analysis in the emerging multi-US$ billion liquid biopsy market. To drive commercialisation, ANGLE has established both a product business and a services business with differing regulatory pathways, routes to market and near and longer-term revenue potential. 1. Product business area ANGLE has developed the Parsortix system including instruments and one-time use cassettes that can be sold to third-party laboratories for their use in research, pharmaceutical development or clinical use. To enable customers to carry out downstream analysis of the Parsortix harvest, ANGLE will also offer assay kits for cell imaging, use protocols and data packets for molecular platforms and algorithms for clinical interpretation of results. 2. Services business area ANGLE has established clinical laboratories in the UK and United States as accelerators and demonstrators that have the capability and required quality systems to process patient samples and offer validated clinical tests using the Parsortix system. The laboratories, in Guildford, UK and Plymouth Meeting, Pennsylvania, United States are being used to provide services to pharma and biotech customers running clinical trials (pharma services) and will be able to offer laboratory developed tests (LDTs) for patient management as a first step towards product roll-out of tests. Both business areas are supported by a growing body of published evidence from leading cancer centres showing the utility of the system through peer-reviewed publications, scientific data and clinical research evidence, highlighting a wide range of potential applications. This includes breakthrough research such as that published in June 2022 by the Molecular Oncology Laboratory at the Swiss Federal Institute of Technology in Zurich, Switzerland. The study revealed the link between cancer metastasis and the circadian rhythm, demonstrating that the spread of breast cancer accelerates during sleep. The research was published in the high-impact journal Nature and provides novel insights and potential targets for drug discovery. Parsortix products On 25 May 2022, FDA granted a De Novo Class II classification for the Parsortix PC1 Clinical System for use in harvesting CTCs, intact living cancer cells, from metastatic breast cancer (MBC) patient blood for subsequent analysis. This means that an entirely new medical device classification has been granted by FDA for the Parsortix PC1 Clinical System. De Novo clearance is extremely challenging and costly and consequently is rare and the Parsortix clearance is the first such medical device classification for a new instrument in oncology for many years. With a view to driving longer-term product revenues, during the year ANGLE expanded its commercial operations team, including product management, logistics, service and maintenance, and, following the FDA clearance and CE marking, has successfully established agreements to build an international network of oncology focused distribution partners. This network covers territories in Europe, including Germany, Austria, Czech Republic, Switzerland, Spain and France, the Middle East, China, India and New Zealand, with other geographies in discussion. These partners will provide valuable sales, implementation and ongoing service and maintenance support in their chosen markets. Parsortix assay development To support adoption of its technology by adding "content", ANGLE has been developing a suite of imaging assays (branded Portrait+) and molecular assays (branded Landscape+) to analyse the cancer cells harvested by the Parsortix system. These assays are designed to build a menu for ANGLE's pharma services business and to be sold as products for third-party customers through the growing distribution network for use with the Parsortix system. ANGLE has made good progress in the in-house development of a pipeline of new products, including a sample-to-answer Portrait+ imaging solution for the identification of epithelial and mesenchymal CTCs as well as CTCs in the process of epithelial mesenchymal transition (EMT). A Portrait+ PD-L1 assay is also in progress, enabling quantitative identification of this key target protein for immunotherapy on CTCs harvested using the Parsortix system. The decision has been taken to focus development of the downstream molecular assays (Landscape+) on third-party platforms which have greatly improved in sensitivity and reduced in cost in recent years and offer an installed base of molecular products, which can be leveraged for new Parsortix applications. Early results for evaluations of third-party systems have been highly encouraging and offer the prospect of combining the Parsortix harvest with platforms that are already widely adopted with a global installed base and where there are targeted sequencing panels already validated and commercially available for a wide range of solid tumour types. Pharma services The pharma services business utilising the Parsortix system offers the potential for substantial revenues in the large cancer drug trials market where ANGLE is clearly differentiated. The FDA clearance has helped open doors to pharma and the pipeline of potential pharma services customers has expanded significantly. There is a growing number of potential new customers and projects in discussion, including major pharma companies. In addition, ANGLE anticipates a high level of repeat business opportunities with existing customers and, during the year, announced it had secured an additional multi-year contract, worth up to US $1.2 million, with its first large-scale pharma services customer. ANGLE made excellent progress during the year with its first bespoke assay development customer. Following validation in ANGLE's clinical laboratories, the customer expects to employ the assays in clinical studies starting in 2023. The assays identify two target proteins on CTCs that are implicated in DNA Damage Repair (DDR), ɣ-H2AX and pKAP1. This is an area of focus for drug companies developing PARP inhibitors for a range of solid tumours and the assays will be added to our "menu" of pre-developed tests that can be offered to other customers. Initial interest in these assays, which were introduced at an industry event in early 2023, has been very encouraging. To support its pharma services business, ANGLE has been seeking regulatory accreditation of its Parsortix clinical laboratories in the United States and UK. ISO 15189 accreditation was received for the United States laboratory towards the end of the year and is expected for the UK laboratory in due course. This is an important achievement and demonstrates that ANGLE's clinical laboratories maintain globally recognised quality standards meeting all the requirements of major pharma customers. This is a key element as pharma services customers require evidence that the laboratories are stable, robust, compliant, and subject to periodic external inspections by recognised organisations. ANGLE believes that longitudinal monitoring of CTCs is a highly attractive proposition for the pharma industry looking for new insights in cancer drug trials and that prospects are very positive for the growth of this business. ANGLE has initiated its roll out of assays with the EMT and DDR assays being offered to pharma services customers from the clinical laboratories. Clinical services ANGLE intends that its Parsortix clinical laboratories will also offer a limited number of laboratory developed tests (LDTs) to physicians for patient management. These tests will act as "accelerators" of clinical commercialisation and also as "demonstrators" of clinical utility to support the product strategy. They will be the first step towards product roll-out of tests. Processing of patient samples for clinical purposes requires the laboratories to be accredited under the appropriate local regulatory regimes. In March 2022, the Centers for Medicare and Medicaid Services (CMS) issued a Certificate of Registration, under the CLIA process, to the Company's United States clinical laboratory. This is a key step towards achieving CLIA accreditation of the laboratory. The process will be completed once the first LDTs are being offered from the laboratories, which is a requirement. Parsortix clinical studies ANGLE is conducting clinical studies in selected high-risk patient groups. Successful studies demonstrate the value of CTC analysis by providing evidence of their predictive power. Successful results will also provide the data required to support the launch of LDTs from ANGLE's own clinical laboratories as accelerators and demonstrators (see above). Once published, results could also encourage third-party laboratories to offer these tests from their own accredited laboratories, enabling the sale of instruments and consumables. Ovarian cancer ANGLE has utilised Parsortix to investigate the diagnosis of ovarian cancer in women with an abnormal pelvic mass. Headline results for the clinical validity study were announced during the year demonstrating exceptional performance with ROC-AUC (accuracy) of 95.4%. This was in-line with the Company's earlier clinical study and achieved the Company's objective of best-in-class results with both sensitivity and specificity of 90% or greater. This result far out-performed standard of care for the detection of ovarian cancer demonstrating the value of the Parsortix system for real-world clinical decision-making and the clinical relevance of investigating CTCs. Following these excellent results, ANGLE has carefully considered the most appropriate commercial route for this test. With a view to maximising commercial potential and recognising the improvement in sensitivity and reduction in costs of other molecular systems with an established installed base, the Landscape+ Ovarian assay will now be optimised utilising a third-party molecular analysis platform. Validation of the optimised assay can be undertaken utilising patient samples stored from the already completed studies. The major advantage of this approach is it will leverage the third-party installed base providing them with "content" and will allow a larger scale product-based commercialisation strategy for ovarian cancer, substantially increasing market potential and the rate of adoption. Prostate cancer During the year, ANGLE announced it had signed a master clinical study agreement with Solaris Health Holdings, LLC (Solaris) and joinder agreements with MidLantic Urology LLC, to collaborate and conduct clinical studies in prostate cancer and as a potential route to market in the United States. MidLantic Urology, an affiliate of Solaris, is one of the largest providers of specialist urology services in the United States with more than 70 physicians operating from 47 dedicated urology centres across the state of Pennsylvania. The Solaris Health network encompasses more than 500 clinical urology providers across 179 locations and nine States with more than 729,000 unique patients annually. Together with MidLantic Urology, ANGLE has initiated a clinical study aimed at investigating the use of the Parsortix system for the detection of prostate cancer and prediction of its severity in patients who present with an elevated prostate specific antigen (PSA) level and/or abnormal digital rectal exam. This study is initially enrolling 100 men scheduled to undergo a prostate tissue biopsy at a minimum of three study sites. Blood samples collected by MidLantic Urology are being shipped to ANGLE's United States clinical laboratory for processing by the Parsortix system to harvest and analyse CTCs and associated immune cells. The Parsortix harvests will be evaluated by both imaging and molecular analysis to assess the potential to predict the presence of clinically significant prostate cancer prior to tissue biopsy and to assess potential correlation with established disease severity scores (e.g., the Gleason score) in those patients found to have prostate cancer. Patient enrolment for the pilot study is on track and ANGLE expects headline results around the year end. Solaris is planned to be ANGLE's first route to market for this test, offering the established test to their extensive patient base and opening up a significant market opportunity for ANGLE. Parsortix corporate partnerships Addressing a large and complex healthcare market with a new technology requires significant resources and ANGLE is seeking long term corporate partnerships on a case-by-case basis to assist in accelerating market access and maximising commercial potential across its business lines. The partnership with Solaris Health in prostate cancer signed during the year provides an example of this approach, fast-tracking clinical studies and providing a valuable first route to market with a substantial patient base. The agreement with BioView to develop a CTC HER2 assay for breast cancer using a combination of ANGLE's FDA cleared Parsortix® PC1 Clinical System and BioView's automated microscopy systems and software to detect and assess the HER2 expression and/or gene amplification in CTCs is another significant development. The changing market dynamics of the HER2 breast cancer marketplace, with the introduction of new drugs targeting low HER2 expression, have provided ANGLE and BioView with a major commercial opportunity to develop a quantitative CTC-based HER2 assay, to assess HER2 protein expression and/or gene amplification levels by analysing fluorescence intensities. This would be the only product-based solution on the market for this purpose, leveraging both companies' previous FDA product clearances. Unlike current standard of care tests developed for use on FFPE tissue, a CTC HER2 assay could be used for longitudinal monitoring of HER2 status throughout disease progression, thereby ensuring the patient is targeted for the most appropriate treatment at every stage. The development phase is estimated to take around a year to complete and will generate revenue of c. £1.2 million. Given the significant third-party interest in a new assay for quantitative HER2 analysis based on CTCs, the agreement allows for the inclusion of third parties in this project and its funding at the commercialisation stage after the initial development work is complete. ANGLE continues to discuss strategic routes to market with potential corporate partners. As described above, following a review of third-party molecular systems, ANGLE has focused its resources on evaluating the performance of Parsortix samples in combination with multiple third-party downstream DNA and RNA sequencing technologies. Initial results from these evaluations have been highly encouraging and ANGLE believes this will open the door to partnering discussions with these technology providers, who are keen to add "content" to their product menus. Summary Despite very challenging macro conditions outside the Company's control, ANGLE is making strong progress building both its products and services businesses. Harvesting intact living cancer cells for analysis, ANGLE's FDA cleared Parsortix PC1 Clinical System is differentiated from all other approaches to liquid biopsy and offers the prospect of cost-effective, non-invasive repeat testing for cancer patients. ANGLE is now well on the way to getting this approach adopted by pharma in the cancer drug trials process and is building the data to drive adoption in the diagnosis and treatment of cancer patients to improve patient outcomes and reduce healthcare costs. Andrew Newland Chief Executive 20 April 2023 ANGLE PLC CONSOLIDATED STATEMENT OF COMPREHENSIVE INCOME FOR THE YEAR ENDED 31 DECEMBER 2022 2022 2021 Note £'000 £'000 Revenue 1,041 1,013 Cost of sales (428) (302) Gross profit 613 711 Other operating income 1 41 Operating costs (24,821) (17,987) Operating profit/(loss) (24,207) (17,235) Finance income 136 29 Finance costs (368) (157) Profit/(loss) before tax (24,439) (17,363) Tax (charge)/credit 5 2,753 2,351 Profit/(loss) for the year (21,686) (15,012) Other comprehensive income/(loss) Items that may be subsequently reclassified to profit or loss: Exchange differences on translating foreign operations (2,023) (175) Other comprehensive income/(loss) (2,023) (175) Total comprehensive income/(loss) for the year (23,709) (15,187) Earnings/(loss) per share attributable to owners of the parent Basic and Diluted (pence per share) 6 (8.79) (6.67) All activity arose from continuing operations. ANGLE PLC CONSOLIDATED STATEMENT OF FINANCIAL POSITION AS AT 31 DECEMBER 2022 2022 2021 Note £'000 £'000 Assets Non-current assets Intangible assets 2,764 3,573 Property, plant and equipment 3,505 2,172 Right-of-use assets 4,971 2,204 Total non-current assets 11,240 7,949 Current assets Inventories 2,059 1,748 Trade and other receivables 1,797 1,269 Taxation 2,876 4,510 Cash and cash equivalents 31,896 31,839 Total current assets 38,628 39,366 Total assets 49,868 47,315 Liabilities Non-current liabilities Lease liabilities (4,339) (1,816) Provisions (157) - Trade and other payables (59) (257) Total non-current liabilities (4,555) (2,073) Current liabilities Lease liabilities (662) (522) Provisions (610) - Trade and other payables (3,978) (4,390) Total current liabilities (5,250) (4,912) Total liabilities (9,805) (6,985) Net assets 40,063 40,330 Equity Share capital 7 26,058 23,514 Share premium 115,918 99,406 Share-based payments reserve 5,321 2,727 Other reserve 2,553 2,553 Translation reserve (5,983) (3,960) Accumulated losses (103,702) (83,808) ESOT shares (102) (102) Total equity 40,063 40,330 ANGLE PLC CONSOLIDATED STATEMENT OF CASH FLOWS FOR THE YEAR ENDED 31 DECEMBER 2022 2022 £'000 2021 £'000 Operating activities Profit/(loss) before tax (24,439) (17,363) Adjustments for: Depreciation of property, plant and equipment 920 701 Depreciation and impairment of right-of-use assets 940 532 (Profit)/loss on disposal of property, plant and equipment 172 4 Amortisation and impairment of intangible assets 978 254 Share-based payment charge 4,386 1,325 Exchange differences (2,072) (170) Net finance (income)/costs 232 128 Operating cash flows before movements in working capital (18,883) (14,589) (Increase)/decrease in inventories (580) (1,015) (Increase)/decrease in trade and other receivables (650) 204 Increase/(decrease) in trade and other payables (978) 1,417 Increase/(decrease) in provisions 594 - Operating cash flows (20,497) (13,983) Research and development tax credits received 4,506 - Overseas tax payments (59) (27) Net cash from/(used in) operating activities (16,050) (14,010) Investing activities Purchase of property, plant and equipment (1,718) (1,666) Purchase of intangible assets (169) (122) Transfer from short-term deposits - 16,538 Interest received 136 24 Net cash from/(used in) investing activities (1,751) 14,774 Financing activities Net proceeds from issue of share capital - placing 18,922 18,765 Proceeds from issue of share capital - share option exercises 123 925 Principal elements of lease payments (814) (614) Interest elements of lease payments (135) (85) Net cash from/(used in) financing activities 18,096 18,991 Net increase/(decrease) in cash and cash equivalents 295 19,755 Cash and cash equivalents at start of year 31,839 12,080 Effect of exchange rate fluctuations (238) 4 Cash and cash equivalents at 31 December 31,896 31,839 ANGLE PLC CONSOLIDATED STATEMENT OF CHANGES IN EQUITY FOR THE YEAR ENDED 31 DECEMBER 2022 --------Equity attributable to owners of the parent------ Share capital £'000 Share premium £'000 Share-based payments reserve £'000 Other reserve £'000 At 1 January 2021 21,540 81,532 1,745 2,553 For the year to 31 December 2021 Consolidated profit/(loss) Other comprehensive income/(loss): Exchange differences on translating foreign operations Total comprehensive income/(loss) Issue of shares (net of costs) 1,974 17,874 Share-based payment charge 1,325 Released on exercise (295) Released on forfeiture (48) At 31 December 2021 23,514 99,406 2,727 2,553 For the year to 31 December 2022 Consolidated profit/(loss) Other comprehensive income/(loss): Exchange differences on translating foreign operations Total comprehensive income/(loss) Issue of shares (net of costs) 2,544 16,512 Share-based payment charge 4,386 Released on exercise (43) Released on forfeiture/lapse (1,749) At 31 December 2022 26,058 115,918 5,321 2,553 ANGLE PLC CONSOLIDATED STATEMENT OF CHANGES IN EQUITY FOR THE YEAR ENDED 31 DECEMBER 2022 (continued) -------- Equity attributable to owners of the parent ---------- Translation reserve £'000 Accumulated losses £'000 ESOT shares £'000 Total equity £'000 At 1 January 2021 (3,785) (69,139) (102) 34,344 For the year to 31 December 2021 Consolidated profit/(loss) (15,012) (15,012) Other comprehensive income/(loss): Exchange differences on translating foreign operations (175) (175) Total comprehensive income/(loss) (175) (15,012) (15,187) Issue of shares (net of costs) 19,848 Share-based payment charge 1,325 Released on exercise 295 - Released on forfeiture 48 - At 31 December 2021 (3,960) (83,808) (102) 40,330 For the year to 31 December 2022 Consolidated profit/(loss) (21,686) (21,686) Other comprehensive income/(loss): Exchange differences on translating foreign operations (2,023) (2,023) Total comprehensive income/(loss) (2,023) (21,686) (23,709) Issue of shares (net of costs) 19,056 Share-based payment charge 4,386 Released on exercise 43 - Released on forfeiture/lapse 1,749 - At 31 December 2022 (5,983) (103,702) (102) 40,063 ANGLE PLC NOTES TO THE PRELIMINARY ANNOUNCEMENT FOR THE YEAR ENDED 31 DECEMBER 2022 1 Preliminary announcement The preliminary results for the year ended 31 December 2022 were approved by the Board of Directors on 20 April 2023. The preliminary announcement set out above does not constitute ANGLE plc's statutory Financial Statements for the years ended 31 December 2022 or 31 December 2021 within the meaning of section 434 of the Companies Act 2006 but is derived from those audited Financial Statements. The auditor's report on the Consolidated Financial Statements for the years ended 31 December 2022 and 31 December 2021 is unqualified and does not contain statements under s498(2) or (3) of the Companies Act 2006. The accounting policies used for the year ended 31 December 2022 are unchanged from those used for the statutory Financial Statements for the year ended 31 December 2021. The 31 December 2022 statutory Financial Statements will be delivered to the Registrar of Companies following the Company's Annual General Meeting. 2 Compliance with accounting standards While the financial information included in this preliminary announcement has been computed in accordance with the measurement principles of UK-adopted international accounting standards, this announcement does not itself contain sufficient information to comply with these accounting standards. Accounting standards adopted in the year No new accounting standards that have become effective and adopted in the year have had a significant effect on the Group's Financial Statements. Accounting standards issued but not yet effective At the date of authorisation of the Financial Statements, there were a number of other Standards and Interpretations (International Financial Reporting Interpretation Committee - IFRIC) which were in issue but not yet effective, and therefore have not been applied in these Financial Statements. The Directors have not yet assessed the impact of the adoption of these standards and interpretations for future periods. 3 Going concern The Financial Statements have been prepared on a going concern basis which assumes that the Group will be able to continue its operations for the foreseeable future. The Group's business activities, together with the factors likely to affect its future development, performance and financial position are set out in the Chairman's Statement. The Directors have considered the uncertainties, risks and potential impact on the business associated with potential negative trading scenarios and market and geopolitical uncertainty (Ukraine-Russia conflict). Discretionary expenditure within the business provides flexibility to scale back operations to address adverse events if required. Mitigation measures to reduce costs could be taken if needed and other potential sources of funding exist such as grants, exclusivity and/or milestone payments for corporate partnerships being developed and equity proceeds. The Directors have prepared and reviewed the financial projections for a period in excess of 12 months from the date of approval of these Financial Statements with discretionary expenditure carefully controlled in line with available resources, as certain projects may be deferred until additional resources are available. Based on the level of existing cash and expected R&D tax credits, the projected income and expenditure (the quantum and timing of some of which is at the Group's discretion) and other potential sources of funding, the Directors have a reasonable expectation that the Company and Group have adequate resources to continue in business for the foreseeable future. Accordingly, the going concern basis has been used in preparing the Financial Statements. 4 Critical accounting estimates and judgements The preparation of the Financial Statements requires the use of estimates, assumptions and judgements that affect the reported amounts of assets and liabilities at the date of the Financial Statements and the reported amounts of revenues and expenses during the reporting year. Although these estimates, assumptions and judgements are based on the Directors' best knowledge of the amounts, events or actions, and are believed to be reasonable, actual results ultimately may differ from those estimates. The estimates that have a significant risk of causing a material adjustment to the carrying amounts of assets and liabilities are described below. Share-based payment charge In calculating the fair value of equity-settled share-based payments the Group uses options pricing models. The Directors are required to exercise their judgement in choosing an appropriate options pricing model and determining input parameters that may have a material effect on the fair value calculated. These key input parameters are expected volatility, expected life of the options and the number of options expected to vest. 5 Tax The Group undertakes R&D activities. In the UK these activities qualify for tax relief and result in R&D tax credits. 6 Earnings/(loss) per share attributable to owners of the parent The basic and diluted earnings/(loss) per share is calculated by dividing the after tax loss for the year attributable to the owners of the parent of £21.7 million (2021: £15.0 million) by the weighted average number of shares in the year. In accordance with IAS 33 Earnings per share, 1) the "basic" weighted average number of Ordinary shares calculation excludes shares held by the Employee Share Ownership Trust (ESOT) as these are treated as treasury shares and 2) the "diluted" weighted average number of Ordinary shares calculation considers potentially dilutive Ordinary shares from instruments that could be converted. Share options are potentially dilutive where the exercise price is less than the average market price during the year. Due to losses in the 2022 and 2021 reporting years, share options are non-dilutive for those years as adding them would have the effect of reducing the loss per share and therefore the diluted loss per share is equal to the basic loss per share. The basic and diluted earnings/(loss) per share are based on 246,579,644 weighted average ordinary £0.10 shares for the year (2021: 225,073,380). 7 Share capital The Company has one class of Ordinary shares which carry no right to fixed income and at 31 December 2022 had 260,580,547 Ordinary shares of £0.10 each allotted, called up and fully paid (2021: 235,143,050). The Company issued 25,162,500 new Ordinary shares with a nominal value of £0.10 at an issue price of £0.80 per share in a placing of shares realising gross proceeds of £20.1 million. Associated costs of £1.2 million were incurred. Shares were admitted to trading on AIM in July 2022. The Company issued 274,997 new Ordinary shares with a nominal value of £0.10 at exercise prices between £0.385 to £0.530 per share as a result of the exercise of share options by employees realising gross proceeds of £0.1 million. Shares were admitted to trading on AIM at various dates across the year. 8 Shareholder communications Copies of this announcement are posted on the Company's website . The Annual General Meeting (AGM) of the Company will be held at 2:00 pm on Wednesday 28 June 2023 at the Holiday Inn Guildford, Egerton Road, Guildford, GU2 7XZ. The Board is looking forward to welcoming shareholders to the AGM in person. Details will be included in the notice of AGM. Notice of the AGM will be enclosed with the audited statutory Financial Statements. The audited statutory Financial Statements for the year ended 31 December 2022 are expected to be distributed to shareholders no later than 1 June 2023 and will subsequently be available on the Company's website or from the registered office, 10 Nugent Road, Surrey Research Park, Guildford, GU2 7AF. This preliminary announcement was approved by the Board of Directors on 20 April 2023. This information is provided by RNS, the news service of the London Stock Exchange. RNS is approved by the Financial Conduct Authority to act as a Primary Information Provider in the United Kingdom. Terms and conditions relating to the use and distribution of this information may apply. For further information, please contact rns@lseg.com or visit . SOURCE: ANGLE plc View source version on accesswire.com:

The biosensor could help optimize cancer treatment, identify DNA damage factors, and elucidate repair mechanisms BUSAN, South Korea, March 14, 2023 /PRNewswire/ -- Scientists from Korea have developed a new biosensor that uses fluorescence resonance energy transfer (FRET) to detect DNA double-strand breaks in living specimens in real time. This biosensor has the potential to revolutionize cancer treatment by helping doctors understand how cells respond to treatment and facilitating the discovery of new DNA repair drugs. It can also aid the development of new treatments for DNA damage-related diseases by revealing how our bodies repair damaged DNA. Double-strand breaks (DSBs) are a type of DNA damage where both strands of DNA break at the same location. They can adversely affect cell growth and functioning. Currently, DSBs are detected by immunostaining techniques, which identify markers that accompany DNA damage, such as the protein γH2AX. However, these methods are tedious, and cannot be used to detect DSBs in real time in living specimens. In a 2023 study, published in Biomaterials Research, researchers describe a fluorescence resonance energy transfer (FRET) biosensor that can detect DSBs in real time, and provide time- and location-based information on yH2AX. "The biosensor we have designed could be useful in areas such as cancer treatment and drug discovery," says Associate Professor Tae-Jin Kim, from Pusan National University, Korea, who led the study. FRET sensors consist of two fluorescent proteins or dyes—a donor and an acceptor—which investigate interactions between biological molecules. The energy transfer, and consequently, the amount of emitted light (the FRET signal) depends on the distance and orientation between the two dyes. The researchers attached the fluorescent dyes with proteins that are involved in the cellular response to DNA damage, namely the H2AX substrate and BRCT1 domain. The H2AX substrate is a target for the H2AX protein to bind and become phosphorylated (forming γH2AX). On the other hand, the BRCT1 domain acts as a site for the accumulation of repair proteins, including γH2AX. Thus, when a DSB occurs, γH2AX is attracted to the BRCT1 domain, leading to a conformational change in the fluorescent proteins, thereby causing a change in the FRET signal. The researchers then confirmed the validity of the sensor by introducing plasmids (DNA that, here, contain instructions to make the FRET sensor inside the cells) encoding the FRET sensor into human embryonic kidney cells (HEK293T) cells. Compared to conventional immunostaining techniques, this biosensor was more sensitive at reacting to the presence of γH2AX, making it more effective at detecting drug- and radiation-induced DSBs. "Moreover, as changes in the FRET signal give useful indications of the extent of the DNA damage, the sensor can also be used to examine DNA damage and repair mechanisms, optimize cancer treatments, discover and assess DNA repair drugs, and identify DNA damaging factors in the environment," concludes Associate Prof. Kim. Reference Title of original paper: A novel DNA double-strand breaks biosensor based on fluorescence resonance energy transfer Journal: Biomaterials Research DOI: *Corresponding author's email: tjkim77@pusan.ac.kr About Pusan National University Website: