Cholera vaccine (inactivated, oral)(Valneva Sweden AB)

摘要：疫苗是控制传染病最有效的工具，这些疾病会导致显著的发病率和死亡率。大多数疫苗通过非肠道途径给药，能够引发强烈的系统性体液免疫反应，但它们诱导的T细胞介导的免疫较弱，且在诱导粘膜保护方面效果不佳。考虑到大多数病原体通过粘膜表面进入体内，一种能在宿主与病原体首次接触点引发保护的疫苗是很有前景的。然而，尽管粘膜疫苗作为在粘膜表面提供保护的良好选择具有优势，目前只有少数粘膜疫苗获得批准。在本篇综述中，我们讨论了疫苗在不同粘膜表面的给药影响；适当的佐剂如何增强保护性粘膜免疫的诱导以及其他可能影响疫苗粘膜免疫反应的因素。 1.引言 疫苗是控制传染病、导致发病率和死亡率的重要原因的最有效工具。根据世界卫生组织的数据，疫苗每年在全球范围内预防超过250万人死亡。因此，疫苗成功地根除了天花，并减少了其他几种疾病如脊髓灰质炎和麻疹的发病率。 疫苗通过在体内模拟病原体感染来发挥作用，这导致免疫系统激活以对抗病原体。疫苗给药后，疫苗中的病原体相关分子模式（PAMPs）与注射部位的先天免疫细胞中的模式识别受体（PRRs）相互作用，并触发免疫级联反应，涉及趋化因子和细胞因子的产生增加，导致注射部位的抗原呈递细胞（APCs）数量增加。APCs主要负责抗原的摄取和随后呈递给激活B细胞的T细胞，导致抗体产生。因此，为了对病原体产生强大和有效的免疫反应，强烈激活B细胞和T细胞是必要的。 消除病原体后，适应性免疫系统会对其发展免疫记忆。这种免疫记忆是长期个体保护的基础，其特点是抗体的持续存在和记忆细胞的产生，这些细胞在再次暴露于同一病原体时会迅速重新激活。因此，疫苗接种的目标是诱导记忆细胞。 大多数疫苗通过非肠道途径给药，通常包括：皮下（SC）、皮内（ID）和肌肉内（IM）。ID免疫接种比IM注射诱导更强的免疫反应，而SC和IM在临床研究中诱导相似的反应。一个可能的解释是，真皮含有更多的树突状细胞（DCs），这有助于抗原的捕获，局部炎症诱导DCs的成熟和它们迁移到引流淋巴结，导致疫苗剂量节省。尽管ID疫苗注射比其他非肠道途径更有效地诱导免疫反应，但它在给药部位引起更严重的不良反应，其次是SC和IM途径。有研究表明，在15个月至5岁儿童中，接种B型流感嗜血杆菌多糖疫苗时，SC/ID注射可能比IM注射更疼痛。尽管有其优势，但使用非肠道途径需要针头，这使得这种途径更具侵入性且成本更高，除了由于使用不当而存在感染风险外，还需要训练有素的人员进行给药。此外，非肠道疫苗可以引发强烈的系统性体液反应，但提供较弱的T细胞介导的免疫和粘膜保护。 与非肠道疫苗相关的另一个问题是，这种途径只保护疾病而不是感染。一些研究发现，接种了针对B型脑膜炎奈瑟菌的疫苗的个体对侵袭性疾病有保护作用，但在随后的12个月内，并没有迹象表明N. meningitidis的携带密度有所减少。 考虑到大多数病原体通过粘膜表面进入体内，因为这些表面直接与空气、水、食物和环境接触，针对宿主与病原体首次接触点的疫苗具有吸引力，因为它们有潜力从首次接触点保护宿主免受病原体侵害。 通过粘膜表面和ID途径给药的疫苗显示出抗原的快速和广泛生物分布，以及诱导保护性粘膜和系统性细胞和体液反应的能力。特别是，粘膜疫苗更安全，因为它们不需要针头。这一方面也导致依从性增加。因此，它们便于在流行病/大流行环境中进行大规模疫苗接种，因为它们易于给药。尽管有所有这些优势，但由于粘膜疫苗需要安全有效的粘膜佐剂来实现预期的免疫反应，此外，抗原必须根据粘膜给药途径免受降解，目前只有少数粘膜疫苗可供商业使用，如表1所示。 鉴于上述情况，在本篇综述中，我们讨论了疫苗在粘膜表面的给药影响，适当的佐剂或载体配方如何促进保护性粘膜免疫的诱导，以及其他可能影响疫苗粘膜免疫反应的因素。 2.粘膜表面的免疫防御 粘膜表面覆盖了身体的400平方米，包括胃肠道、泌尿生殖道和呼吸道。为了防御感染，粘膜表面配备了由粘膜上皮细胞提供的物理、化学和免疫机制（图1）。物理屏障由细胞间的紧密连接和浓密的粘液层组成，防止外来物质的进入和/或粘附。生化屏障由pH差异和微生物群产生的抗菌肽提供。最后，免疫防御包括先天和适应性免疫反应。 图1 粘膜表面和防御机制。上皮细胞之间的紧密连接是抵御病原体的物理屏障。微生物群和抗菌肽分别是病原体的生物和生化屏障。为了引发特异性免疫反应，微褶(M)细胞负责在腔内摄取抗原并将其呈递给树突状细胞，树突状细胞通过MHC-II将其呈递给T淋巴细胞。T细胞分泌细胞因子，调节细胞反应的模式（Th1、Th2、Th17、Treg），并促进B细胞分化为浆细胞，这些浆细胞产生分泌型IgA（SIgA），能够在粘膜中和病原体。 粘膜组织拥有一个被称为粘膜相关淋巴组织（MALT）的有组织的淋巴组织团块。MALT包括被滤泡相关上皮覆盖的淋巴滤泡，其中含有微褶（M）细胞，它们负责在腔内摄取抗原并呈递给树突状细胞（DCs）和巨噬细胞，在那里它们被处理并呈递给淋巴细胞。DCs将抗原呈递给初始T细胞，以克隆扩增并分化为T辅助（Th）细胞亚群（Th1、Th2、Th17或调节性T细胞）。每个亚群将决定细胞因子分泌的轮廓，之后，T细胞与B细胞相互作用，促进它们在多个粘膜部位的分化和抗体产生。这些B细胞将产生抗原特异性的分泌型免疫球蛋白A（SIgA），它防止病原体在粘膜表面的侵袭，以及IgG抗体。Th细胞在粘膜免疫中扮演重要角色。一些报告表明，主要由Th17细胞分泌的白介素（IL）-17是疫苗诱导的对肺炎链球菌、结核分枝杆菌和流感的保护所必需的。此外，IL-17可以通过上调粘膜上皮中的多聚Ig受体水平来增加SIgA水平，并促进B细胞不依赖T细胞分化成SIgA。 在肠道中，免疫系统级联反应发生在有组织的肠道相关淋巴组织（GALT）中，主要在Peyer氏斑中，这是IgA类别转换的主要部位。在上呼吸道中，它发生在小鼠的鼻相关淋巴组织中，由位于鼻咽管入口的硬腭上的一对有组织的淋巴聚集体组成。这种鼻组织在功能上似乎与人类的Waldeyer环扁桃体和腺样体相当。在下呼吸道中，发现了支气管相关淋巴组织。它是肺淋巴系统的一部分，通常在健康成人中不存在，但在抗原刺激下出现，包括炎症、吸烟习惯、胶原血管疾病、感染和艾滋病。 2.1.分泌型IgA：粘膜武器 如前所述，SIgA通过控制与宿主的首次接触以来的病原体定植，从而预防传染病，这是粘膜免疫的一个主要特征。IgA抗体，以其α重链为特征，存在于血清和粘膜中，有两种亚型：通常在血清中发现的IgA1，以及在粘膜中经常出现的IgA2。虽然IgA1主要由蛋白质抗原诱导，但IgA2在抗脂多糖（LPS）反应中占主导地位。 SIgA和血清IgA在位置、结构和功能上有所不同。SIgA通常呈现二聚体结构，并具有分泌成分，这为分子提供了稳定性，保护免疫球蛋白不被降解（鉴于粘膜环境的侵略性），从而帮助抗体与病原体蛋白结合。此外，SIgA是局部产生的，分泌量高，能够中和病原体。有证据表明粘膜中B细胞的快速周转以及长寿浆细胞的存在。最有可能的情况是，浆细胞会根据微生物群组成的变化而持续存在，微生物群的组成受到饮食、感染和抗生素治疗的影响，因此建立了一个动态平衡。 相反，血清IgA通常呈现单体结构，并且分泌滴度较低。有证据表明它有助于控制炎症，但关于其激活补体系统和促进病原体吞噬的能力存在矛盾的研究。SARS-CoV-2感染的粘膜性质导致了几项针对血清IgA的研究。早在2020年，就已验证人类单克隆IgA抗体能够有效地与SARS-CoV-2结合，并阻断血管紧张素转换酶2（ACE2）和受体结合域（RBD）的相互作用，表现出中和活性。因此，当转化为SIgA和IgG结构时，只有前者能够中和SARS-CoV-2。血清IgA也能够中和SARS-CoV-2，如Sterlin等人的研究，而Mazzini等人发现ELISA-IgA与微量中和试验有很好的相关性，因此，从血清样本中去除IgA显著降低了中和活性。比较表明，二聚体IgA比单体IgA表现出更强的中和能力，以及单体IgA和IgG。最近，Fox等人验证了COVID-19患者的牛奶样本在假病毒试验中具有中和作用。此外，粘膜免疫可以改善血清IgA的诱导，可能是因为B细胞在粘膜激活后归巢到脾脏边缘区。 目前的SARS-CoV-2疫苗也可以诱导IgA的产生，这一点已经对Moderna（mRNA1273）和辉瑞/BioNTech（BNT162b2）的mRNA疫苗以及牛津/阿斯利康（ChAdOx1）的病毒载体疫苗进行了验证。未来的粘膜疫苗可能会从异源初免-加强方法中受益。虽然粘膜免疫会诱导SIgA抗体，但非肠道剂量会改善系统性反应。啮齿动物的数据显示鼻内免疫增强了血清中的IgA以及血清和呼吸道粘膜中的IgA。然而，来自人类研究的证据很少。以前感染过COVID-19的疫苗接种志愿者，可能已经诱导了IgA反应，表现出比没有COVID-19病史的受试者更高的IgA滴度和更持久的IgA反应。我们可以推测，如果自然感染被视为鼻内初免，随后的非肠道疫苗将提供更好的免疫反应。另一项研究验证了mRNA疫苗在人乳中增加了IgA和IgG，尽管IgA比IgG更早减少。 有建议认为非肠道部位的淋巴引流可以作为系统性和粘膜免疫之间的交叉路径。这种观察可能让我们对通常分开的粘膜和系统免疫的观点产生疑问，并假设疫苗策略将从初免-加强方法中受益。尽管证据有限，但SIgA的贡献和血清IgA的前景作用应在疫苗研究中进行评估。 3.给药途径 粘膜疫苗能够在系统和粘膜部位诱导抗原特异性的体液和细胞介导的免疫反应。因此，粘膜免疫反应的诱导取决于疫苗的给药部位。口服免疫通常刺激胃肠道、唾液腺和乳腺的免疫反应和抗原特异性SIgA，而鼻内免疫诱导上呼吸道、下呼吸道、唾液腺和生殖器粘膜的抗体产生，舌下疫苗接种诱导胃肠道以及上下呼吸道的抗原特异性免疫反应，如图2所示。上述所有途径以及其他途径，例如阴道内和直肠途径，已在实验动物模型中应用，但只有口服和鼻内途径已在许可的人类疫苗中使用。 图2 粘膜部位的免疫接种不仅引发局部和系统性免疫反应，还在不同部位诱导粘膜反应。(a) 口服免疫刺激胃肠道、唾液腺和乳腺，考虑到母体-胎儿抗体转移，这一点尤其重要。(b) 鼻内途径在上呼吸道、下呼吸道、唾液腺和生殖器粘膜中诱导免疫。(c) 舌下疫苗接种在胃肠道以及上下呼吸道中引发反应。 尽管粘膜疫苗具有已知的潜力，但每种途径都有其含义。口服疫苗的开发代表了最大的挑战，这不仅是由肠道环境的侵略性所构成的——它降解了在其中传递的大多数抗原表位，影响疫苗的生物利用度——而且还由粘膜耐受性所构成，粘膜耐受性保护了对消化抗原的不希望的免疫反应。鼻内途径是一个有吸引力的部位，因为它所需的抗原和佐剂剂量比口服途径少，但它也面临着疫苗开发的挑战。鼻内疫苗配方需要到达呼吸道腔并穿过粘液层，其吸收就在那里发生；为此，疫苗配方需要考虑鼻腔的解剖和生理特征，如前庭区域的通透性差，以及毛发和粘液的快速周转，这些都干扰了成分的吸收。此外，鼻腔酶和局部pH可能影响疫苗的稳定性。 理想的粘膜疫苗在物理和化学屏障（pH和酶）方面是稳定的，将抗原传递到特定的免疫诱导部位，适应粘液周转，能够穿过上皮屏障，并且最终被APCs捕获，并且不诱导粘膜耐受性。使用佐剂是一种改善疫苗稳定性和吸收的策略，由于粘膜的生理和化学屏障，提供了更好的抗原传递。 4.粘膜佐剂 “佐剂”一词源于拉丁语adjuvare，意为帮助或援助。佐剂被定义为添加到疫苗配方中的功能性辅料，以增加其抗原的免疫原性。它们允许使用较低剂量的抗原和较少剂量的疫苗免疫方案。因此，佐剂通过增强其在粘膜部位的生物利用度来帮助增加配方的稳定性，其中自然防御机制，如pH、粘液层和运动可能会限制与APCs的相互作用，甚至降解抗原。这一方面影响了免疫反应的强度和持续时间。佐剂构成了一个异质化合物群，可以分为传递系统或免疫刺激分子。 4.1.免疫刺激佐剂 免疫刺激佐剂通过三种机制启动免疫激活：(1) 与非Toll样受体（非TLR）结合后的毒素介导的免疫刺激，例如宿主细胞表面存在的糖鞘脂。这一类别的最好代表是霍乱毒素（CT）和大肠杆菌产生的热敏毒素（LT）；(2) 是PRR配体的物质，例如TLR激动剂；(3) 天然免疫调节剂，如细胞因子、补体片段和信使分子。 研究最深入和最有效的粘膜佐剂是CT、LT和白喉毒素。这些毒素具有佐剂效应，被认为是粘膜佐剂的黄金标准；然而，由于它们的有害毒性效应，它们并未用于临床应用。为了避免这些毒性效应，已经创造了较少毒性的突变体，并连接到其他部分或肽以破坏毒素的活性位点，例如，重组霍乱毒素亚单位B已在Dukoral®疫苗中使用。 另一种使用的佐剂是PRRs配体。这些佐剂基于PAMPs激活，并通过TLR刺激作用。一个有前景的粘膜佐剂是单磷酸脂质A（MPL），源自大肠杆菌的LPS，其中MPL通过与TLR4相互作用刺激免疫反应。单独使用MPL不会引起系统性抗体或T细胞反应。因此，MPL已在包括脂质体和乳液的复合配方中使用，以及与Quillaja saponaria树皮的活性部分（QS-21）结合，形成AS01，它被包括在最近的疟疾疫苗RTS,S（Mosquirix）和目前正在开发的其他疫苗中，以诱导抗原特异性CD4+ T细胞反应，或与氢氧化铝结合形成AS04，由Glaxo-SmithKline在Cervarix™（人类乳头瘤病毒疫苗）和Fendrix™（乙型肝炎疫苗）中使用。 含有未甲基化CpG的合成寡脱氧核苷酸（ODNs）是另一种TLR激动剂。其基序触发表达TLR9的细胞，引发以Th1和促炎细胞因子产生为特征的先天免疫反应。CpG ODN诱导强烈的Th1反应，支持其诱导细胞毒性T淋巴细胞（CTL）和干扰素（INF）-γ分泌的能力。它促进抗原的免疫调节效应，激活APCs，并加快免疫反应。CpG促进浆细胞样DCs上MHC、CD40和CD80的表达，并增强抗原处理和呈递。此外，存在于人类肠道粘膜的B淋巴细胞表达TLR9并响应CpG刺激，导致IgA水平增加。2017年FDA批准的乙型肝炎疫苗HEPLISAV-B是第一个使用CpG ODN作为佐剂的疫苗。 人类TLR5的天然激动剂鞭毛蛋白是细菌鞭毛的关键组成部分，也是另一种粘膜佐剂。鞭毛蛋白与TLR5结合，动员核因子（NF）-κB并通过MyD88依赖途径刺激肿瘤坏死因子（TNF）-α的产生。鞭毛蛋白也可以被NOD样受体（NLR）家族的另外两个受体NLRC4和NAIP5识别；然后，它激活炎症体途径。鞭毛蛋白是先天和适应性免疫系统的有效激活剂。已经证明，促进最大抗原特异性抗体反应所需的鞭毛蛋白剂量低于刺激最大先天免疫反应所需的剂量，因为鞭毛蛋白能够诱导包括DCs、自然杀伤细胞、上皮细胞和淋巴结基质细胞在内的许多先天和非免疫细胞产生促炎细胞因子和趋化因子。 细胞因子和趋化因子已被证明在粘膜部位作为佐剂发挥作用。包括干扰素（INF）-γ、INF-α、IL-12、IL-2、IL-15、IL-18、IL-21、粒细胞-巨噬细胞集落刺激因子和Flt-3配体在内的重组细胞因子类似物已被报道具有佐剂活性。IL-1家族细胞因子能够诱导IgA和IgG抗体，而IL-6、IL-12和IL-15诱导CTL反应和疫苗特异性IgA抗体。然而，剂量相关的毒性和稳定性问题等限制阻碍了它们在疫苗中的应用，尽管通过将其包裹在脂质体或微/纳米颗粒中已用来克服这些问题。 4.2.传递系统 传递佐剂包括微球、纳米或微粒、聚合物、乳液、脂质体和免疫刺激复合物（ISCOM），它们被用来包裹抗原并将其传递给特定细胞。传递系统旨在改善抗原在粘膜表面的运输，同时保护它不受降解和/或在局部粘膜环境中的清除，以改善疫苗分布。 脂质体是具有两亲性质的球形脂质双层结构。由于一些优势，它们已被用作疫苗抗原的传递载体，例如防止抗原降解、组织储存效应或抗原的缓慢释放以及促进APCs对抗原的摄取，除了它们的生物相容性、生物可降解性、低毒性和两亲性质。此外，可以在脂质体表面添加：配体，用于特定细胞靶向，粘膜粘附剂和甘露糖衍生物，除了纳入TLR配体或其他PRR配体和免疫球蛋白以有效传递包裹或结合的抗原。阳离子脂质体比阴离子和中性脂质体更具免疫原性；此外，带正电荷的脂质体更好地与带负电荷的抗原结合，并影响它们在给药部位的保留，从而产生储存效应。二甲基二十八烷基铵（DDA）是一种广泛使用的阳离子脂质体，它主要由于抗原负载脂质体和单独给药的抗原之间的大小特征，在注射部位促进储存形成。阳离子佐剂配方01（CAF01）由DDA和海藻糖二油酸酯组成，这是一种合成变体，位于结核分枝杆菌细胞壁的绳索因子。这种配方已在动物模型中与不同病原体一起使用，如结核分枝杆菌、沙眼衣原体、链球菌等，以及基于重组蛋白亚单位（CTH522）的沙眼疫苗的1期临床试验，该疫苗用CAF01佐剂。CAF01被观察到是一种良好的粘膜佐剂；小鼠研究表明，它诱导Th1/Th17谱并增加IgA抗体反应。 粘膜粘附和粘液穿透成分的结合补偿了疫苗配方在粘膜液体中的稀释以及它们在粘膜更替中的损失，通过它们与粘膜上皮表面粘液层和粘液分子的相互作用，增加了剂量配方在吸收部位的抵抗时间。聚乙二醇是一种亲水成分，可以与粘液形成氢键，导致粘膜粘附，并帮助颗粒穿透粘膜，防止聚集，从而促进其穿过粘膜屏障。 另一种广泛研究的粘膜粘附剂是壳聚糖，它是一种低毒性的亲水、生物相容性和生物可降解的聚合物。壳聚糖具有一些对粘膜疫苗接种相关的属性，例如其带正电荷的分子与带负电荷的粘液相互作用以改善粘附；它增强了由于其能够短暂打开上皮紧密连接而增强的穿透性，从而改善了抗原取样及其促进INF-γ、IgG和SIgA分泌的佐剂属性。一项研究评估了一种基于壳聚糖功能化的铁氧化物纳米酶的鼻内流感疫苗，该研究发现壳聚糖增加了抗原对鼻粘膜的粘附，促进了DC的招募和跨上皮树突的形成以进行病毒摄取，并增加了小鼠的IgA-粘膜适应性免疫，提供了针对病毒的保护。 ISCOM是一种直径为30-40纳米的带负电荷颗粒，用作抗原的传递系统。ISCOM是在将抗原与胆固醇、磷脂（通常是磷脂酰乙醇胺和磷脂酰胆碱）以及从Quillaja saponaria Molina树树皮提取的皂苷Quil A混合后形成的。ISCOM的物理特性有助于抗原稳定性，减少与皂苷相关的溶血作用，并改善与DCs的相互作用，从而增强了所结合抗原的交叉呈递，产生抗体和CD4+和CD8+ T细胞反应。ISCOM上调Th1和Th2反应，并刺激强烈的体液反应（IgG1、IgG2a和IgG2b）以及细胞毒性T细胞诱导。已证明ISCOM佐剂是一种良好的粘膜佐剂，因为它诱导强烈的粘膜免疫反应，包括在鼻内接种ISCOM复合多种抗原后在鼻洗液和肺部产生大量IgA。使用与CT和金黄色葡萄球菌蛋白A（CTA1-DD）融合的ISCOM通过口服或鼻内给药诱导强烈的细胞介导和体液免疫反应，包括粘膜IgA。 5.母体-胎儿抗体转移 母体IgG抗体在怀孕期间通过胎盘被动转移到胎儿体内，这对出生后第一年系统免疫至关重要，因为此时免疫系统正在适应和成熟。推荐孕妇接种一些疫苗加强剂，例如针对流感、破伤风、百日咳和白喉的疫苗，以确保母亲和婴儿都受到保护。 然而，SIgA也通过母乳喂养传递给新生儿，并保护胃肠道——考虑到儿童经常遭受腹泻性感染，这是一个重要的问题。如本综述（给药途径部分/图2）所述，粘膜免疫可以增强乳腺中的SIgA，这将有利于通过母乳喂养传递SIgA。研究发现，从初乳样本中分离的SIgA在体外抑制了肠致病性大肠杆菌对Hep-2细胞的聚集，而从IgA中耗尽的初乳则无法做到这一点。因此，母亲的初乳和她婴儿的粪便在免疫印迹分析中识别了相同的大肠杆菌抗原，表明这些抗体在整个胃肠道中的持久性。母乳喂养的新生儿自出生后第一周起粪便中就含有SIgA，而配方奶喂养的新生儿直到第三周才含有。鉴于胃肠道感染对儿童的负担，应考虑在怀孕期间进行粘膜免疫。下一节将讨论微生物群对免疫反应调节的重要性。值得注意的是，母乳喂养可能通过传递微生物和作为益生元来塑造微生物群，这表明这些主题是多么复杂。 6.影响粘膜免疫反应的其他因素 6.1.维生素 维生素A（VA）是一种脂溶性维生素，包括视黄醇、视醛和视黄酯。VA通过食物途径从植物和水果中的前维生素A类胡萝卜素（如β-胡萝卜素）中获取。VA通过一系列酶，包括醇脱氢酶和视醛脱氢酶，代谢成其生物活性形式视黄酸（RA）。RA对激活的淋巴细胞上肠归巢分子α4β7和CCR9的诱导至关重要，并且对依赖于RA和TGF-β结合的FoxP3 Treg细胞的诱导至关重要。因此，RA被报道指导B细胞中免疫球蛋白类别转换，增加IgA的产生，这有助于宿主抵御肠道中的微生物。 维生素A缺乏症（VAD）与疾病易感性增加、疫苗反应差和死亡率增加有关。VAD导致大鼠和小鼠的肠粘膜以及雏鸡的气管上皮中IgA的产生减少。此外，VAD损害疫苗接种后的免疫反应：研究表明，在VAD小鼠中FluMist疫苗接种损害了免疫反应，而接种疫苗时口服RA或视黄棕榈酸酯增加了VAD小鼠中IgA的产生。在接种肺炎球菌疫苗（Prevnar-13）时，通过腹膜内途径给VAD小鼠口服VA补充剂（VAS），结果显示肺炎球菌（T4）特异性抗体（IgM和IgG1）得到改善。另一项研究表明，VAS和重组沙眼衣原体抗原的非肠道疫苗在小鼠中增加肠道IgA。因此，所有这些研究表明VAD损害了对疫苗的粘膜免疫反应，但VAS可以逆转这种效应，即使在非肠道免疫中也能增加IgA的产生。 补充全反式视黄酸（ATRA），VA的活性代谢物，导致恒河猴、黑猩猩和人类血液样本中DCs的α4β7整合素表达增加，这对肠道归巢运输很重要。这种治疗还改善了人类B细胞在体外培养中向浆细胞的分化和成熟。 研究表明，维生素A和D补充剂改善了儿童对流感疫苗的体液反应，这些儿童在基线时这些营养素的水平不足。在儿童和成人的混合队列中，评估了VA、抗流感抗体和炎症相关细胞因子。VA水平较高与IgA抗体和更高滴度的中和抗体相关。有趣的是，VA水平低的个体的鼻腔冲洗样本表达了更多的细胞因子和生长因子，使作者建议VA可能在控制粘膜组织中的炎症中发挥作用。 在婴儿中，一些研究表明VAS可以改善对某些疫苗（如破伤风、白喉类毒素、麻疹和乙型肝炎）的抗体反应。 尽管实验模型的证据非常充分，但关于VAS和疫苗反应的人类研究仍然很少，这使其成为一个有前景的研究领域。 6.2.微生物群 微生物群在许多身体功能中发挥关键作用，如食物消化、合成必需维生素和抵御病原体入侵。微生物群落稳态的改变导致肠道微生物失衡和肠道屏障异常。 已经表明微生物群影响疫苗的免疫反应，微生物的改变影响对注射和口服疫苗的免疫反应。无菌（GF）小鼠缺乏微生物群，显示Peyer氏斑的数量和大小减少，更少的固有层CD4+ T细胞，除了降低类别转换抗体IgA和IgG的水平；它们还缺乏发达的GALT。此外，小鼠早期生活中的抗生素驱动的失衡导致对全球通常给婴儿接种的五种不同疫苗的抗体反应受损——B群脑膜炎球菌疫苗（Bexsero）、C群脑膜炎球菌疫苗（NeisVac-C）、13价肺炎球菌结合疫苗（Prevenar）、六联疫苗（对抗乙型肝炎、白喉、破伤风、百日咳、B型流感嗜血杆菌和脊髓灰质炎病毒，INFANRIX Hexa），以及结核BCG疫苗。另一项研究还表明，抗生素治疗导致微生物组失衡，并影响健康成人对H1N1疫苗的免疫反应。 在中国婴儿中，梭菌科的富集与口服脊髓灰质炎疫苗最后一剂后抗脊髓灰质炎粘膜IgA的阴性检测有关，而放线菌的丰富与抗脊髓灰质炎粘膜IgA呈正相关，多样的肠道微生物群与口服脊髓灰质炎疫苗的粘膜免疫反应呈负相关。 加纳的一项研究报告称，口服轮状病毒疫苗（ORVV）的应答者在接种疫苗前6周大的婴儿中，Bacilli类成员（特别是Streptococcus bovis）的丰度较高，而非ORVV应答者在Bacteroidetes门中，特别是Bacteroides和Provotella种的丰度较高。 另一项研究分析了微生物群对成人接种口服伤寒沙门氏菌疫苗前1周的免疫反应的影响。研究发现，具有更多样化、复杂细菌群落的参与者具有多相细胞介导的免疫反应，这与对疫苗的更好反应相关；然而，该研究没有显示体液应答者和非应答者之间的群落多样性差异。 益生菌对先天和适应性免疫反应有影响。它们通过与肠道免疫细胞和上皮细胞的相互作用或间接通过肠道微生物组调节来介导免疫效应。一项研究表明，每天服用动物双歧杆菌（BB-12®）和副干酪乳杆菌（L. casei 431®）的志愿者，连续6周，并在接种三价灭活流感疫苗后，流感特异性IgG、IgG1、IgG3水平增加，特异性IgA水平更高。 另一项研究中，志愿者连续5周服用L. acidophilus和L. rhamnosus，结果显示接种疫苗后，脊髓灰质炎病毒中和抗体水平和脊髓灰质炎病毒特异性IgA和IgM水平增加。此外，一项比较7种不同益生菌菌株（B. lactis、L. acidophilus、L. plantarum和L. paracasei）连续3周的研究表明，在口服霍乱疫苗接种后，霍乱特异性IgG水平提高，接种B. lactis和L. acidophilus的个体诱导了高滴度的霍乱特异性IgA和IgM。 6.3.在发展中国家口服疫苗的问题 当分析现实世界证据时，口服疫苗在高收入国家的效能高于中低收入国家。值得注意的是，这些国家胃肠道感染的负担更为突出。 虽然在美国和芬兰等国家，活体口服轮状病毒疫苗的效能超过70%，但在非洲国家的效能为61%——值得一提的是，个体评估在南非为76.9%，而在马拉维为49.4%。在美国与印度相比，口服脊髓灰质炎疫苗后的血清转换率更高。在接种口服霍乱疫苗后，来自瑞典斯德哥尔摩的儿童比来自尼加拉瓜莱昂的儿童表现出更高的IgA和弧菌杀菌滴度。这样的方面引发了一个问题：是什么损害了这些疫苗的免疫原性？ 正如本综述前面所讨论的，VAD和微生物群的损害影响免疫反应。食物不安全导致微量营养素营养不良；缺乏清洁水源、适当的卫生设施和卫生教育可能会破坏微生物群的构成，增加肠道病原体的定植。这些问题在发展中国家更为常见，妨碍了口服疫苗的效能。另一个相关问题是新生儿肠道中存在高滴度的抗体，可能通过母乳喂养从不断接触肠道病原体的母亲那里转移。因此，应该研究疫苗接种时间表。有报告称，最近的脊髓灰质炎疫苗接种限制了轮状病毒疫苗的效果，可能是因为口服脊髓灰质炎疫苗可能与口服轮状病毒疫苗竞争，妨碍了强大的体液反应。 总之，一系列因素妨碍了贫困国家口服疫苗的效能。建议使用加强剂量作为克服这些障碍的方法。然而，造成这个问题的社会经济差异应该是讨论的话题，需要政府、科学家和私营部门的参与来解决——就像公共卫生的任何话题一样。 6.4.我们应该针对SARS-CoV-2开发粘膜疫苗吗？ SARS-CoV-2是一种呼吸道病原体——累积证据表明也涉及胃肠道。有效的粘膜免疫被认为是轻度和重度患者之间的差异性方面，提供对上呼吸道病原体的控制。到目前为止，证据已经指出，尽管肌肉内疫苗激发了有效的系统性免疫，但它们未能激活粘膜免疫，这将解释为什么SARS-CoV-2的传播仍然是一个问题。鼻微生物组也被认为是COVID-19临床表现的决定性方面。如前所述，粘膜疫苗易于给药并诱导局部免疫，这两个方面在考虑SARS-CoV-2的粘膜性质和在大流行情况下迫切需要大规模免疫的情况下都是有趣的。 确实，目前有多种粘膜COVID-19疫苗正在进行临床试验，主要是鼻内给药。然而，非肠道疫苗与粘膜疫苗的数量差异——分别是118种和16种，更不用说目前有20多种非肠道疫苗在使用中——留下了一个未解的问题：粘膜免疫是一种被低估的技术，还是前面讨论的障碍阻碍了这种有前途的工具的开发，以控制COVID-19？ 7.结论 粘膜疫苗可以在粘膜表面保护宿主免受病原体的初次接触，并且由于其给药方便和患者接受度高，其应用成为流行期间的一个良好选择。然而，尽管有所有这些优势，但并没有很多粘膜疫苗被批准用于人类使用，因为许多疫苗配方需要根据粘膜给药途径保护抗原免受降解。因此，在疫苗配方中应用佐剂是克服问题的一个好策略。然而，选择一个好的佐剂并不足以改善粘膜疫苗，因为微生物群和维生素可以影响粘膜免疫反应。 一些疾病对微生物群落和维生素吸收有显著影响，如肥胖、糖尿病、炎症性肠病等，这可能妨碍粘膜疫苗的效能。此外，在疫苗接种时间表中应用VA补充剂和益生菌似乎是一个调节粘膜免疫和增加某些个体对疫苗免疫反应的好策略。 因此，开发理想的粘膜疫苗不能仅仅归结为选择完美的佐剂；有必要考虑患者特定的因素，并且，这样分析可能影响疫苗粘膜免疫反应的所有方面。 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。

First-Half Sales Performance in Line with Full-Year 2024 Guidance Total revenues of €70.8 million, including product sales of €68.3 million, in line with anticipated supply and sales phasingNet Profit of €34.0 million, including proceeds from PRV sale Operating profit of €46.7 million compared to an operating loss of €35.0 million in the first half of 2023 Cash position of €131.4 million Substantially lower cash burn expected in the second half of 2024 as Valneva completed its cost contributions to the agreed R&D budget for its partnered Lyme disease program in the second quarterSignificantly extended cash runway with update of debt financing agreement1 Full-year 2024 Financial Guidance Confirmed Expected total revenues between €170 million and €190 million, including €160 million to €180 million of product sales Expected R&D investments between €60 million and €75 millionExpected Other income between €100 million and €110 million, including €95 million from the PRV sale Strategic Pipeline Expansion, Strong Clinical and Regulatory Execution Exclusive worldwide license for S4V Shigella vaccine candidate, adding an attractive Phase 2 clinical asset to Valneva’s R&D pipeline without impacting full-year or mid-term financial guidance2Additional marketing authorizations for single-shot IXCHIQ® granted in Europe and Canada ahead of initial guidance; Ongoing regulatory reviews in the UK and Brazil; Recommended by ACIP and adopted by U.S. CDC3New CEPI grant4 of $41.3 million contributes significantly to Phase 4 costs and other studies supporting broader access to the world’s first chikungunya vaccineReported positive six-month data for Phase 3 adolescent study of IXCHIQ®5; expects to submit label extensions for 12 to 17 years old in the U.S., Europe and Canada in the second half of 2024 IXCHIQ® two-year antibody persistence data published in the Lancet Infectious DiseasesIXCHIQ® pediatric Phase 2 study fully enrolledCompleted primary vaccinations (three doses) of VALOR Lyme disease Phase 3 trial participants6Initiated Phase 1 clinical trial for second-generation Zika vaccine candidate7 Financial Information(Unaudited results, consolidated per IFRS) € in million 6 months ended June 30, 2024 2023 Total revenues 70.8 73.7 Product sales 68.3 69.7 Net profit / loss 34.0 (35.0) Adjusted EBITDA8 56.2 (28.3) Cash 131.4 204.4 Saint-Herblain (France), August 13, 2024 – Valneva SE (Nasdaq: VALN; Euronext Paris: VLA), a specialty vaccine company, today reported its consolidated financial results for the first half of the year, ended June 30, 2024. The half year financial report, including the condensed consolidated interim financial report and the half year management report, is available on the Company’s website (Financial Reports – Valneva). Valneva will provide a live webcast beginning at 3 p.m. CEST / 9 a.m. EDT today. This webcast will also be available on the Company’s website. Please refer to this link: https://edge.media-server.com/mmc/p/mmuf83o5 Peter Bühler, Valneva’s Chief Financial Officer, commented, “Our first half sales performance is in line with our expectations. We aim to further capitalize on the travel industry recovery as we focus on ramping up sales for IXCHIQ® to support our commercial growth, while continuing to execute on our key R&D and regulatory milestones. The successful sale of our PRV and deferral of our loan reimbursement allow us to maintain a solid cash position and, with completion of our payments for the Lyme disease program in the second quarter, we anticipate a significantly lower cash burn in 2024.”Commercial Portfolio Valneva’s commercial portfolio is composed of three travel vaccines, IXIARO®/JESPECT®, DUKORAL® and recently launched IXCHIQ®. The Company also distributes certain third-party products in countries where it operates its own marketing and sales infrastructure. Valneva’s sales in the first half of 2024 were €68.3 million compared to €69.7 million (€64.0 million excluding final COVID-19 vaccine sales) in the first half of 2023. While product sales grew meaningfully in the second quarter and included first sales for IXCHIQ®, first half 2024 sales were affected by previously reported supply constraints for IXIARO® and third-party products. JAPANESE ENCEPHALITIS VACCINE IXIARO®/JESPECT® In the first half of 2024, IXIARO®/JESPECT® sales increased by 38% to €41.9 million compared to €30.3 million in the first half of 2023. The increase primarily reflects sales to the U.S. military, which were minimal in the first half of 2023, as well as increased sales to travelers. CHOLERA / ETEC9-DIARRHEA VACCINE DUKORAL® In the first half of 2024, DUKORAL® sales were €14.9 million compared to €17.1 million in the first half of 2023. The decrease was due to reduced marketing investments while Valneva’s new manufacturing site in Sweden underwent regulatory evaluation and approval. CHIKUNGUNYA VACCINE IXCHIQ® IXCHIQ® is the world’s first and only licensed chikungunya vaccine available to address this significant unmet medical need. The vaccine is now approved in the U.S. 10, Europe11, and Canada12 for the prevention of disease caused by the chikungunya virus in individuals 18 years of age and older. The U.S. launch is underway while first sales in Canada and Europe are anticipated in the fourth quarter of 2024. Following adoption of the U.S. Advisory Committee on Immunization Practices (ACIP)’s recommendations by the U.S. Centers for Disease Control and Prevention (CDC)13 at the beginning of March 2024, Valneva recognized initial sales of €1.0 million in the first half of 2024. Key launch metrics, including stocking and re-stocking across all sales channels, active customer accounts, as well as reimbursement for IXCHIQ® by commercial and MediCare insurance plans continue to track in line with expectations. In addition to ramping up sales, Valneva is focusing on expanding the vaccine’s label and access. The Company expects marketing authorizations in the UK and Brazil in the second half of 2024 and recently expanded its partnership with CEPI14 to support broader access to the vaccine in Low Middle-Income Countries (LMICs), post-marketing trials and potential label extensions in children, adolescents and pregnant women. CEPI will provide Valneva up to $41.3 million of additional funding over the next five years, with support from the European Union’s (EU) Horizon Europe program. Based on positive pivotal six-month Phase 3 data reported in May 202415, Valneva expects to file for potential label extensions for use in adolescents aged 12 to 17 years in the second half of 2024. These data show that a single-dose vaccination with IXCHIQ® induces a high and sustained immune response in 99.1% of adolescents, and that the vaccine was generally well tolerated. Conducted in Brazil in collaboration with Instituto Butantan, the trial is also expected to support licensure of the vaccine in Brazil, which would be the first potential approval for use in endemic populations. Additionally, Valneva recently completed enrollment of a Phase 2 pediatric trial, VLA1553-221, in children aged 1 to 11 years16, designed to support a Phase 3 pivotal pediatric study and potential future label extension to this age group. The peer-reviewed medical journal, The Lancet Infectious Diseases, also just published the vaccine’s Phase 3 antibody persistence results two years after vaccination with a single dose. The data show that IXCHIQ®’s robust immune response was sustained for two years by 97% of participants and was equally durable in younger and older adults17. These data, which further support the anticipated long-term durability of the immune response, will also be used to potentially expand the vaccine’s current label. Valneva will continue to measure antibody persistence for a period of at least five years and expects to report three-year durability results later this year. THIRD-PARTY DISTRIBUTION Valneva distributes certain third-party vaccines in countries where it operates its own marketing and sales infrastructure. During the first half of 2024, third-party sales decreased by 37% to €10.5 million compared to €16.5 million in the first half of 2023 as a result of anticipated supply constraints. Valneva expects third-party sales to gradually wind down to less than 5% of overall product sales by 2026/2027, allowing the Company to improve gross margins. Clinical Vaccine Candidates LYME DISEASE VACCINE CANDIDATE – VLA15 Phase 3 primary vaccination completed Valneva and Pfizer are developing VLA15, a Phase 3 vaccine candidate targeting Borrelia, the bacterium that causes Lyme disease. VLA15 is the only Lyme disease program in late-stage clinical development today and has received Fast Track designation from the FDA. VLA15 is a multivalent recombinant protein vaccine that targets six serotypes of Borrelia representing the most common strains found in the United States and Europe. Valneva and Pfizer reported results for the Phase 2 clinical trials of VLA15 in both adult and pediatric populations, in which high levels of antibodies against all six serotypes were observed. Results of Phase 2 trials VLA15-201 and VLA15-202 were published in the peer-reviewed medical journal, the Lancet Infectious Diseases, in June 202418. Additional 18-month booster results from Study VLA15-202 were also published in the same journal in July 202419. In July 2024, Valneva and Pfizer announced completion of primary vaccinations (three doses) of over 9,000 participants in the Phase 3 trial “Vaccine Against Lyme for Outdoor Recreationists” (VALOR)20. Completion of the VALOR trial is still expected by the end of 2025, with the aim for Pfizer to submit a Biologic License Application (BLA) to the FDA and a Marketing Authorization Application (MAA) to the EMA in 2026, subject to positive data. Based on the agreement with Pfizer, Valneva’s agreed-upon cost contributions for the Lyme disease program were completed in the second quarter of 2024, contributing to a substantially lower expected cash burn in the second half of 2024. SHIGELLA VACCINE CANDIDATE – S4V Valneva recently entered into a strategic partnership and exclusive licensing agreement with LimmaTech Biologics AG for the development, manufacturing and commercialization of Shigella4V (S4V), a tetravalent bioconjugate vaccine candidate against shigellosis21. Shigellosis, caused by Shigella bacteria, is the second leading cause of fatal diarrheal disease worldwide. It is estimated that up to 165 million cases of disease and an estimated 600,000 deaths are attributed to Shigella each year22, particularly among children in Low- and Middle-Income Countries (LMICs). No approved Shigella vaccine is currently available and the development of Shigella vaccines has been identified as a priority by the World Health Organization (WHO)23. Shigellosis also affects international travelers from high-income countries and deployed military personnel in endemic regions. The global market for a vaccine against Shigella is estimated to exceed $500 million annually24. Under the terms of the agreement with Valneva, LimmaTech will receive an upfront payment of €10 million and be eligible to receive additional regulatory, development and sales-based milestone payments of up to €40 million as well as low double-digit royalties on sales. LimmaTech will conduct a Phase 2 Controlled Human Infection Model study (CHIM) in the U.S. and a Phase 2 pediatric study in LMICs. Both clinical trials are expected to begin in the second half of 2024. Valneva will assume all further development, including CMC (chemistry, manufacturing and controls) and regulatory activities, and be responsible for its commercialization worldwide if approved. The anticipated development path follows a staggered and risk-mitigated strategy, allowing an efficient capital allocation in line with Valneva’s communicated plan of having a new R&D program in Phase 3 by 2027. ZIKA VACCINE CANDIDATE – VLA1601Phase 1 ongoing with second-generation vaccine candidate VLA1601 is a second-generation adjuvanted inactivated vaccine candidate against the mosquito-borne disease caused by the Zika virus (ZIKV). In March 2024, Valneva initiated a Phase 1 clinical trial to investigate the safety and immunogenicity of VLA160125. The randomized, placebo-controlled, Phase 1 trial, VLA1601-102, is planned to enroll approximately 150 participants aged 18 to 49 years in the United States. Participants will receive a low, medium or high dose of VLA1601. In addition, the low dose of VLA1601 will be evaluated with an additional adjuvant. Topline data from the trial are expected in the first half of 2025. Zika disease outbreaks have been reported in tropical Africa, Southeast Asia, the Pacific Islands, and, since 2015, in the Americas. Zika virus transmission persists in several countries in the Americas and in other endemic regions. To date, a total of 89 countries and territories have reported evidence of mosquito transmitted Zika virus infection26; however, surveillance remains limited globally. There are no preventive vaccines or effective treatments available and, as such, Zika remains a public health threat and is included in the FDA’s Tropical Disease Priority Review Voucher Program27. A vaccine against ZIKV could be a valuable addition to Valneva’s portfolio against mosquito-borne diseases, which already includes IXCHIQ® and IXIARO®. First Half 2024 Financial Review (Unaudited, consolidated under IFRS) RevenuesValneva’s total revenues were €70.8 million in the six months ended June 30, 2024 compared to €73.7 million in the six months ended June 30, 2023. Valneva’s total product sales reached €68.3 million in the six months ended June 30, 2024 compared to €69.7 million in the same period of 2023. The impact of currency fluctuations of €0.1 million was minimal.Excluding final COVID-19 vaccine sales in the six months ended June 30, 2023, travel vaccine sales show a growth of €4.3 million or 7% year-over-year.IXIARO®/JESPECT® sales were €41.9 million in the six months ended June 30, 2024 compared to €30.3 million in the six months ended June 30, 2023. The 38% increase primarily reflects sales to the U.S. military, which were minimal in the first half of 2023, as well as increased sales to travelers. The impact of foreign currency movements in IXIARO®/JESPECT® sales was negligible. DUKORAL® sales were €14.9 million in the six months ended June 30, 2024 compared to €17.1 million in the comparative period of 2023. This 13% decrease was due to reduced marketing investments while Valneva’s new manufacturing site in Sweden underwent regulatory evaluation and approval. Foreign currency fluctuations had an immaterial impact on DUKORAL® sales.Following adoption of the U.S. Advisory Committee on Immunization Practices (ACIP)’s recommendations by the U.S. Centers for Disease Control and Prevention (CDC) at the beginning of March 2024, Valneva recognized initial sales for IXCHIQ® of €1.0 million in the first half of 2024.Third Party product sales were €10.5 million in the six months ended June 30, 2024 compared to €16.5 million in the six months ended June 30, 2023. This 37% decrease was mainly driven by lower sales of Rabipur®/RabAvert® and Encepur®, under the distribution agreement with Bavarian Nordic, due to supply shortages. Other revenues, including revenues from collaborations, licensing and services amounted to €2.5 million in the six months ended June 30, 2024 compared to €4.1 million in the same period of 2023. The reduction mainly resulted from lower revenue recognition related to the R&D collaboration activities for chikungunya with Instituto Butantan and the divestment of the CTM unit in Sweden in July 2023. Operating Result and adjusted EBITDA Costs of goods and services sold (COGS) were €45.6 million in the six months ended June 30, 2024. The gross margin on commercial product sales, excluding IXCHIQ®, amounted to 47.7% compared to 40.0% in the six months ended June 30, 2023. COGS of €17.8 million related to IXIARO® product sales, yielding a product gross margin of 57.5%. COGS of €9.7 million related to DUKORAL® product sales, yielding a product gross margin of 34.8%. Product gross margins are expected to improve in the second half of the year as the supply shortages during the first half have been resolved. Of the remaining COGS in 2024, €7.7 million related to the third-party products distribution business, €4.0 million to IXCHIQ®, €2.1 million to idle capacity costs and €4.6 million to cost of services. In 2023, overall COGS were €53.8 million, of which €48.4 million related to cost of goods and €5.5 million related to cost of services. Research and development expenses amounted to €29.7 million in the six months ended June 30, 2024, compared to €26.0 million in the six months ended June 30, 2023. This increase was mainly driven by higher costs related to the ongoing transfer of operations into the new Almeida manufacturing facility and higher R&D costs for IXCHIQ®. Marketing and distribution expenses in the first six months of 2024 amounted to €23.2 million compared to €20.0 million in the first six months of 2023. The increase is mainly related to €9.8 million of expenses associated with launch activities for IXCHIQ® (first half of 2023: €7.8 million). In the six months ended June 30, 2024, general and administrative expenses remained stable at €22.8 million after €22.9 million in the same period of 2023. The largest expense categories were employee-related expenses of €10.5 million and consulting and other services of €9.6 million.During the first half of 2024, a net gain of €90.8 million from the sale of the PRV was recorded. The gross proceeds of $103 million were reduced by transaction costs as well as contractual payment obligations related to the sale of the PRV.Other income, net of other expenses decreased to €6.4 million in the six months ended June 30, 2024 from €14.0 million in the six months ended June 30, 2023. In the first half of 2023, Valneva recorded income from grants and tax credits for research and development totaling €14.9 million, of which €8.7 million were awarded by Scottish Enterprise (SE) for non-COVID-19 vaccine development (IXCHIQ® and IXIARO®).Valneva recorded an operating income of €46.7 million in the six months ended June 30, 2024 compared to an operating loss of €35.0 million in the comparative period of 2023. The increase was mainly the result of the PRV sale.Adjusted EBITDA (as defined below) profit in the six months ended June 30, 2024 was €56.2 million, whereas in the six months ended June 30, 2023 an adjusted EBITDA loss of €28.3 million was recorded. Net Result In the six months ended June 30, 2024, Valneva generated a net profit of €34.0 million, mainly resulting from the sale of the PRV in February 2024. This compared to a net loss of €35.0 million in the first half of 2023.Finance expense and currency effects in the first half of 2024 resulted in a net finance expense of €12.8 million, compared to a net finance expense of €3.9 million in the first half of 2023. This increase was mainly due to €5.7 million higher interest expenses on loans resulting from the amendment of the Deerfield Management Company and OrbiMed (D&O) loan facility. Additionally foreign exchange losses of €1.7 million were recorded in the first half of 2024 compared to gains of €4.5 million observed in the first half of 2023, primarily related to the development of the USD and GBP exchange rates to the EUR. Cash Flow and Liquidity Net cash used in operating activities amounted to €66.3 million in the six months ended June 30, 2024 compared to €65.4 million of cash used in operating activities in the same period of 2023. Cash outflows in the first half of 2024 were largely derived from the operating loss for the period (net of gains from PRV sale) amounting to €56.9 million and from working capital in the amount of €31.2 million, which includes all payments to the Lyme disease clinical program as per the R&D budget agreed between Pfizer and Valneva. In 2023, changes in working capital were higher, mainly related to higher payments to Pfizer in conjunction with the Lyme disease program, reducing the refund liability. Cash inflows from investing activities amounted to €87.6 million in the six months ended June 30, 2024 compared to cash outflows of €6.6 million in the six months ended June 30, 2023. While both years include outflows from construction activities across production sites in Scotland and Sweden, the sale of the PRV positively impacted 2024 by €90.8 million.Net cash used in financing activities increased to €16.6 million in the six months ended June 30, 2024 from €9.5 million in the six months ended June 30, 2023. This increase was primarily due to €5.4 million higher interest payments resulting from the increase in the D&O loan facility. Cash and cash equivalents were €131.4 million as at June 30, 2024, compared to €126.1 million at December 31, 2023. Non-IFRS Financial Measures Management uses and presents IFRS results as well as the non-IFRS measure of Adjusted EBITDA to evaluate and communicate its performance. While non-IFRS measures should not be construed as alternatives to IFRS measures, management believes non-IFRS measures are useful to further understand Valneva’s current performance, performance trends, and financial condition. Adjusted EBITDA is a common supplemental measure of performance used by investors and financial analysts. Management believes this measure provide additional analytical tools. Adjusted EBITDA is defined as net profit / (loss) for the period before income tax, finance income/expense, foreign exchange (gain)/loss, amortization, depreciation, and impairment (excluding impairment loss of disposal). A reconciliation of Adjusted EBITDA to operating loss, which is the most directly comparable IFRS measure, is set forth below: € in million 6 months ended June 30 (unaudited results, consolidated per IFRS) 2024 2023 Net profit / (loss) 34.0 (35.0) Add: Income tax benefit (0.2) (3.8) Total Finance income (0.8) (0.5) Total Finance expense 12.0 8.9 Foreign exchange (gain)/ loss – net 1.7 (4.5) Amortization 2.5 3.2 Depreciation 7.0 5.4 Impairment, excluding impairment loss of disposal - (1.9) Adjusted EBITDA 56.2 (28.3) About Valneva We are a specialty vaccine company that develops, manufactures, and commercializes prophylactic vaccines for infectious diseases addressing unmet medical needs. We take a highly specialized and targeted approach, applying our deep expertise across multiple vaccine modalities, focused on providing either first-, best- or only-in-class vaccine solutions. We have a strong track record, having advanced multiple vaccines from early R&D to approvals, and currently market three proprietary travel vaccines, including the world’s first and only chikungunya vaccine, as well as certain third-party vaccines. Revenues from our growing commercial business help fuel the continued advancement of our vaccine pipeline. This includes the only Lyme disease vaccine candidate in advanced clinical development, which is partnered with Pfizer, the world’s most clinically advanced Shigella vaccine candidate, as well as vaccine candidates against the Zika virus and other global public health threats. More information is available at www.valneva.com. Valneva Investor and Media ContactsLaetitia Bachelot-FontaineVP, Global Communications and European Investor RelationsM +33 (0)6 4516 7099Laetitia.bachelotfontaine@valneva.com Joshua Drumm, Ph.D.VP, Global Investor Relations M +001 917 815 4520joshua.drumm@valneva.com Forward-Looking StatementsThis press release contains certain forward-looking statements relating to the business of Valneva, including with respect to guidance for certain financial results in fiscal year 2024 and mid-term outlook on financial results, cash position, and other business developments, including results of ongoing clinical trials, the timing and possible occurrence of further or initial regulatory approvals of its product candidates, the anticipated size of markets for approved products and sales of those products, receipt of funding from external sources, supply of products sold by Valneva, and relationships with current business partners. In addition, even if the actual results or development of Valneva are consistent with the forward-looking statements contained in this press release, those results or developments of Valneva may not be indicative of future results. In some cases, you can identify forward-looking statements by words such as "could," "should," "may," "expects," "anticipates," "believes," "intends," "estimates," "aims," "targets," or similar words. These forward-looking statements are based on the current expectations of Valneva as of the date of this press release and are subject to a number of known and unknown risks and uncertainties and other factors that may cause actual results, performance or achievements to be materially different from any future results, performance or achievement expressed or implied by these forward-looking statements. These risks and uncertainties include those developed or identified in any public documents filed with the French financial markets authority (Autorité des marchés financiers) and the U.S. Securities and Exchange Commission made or to be made by Valneva. In particular, the expectations of Valneva could be affected by, among other things, uncertainties involved in the development and manufacture of vaccines (including in relation to organic or strategic expansion of Valneva’s clinical pipeline), unexpected clinical trial results, unexpected regulatory actions or delays, competition in general, currency fluctuations, the impact of the global and European financial crisis and other global economic or political events, the ability to obtain or maintain patent or other proprietary intellectual property protection, the cancellation of existing contracts, the impact of a pandemic, and changes in the regulatory environment in which Valneva operates. The occurrence of any of these risks and uncertainties could substantially harm Valneva’s business, financial condition, prospects and results of operations. In light of these risks and uncertainties, there can be no assurance that the forward-looking statements made during this presentation will in fact be realized. Valneva is providing the information in this press release as of the date hereof and disclaims any intention or obligation to publicly update or revise any forward-looking statements, whether as a result of new information, future events, or otherwise. 1 Valneva Announces Extension of the Interest-Only Period of Its Debt Facility with Deerfield and OrbiMed - Valneva2 Valneva and LimmaTech Enter into a Strategic Partnership to Accelerate the Development of the World’s Most Clinically Advanced Tetravalent Shigella Vaccine Candidate - Valneva3 ACIP Vaccine Recommendations and Schedules | CDC4 CEPI Expands Partnership with Valneva with a $41.3 Million Grant to Support Broader Access to the World’s First Chikungunya Vaccine - Valneva5 Valneva Reports Further Positive Pivotal Phase 3 Data in Adolescents for its Single-Shot Chikungunya Vaccine - Valneva6 Phase 3 VALOR Lyme Disease Trial: Valneva and Pfizer Announce Primary Vaccination Series Completion - Valneva7 Valneva Initiates Phase 1 Trial of Second-Generation Zika Vaccine Candidate - Valneva8 For additional information on Adjusted EBITDA, please refer to the “Non-IFRS Financial Measures” section at the end of the PR9 Indications differ by country - Please refer to Product / Prescribing Information (PI) / Medication Guide approved in your respective countries for complete information, incl. dosing, safety and age groups in which this vaccine is licensed, ETEC = Enterotoxigenic Escherichia coli (E. Coli) bacterium.10 Valneva Announces U.S. FDA Approval of World’s First Chikungunya Vaccine, IXCHIQ® - Valneva11 Valneva Receives Marketing Authorization in Europe for the World’s First Chikungunya Vaccine, IXCHIQ® - Valneva12 Valneva Announces Health Canada Approval of the World’s First Chikungunya Vaccine, IXCHIQ® - Valneva13 ACIP Vaccine Recommendations and Schedules | CDC14 CEPI Expands Partnership with Valneva with a $41.3 Million Grant to Support Broader Access to the World’s First Chikungunya Vaccine - Valneva15 Valneva Reports Further Positive Pivotal Phase 3 Data in Adolescents for its Single-Shot Chikungunya Vaccine - Valneva16 Valneva Vaccinates First Participant in Pediatric Trial of Single-Shot Chikungunya Vaccine - Valneva17 https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(24)00357-8/fulltext 18 Valneva Announces Publication of Lyme Disease Phase 2 Trials in the Lancet Infectious Diseases - Valneva19 Immunogenicity and safety of an 18-month booster dose of the VLA15 Lyme borreliosis vaccine candidate after primary immunisation in healthy adults in the USA: results of the booster phase of a randomised, controlled, phase 2 trial - The Lancet Infectious Diseases20 Phase 3 VALOR Lyme Disease Trial: Valneva and Pfizer Announce Primary Vaccination Series Completion - Valneva21 LimmaTech Biologics AG22 Shigellosis | CDC Yellow Book 202423 Immunization, Vaccines and Biologicals (who.int)24 Valneva’s Initial internal assessment 25 Valneva Initiates Phase 1 Trial of Second-Generation Zika Vaccine Candidate - Valneva26 Zika virus disease (who.int)27 Tropical Disease Priority Review Voucher Program | FDA Full-year 2024 Financial Guidance Confirmed Attachment 2024_08_13_VLA_H1_Results_EN_Final