Discussion 讨论
The emergence of antibiotic resistance, with the associated worldwide morbidity and mortality, has renewed interest in phage therapy. Despite Felix d’Hérelle’s first publication on the clinical use of phage therapy in 19213,7,8,9, over 100 years later phage therapy is only available in the U.S. by investigational new drug (IND) application approval, which is typically a single patient IND (SPIND) reviewed by the Food and Drug Administration (FDA) under the FDA’s expanded access IND (eaIND) program. In other countries, access to phage therapy may be regulated, for example, by a temporary use authorization (in France by the French National Agency for Medicines and Health Products Safety) or by special access schemes (Therapeutic Goods Administration) in Australia17.
抗生素耐药性的出现以及与之相关的全球发病率和死亡率，重新激发了人们对噬菌体疗法的兴趣。尽管费利克斯-德-赫雷勒（Felix d’Hérelle）于 1921 年首次发表了关于噬菌体疗法临床应用的文章 3,7,8,9 ，但 100 多年后的今天，噬菌体疗法在美国也只能通过研究性新药的形式获得。但 100 多年后的今天，噬菌体疗法在美国只能通过研究性新药（IND）申请获得批准，通常是由食品药品管理局（FDA）根据 FDA 的扩大准入 IND（eaIND）计划审查的单病种 IND（SPIND）。在其他国家，噬菌体疗法的使用可能会受到监管，例如，法国的临时使用授权（由法国国家药品和保健品安全局负责）或澳大利亚的特殊使用计划（治疗用品管理局） 17 。
This study uses a phage targeting PSA to provide an example of the modular approach used at Yale’s Center for Phage Biology & Therapy to evaluate and prepare a phage for phage therapy for a SPIND “compassionate case”. In addition to ensuring that phage preparations meet safety and efficacy requirements, a biological understanding of phage-driven bacterial evolution is important to effectively complete such requirements. Previously published protocols or pipelines for phage therapy preparation have detailed individual steps that focused on options for phage amplification70, purification37, design of phage cocktails71,72, quality and safety controls for personalized phage products73, testing of phages within a phage bank74, and cGMP production guidelines75. Building upon this experience, this study focuses on phage isolation, characterization, PCB and PVS production, sterility, potential trade-offs of phage-resistant mutants, and phage amplification to have a product that is ready for phage therapy.
本研究使用一种针对 PSA 的噬菌体，举例说明耶鲁大学噬菌体生物学与治疗中心采用模块化方法评估和制备用于 SPIND “同情病例 “噬菌体疗法的噬菌体。除了确保噬菌体制剂符合安全性和有效性要求外，对噬菌体驱动的细菌进化的生物学理解对于有效完成这些要求也非常重要。纯化 {{1}噬菌体鸡尾酒的设计 71,72 个性化噬菌体产品的质量和安全控制 73 噬菌体库内的噬菌体检测 74 以及 cGMP 生产指南 75 。在此经验的基础上，本研究重点关注噬菌体的分离、表征、PCB 和 PVS 生产、无菌性、噬菌体抗性突变体的潜在权衡以及噬菌体扩增，以获得可用于噬菌体疗法的产品。
A prior publication reported that phage production requires 18.5 to 20.5 days from phage isolation to final phage solution without three times plaque picking, phage characterization, analysis of a phage-resistant mutant, or sterility testing37. In Belgium, phage preparation, which may be the most standardized for phage therapy in the world, includes special accreditation for pharmacies that reportedly takes ≥ 2 weeks for full characterization that results in phage designated as an Active Pharmaceutical Ingredient24,76,77. A pipeline set up in Germany during the peak of the COVID-19 pandemic required 2 weeks for phage isolation to lead to phage production in an innovative cell-free phage production platform78. However, these studies37,78 did not require sterility testing. In the U.S. “gold standard” sterility testing is USP < 71 > , which typically requires 14 days79, while future new methods may become even more rapid40,73,80. In addition, these protocols did not include testing for phage induced “trade-offs” or “trade-ups”22. It is possible to create a more time-efficient platform by completing genomic analyses, phage characterization, and testing for “trade-offs” or “trade-ups” concurrently with sterility testing. In our experience, this overlap allows completion of the pipeline in less than 3 weeks, and in some emergent cases more rapidly. Ideally, this efficiency continues to improve, especially for emergent phage therapy cases. In our experience, the U.S. FDA has been incredibly helpful by providing very timely responses to SPIND applications.
之前的一份出版物报告称，噬菌体生产从噬菌体分离到最终噬菌体溶液需要 18.5 到 20.5 天，其中不包括三次斑块挑选、噬菌体特征描述、噬菌体抗性突变体分析或无菌测试 37 。在比利时，噬菌体制备可能是世界上最规范的噬菌体疗法，包括对药房的特别认证，据报道，药房需要≥ 2 周的时间进行全面特征描述，从而将噬菌体指定为活性药物成分 24,76,77 。在 COVID-19 大流行的高峰期，德国建立了一条噬菌体生产线，从分离噬菌体到在创新的无细胞噬菌体生产平台上生产噬菌体需要 2 周时间 78 。然而，这些研究 37,78 并不要求进行无菌测试。在美国，无菌测试的 “黄金标准 “是 USP < 71 >，通常需要 14 天 79 ，而未来的新方法可能需要更长的时间 40,73,80 。未来的新方法可能会更加快速 40,73,80 。此外，这些方案不包括噬菌体诱导的 “取舍 “或 “交换 “检测 22 。在进行无菌检测的同时完成基因组分析、噬菌体鉴定和 “权衡 “或 “取舍 “检测，可以创建一个更省时的平台。根据我们的经验，这种重叠可在 3 周内完成整个流程，在某些紧急情况下甚至更快。理想情况下，这种效率会继续提高，特别是对于紧急噬菌体疗法病例。根据我们的经验，美国 FDA 对 SPIND 申请的回复非常及时，提供了极大的帮助。
One important consideration for phage therapy that affects production is the choice of an empiric vs. targeted approach. An empiric, pre-made approach may allow for faster production but is limited by potential lack of efficacy against the target bacteria if phage sensitivity screening is not included in the pipeline24,37,71,74,78. To improve the production time of the targeted approach, a strategy has been proposed to develop large collections of fully characterized and ready-to-use phages for phage therapy (aka phage banks)17,81,82. Yerushalmy et al.81 previously compiled an overview of worldwide phage banks including some banks possibly suitable for human use in the future after characterization and purification of their phages [e.g., American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ)]. The Israeli phage bank, with 1/5 of the phages characterized by genome sequencing, is well equipped for phage therapy81. Given the growing interest in phage therapy, the number of phage banks will likely increase. The phage bank approach requires monitoring for phage titer(s) and sterility, which will require continual testing and updating71. Our experience83, and others74, suggests that such characterization, monitoring, and re-amplification provides phages that target relevant clinical pathogens. For some bacteria where phages have high host specificity, such a library may require many phages to maintain clinical relevance. An example is Klebsiella pneumoniae, which has been predicted to require > 500 phages78,84,85,86. Continued efforts are required to maintain a diverse phage library for clinically relevant bacteria, which will be facilitated by collaborations within the phage community.
噬菌体疗法影响生产的一个重要考虑因素是选择经验性方法还是靶向性方法。经验性的预制方法可能会加快生产速度，但如果噬菌体敏感性筛选不包括在生产流水线中，则可能会因缺乏对目标细菌的疗效而受到限制 24,37,71,74,78 。为了缩短靶向方法的生产时间，有人提出了一种策略，即为噬菌体疗法开发大量特征完全、随时可用的噬菌体（又称噬菌体库） 17,81,82 。Yerushalmy 等人 81 此前汇编了全球噬菌体库的概况，其中包括一些在表征和纯化噬菌体后可能适合人类使用的噬菌体库[如美国模式培养物保藏中心（ATCC）或德国微生物和细胞培养物保藏中心（DSMZ）]。以色列噬菌体库有 1/5 的噬菌体通过基因组测序鉴定，完全具备噬菌体疗法的条件 81 。鉴于人们对噬菌体疗法的兴趣与日俱增，噬菌体库的数量很可能会增加。噬菌体库方法需要对噬菌体滴度和无菌性进行监测，这就需要不断进行测试和更新 71 。我们的经验 83 我们和其他人的经验 74 我们的经验{{5}和其他人的经验{{6}表明，这种特征描述、监测和再扩增可以提供针对相关临床病原体的噬菌体。对于某些噬菌体具有高度宿主特异性的细菌，这样的噬菌体库可能需要许多噬菌体才能保持临床相关性。肺炎克雷伯氏菌就是一个例子，据预测，它需要 500 个以上的噬菌体 78,84,85,86 。我们需要继续努力，为临床相关细菌维持一个多样化的噬菌体文库，而噬菌体社区内部的合作将有助于实现这一目标。
The pipeline reported here includes additional phage characterization such as investigating the phage receptor. This can assist to increase sophistication of phage cocktail(s)71,87, but to date has not been addressed in prior phage therapy pipelines37,70,73,74. In our study, we used spot-based screening of a knock-out library, phage resistance to ΔpilQ in liquid culture, and mutations found in PA4.6C-R that suggested ΦSB adheres to Type-IV pili (TIVP), which are common receptors for PSA phages62,63. However, we did not confirm this finding by complementation of TIVP88. While the consequences of all non-synonymous mutations between phage-resistant mutant strain and phage-sensitive parent strain are not entirely understood, they are sufficient to both cause phage resistance and—regarding pilQ—a trade-off similar to a transposon knockout mutant. Therefore, while all possible effects of these mutations are beyond the scope of this pipeline manuscript, the outcome is consistent with disruption of TIVP function. Although not directly pertinent to emergency phage preparation, we are actively investigating the specific roles of each mutation as part of our ongoing laboratory projects.
本文报告的流程包括噬菌体的其他特征描述，如研究噬菌体受体。这有助于提高噬菌体鸡尾酒 71,87 的复杂性，但迄今为止，噬菌体疗法管道 37,70,73,74 尚未涉及这一点。但迄今为止，以前的噬菌体疗法管道尚未涉及这一问题 37,70,73,74 。在我们的研究中，我们利用基因敲除文库的定点筛选、液体培养中噬菌体对ΔpilQ的抗性，以及在PA4.6C-R中发现的突变，表明ΦSB粘附在IV型纤毛（TIVP）上，而TIVP是PSA噬菌体的常见受体 62,63 。然而，我们没有通过对 TIVP 的互补来证实这一发现 88 。虽然噬菌体抗性突变株与噬菌体敏感亲本株之间的所有非同义突变的后果尚不完全清楚，但它们足以导致噬菌体抗性，而且与转座子敲除突变株类似，在 pilQ 方面也有权衡。因此，虽然这些突变可能产生的所有影响都超出了本报告的范围，但其结果与 TIVP 功能的破坏是一致的。虽然与应急噬菌体制备并不直接相关，但作为我们正在进行的实验室项目的一部分，我们正在积极研究每个突变的具体作用。
Further phage characterization in this pipeline includes clinically important information such as phage growth kinetics at different titers, pH and thermal stability, and stability in saline. As noted by Gelman et al.74, we agree that phage characterization can be reduced to meet time limits of patients: electron microscopy, host range testing, adsorption assay, and the one-step-growth curves are not essential information for phage therapy in individual patients. In addition, the clearance capability of the phage could be roughly estimated by growth kinetics instead of short latent periods or large burst sizes71. In this study, ΦSB suppressed PSA growth at a low concentration of 1e3 PFU/mL in liquid co-incubation. Specific experiments tailored to the possible clinical application included biofilm inhibition and disruption assays in this study. Gelman et al.74 recommended characterizing biofilm inhibition by crystal violet staining, confocal microscopy, and electron microscopy. We propose to also use an assay indicating cell viability by CFUs. To efficiently target multiple PSA bacteria in patients with a single phage, we recommend conducting a comprehensive EOP analysis across a broad spectrum of host bacteria.
该管道中噬菌体的进一步特征描述包括临床上重要的信息，如不同滴度下噬菌体的生长动力学、pH 值和热稳定性以及在生理盐水中的稳定性。正如 Gelman 等人 74 所指出的，我们同意噬菌体特征描述的时间可以缩短，以满足临床研究的需要。我们同意，噬菌体的特征描述可以减少，以满足患者的时间限制：电子显微镜、宿主范围测试、吸附试验和一步生长曲线并不是对个别患者进行噬菌体治疗的必要信息。此外，噬菌体的清除能力可以通过生长动力学来粗略估算，而不是通过短潜伏期或大爆发量来估算 71 。在这项研究中，ΦSB在液体共培养中以1e3 PFU/mL的低浓度抑制了PSA的生长。针对临床应用的具体实验包括生物膜抑制和破坏试验。Gelman 等人 74 建议通过水晶紫染色、共聚焦显微镜和电子显微镜鉴定生物膜抑制作用。我们还建议使用一种以细胞存活率（CFUs）表示细胞存活率的检测方法。为了用一种噬菌体有效靶向患者体内的多种 PSA 细菌，我们建议对广泛的宿主细菌进行全面的 EOP 分析。
Another consideration for orthopedic treatments is more in-depth biofilm assays that use human bone material or metal implants, by varying MOIs, and by pre-forming biofilm for various timepoints before adding phages. In preparation for i.v. treatments, phage titer can be determined after 30- and 60-min incubation with the patient serum as described in the Belgian Standardized Multidisciplinary Treatment Protocol by Onsea et al.77. For certain bacterial infections, particularly polymicrobial infections, phage interactions in a community context may provide useful information for treatment approaches, although these are complicated experiments to design and to execute89.
骨科治疗的另一个考虑因素是利用人体骨骼材料或金属植入物进行更深入的生物膜检测，方法是在加入噬菌体之前改变 MOI，并在不同时间点预先形成生物膜。在准备静脉注射治疗时，噬菌体滴度可在与患者血清孵育 30 分钟和 60 分钟后测定，具体方法见 Onsea 等人的比利时标准化多学科治疗方案 77 。对于某些细菌感染，尤其是多微生物感染，噬菌体在群落环境中的相互作用可能会为治疗方法提供有用的信息，尽管这些实验的设计和执行都很复杂 89 。
We argue that the phage therapy pipelines should include, at least briefly, an analysis of phage-resistant bacteria that results from evolutionary selection pressure by phages. Similar to antibiotics, phages exert an evolutionary selection pressure on bacteria3,22,33,35,71. The resulting trade-offs or trade-ups of phage-driven escape mutant bacteria have to be considered prior to personalized use to prevent treatment with a phage that might foster the generation of more virulent bacteria3,22,35. Our phage therapy approach takes advantage of PSA evolved resistance by choosing phages that target bacterial cell surface receptors that contribute to antibiotic resistance or virulence3,34,35,83. In addition to killing PSA, this strategy directs evolutionary selection toward trade-offs that compromise bacterial virulence in surviving bacterial mutants35. However, the potential for increased virulence, or trade-up, exists. Therefore, we studied phage-resistant mutant PA4.6C-R and found trade-offs that include changes in growth rate, pyocyanin production, motility, and biofilm formation, which suggest attenuated virulence in surviving bacteria.
我们认为，噬菌体疗法流水线至少应简要分析噬菌体进化选择压力导致的噬菌体抗性细菌。与抗生素类似，噬菌体也会对细菌 3,22,33,35,71 施加进化选择压力。在个性化使用噬菌体之前，必须考虑噬菌体驱动的逃逸突变细菌所导致的权衡或取舍，以防止噬菌体治疗可能会促进产生毒性更强的细菌 3,22,35 。我们的噬菌体疗法利用了PSA进化出的抗药性，选择了针对细菌细胞表面受体的噬菌体，这些受体有助于提高抗生素抗药性或毒力 3,34,35,83 。除了杀灭 PSA 外，这种策略还引导进化选择进行权衡，以削弱存活细菌突变体的细菌毒力 35 。然而，也存在提高毒力或权衡的可能性。因此，我们研究了抗噬菌体的突变体 PA4.6C-R，发现其中的权衡包括生长速度、脓青素产量、运动性和生物膜形成的变化，这表明存活细菌的毒力有所减弱。
Twitching motility depends on orchestrated pilus motility, which allows PSA to migrate into poorly accessible body spaces62,90,91,92. PA4.6C-R showed decreased twitching motility that is consistent with four point mutations identified in its genome in the gene coding for pilQ, which is a component of TIVP62,90. The gene of pilD only exhibited one point mutation, which may not affect its phenotype. The eight point mutations in the gene for fucose-specific lectin, lecB, might impair adherence and biofilm stability65,66. Other point mutations in PA4.6C-R were identified by the breseq pipeline in domains of unknown function and prophage genes. Additionally, three single point mutations were found in three genes of the two-partner secretion (Tps) systems of TpsA and TpsB, one point mutation in a gene coding for the basic-amino-acid-specific porin OprD93, and three single point mutations in three different genes for DNA modification. In the case of genomic analyses pointing towards increased virulence of phage-resistant mutants compared to the ancestral patient strain, we recommend reassessing the phenotypic implications and reconsidering the use of the phage in question for phage therapy. Additional assays studying the effect of PA4.6C-R to increase cytokine and chemokine production on relevant cells may help to ensure that no trade-up is present. In our experiments, this included measuring IL-8, which is an important chemokine for neutrophil recruitment.
抽动运动依赖于协调的柔毛运动，这使得 PSA 能够迁移到难以进入的身体空间 62,90,91,92 .PA4.6C-R的抽动运动能力下降，这与在其基因组中发现的四点突变是一致的，突变基因编码pilQ，而pilQ是TIVP的一个组成部分 62,90 。pilD 的基因只有一个点突变，这可能不会影响其表型。岩藻糖特异性凝集素 lecB 基因的 8 个点突变可能会损害粘附性和生物膜稳定性 65,66 。breseq 管道在功能未知域和噬菌体基因中发现了 PA4.6C-R 中的其他点突变。此外，在 TpsA 和 TpsB 的双伙伴分泌（Tps）系统的三个基因中发现了三个单点突变，在编码碱性氨基酸特异性孔蛋白 OprD 的一个基因中发现了一个点突变 93 。以及三个不同的 DNA 修饰基因中的三个单点突变。如果基因组分析表明，噬菌体抗性突变体的毒力比患者祖先菌株更强，我们建议重新评估表型的影响，并重新考虑是否使用相关噬菌体进行噬菌体治疗。对 PA4.6C-R 在相关细胞中增加细胞因子和趋化因子分泌的效果进行额外的检测，可能有助于确保不存在交换效应。在我们的实验中，这包括测量 IL-8，它是中性粒细胞募集的重要趋化因子。
Such a pipeline may also affect the approach to phage cocktails, whose use has been re-evaluated in the past few years94,95,96. We have learned that bacterial load reduction is not necessarily enhanced by a higher number of phages in the cocktail96, and that a combination of phages in a cocktail might be neutral, beneficial or detrimental to bacterial eradication94 with the biggest concern that phage cocktails might result in broadly phage-resistant bacteria 74. Thus, we recommend designing these phage cocktails according to different receptors and to take into account synergies and antagonisms by a systematic phage design approach71,95,96,97,98,99,100.
这种流水线还可能影响噬菌体鸡尾酒的使用方法，在过去几年中，噬菌体鸡尾酒的使用已被重新评估 94,95,96 。我们了解到，鸡尾酒中噬菌体的数量越多，减少细菌负荷的效果不一定越好 96 ，鸡尾酒中噬菌体的组合可能是中性的、有益的或有害的 94 。鸡尾酒中的噬菌体组合对消灭细菌可能是中性的、有益的或有害的94，最大的担忧是噬菌体鸡尾酒可能会导致细菌对噬菌体产生广泛的抗药性 74 。因此，我们建议根据不同的受体设计这些噬菌体鸡尾酒，并通过系统的噬菌体设计方法考虑协同作用和拮抗作用 71,95,96,97,98,99,100 。
The pipeline presented here has several limitations. First, for PSA we have extensive knowledge about phage-bacteria interaction that may not be present for other bacteria considered for phage therapy. While we added assays for trade-offs and trade-ups, our pipeline did not include antibiotic synergy testing101. We have not routinely performed this test for PSA due to various multidrug- and pandrug-resistant bacteria in our phage therapy cases. The evaluation of these approaches, which also depends on the location of the infection being treated, needs to be refined in the future. Innovative methods of safe and high-titer phage production such as the cell-free phage platform were not applied in this study but are detailed in a previous phage preparation pipeline78,102. Pharmaceutical formulations, e.g., ointments, encapsulations for oral administration, or hydrogels are not covered here.
本文介绍的方法有几个局限性。首先，对于 PSA，我们掌握了大量噬菌体与细菌相互作用的知识，而对于噬菌体疗法所考虑的其他细菌，这些知识可能并不存在。虽然我们增加了权衡和取舍的检测方法，但我们的流程并不包括抗生素协同作用测试 101 。由于我们的噬菌体疗法病例中存在多种耐多药和耐潘生丁药细菌，因此我们没有对 PSA 进行常规检测。对这些方法的评估也取决于所治疗感染的部位，今后需要进一步完善。本研究没有采用安全、高滴度噬菌体生产的创新方法，例如无细胞噬菌体平台，但在之前的噬菌体制备管道 78,102 中有详细介绍。药物制剂，如软膏、用于口服的封装或水凝胶在此未涉及。
Future studies of phage pipelines or protocols for personalized therapy could explore specific steps required for particular phage applications, such as inhaled therapy for cystic fibrosis patients, biofilms on aortic prostheses, foot ulcers with polymicrobial infections, or phage therapy targeting the gut microbiome. All of this will be informed by an increasing number of clinical trials on phage therapy.
未来对噬菌体管道或个性化疗法方案的研究可以探索特定噬菌体应用所需的具体步骤，如囊性纤维化患者的吸入疗法、主动脉假体上的生物膜、多微生物感染的足部溃疡或针对肠道微生物组的噬菌体疗法。越来越多的噬菌体疗法临床试验将为所有这些研究提供信息。
In summary, we present an efficient modular pipeline for individualized, timely, safe and high-quality phage solutions for SPINDs, including a thorough characterization of the phage and a representative phage-resistant mutant, allowing for rational decision for the most appropriate phage candidate(s) for each patient. A novel PSA phage is characterized, and studies are used to identify phage-induced trade-offs, while minimizing phage-induced trade-ups. Overall, phage characterization should be tailored to the specific use and time constraints of each patient.
总之，我们提出了一种高效的模块化方法，用于为 SPINDs 提供个性化、及时、安全和高质量的噬菌体解决方案，包括对噬菌体和具有代表性的抗噬菌体突变体进行全面鉴定，从而为每位患者合理选择最合适的候选噬菌体。对新型 PSA 噬菌体进行表征，并通过研究确定噬菌体引起的权衡，同时尽量减少噬菌体引起的权衡。总之，噬菌体的特征描述应适合每位患者的具体用途和时间限制。