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噬菌体疗法可以战胜耐药性疾病

Phage Therapy Could Beat Drug-Resistant Illnesses
噬菌体疗法可以战胜耐药性疾病
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2019-11-07 22:56
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火星译客

Bobby Burgholzer has cystic fibrosis, a genetic disease that throughout his life has made him vulnerable to bacterial infections in his lungs. Until a few years ago antibiotics held his symptoms mostly at bay, but then the drugs stopped working as well, leaving the 40-year-old medical device salesman easily winded and discouraged. He had always tried to keep fit and played hockey, but he was finding it harder by the day to climb hills or stairs. As his condition worsened, Burgholzer worried about having a disease with no cure. He had a wife and young daughter he wanted to live for. So he started looking into alternative treatments, and one captured his attention: a virus called a bacteriophage.

鲍比·伯格尔泽患有囊性纤维症,这是一种遗传性疾病,这种疾病使他一生都很容易受到肺部细菌感染。直到几年前,因为注射抗生素才使他的症状基本得到控制,但后来这些药物也失效了,这让这位40岁的医疗设备销售员会经常性地喘不过气,心情也持续低落。他一直努力保持身体健康,打曲棍球,但他发现自己在爬小山或楼梯时愈加吃力。随着病情的恶化,伯格尔泽担心自己的病无法治愈。他想为他的妻子和小女儿而活。于是他开始寻找替代疗法,其中一种叫做噬菌体的病毒引起了他的注意。

Phages, as they are known, are everywhere in nature. They replicate by invading bacteria and hijacking their reproductive machinery. Once inside a doomed cell, they multiply into the hundreds and then burst out, typically killing the cell in the process. Phages replicate only in bacteria. Microbiologists discovered phages in the 1910s, and physicians first used them therapeutically after World War I to treat patients with typhoid, dysentery, cholera and other bacterial illnesses. Later, during the 1939–1940 Winter War between the Soviet Union and Finland, use of the viruses reportedly reduced mortality from gangrene to a third among injured soldiers.

众所周知,噬菌体在自然界中无处不在。它们通过入侵细菌并劫持生殖细胞进行DNA复制。一旦侵入某个细胞,这通常注定该细胞无法存活,之后噬菌体就会繁殖到数百个最终全面爆发,通常在这个过程中会杀死细胞。噬菌体只在细菌中进行复制。微生物学家在20世纪10年代发现了噬菌体,第一次世界大战后,医生首次将其用于治疗伤寒、痢疾、霍乱和其他细菌性疾病。后来,在1939-1940年苏芬战争(冬季战争)期间,据报道,使用这种病毒使伤兵的坏疽死亡率降低了三分之一。

Treatments are still commercially available in former Eastern Bloc countries, but the approach fell out of favor in the West decades ago. In 1934 two Yale University physicians—Monroe Eaton and Stanhope Bayne-Jones—published an influential and dismissive review article claiming the clinical evidence that phages could cure bacterial infections was contradictory and inconclusive. They also accused companies that manufactured medicinal phages of deceiving the public. But the real end of phage therapy came in the 1940s as doctors widely adopted antibiotics, which were highly effective and inexpensive.

在前东欧国家,这种治疗性的毒剂仍可在市场上买到,但几十年前这种方法在西方国家就不受欢迎了。1934年,耶鲁大学的两位物理学家门罗·伊顿和斯坦霍普·拜恩-琼斯共同发表了一篇颇具影响力且带轻蔑态度的评论性文章,声称证明噬菌体可以治愈细菌感染的临床成果不成立,因为该依据与现实存在很大的矛盾性,而且至今未成定论。他们还指控制造公司利用药用噬菌体欺骗公众。但噬菌体疗法直到20世纪40年代才真正终结,当时抗生素因其高效便宜而受到医生们的广泛认可。

Phage therapy is not approved for use in humans in any Western market today. Research funding is meager. And although human studies in Eastern Europe have generated some encouraging results—particularly those from the Eliava Institute in Tbilisi, Georgia, the field's research epicenter—many Western scholars say the work does not meet their rigorous standards. Furthermore, a smattering of clinical trials in Western Europe and the U.S. have produced some high-profile failures.

因研究经费不足,目前噬菌体疗法用于人体试验的建议在所有西方市场都未得到批准。尽管东欧国家在人类研究方面已经取得了一些令人鼓舞的成果,其中格鲁吉亚第比利斯的埃利亚瓦研究所的研究成果尤其突出。尽管该研究所是该领域的研究中心,但许多西方学者认为这项工作没有达到其严格的规定标准。此外,西欧和美国也进行了几次临床试验,但都以轰动一时的失败告终。

Yet despite the historical skepticism, phage therapy is making a comeback. Attendance at scientific conferences on the treatment is skyrocketing. Regulators at the U.S. Food and Drug Administration and other health agencies are signaling renewed interest. More than a dozen Western companies are investing in the field. And a new wave of U.S. clinical trials launched this year. Why the excitement? Phage treatments have been curing patients with multidrug-resistant (MDR) infections that no longer respond to antibiotics. The FDA has allowed petitioning doctors to administer these experimental treatments on a “compassionate use” basis when they could show that their patients had no other options—exactly what Burgholzer was hoping to prove.

然而,尽管过去的学者对噬菌体疗法的怀疑不减,但它正试图卷土重来。参加有关这种治疗方法的科学会议的人数直线上升。美国食品和药物管理局(fda)和其他卫生机构的监管机构也再度表示对此很感兴趣。十几家西方公司正在该领域寻找投资机会。今年美国开始了新一轮临床试验。为什么兴奋?噬菌体治疗已经治愈了耐多药(MDR)感染的患者,抗生素对这些患者已不再产生效用。食品和药物管理局已经允许提交申请的医生在“体恤使用”的基础上进行这些实验性的治疗,条件是他们能够证明其病人没有其他治疗选择,而这正是伯格尔泽一直以来力求证明的一点。

MDR infections are a rapidly growing public health nightmare. At least 700,000 people worldwide now die from these incurable maladies every year, and the United Nations predicts that number could rise to 10 million by 2050. In the meantime, the drug industry's antibiotic pipeline is running dry.

耐多药感染正迅速发展为公共卫生的噩梦。全世界每年至少有70万人死于这些无法治愈的疾病,联合国预测,到2050年,该数字可能会上升到1000万。与此同时,制药行业的抗生素供应渠道日渐枯竭。

Like all viruses, phages are not really alive—they cannot grow, move or make energy. Instead they drift along until by chance they stick to bacteria. Unlike antibiotics, which kill a range of helpful bacteria as they kill the strains making a person sick, a phage attacks a single bacterial species, and perhaps a few of its closest relatives, and spares the rest of the microbiome. Most phages have an icosahedral head—like a die with 20 triangular faces. It contains the phage's genes and connects to a long neck that ends in a tail of fibers, which bind to receptors on a bacterium's cell wall. The phage then plunges a kind of syringe through the wall and injects its own genetic material, which co-opts the bacterium into making more phage copies. Other types of phages, not used medically, enter the same way but live dormantly, reproducing only when the cell divides.

像所有的病毒一样,噬菌体并不是真实存在的活体——它们不能生长、移动或产生能量。相反,它们随波逐流,直到偶然地附着在细菌上。与抗生素不同的是,噬菌体只攻击一种细菌,或许还会攻击与之亲缘关系最近的几种细菌,而不攻击微生物群落的其他部分。大多数噬菌体的头部接近于二十面体,看上去像有20个三角形面。它包含噬菌体的基因,并与长颈相连,长颈末端有纤维,纤维与细菌细胞壁上的受体相结合。然后,噬菌体将通过注射器穿过细胞壁,注入自己的遗传物质,从而协同细菌制造更多的噬菌体副本。其他类型的噬菌体,不用于医学,但侵入细胞的方式是相同的,平常就处于休眠状态,只有在细胞分裂时才开始繁殖。

Phages have co-evolved with bacteria for billions of years and are so widespread that they kill up to 40 percent of all the bacteria in the world's oceans every day, influencing marine oxygen production and perhaps even Earth's climate. The spotlight on phages as medical tools is getting brighter as technological advances make it possible to match the viruses to their targets with better accuracy. The few facilities that are technically able to provide phage therapy, under strict regulatory protocols, are being overwhelmed with requests.

噬菌体与细菌相成相生,这一过程已经持续了数十亿年。噬菌体分布如此广泛,以至于每天会杀死世界海洋中40%的细菌,从而影响海洋氧气的产生,甚至可能影响全球气候。随着技术的进步,病毒能够更准确地与目标匹配,噬菌体作为医疗工具的前景越来越光明。在严格的监管协议下,在超负荷的需求下,技术上能够提供噬菌体治疗的设施却屈指可数。

Clinical trials underway are beginning to generate the high-quality data needed to convince regulators that phage therapy is viable, but considerable questions remain. The biggest is whether phage therapy can tackle infections on a large scale. Clinicians have to match phages to the specific pathogens in a patient's body; it is not clear whether they can do that cost-effectively and with the speed and efficiency needed to bring phages into routine use. Also problematic is a shortage of regulatory guidelines governing the production, testing and use of phage therapy. “But if it has the potential to save lives, then we as a society need to know whether it will work and how best to implement it,” says Jeremy J. Barr, a microbiologist at Monash University in Melbourne, Australia. “The antibiotic-resistance crisis is too dire to not embrace phage therapy now.”

因为目前进行的临床试验所产生的数据的质量越来越高,监管机构也更加相信噬菌体疗法的可行性。但这一领域还存在着相当多的问题。其中最严重的问题是噬菌体疗法能否大规模地解决感染问题。临床医生必须使噬菌体与病人体内的特定病原体相匹配;在确保成本效益的条件下,能够达到使噬菌体投入日常使用的速度和效率,关于他们是否能做到这一点目前还不清楚。同样存在问题的是,缺乏规范噬菌体治疗的生产、检测和使用的管理准则。澳大利亚墨尔本莫纳什大学的微生物学家杰里米·巴尔(Jeremy J. Barr)说:“但如果它有可能拯救生命,那么就社会层面来看,我们需要知道它是否有效,以及如何找到最优实施方法。”“抗生素耐药性危机太可怕了,现在不能不接受噬菌体疗法。”

Trading Vulnerabilities

交换漏洞

Burgholzer learned about phages by talking to other people with cystic fibrosis around the country. While scouring the Internet for more information, he came on a YouTube video made by phage researchers at Yale University. Soon he was corresponding with Benjamin Chan, a biologist in Yale's department of ecology and evolutionary biology. Since arriving there in 2013, Chan has accumulated a “library” of phages, harvested from sewage, soil and other natural sources, that he makes available to doctors at Yale New Haven Hospital and elsewhere.

伯格尔泽通过与全国其他囊性纤维化患者交谈了解了噬菌体。当他在互联网上搜寻更多信息时,他看到了一个由耶鲁大学噬菌体研究人员制作的YouTube视频。很快,他就和耶鲁大学生态与进化生物学系的生物学家本杰明·陈取得了联系。自2013年抵达那里以来,陈安道建立了一个贮藏了众多噬菌体的“图书馆”,在馆内收集了来自污水、土壤和其他自然资源的噬菌体,供耶鲁纽黑文医院(Yale New Haven Hospital)和其他地方的医生使用。

Chan's first case, in 2016, was a resounding success. He isolated a phage from pond water, and doctors used it to cure Ali Khodadoust, a prominent eye surgeon. Khodadoust had been suffering from a raging MDR infection in his chest, a complication from open-heart surgery four years earlier. He was taking massive daily doses of antibiotics to try to fight his invading pathogen, the tenacious bacterium Pseudomonas aeruginosa. The virus Chan selected latches on to what is known as an efflux pump on the bacterial cell wall. The pumps expel antibiotics and are frequently found in drug-resistant bacteria. Most of the P. aeruginosa in Khodadoust's body had the pumps, and the phage killed them. The relatively few remaining P. aeruginosa faced an evolutionary trade-off: their lack of efflux pumps meant they survived the virus attack, but it made them defenseless against antibiotics. By taking the phages and antibiotics together, Khodadoust gradually recovered in just a few weeks. He died two years later, at age 82, from noninfectious illnesses.

陈2016年的第一个案例获得了巨大的成功。他从池塘的水中分离出一种噬菌体,医生们使用它治愈了著名的眼科医生阿里·霍达杜斯特。霍达杜斯特的胸腔内存在严重的多药耐药感染,这是四年前开胸手术的并发症。他每天都服用大量的抗生素来对抗入侵体内的病原体,即顽固的铜绿假单胞菌。陈选择的病毒附着在细菌细胞壁上的一个所谓的流出泵上。这种泵能排出抗生素,而且经常在耐药细菌中发现。霍达杜斯特体内的大多数铜绿假单胞菌都有水泵,噬菌体杀死了水泵。剩下的相对较少的铜绿假单胞菌在繁殖过程中需要进行权衡:它们缺少外排泵,这意味着尽管它们在病毒攻击中幸存了下来,但却对抗生素毫无抵抗力。通过同时使用噬菌体和抗生素,霍达杜斯特在短短几周内逐渐康复。两年后,他死于非传染性疾病,享年82岁。

After that first case, Chan supplied phages for nearly a dozen more experimental treatments at Yale, most involving cystic fibrosis patients with P. aeruginosa lung infections. He asked Burgholzer to send a sputum sample by overnight delivery so he could identify phages that might help.

在该病例获得首次成功后,陈医生为耶鲁大学的十几项实验治疗提供了噬菌体,其中大多数是患有铜绿假单胞菌肺部感染的囊性纤维化患者。他让伯格尔泽隔夜用快递送去一份痰液样本,这样他就能鉴别出可能有帮助的噬菌体。

I visited Chan at Yale last December, after the screening had begun. He was wearing a checkered oxford shirt, khakis and loafers, and before long he was calling me “dude,” his preferred moniker. After chatting briefly in his office, we headed for an adjacent laboratory, where Chan showed me a petri dish. Burgholzer's bacteria had developed into a gray lawn spanning the dish, but two thin, clear rows cut across it. The bacteria that had been in those rows were all dead, Chan told me, killed by drips of a phage solution Burgholzer would soon be treated with. Burgholzer's infection was caused by three species of the bacterial genus Achromobacter, and Chan planned to select individual phages that could pick them off one by one—an approach known as sequential monophage therapy. “We're essentially playing chess in an antimicrobial war,” Chan said. “We need to make calculated moves.”

去年12月,筛选开始后,我去耶鲁大学看望了陈。他穿着格子牛津衬衫、卡其裤和乐福鞋,没过多久,他就以“哥们”称呼我,因为这是他喜欢的绰号。在他的办公室简短地聊了几句之后,我们去了隔壁的实验室,陈给我看了一个培养皿。伯格尔泽的体内细菌已布满了整个培养皿,分化成了一片“灰色草坪”,但是有两排细细的、清晰的细菌横排在培养皿之中。陈告诉我,那些一排排的细菌都死了,它们是被很快侵入伯格尔泽体内的噬菌体溶液一点点杀死的。伯格尔泽的感染病是由三种细菌属的无色细菌引起的,陈计划选择单个的噬菌体,一个一个地将它们消灭——这种方法被称为顺序单噬菌体疗法。“我们本质上是在进行一场抗菌博弈,”陈说。“我们需要精心策划才有希望战胜它们。”

Chan hoped to induce an evolutionary trade-off similar to the one he believes worked for Khodadoust. Unable to find a phage that targets efflux pumps on Achromobacter bacteria, he instead selected one that targets a large protein called lipopolysaccharide (LPS) in the microbe's cell wall. LPS has side chains of molecules known as O antigens, which vary in length. The longer the chain, the better the bacteria's ability to resist not only antibiotics but also the host's immune system. Chan planned to kill the hardy long-chain strains with phages, leaving the weaker short-chain pathogens behind. In the best scenario, he said, a succession of phages would shift the bacterial population toward short-chain strains that might be more easily controlled by drugs and Burgholzer's own immune defenses. “Bacteria compete for real estate in the body,” Chan said. “After large numbers of one species are suddenly killed by phage, in many cases, others move in.” He wanted the new occupants to be less virulent than their predecessors.

陈希望在细菌进化上做出取舍,他认为这种取舍与霍达杜斯特的做法类似。由于无法找到一种针对无色细菌的外移泵的噬菌体,他转而选择了一种针对微生物细胞壁中一种名为脂多糖(LPS)的大型蛋白质的噬菌体。LPS有称为O抗原的侧链分子,其长度各不相同。链越长,细菌对抗生素和宿主免疫系统的抵抗力就越强。陈计划用噬菌体杀死耐寒的长链菌株,留下较弱的短链病原体。他说,在最好的情况下,一系列噬菌体将使细菌种群转向短链菌株,这种菌株可能更容易被药物和伯格尔泽自身的免疫防御所控制。“细菌在体内争夺地盘,”陈说。“在一种生物突然被噬菌体大规模杀死后,在很多情况下,其他的物种会侵入体内。他希望新的寄居物种对人体的侵害性比之前的物种有所降低。

Chan's boss, Paul Turner, has devoted his career to studying evolutionary trade-offs in the microbial world. A professor in Chan's department, he explained later on the day of my visit that phage treatments can work without completely ridding the body of a disease-causing bacteria. Especially when treating chronic conditions, doctors can use phages to selectively shape the population of the bad bacteria so it develops other vulnerabilities. “Should those vulnerabilities be toward antibiotics, then so much the better,” he told me. Combining antibiotics with phages to achieve optimal effects for patients, he says, “makes it easier to move forward with phage therapy quickly.”

陈的老板保罗·特纳(Paul Turner)毕生致力于研究微生物世界的进化权衡。陈教授所在系的一位教授在我到访当天晚些时候解释说,噬菌体疗法可以在不完全清除体内致病细菌的情况下奏效。特别是在治疗慢性疾病时,医生可以使用噬菌体来选择性地控制有害细菌的数量,从而使其暴露出其他的弱点。“如果这些弱点是针对抗生素的,那就更好了,”他告诉我。他说,将抗生素与噬菌体相结合,可达到最佳效果,“同时大大推进噬菌体的医疗进程。”

I drove with Chan to Yale New Haven Hospital to watch as Burgholzer's phage treatment got underway. We took an elevator to the second floor, where we waited for Chan's clinical collaborator, Jonathan Koff, to arrive. A pulmonologist and director of the Adult Cystic Fibrosis Program, Koff soon came bounding in, a knapsack slung over his shoulder. Burgholzer met the three of us in a treatment room and spoke with a rasp—the only outward sign of his disease. As Koff and Chan compared notes, he told me he wanted to stay healthy for his three-year-old daughter. When treatment time arrived, he tossed his cell phone to his wife. “Here, take a photo for my mother,” he said with a grin. Then he raised a nebulizer over his mouth and nose and began inhaling a vaporized phage solution into his lungs.

我和陈一起开车去耶鲁纽黑文医院拜访伯格尔泽,观看其接受的噬菌体治疗过程。我们乘电梯到了二楼,陈的临床研究伙伴乔纳森·考夫就在那里等着。科夫是一名肺科医生,也是负责成人囊性纤维化项目的主任。伯格尔泽在一间治疗室里与我们三个人见面,说话的声音很刺耳——这是唯一可以表明他处于生病状态的外部特征。当考夫和陈交流意见时,他告诉我他想为他三岁的女儿女努力恢复健康。治疗时间到了,他把手机扔给了妻子。“来,给我妈妈照张相,”他笑着说。然后他拿起喷雾器盖住嘴巴和鼻子,开始往肺里吸入一种蒸发的噬菌体溶液。

Phage Cocktails

噬菌体鸡尾酒制剂

According to Koff, sequential monophage therapy makes sense for treating cystic fibrosis and certain other chronic diseases that sequester bad bacteria in the body. When there is no proven way to eliminate the pathogens completely, he says, the tactic is to chip away at the harmful strains.

考夫认为,序贯单噬菌体疗法对于治疗囊胞性纤维症和其他一些将有害细菌隔离在体内的慢性疾病是有意义的。他说,因为可以完全消灭病原体的治疗方案还未被证实,我们可以转而清除有害的菌株。

Some clinicians are choosing a different approach: They give patients multiple phages in a therapeutic cocktail, trying to knock out an infection completely by targeting a variety of bacterial resistance mechanisms simultaneously. Ideally, each phage in a cocktail will glom on to a different receptor, so if bacteria evolve resistance to one virus in the mixture, other viruses will keep up the attack.

一些临床医生正在选择一种不同的方法:他们给病人提供存在多种噬菌体的鸡尾酒治疗制剂,试图通过同时瞄准多种细菌抵抗机制来彻底根除感染问题。理想情况下,鸡尾酒中的每个噬菌体会附着在不同的受体上,所以如果细菌进化出对混合物中的一种病毒的抗药性,其他病毒就会继续攻击。

Chan and Koff argue that phage interactions with bacteria are unpredictable and that when exposed to cocktails, pathogens might develop resistance to all the viruses in the mixture at once, which could limit future treatment options. “Splitting the cocktail into sequential treatments allows you to treat patients for longer durations,” Koff says.

陈和考夫认为噬菌体与细菌之间的相互作用是不可预测的,当暴露在混合物中时,病原体可能会同时对混合物中的所有病毒产生耐药性,这可能会限制未来的治疗选择。考夫说:“把鸡尾酒分成几份治疗制剂持续提供给病人,会延长治疗时间。”

Jessica Sacher, co-founder of the Phage Directory, an independent platform for improving access to phages and phage expertise, says convincing arguments can be made for either method. “The science isn't there yet to say one is necessarily better than the other.” She notes that cocktails might be more appropriate for acutely ill patients, who cannot always wait for doctors to develop a sequential strategy.

杰西卡·萨赫是Phage Directory公司的联合创始人,这是一个旨在扩大噬菌体使用范围和提供用户有关噬菌体的专业知识的独立平台。“关于这两者的比较优势,科学还未形成定论。她指出,鸡尾酒疗法可能更适合重症患者,因为他们无法长期等着医生制定出一个循序渐进的治疗方案。

Urgency was paramount in the now famous case of Tom Patterson, a professor at the University of California, San Diego, who in 2016 was saved by phage cocktails after being stricken by an MDR infection during a trip to Egypt. The invader was Acinetobacter baumannii, a notoriously drug-resistant microbe that is common in Asia and is spreading steadily toward the West. Patterson was in multiorgan failure by the time doctors delivered mixtures of four viruses through a catheter into his abdomen and a fifth intravenously. The physicians treated him twice a day for four weeks, and he was cleared of infection within three months. He still needed extensive rehabilitation, but he remains healthy today.

加州大学圣迭戈分校(University of California, San Diego)教授汤姆·帕特森(Tom Patterson)在2016年的埃及之行中感染了多药耐药性,之后他在注射噬菌体鸡尾酒后治愈了。入侵细菌是鲍曼不动杆菌(Acinetobacter baumannii),这是一种出了名的耐药微生物,在亚洲很常见,而且正在向西方稳步蔓延。当医生通过一根导管将四种病毒的混合物注入帕特森的腹部,并通过静脉注射第五种病毒时,帕特森已经出现了多器官衰竭。医生给他治疗了四个星期,每天两次,三个月后就完全清除了感染细菌。他仍然需要进行长久的康复过程,但他今天仍然健康。

The case drew worldwide media attention. The treating physicians were Robert Schooley, a friend of Patterson's and chief of infectious diseases at U.C. San Diego, and Patterson's wife, Steffanie Strathdee, then director of the university's Global Health Institute. Two years later, with an initial investment of $1.2 million, Schooley and Strathdee launched the Center for Innovative Phage Applications and Therapeutics at U.C. San Diego to fund clinical research and promote the field.

这起案件引起了全球媒体的关注。负责治疗的医生有帕特森的朋友、加州大学圣迭戈分校传染病科主任罗伯特·斯库利(Robert Schooley),以及帕特森的妻子、时任该校全球卫生研究所(Global Health Institute)所长的斯特拉斯迪(Steffanie Strathdee)。两年后,斯库利和斯特拉斯迪以120万美元的初始投资,在加州大学圣地亚哥分校成立了创新噬菌体应用与治疗中心,为临床研究提供资金支持,并促进该领域的发展。

Each phage Patterson was treated with was screened for its ability to kill A. baumannii in infectious samples obtained from his body, using assays at the Naval Medical Research Center at Fort Detrick, Md., and at Texas A&M University. The assays can test hundreds of phages against bacterial pathogens simultaneously in just eight to 12 hours, according to Biswajit Biswas, chief of the bacteriophage division at the center, which supplied some of the phages used in Patterson's treatment. Biswas, who developed the assay and created the center's phage bank, says the assay allows new viruses to be easily swapped in to counter the onset of resistance. Patterson did develop resistance to his first cocktail within two weeks, prompting the navy to prepare a second one with longer-lasting effects. A company called Adaptive Phage Therapeutics in Gaithersburg, Md., has since licensed the navy's assay and its phage bank and will soon take them both into clinical trials in patients with urinary tract infections.

使用马里兰州德特里克堡海军医学研究中心(Naval Medical Research Center at Fort Detrick, Md.)和得克萨斯农工大学(Texas A&M University)的分析用试样对每一种用于治疗帕特森的噬菌体进行筛选,以确定其在从鲍曼尼氏杆菌身上获得的感染样本中的杀灭能力。该中心噬菌体部负责人比斯瓦吉特·比斯瓦斯(Biswajit Biswas)表示,这种检测方法可以在8到12个小时内同时检测数百种噬菌体,以对抗致病菌。比斯瓦斯开发了这种分析方法,并创建了该中心的噬菌体库。帕特森确实在两周内对他的第一杯鸡尾酒制剂产生了抗药性,这使得研究中心不得不准备了第二杯效果更持久的鸡尾酒。马里兰州盖瑟斯堡(Gaithersburg)一家名为Adaptive Phage Therapeutics的公司已经批准了改研究中心的分析方法及其噬菌体库,并将很快把它们用于尿路感染患者的临床试验。

The navy assay checks only for bacterial cell death; it does not reveal which receptors are targeted. Whether cocktails should target known receptors is in debate. Ry Young, a phage geneticist at Texas A&M, who supplied viruses for Patterson, argues they should. “We don't even know if phages were responsible for his successful outcome,” he says. “Our best guess is that phage treatment lowered his infectious load to a level where his immune system took over.” The better approach to cocktails, Young says, is to combine three or four viruses targeting distinct receptors on the same bacterial strain. The odds of a bacterium evolving resistance to a single phage are about a million to one, he says, whereas the odds of it losing or developing mutant forms of receptors targeted by all the phages in a cocktail “are essentially zero.” Furthermore, the identification of important receptors is critical if clinicians hope to make bacteria sensitive to antibiotics again.

该研究中心的分析用试样只用于核实细菌细胞的死亡;并无法显示透露目标受体。关于鸡尾酒是否应该针对已知受体还存在争议。为帕特森提供病毒的德州农工大学噬菌体遗传学家Ry Young也表示认同。他说:“我们甚至不知道噬菌体是否是导致他成功的原因。”“我们可预知的最佳情况是,噬菌体治疗降低了他的感染负荷,完全控制了其免疫系统。Young说,更好的鸡尾酒疗法是将三到四种针对同一菌株不同受体的病毒结合起来。他说,一个细菌进化出对单一噬菌体的耐药性的几率大约是百万分之一,而它失去或发展被鸡尾酒中的所有噬菌体靶向的突变受体的几率“基本上为零”。此外,如果临床医生希望使细菌再度对抗生素产生敏感性,识别重要的受体是至关重要的。

Barr says scientists are working to identify the receptors targeted by Patterson's cocktails, but he disagrees on the need to identify the receptors prior to use. “It's an understandable viewpoint and a hot topic in the field,” he says. “We know very little about these phages, and we need checks and balances before using them in therapy. Does that mean we need to identify host receptors? That is a huge amount of work currently, so I would say it's not required but definitely desirable.”

巴尔说,科学家们正在努力识别帕特森鸡尾酒所针对的受体,但他不同意在使用之前必须先识别受体。“这一观点是可以理解的,也是该领域的一个热门话题,”他说。“我们对这些噬菌体知之甚少,在将它们用于治疗之前,我们需要相互制衡。这是否意味着我们需要识别宿主受体?这是一个巨大的工作量,所以我认为这不是必需的,但绝对是可取的。”

Engineered Phages

工程噬菌体

Given the vagary of cocktails, some researchers say phages should be genetically engineered to bind to specific receptors and also to kill bacteria in novel ways. The vast majority of phages used thus far have been natural, harvested from the environment, but phage engineering is an emerging frontier with a new success story under its belt. Isabelle Carnell, a British teenager with cystic fibrosis, was suffering from life-threatening infections in her liver, limbs and torso after undergoing a double lung transplant in 2017. Her bacterial nemesis—Mycobacterium abscessus—was not responding to any antibiotics. Yet this year, in a first for the field, researchers from several institutions successfully treated the girl with an engineered cocktail of three phages. One naturally rips apart M. abscessus as it replicates. The other two also kill bacteria but not as completely, leaving 10 to 20 percent surviving the process. So the team, led by Graham Hatfull, a professor of biological sciences at the University of Pittsburgh, deleted a single gene from each of those two phages, turning them into engineered assassins. The cocktail of three phages cleared Carnell's infection within six months.

一些研究人员说,考虑到鸡尾酒的变幻莫测,噬菌体应该通过基因工程与特定的受体结合,并以新的方式杀死细菌。迄今为止使用的噬菌体绝大多数是自然的,从环境中获取的,但噬菌体工程是一个新兴的前沿领域,必将带来新的成功案例。患有囊性纤维症的英国少女伊莎贝尔·卡内尔(Isabelle Carnell)在2017年接受了双肺移植手术后,她的肝脏、四肢和躯干出现了危及生命的感染。她的细菌线虫——脓肿分枝杆菌——对任何抗生素都没有反应。然而,今年,来自几个机构的研究人员首次用三种噬菌体混合的鸡尾酒成功治愈了这个女孩。当M. essus复制时,它会被自然地撕开。另外两种方法也可以杀死细菌,但效果不那么彻底,剩下10%到20%的细菌可以存活下来。因此,由匹兹堡大学(University of Pittsburgh)生物科学教授格雷厄姆哈特费尔(Graham Hatfull)领导的研究小组,从这两种噬菌体中各删除了一个基因,把它们变成了工程刺客。三个噬菌体的混合物在六个月内清除了卡内尔的感染。

Researchers at Boston University first developed engineered phages in 2007. They coaxed one into producing an enzyme that more effectively degrades the sticky biofilms secreted by certain infectious bacteria for protection. Scientists have since modified phages to kill broader ranges of harmful bacteria or potentially to deliver drugs and vaccines to specific cells. These lab-designed viruses are also more patentable than natural phages, which makes them more desirable to drug companies. As if to underscore that point, a division of the pharmaceutical giant Johnson & Johnson struck a deal in January with Locus Biosciences, worth up to $818 million, to develop phages engineered with the gene-editing tool CRISPR.

2007年,波士顿大学的研究人员首次开发出了工程噬菌体。他们诱导一个细菌产生一种酶,这种酶能更有效地降解某些感染性细菌分泌的用于保护的粘性生物膜。自那以后,科学家们对噬菌体进行了改造,以杀死更广泛的有害细菌,或将药物和疫苗输送到特定的细胞。这些实验室设计的病毒也比天然噬菌体更容易获得专利,这使它们更受制药公司的欢迎。似乎是为了强调这一点,制药巨头强生(Johnson & Johnson)的一个部门在今年1月与基因位点生物科学公司(Locus Biosciences)达成了一项协议,开发利用基因编辑工具CRISPR设计的噬菌体,交易金额最高可达8.18亿美元。

Developing a phage therapy that is commercially viable will not be easy. Barr and other scientists point out that it takes a tremendous amount of time, money and effort to engineer a phage, and after all that the target bacteria might soon evolve resistance to it. Furthermore, regulatory approval for an engineered phage “could be a tough sell,” says Barr, echoing the view of several scientists interviewed for this story. But FDA spokesperson Megan McSeveney, in an e-mail, claimed the agency does not distinguish between natural and engineered phages as long as therapeutic preparations are deemed safe.

开发一种商业上可行的噬菌体疗法并不容易。巴尔和其他科学家指出,设计噬菌体需要大量的时间、金钱和努力,毕竟目标细菌可能很快就会进化出对噬菌体的抗性。此外,巴尔表示,监管机构批准转基因噬菌体“可能很难让人接受”,这与为本文采访的几位科学家的观点一致。但是FDA发言人Megan McSeveney在一封电子邮件中声称,只要治疗制剂被认为是安全的,FDA就不会区分天然噬菌体和工程噬菌体。

Future Prospects

未来前景

Companies are now testing different ways to bring phages to broader markets. Some companies want to supply patients with personalized therapies matched specifically to their infections. That is the strategy at Adaptive Phage Therapeutics. The company's chief executive officer, Greg Merril, says assays used to screen the navy's phages against infectious samples could be offered at diagnostic labs and major medical centers worldwide. Phages effective against locally prevalent bacteria in each region could be supplied in kiosks, bottled in FDA-approved, ready-to-use vials. Merril says doctors could continually monitor treated patients for resistance, swapping in new phages as needed until the infections are under control. He estimates that the per-patient cost under the current compassionate-use system is approximately $50,000, an expense that should fall with economies of scale.

各家公司现在都在尝试不同的方法为噬菌体创造更广阔的市场。一些公司希望为患者提供专门针对其感染的个性化治疗。这就是适应性噬菌体疗法的策略。该公司首席执行官格雷格·梅里尔(Greg Merril)表示,可以在世界各地的诊断实验室和主要医疗中心提供用于筛选海军噬菌体以对抗感染样本的化验。对每个地区流行的细菌有效的噬菌体可以在售货亭中提供,装在fda批准的随时可用的小瓶中。梅里尔说,医生可以继续监测病人的耐药性,根据需要更换新的噬菌体,直到感染得到控制。他估计,在当前的同情使用制度下,每个病人的成本约为5万美元,这一费用应该随着规模经济的下降而下降。

Other companies reject this personalized strategy in favor of fixed phage products more akin to commercial antibiotics. Armata Pharmaceuticals' lead product is a cocktail of three natural phages targeted at Staphylococcus aureus bacteria, the cause of common staph infections often contracted at hospitals. It is in clinical trials in patients who have infected mechanical heart pumps. Armata's plan is to monitor for treatment-resistant staph in the general population, then introduce new cocktails as needed, in much the same way that influenza vaccines are tuned every year to match the latest circulating strains. Pharmaceutical executives said it was too soon to estimate what the costs would be.

其他公司则反对这种个性化的策略,更倾向于固定噬菌体产品,更类似于商业抗生素。阿玛塔制药公司的主要产品是三种针对金黄色葡萄球菌的天然噬菌体的混合物,金黄色葡萄球菌是在医院感染常见葡萄球菌的原因。它正在对感染了机械式心脏泵的患者进行临床试验。Armata的计划是在普通人群中监测难治性葡萄球菌,然后根据需要引入新的鸡尾酒疗法,就像每年调整流感疫苗以适应最新的流行毒株一样。制药公司高管表示,现在估计成本还为时过早。

Experts still cannot say which of the current strategies—sequential monotherapy, cocktails, engineered phages, and general or personalized treatments—may ultimately win out, assuming any do. An optimal approach “might not even exist,” says Barr, considering that “phage treatments in each case could depend on complicating issues, such as the target pathogen, the disease and the patient's medical history.”

专家们仍然不能确定目前的策略——序贯单药疗法、鸡尾酒疗法、工程噬菌体疗法,以及一般或个性化治疗——是否最终会被采用。巴尔说,一种最佳的方法“可能甚至不存在”,因为“每种情况下的噬菌体治疗可能取决于复杂的问题,比如目标病原体、疾病和病人的病史。”

Phage therapy is still saddled by geopolitical biases, too, says Strathdee. What is really needed now, she says, are positive results from well-controlled clinical trials that can help overcome residual skepticism. Alan Davidson, a biochemist at the University of Toronto, speculates that within a decade phage therapy might be cheaper, easier and faster than it is today. He leans toward the engineering approach, saying sequencing the whole genome of a patient's bacteria and then synthesizing a phage to cure an infection could be quicker and less expensive “than screening the pathogens against a battery of viruses drawn from nature.”

斯特拉斯迪说,噬菌体疗法仍然受到地缘政治偏见的束缚。她说,现在真正需要的是控制良好的临床试验的积极结果,这有助于克服残留的怀疑。多伦多大学的生物化学家艾伦·戴维森推测,十年内噬菌体疗法可能会比现在更便宜、更容易、更快。他倾向于工程方法,他说,对病人的细菌进行全基因组测序,然后合成噬菌体来治愈感染,可能比“从大自然中提取一组病毒来筛选病原体”更快、更便宜。

Meanwhile Burgholzer, who was self-administering phage therapy with a nebulizer at home until March 2019, has not yet experienced the clinical improvements he was hoping for. In March, Chan and Koff introduced a second phage targeted at another Achromobacter strain. By April the bacterial counts in Burgholzer's lungs had fallen by more than two orders of magnitude since the initial treatment began. “So it does appear we can pick off those strains successively,” Koff told me. Yet Koff acknowledged that Burgholzer was not noticing a dramatic change in lung function. When I asked why, Koff responded, “We know a lot more about the phage we use against P. aeruginosa than we do about phages targeting Achromobacter.” The ability to manipulate the infection “is less informed.”

与此同时,直到2019年3月,伯格尔泽还在家中使用喷雾器进行噬菌体治疗,但他还没有体验到他所希望的临床改善。今年3月,陈冯富珍和考夫推出了针对另一株无色杆菌的第二种噬菌体。到4月,自最初的治疗开始以来,伯格尔泽肺部的细菌计数下降了两个多数量级。“这样看来,我们确实可以连续地清除这些菌株,”考夫告诉我。然而,考夫承认伯格尔泽并没有注意到肺功能的巨大变化。当我问为什么时,考夫回答说:“我们对用来对付铜绿假单胞菌的噬菌体了解得比针对无色杆菌的噬菌体多得多。”“控制感染的能力”信息较少。

The next step, Koff says, will be to genetically sequence mucus samples from Burgholzer's lungs. “We really need to understand what's happening with his bacteria so we can get to the high level of sophistication we have with P. aeruginosa. Bobby is letting us take a chance to see if, at a minimum, we can help.” Frustrated but still eager, Koff says, “Some patients respond better than others. We need to understand those dynamics.”

考夫说,下一步将对伯格尔泽肺部的粘液样本进行基因排序。“我们真的需要了解他的细菌发生了什么,这样我们才能达到对铜绿假单胞菌所要求的的高度复杂程度。鲍比让我们碰碰运气,至少看看我们能不能帮上忙。考夫说,有些病人的反应比其他人好。我们需要了解这些动态变化。”

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