Health Care Science | 异种移植中的猪巨细胞病毒：人类移植的新前沿？

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The first gene-edited pig heart transplantation taking place in March this year in Maryland, USA, was hailed as a step into a new world, a promise to finally serve those who are waiting for organs on the heart transplant waitlist. Much effort had been put into this endeavor in the past decades, first to deal with the issue of complement activation and hyperacute humoral rejection, second to deal with PERV by inactivating the main enzyme reverse transcriptase, thus cells still produce virus particles which are able to infect new human cells, but they cannot integrate in the genome of the new target cell and third to control unwanted growth of the organ after transplantation using e.g. mTOR inhibitors.

今年3月在美国马里兰州进行的首例基因编辑猪心脏移植手术被誉为迈向新世界的一步，承诺最终为那些在心脏移植等待名单上等待器官的人服务。在过去的几十年中，人们在这方面做了很多努力，首先是解决补体激活和超急性体液排斥的问题，其次是通过灭活主要的逆转录酶来处理PERV，因此细胞仍然产生能够感染新的人类细胞的病毒颗粒，但它们不能整合到新的靶细胞的基因组中，第三是使用例如mTOR抑制剂来控制移植后不需要的器官生长。

In addition, early on, the focus had been already directed on porcine cytomegalovirus (PCMV) as a potential contributor of harm to both patient and organ. It was known, that almost 100% of the pigs were infected, it was known, that human fibroblasts could be infected, and it was known that humans working close to pigs (butchers, labors in the meat industry) displayed antibodies against PCMV indicating either an infection without symptoms or harbor antibodies which are directed against HHV-6, and which are cross-reacting with PCMV. It was known that PCMV more resembles HHV-6/HHV-7 and not HHV-5 (human cytomegalovirus HCMV) and it was known that particularly the main drug against HCMV, Ganciclovir Is ineffective against PCMV at clinical doses for humans but pharmacological control of PCMV with Cidofovir or Foscarnet at the cost of significant toxicity might be achieved. And it was known that PCMV in pig to baboon xenotransplantation not only leads to graft loss, but to a general infection of baboon organs eliciting via upregulation of adhesion molecules a consumptive coagulopathy. In addition, it was known, that the diagnosis of PCMV in pigs is difficult, and so far, needs tissue specimens to rule out a dormant infection controlled by the immune system as we know it from Herpesvirus infected humans. Hence a PCMV-free donor pig or population was deemed to be a prerequisite to provide a safe organ for transplantation.

此外，早前人民已经把重点放在猪巨细胞病毒（PCMV）上，认为它是对患者和器官造成伤害的潜在因素。已知几乎100%的猪被感染，且人成纤维细胞也可以被感染，并且已知从事与猪密切接触工作的人（屠夫、肉类行业的工人）有抗PCMV的抗体，表明无症状感染或携带与PCMV交叉反应的针对HHV-6的抗体。已知PCMV更类似于HHV-6/HHV-7而不是HHV-5（人巨细胞病毒HCMV），且尤其是抗HCMV的主要药物Ganciclovir在人类临床剂量下对PCMV无效，但用Cidofovir或Foscarnet对PCMV进行药物控制有明显毒性。PCMV在猪到狒狒的异种移植中不仅导致移植物损失，而且导致狒狒器官的普遍感染，通过粘附分子的上调引起消耗性凝血病。此外，在猪中诊断PCMV困难，且目前为止，需要组织标本来排除由免疫系统控制的休眠感染，因为我们知道它来自疱疹病毒感染的人类。因此，无PCMV的供体猪或群体被认为是为移植提供安全器官的先决条件。

Griffith et al. report in the NEJM from June 22, 2022, that “Testing for mcfDNA was positive for PCMV at low levels on day 20, and the levels increased over subsequent weeks. The detection of PCMV was unexpected, given the husbandry practices, negative surveillance PCR testing of nasal swab specimens from the donor animal before organ transplantation, and the use of antiviral prophylaxis. It is uncertain whether the detection of PCMV through plasma mcfDNA or PCR testing represents replicating virus in the xenograft, replicating virus in the recipient, or shedding of genetic material from the xenograft. The presence of PCMV in explanted xenografts from nonhuman primate recipients has been correlated with worse outcomes than an absence of PCMV, for reasons that are unclear.” Next to the PCMV findings they reported: “Further viral testing is warranted because human herpesvirus 6 (HHV-6) was also detected in a lung-lavage specimen from this patient. HHV-6 has been shown to cross-react with PCMV and has been associated with allograft rejection. No obvious viral cytopathic changes were identified on preliminary hematoxylin–eosin examination of thoracic or abdominal organs.” The findings thus far were not conclusive with typical findings of rejections and further studies are underway to identify the pathophysiologic mechanisms observed.

格里菲斯等人在2022年6月22日的《NEJM》上的报道中指出：“在第20天检测到低水平阳性的mcfDNA，并且在随后的几周内水平上升。鉴于饲养方式、器官移植前对供体动物的鼻拭子标本进行阴性PCR检测，以及使用抗病毒预防措施，检测到PCMV是意料之外的”。尚不能确定通过血浆mcfDNA或PCR测试检测到的PCMV是否代表异体移植中的复制病毒、受体中的复制病毒或异体移植中的遗传物质脱落。在非人灵长类受体切除的异种移植体中存在PCMV与比不存在PCMV的更差的结果相关，原因尚不清楚。除了PCMV结果外，他们还报告：“需要进行进一步的病毒检测，因为在该患者的肺灌洗标本中也检测到了人类疱疹病毒6（HHV-6）。HHV-6可与PCMV发生交叉反应，并与同种异体移植排斥反应有关。胸部或腹部器官的初步苏木精-伊红染色检查未发现明显的病毒细胞病变”。迄今为止的研究结果与典型的排斥反应结果尚无定论，进一步的研究正在进行中，以确定所观察到现象的病理生理机制。

As a clinician, I would completely agree with these findings though the hottest lead in the moment seems to be PCMV. As we know, do zoonotic viruses infect humans such as HEV, for example, consuming undercooked meat. In general, harmless to humans, it might cause Hepatitis and there are only a few reports on severe courses of the disease, unless you are a recipient of an organ transplant under immunosuppression, under these circumstances, courses can become ugly and might end in organ failure. As a matter of fact, undercooked meat or ground meat from pork is consumed, for example, in Germany, it is called “Mettbrötchen” and widely appreciated, at least in Germany. Do we need a black box warning for eating undercooked pork meat in the future for xenotransplant recipients?

作为一名临床医生，我完全同意这些发现，尽管目前最热门的线索似乎是PCMV。正如我们所知，人畜共患病毒（如戊型肝炎病毒）是否会感染人类，例如食用未煮熟的肉类。一般来说，它对人类无害，但可能会导致肝炎，而且只有少数关于严重病程的报道，除非是免疫抑制下的器官移植接受者，在这种情况下，病程可能会变得很不乐观，并可能以器官衰竭告终。事实上，人们会去食用未煮熟的肉或猪肉碎肉，例如在德国，它被称为“ Mettbrötchen ”，并受到广泛赞赏，至少在德国是这样。我们是否需要在未来为异种移植受体食用未煮熟的猪肉设置黑框警告？

In the reported case the donor animal had been going through husbandry practices to provide a PCMV-negative population. Nasal swabs had been negative, but after xenotransplantation and the finding of PCMV in the recipient (patient's PBMC were positive for PCMV) tissue of the donor's spleen tested positive for PCMV, indicating that the animal was likely to have a latent PCMV infection. Hence the question is open, did the virus came with the pig's heart, or came it with food? Finally, how about the butchers working in the meat industry? Did they get infected at all or where did the antibodies come from. And how about the PCMV antibodies found in the normal population? There are a couple of open questions that certainly need to be addressed before boldly moving ahead.

在报告的案例中，供体动物一直在进行饲养实践，以提供PCMV阴性种群。鼻拭子呈阴性，但在异种移植和受体中发现PCMV（患者的PBMC对PCMV呈阳性）后，供体脾脏组织对PCMV检测呈阳性，表明该动物可能有潜在的PCMV感染。因此，问题是开放的，病毒是来自猪的心脏，还是来自食物？最后，在肉类行业工作的屠夫怎么样？他们到底有没有被感染，或者抗体是从哪里来的。那么在正常人群中发现的PCMV抗体呢？在大胆向前迈进之前，有几个悬而未决的问题肯定需要解决。

Let us have a look at the “cost of significant toxicity” that might come with the treatment of a PCMV-infected heart xenotransplant. Ganciclovir, Cidofovir, and Foscarnet in combination with Calcineurin Inhibitors are nephrotoxic, which might cause end-stage renal disease which is a frequent complication after heart transplantation and is associated with poor survival [22]. In the reported case, renal function was normal at the time of death, but that does not exclude in general a potential risk in the future in a larger study population. In summary, we do not know the answer yet, but the question is: are we getting from bad to worse?

让我们来看看治疗PCMV感染的心脏异种移植可能带来的“显著毒性成本”。Ganciclovir、Cidofovir和Foscarnet与Calcineurin抑制剂联合使用有肾毒性，可能会导致终末期肾病，而终末期肾病是心脏移植后的常见并发症，与生存率低有关。在报告的病例中，死亡时肾功能正常，但在更大的研究人群中，这并不能排除未来的潜在风险。总之，我们还不知道答案，但问题是：情况是否每况愈下？

So, the question is, are we back to square one? We have to keep in mind, that if the recipient becomes infected with PCMV the virus will meet an immunocompromised naive immune system, it will most likely infect the pig heart (endothelium and fibroblasts), it might infect other organs of the recipient as well and it might trigger a consumptive coagulopathy by upregulation of adhesion molecules on the infected endothelium, it might cause a cytokine storm leading to a SIRS (Systemic Inflammatory Response Syndrome) given the recipients immune systems naivety to the virus as it already happened in baboons.

所以问题是，我们又回到原点了吗？须谨记如果受体感染了PCMV，病毒将遇到受损的初始免疫系统，将很可能感染猪心脏（内皮和成纤维细胞），也可能感染受体的其他器官，病毒可能通过上调受感染内皮上的粘附分子而引发消耗性凝血病，可能因受体免疫系统对病毒的幼稚性引起细胞因子风暴，导致SIRS（全身炎症反应综合征），就像在狒狒身上已经发生的那样。

To prevent uncontrolled growth of the pig heart, baboons had been early weaned from steroids, and temsirolimus, a prodrug of sirolimus, had been given. Since mTOR inhibitors block TORC1 (Target of Rapamycin Complex 1) the intracellular target structure of HCMV for late replication the antiviral effect of mTOR inhibitors (e.g., Sirolimus and Everolimus) had been widely and prospectively demonstrated in human kidney transplantation. Given the preclinical data in the baboon studies, PCMV most likely uses a different molecular signal than HCMV, stressing again its difference to HCMV.

为了防止猪心脏不受控制地生长，狒狒很早就停用了类固醇，并服用了Sirolimus的前体药物Temsirolimus。由于mTOR抑制剂阻断HCMV晚期复制的细胞内靶结构TORC1（Rapamycin复合物1的靶点），mTOR抑制剂（如Sirolimus和Everolimus）的抗病毒作用已在人类肾移植中得到广泛和前瞻性的证实。鉴于狒狒研究中的临床前数据，PCMV的分子信号很可能与HCMV不同，再次强调其与HCMV的差异。

The solution to the current problem might be (1) PCMV-negative donor population (watch the dormant PCMV infection!), (2) development of an effective antiviral drug against PCMV, and (3) potential development of a vaccination against PCMV, something that might become arduous since we know that vaccinations under immunosuppression barely work, but it could be delivered before transplantation, because, if xenotransplantation becomes available, the surgery can be planned and so can a vaccination be given in a timely manner.

当前问题的解决方案可能是（1）PCMV阴性供体群（注意休眠期的PCMV感染！），（2）开发针对PCMV的有效抗病毒药物，以及（3）针对PCMV的疫苗接种的潜在开发，这可能会变得很艰巨，因为我们知道免疫抑制下的疫苗接种几乎不起作用，但它可以在移植前提供，因为如果异种移植成为可能，手术可以计划，所以可以及时进行疫苗接种。

In summary, xenotransplantation comes closer, a drug to treat PCMV infections will be one of the next prerequisites, though once we have it, it was only tested in animal models since the large human population does not exist for clinical trials. Hence, we are facing another problem, I am convinced we will find a solution to deal with this as well.

总之，异种移植越来越近，治疗PCMV感染的药物将是下一个先决条件之一，尽管一旦有了药物，只能在动物模型中进行测试，因为不存在用于临床试验的大量人类群体。因此，我们正面临着另一个问题，我相信我们也会找到处理这个问题的解决方案。

注:译文仅供参考，若与原文相较不尽完整或有歧异等疑义，请以原文为准。

原文链接

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