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  Title	HHV-6 and HHV-7 reactivation in allogeneic CAR-T cell therapy
  ID	8585248
  External ID	7720532140
  Author	Hiu Tung
  Published Date	April 22, 2025, 9:49 a.m.
  Summary	Human herpesviruses 6 and 7 (HHV-6 and HHV-7) pose notable and significant safety threats in chimeric antigen receptor (CAR)-T cell therapy. In 2024, the Food and Drug Administration (FDA) released se
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  URL	https://www.sciencedirect.com/science/article/pii/s0167779925001246
  Sentiment	Negative: 0.02301    Neutral: 0.88923    Positive: 0.08776
  Relevance	0.01429

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  Content ID	5890494
  Key	05789b79-501c-43d0-acbf-24a1cc6e9e30
  Content	Human herpesviruses 6 and 7 (HHV-6 and HHV-7) pose notable and significant safety threats in chimeric antigen receptor (CAR)-T cell therapy. In 2024, the Food and Drug Administration (FDA) released several recommended guidances for the industry to address the safety and testing of manufacturing cell-based medical products. The combination of DNA and RNA technologies provides a powerful tool to detect and monitor viral presence and infection status at various stages of the process, allowing for the investigation of the mechanisms of HHV-6/7 reactivation in CAR-T cell manufacturing. Genetic engineering and chemical treatments offer significant promise for mitigating the risks associated with these viruses during CAR-T cell manufacturing. Abstract Autologous chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment and allogeneic CAR-T cell therapy is poised to advance this revolution. CAR-T cell therapy faces some concerns regarding adventitious agents, which can threaten the safety of patients. Human herpesviruses 6 and 7 (HHV-6 and HHV-7) have become increasingly notable in this context, as they carry a risk with severe health consequences. This review explores these virus reactivations in CAR-T cell therapy and discusses mitigation strategies during allogeneic CAR-T cell manufacturing. We provide an overview of prevention and testing strategies, genetic engineering applications, and chemical substances with potential for interventions. This review aims to enhance understanding of HHV reactivation and improve the safety of allogeneic CAR-T cell therapies. Keywords CAR-T cell therapy manufacturing HHV-6 HHV-7 virus safety virus reactivation Prevalent and life-long latent HHV infections threaten CAR-T cell therapy HHV-6 and HHV-7 are highly prevalent in the human population ( Box 1 ). Initial infections with these viruses usually occur early in life, often presenting as asymptomatic or self-limiting conditions that resolve without medical intervention. Although these initial infections typically resolve within a week or less, they establish a life-long latent infection ( Box 1 ). Reactivation of these viruses can cause acute infections that are associated with severe symptoms and may even be fatal ( Box 1 ). Currently, there are no therapies specifically approved for the treatment of HHV-6 or HHV-7 infections, while antiviral drugs used for cytomegalovirus (CMV) infection may be prescribed to help manage the symptoms associated with HHV-6 and HHV-7 [ 1 ]. Box 1 HHV-6 and HHV-7 HHV-6 and HHV-7 are closely related members of the Beta-herpesviridae subfamily within the Roseolovirus genus. They are enveloped double-stranded DNA viruses whose genomes are encapsulated in a lipid bilayer envelope. The entry of HHV-6 and HHV-7 into host cells is highly specific, determined by interactions between viral envelope glycoproteins and surface receptors on the host cells. HHV-6 primarily infects CD4+ T lymphocytes through OX40/CD134 as an entry receptor, but can also infect a wide range of human cell types, including CD46+ hematopoietic progenitor cells, epithelial cells, and neurons [ 57 ]. Similarly, HHV-7 can infect CD4+ T lymphocytes, but it is less understood compared with HHV-6 due to the numerous unknowns about its viral ligands and unidentified cellular receptors mediating its entry [ 58 ]. Specifically, HHV-7 is capable of infecting CD4+ T lymphocytes independent of CD4+ expression, suggesting that HHV-7 may use other unidentified viral glycoproteins and cellular receptors to enter host cells [ 59 ]. The ability of HHV-7 to infect other cells that lack CD4+, such as neuronal cells, further highlights the significant gaps in our understanding of the virus. Seroprevalence data for these viruses can vary significantly depending on geographic location, age group, and the sensitivity and specificity of the serological tests used. For HHV-6, seropositivity rates are generally near 90% in children and range from 70% to 90% in adults, while the seroprevalence of HHV-7 among humans varies from 60% to 98% across different reports [ 60., 61., 62., 63., 64. ]. Symptoms can vary depending on the specific strain of HHV-6, which is classified into two distinct variants: HHV-6A and HHV-6B, with each variant predominantly found in different geographic regions [ 65 ]. HHV-6A is less well-understood compared with HHV-6B; its primary infections are often asymptomatic but may sometimes present with mild symptoms such as fever and rash. HHV-6B can cause roseola infantum in young children [ 57 ]. Primary infections with HHV-7 often manifest with symptoms akin to those of other common viral infections [ 58 ]. CAR-T cell therapy (see Glossary ) represents a significant leap forward in cancer treatment [ 2 ]. The traditional autologous approach involves collecting and genetically modifying a patient’s own T cells, and then re-infusing them back into the patient to target and destroy cancer cells [ 3 ]. Recent innovations in the allogeneic CAR-T cell approach, which utilizes T cells from healthy donors and produces pre-manufactured cells that are readily available for immediate use, have overcome several limitations inherent in the traditional CAR-T cell therapy ( Table 1 ). This advancement addresses limitations such as the time-consuming cell collection and costly preparation process, as well as the potential for insufficient T cell quality in patients with compromised immune systems [ 4 ]. Although allogeneic CAR-T cell therapy offers numerous advantages, the nature of these cells derived from non-patient sources requires a careful management of associated risks. Table 1. Comparative summary of autologous and allogeneic CAR-T therapy Empty Cell Autologous Allogeneic Source Patient-derived Healthy donor-derived Manufacturing processIndividualized Off-the-shelf Commercial scalabilityNo possibility to scale-up manufacturing as each run produces a single batch of CAR-T cells for one patient. For commercial manufacturing multiple batches have to be produced in parallel (scale out)One production batch can be used in multiple patients. Large-scale batches can be produced from a single donor, increasing the efficiency of the manufacturing process (scale-up) Treatment available to patientsPatients have to wait for their individualized drug to be manufacturedThe product is pre-manufactured and stored and can be administered to patients immediately Source of risk of transferring pathogenOnly manufacturing processFrom donor and manufacturing process Pros Lower risk of immunological rejectionLower cost Almost immediate availability (depends on the supply) Cons Longer turnaround time Higher cost The quality of cells depend on the patient More safety risk associated with non-patient derived source of cells There have been multiple reported cases of unusual HHV-related complications in patients after receiving Food and Drug Administration (FDA)- approved CAR-T cell therapy [ 5., 6., 7. ]. Although some cases have also been reported for other ubiquitous viruses, including CMV and Epstein-Barr virus, HHVs are of critical concern due to their strong preference for infecting and replicating in T cells, which is particularly relevant to CAR-T cell therapy [ 8, 9 ]. In this article, we review documented reactivation incidents of HHV in the context of CAR-T cell therapy and discuss strategies for mitigating associated risks during the allogeneic CAR-T cell manufacturing process. HHV reactivation in CAR-T cell therapy As of Jan 2025, HHV-6-associated inflammatory conditions in the nervous system have been reported during the clinical trials of two FDA-approved autologous CAR-T cell therapies [ 5., 6., 7. ]. These conditions appeared to develop around day 28 and day 30 post-CAR-T cell infusion in both cases. It is unclear whether the reactivation of HHV-6 was caused by exogenous origin, such as viruses introduced during the CAR-T cell manufacturing process, or by endogenous reactivation in the CAR-T cell recipient patients. Research conducted using cells collected from patients who received either the FDA-approved or clinical trial CAR-T cell therapies shows that none of the CAR-T cells showed the presence of HHV-6 before infusion, but some of those cells were found to express HHV-6 after the infusion [ 10 ]. However, the authors also extended the expansion time of CAR-T cells from the 19-day allogeneic CAR-T cell manufacturing process to up to 27 days, where they detected a significant increase in HHV-6B transcript levels during the extended culture period [ 10 ]. Taken together, these data support the notion that HHV-6 can be reactivated during CAR-T cell therapy. However, further studies are required to determine whether the reactivation is triggered by the CAR-T cell manufacturing process, in the patients after receiving CAR-T cells, or a combination of both. Although HHV-7 reactivation has not been reported in CAR-T cell therapy, both HHV-7 and HHV-6 have been observed to reactivate in organ transplant patients [ 11, 12 ]. A common aspect of these therapies is that they involve patients who have undergone immunosuppression. Other than their common occurrence in immunocompromised conditions, the specific factors that trigger HHV-6 and HHV-7 reactivation remain unclear, with much of the current understanding derived from studies of other herpesviruses. Research using in vitro models has demonstrated that herpesviruses reactivation can be induced by various environmental triggers, including trauma, ultraviolet exposure, and other stressors [ 13 ]. Additionally, in vitro systems have shown that chemical treatments can induce reactivation by disrupting the silencing mechanisms of the host that regulate viral latent gene expression [ 14 ]. Further research is needed to determine whether HHV-6 and HHV-7 reactivation is triggered by the immunocompromised state itself or by the CAR-T cell manufacturing process. The impact of HHV on the allogeneic CAR-T therapy The association between CAR-T cell therapy and occurrence of HHV reactivation raises concerns about patient safety. Therefore, CAR-T cell therapy manufacturers must ensure that final therapeutic products test negative for these viruses. Unlike protein biologics produced from cell culture, which can undergo virus clearance in the downstream purification process such as viral inactivation and filtration, CAR-T cell products rely mainly on contamination prevention, in-process detection, and lot rejection [ 15 ]. Once a virus is detected during the CAR-T cell manufacturing process, the only course of action is to discard the entire batch. The rejection events involve significant financial losses, including the direct costs of materials and manufacturing used for the production of the rejected batch, operational expenses related to investigation and downtime, and costs associated with the disposal of the rejected batch. Apart from the financial burden on manufacturers, any debacle will potentially lead to more patients being unable to receive therapy in a timely manner [ 15, 16 ]. HHV mitigation strategies for CAR-T cell manufacturing In other therapies where cases of HHV-6 or HHV-7 reactivation have been observed, routine testing of donors for HHV6 and HHV-7 is not standard practice. Instead, most recent guidelines established by relevant professional societies and associations focus on the aspects of diagnosis and management of HHV-6 and HHV-7 in patients. The American Society of Transplantation Infectious Diseases Community of Practice provides recommendations for the diagnosis and management of HHV-6 and HHV-7 [ 17 ]. The American Society for Transplantation and Cellular Therapy Cord Blood Special Interest Group recommends symptom-based testing for HHV-6B in the early stages after transplantation [ 18 ]. The European Conference on Infections in Leukaemia offers guidelines for assessing and managing HHV-6B following transplantation [ 1 ]. However, these approaches, which primarily rely on clinical management in patients after receiving therapy, do not entirely pertain to CAR-T cell therapy. Emerging evidence indicates that HHV-6 DNA levels may be elevated during the manufacturing process, suggesting that manufacturing should be included within the scope of control in this case [ 10 ]. Therefore, we review a set of practices and controls specifically applicable to allogeneic CAR-T cell manufacturing that should be considered to ensure the safety of the product in the possible event of HHV-6 and HHV-7 reactivation. While we are aware of ongoing discussions and developments regarding antiviral prophylaxis and preemptive therapy for CAR-T cell recipients to mitigate HHV-6 and HHV-7 reactivation, these topics are outside the purview of this review [ 19, 20 ]. The following sections will focus on key considerations for manufacturing CAR-T cell products, beginning with donor selection and continuing through to lot release. Preventing HHVs in CAR-T cell manufacturing Generally, the manufacturing process for CAR-T cell includes the following steps: isolation of T cells from healthy donor or patient leukopaks (leukapheresis), activation of T cells, genetic modification to express CARs on T cell surface, cell selection, cell expansion, harvest, final drug product formulation, and cryopreservation ( Figure 1, Key figure) [ 21 ]. There are two main routes by which adventitious viruses can enter the allogeneic CAR-T cell manufacturing system: viruses that are carried from the donor and those introduced during the manufacturing process. The risk from donor-derived viruses can be mitigated through donor screening and testing ( Figure 1 ). Specifically, leukopak materials intended for CAR-T cell product manufacture must be qualified by testing for communicable disease agents, with a ‘negative’ result being required for use in manufacturing. In the USA, blood products from donors must be tested using FDA-licensed, approved, or cleared donor tests, as per the FDA regulations [21 CFR § Part 1271 Subpart C (2004)] i. Additionally, testing must be performed by a laboratory certified under the Clinical Laboratory Improvement Amendments [42 U.S.C. § 263a (2012)] ii and FDA regulations [42 CFR § Part 493 (2014)] iii, or one meeting equivalent standards set by the Centers for Medicare and Medicaid Services. In the EU, testing should use CE-marked test kits when feasible and may also require additional laboratory certifications. Download: Download high-res image (480KB) Download: Download full-size image Figure 1. Key figure. Major events and proposed strategies to prevent viral risks in the allogeneic chimeric antigen receptor (CAR)-T manufacturing process. The schematic diagram outlines the allogeneic CAR-T manufacturing process. Text below the diagram represents the corresponding proposed testing or mitigation strategies to address safety concerns related to human herpesviruses 6 and 7 (HHV-6 and HHV-7). The manufacturing process starts once a healthy donor is selected and undergoes leukapheresis, a procedure that extracts blood from the bloodstream and separates white blood cells from other blood components. The collected white blood cell fraction is referred to as a leukopak. A specific population of T cells is then isolated from the leukopak, activated, and genetically modified to express a CAR gene which encodes a receptor protein on their surface. The desired genetically modified CAR-expressing cells are selected and expanded. The process then includes product formulation, harvest, cryopreservation, and, finally, lot release. Strategies including testing, contamination control, genetic engineering, and chemical inhibition are proposed to prevent viral risks in the allogeneic CAR-T manufacturing process. Testing can be conducted as early as donor selection (donor testing) and throughout the manufacturing process, including in-process samples and the lot of expanded cells (in-process testing), and the final drug product (DP release testing). Contamination control strategies to prevent viral entry into the manufacturing system should be implemented throughout the entire process. These control strategies include ensuring that reagents, chemicals, and raw materials are free of adventitious viruses, and maintaining quality, safety, and compliance during operations [Current Good Manufacturing Practice (CGMP) and standard operating procedure (SOP)]. Genetic engineering can be integrated during T cell modification to modulate viral and host immunity, while chemical inhibition can be applied at any stage of manufacturing, provided it is optimized. The risk of contamination in production processes can be managed by implementing Current Good Manufacturing Practice (CGMP) principles that adhere to regulations ( Figure 1 ). In summary, these strategies encompass all aspects of qualification and validation of production, including but not limited to facility design and maintenance, equipment and raw materials, as well as personnel qualification in contamination control, hygiene practices, and standard operating procedures (SOPs) ( Figure 1 ) [ 18, 22 ]. In the latest (April 2024) FDA draft guidance for industry, titled ‘Considerations for the use of human and animal-derived materials in the manufacture of cellular and gene therapy and tissue-engineered medical products’ iv, the types of raw materials addressed for use in manufacturing cell therapy products are outlined. The scope of these materials includes human- and animal-derived materials used for reagents, feeder cells, excipients, and other inactive ingredients that come into contact with starting materials, intermediates, and final drug products. Concerns related to the usage of these materials, including the potential transmission of adventitious agents in the production of cellular therapy products, and the corresponding recommendations, are discussed. This draft guidance supplements the existing FDA CGMP regulations and further includes recommendations about special considerations for these materials, including adventitious agent testing and screening, risk assessment, and materials management. Testing in-process samples, the lot of expanded cells, and the final drug products Although donor testing for HHV-6 and HHV-7 is essential for detecting the presence of the virus, it cannot guarantee that an individual or the donor sample is entirely free of HHV-6 and HHV-7. This challenge arises due to several reasons. Firstly, there are no FDA-approved kits for HHV-6 and HHV-7 donor testing, leaving us without a standardized cutoff to determine the acceptance of donor materials for cell therapy products. Secondly, certain variations within the tested population may not be represented. The peripheral blood mononuclear cells (PBMC) population shows that HHV-6 and HHV-7 positive cells are highly heterogeneous, which increases the risk of missing some positive cells and leads to challenges in detecting positive results [ 23, 24 ]. If a leukopak tests ‘negative’ for HHV-6 or HHV-7 but is used in CAR-T cell manufacturing, it will pose a risk that any undetected HHV-positive cells may be introduced into the manufacturing process. Furthermore, the CAR-T cell therapy process itself can potentially trigger the reactivation of latent viruses. Therefore, it is worth considering the implementation of in-process testing ( Figure 1 ) to ensure quality control and management of HHV-6 and HHV-7 throughout the manufacturing process. Evidence indicates that HHV-6 DNA can become more abundant over time during manufacturing, suggesting potential reactivation [ 10 ]. While the exact cause of this reactivation remains unknown, comprehensive testing for HHV-6 and HHV-7 at some stages can provide valuable insights, helping to improve the control of adventitious agents and prevent similar issues in the future. According to the same study, the DNA replication and transcription dynamics of HHV-6B occur over a shorter timespan than the standard CAR-T cell manufacturing window [ 10 ]. This shorter timeframe is advantageous for detecting changes or issues through monitoring DNA and RNA kinetic levels. Early detection and intervention can minimize waste and reduce impacts before they affect the final products. In the FDA’s April 2024 draft guidance for the industry, titled ‘Safety testing of human allogeneic cells expanded for use in cell-based medical products’ v, the potential risks associated with extensive cell culturing during the manufacturing process are addressed. The guidance indicates that the level and scope of cell safety testing necessary to ensure product safety are contingent upon the potential for cell expansion and the number of patients the cell-based medical product is designed to treat. Regarding the mode of expansion and product use in allogeneic CAR-T cells, the draft guidance specifies that for products prepared from a single batch of genetically engineered cells with limited expansion capacity, testing should include the lot of expanded cells or the cells at the end of the production process (drug product) ( Figure 1 ). The draft guidance recommends safety testing that includes polymerase chain reaction (PCR) testing for human pathogens, with HHV-6 and HHV-7 specifically listed. Optimizing HHV molecular testing used in CAR-T cell manufacturing Compared with other traditional virology tests, such as culture tests and serology-based methods, molecular assays provide a more sensitive and specific method for detecting the presence of HHV-6 or HHV-7 [ 25., 26., 27. ]. Molecular assays are techniques used to examine and quantify viral nucleic acids – both DNA and RNA. The knowledge in the field regarding the molecular mechanisms of HHV-6 and HHV-7 infection has made the development and implementation of molecular tests feasible. By analyzing these nucleic acids, we can determine the extent of HHV-6 and HHV-7 presence in the samples and possibly assess the infection status. Since HHV-6 and HHV-7 are DNA viruses, DNA assays, such as PCR and quantitative PCR (qPCR), are effective for directly amplifying and detecting the presence of their genetic material in sample types associated with CAR-T cell manufacturing [ 28 ]. While the sensitivity of a DNA assay for HHV-6 or HHV-7 testing can depend on the type of samples used, various sample types associated with CAR-T cell manufacturing have been reported where DNA assays successfully detected HHV-6 and HHV-7. Optimal assays for detection in whole blood, PBMC, leukopak, and T cell samples have also been reported [ 10, 25, 26, 29, 30 ]. However, a limitation of DNA testing is that they do not directly indicate the infection status [ 30 ]. To determine infection status, a more complex experimental design is required, which may involve combining results from different sample types, time points, or tests [ 31 ]. Figure 2 A provides a summary of this approach. For cellular samples such as whole blood, leukopak, or T cell culture, a positive DNA result does not differentiate between active and latent infection. HHV-6 can integrate its genetic material in the form of DNA into the terminal regions of human chromosomes, a phenomenon referred to as c hromosomally i ntegrated HHV-6 (ciHHV-6). When this integrated viral DNA is transmitted across generations, it is known as inheritable chromosomally integrated HHV-6 (iciHHV-6), whereas evidence for a similar phenomenon in HHV-7 is limited ( Box 2 ). In cases of iciHHV-6, DNA levels can be higher than those observed in individuals with active infections [ 32 ]. The detection of iciHHV-6 (and possibly iciHHV-7) can be achieved through droplet digital PCR (ddPCR), which measures and compares the relative number of HHV-6 DNA copies per cell [ 33, 34 ]. Alternatively, since the chromosome integration site of iciHHV-6 is characterized, fluorescent in situ hybridization (FISH) can be used to identify the chromosomal locations of iciHHV-6 by using labeled DNA probes that stain the viral genome. Alternatively, an RNA assay, which will be discussed in a later paragraph, can be used to differentiate between active and latent infections. Download: Download high-res image (438KB) Download: Download full-size image Figure 2. Interpretation of DNA or RNA assay results for detecting human herpesviruses 6 and 7 (HHV-6 and HHV-7) infection status. (A) DNA assays, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), can be used to detect HHV-6 and HHV-7 DNA in both cellular or non-cellular samples. In cellular samples, during HHV active infection, HHV DNA (a green coil) is transcribed into RNA (I), and replicates new viral DNA (II). The RNA is then translated into proteins, which along with the replicated DNA are assembled into new virus particles. A positive result from cellular samples indicates the presence of HHV DNA and might suggest an active infection. However, a positive result may also indicate a latent infection including inheritable chromosomally integrated HHV-6 and -7 (iciHHV-6 and iciHHV-7), where the HHV DNA is present but not actively replicating. The case cannot be resolved without further investigation using additional assays, such as quantitative measurement of viral loads using droplet digital PCR (ddPCR), viral genome localization by fluorescent in situ hybridization ( FISH), or transcriptomic differentiation of infection stages using RNA sequencing (RNA-seq). When the assay is optimized, the absence of detectable DNA (a negative result) in cellular samples indicates no infection. Non-cellular samples can be used to test for the presence of viral DNA. This method relies on detecting viral particles in the solute which contain viral DNA released from infected cells. A positive result can be interpreted as an active infection, whereas a negative result can indicate no infection. However, the absence of detectable viral DNA cannot rule out a latent infection that does not produce detectable levels of viral DNA in non-cellular samples, or an active infection that does not localize and produce enough detectable DNA in the tested samples (false negative). (B) RNA assays, such as qRT-PCR and RNA-seq, can be used only in cellular samples to detect viral RNA. A positive result, indicating the presence of detectable RNA, may suggest an active infection or, in some cases, a latent infection, as observed with iciHHV-6 if the virus is producing detectable transcripts. Conversely, a negative result, indicating the absence of detectable RNA, could imply the absence of infection, a latent infection in some cases, or a false negative due to issues with suboptimal sample collection or experimental design, such as the choice of inappropriate transcript targets, inadequate time points, or unsuitable tissue types. Additionally, combining RNA assays with other testing methods, such as DNA assays, may enhance the accuracy of infection status determination. Box 2 Latency of HHV-6 and HHV-7 During the latent phase, the virus persists in a dormant state without actively producing mature virions. During latency, HHV-6 and HHV-7 preserve their genetic materials as DNA in their host. In most herpesviruses, the DNA exists as an episome: genetic material that remains separate from the host’s chromosomes in the nucleus [ 66 ]. Conversely, molecular and genetic studies indicate that HHV-6 DNA can integrate into the subtelomeric regions of host chromosomes in infected cells, establishing a condition known as ciHHV-6 [ 67, 68 ]. ciHHV-6 is found in nearly all nucleated cells throughout the body, including germline cells, which suggests that this condition can be inherited (iciHHV-6) [ 68., 69., 70., 71., 72., 73. ]. The prevalence of iciHHV-6 in the global population is estimated to range from approximately 0.21% to 1.5% [ 74 ]. By contrast, the full extent mechanism by which HHV-7 maintains latency is not yet elucidated. The only documented instance suggesting inherited ciHHV-7 was observed in the telomeric regions of a few follicular cells [ 75 ]. This condition is rare and is not typically detected in PBMCs [ 75, 76 ]. Reactivation of HHV-6 and HHV-7 Although these viruses can establish life-long latency, they may reactivate under certain conditions, leading to the production of infectious viral particles and progeny. Reactivation of HHV-6 and HHV-7 is frequently observed in individuals with compromised immune systems or those undergoing immunosuppressive therapy. The clinical consequences of reactivation can vary among different HHV types. HHV-6 reactivation, in particular, poses substantial risks for immunocompromised individuals. Reactivation of HHV-6A has been associated with severe neurological complications such as multiple sclerosis and encephalitis [ 77 ]. Conversely, HHV-6B reactivation is more commonly linked to encephalitis, which often carries a high mortality rate [ 78., 79., 80. ]. By contrast, while HHV-7 is less well-understood compared with HHV-6A and HHV-6B, its reactivation is still associated with a range of serious complications in immunocompromised individuals. These complications include dermatological diseases, neurological disorders, and organ-specific diseases such as hepatitis, colitis, pneumonitis, and encephalitis, particularly in organ transplant patients [ 58, 81 ]. Transcriptional dynamics during HHV-6 and HHV-7 infections During active infection, HHV-6 and HHV-7 sequentially transcribe immediate early, early, and late genes (collectively called lytic genes) at the transcript level [ 82, 83 ]. Conversely, during latent infection, they can silence genes that are typically expressed during the active phase through relying on the host’s epigenetic mechanisms, resulting in the expression of only a restricted subset of latency-associated transcripts [ 84., 85., 86. ]. Additionally, HHV-6 encodes microRNAs (miRNAs) during latency, which function to degrade or inhibit the expression of both viral lytic genes and host cell genes, thereby modulating host immunity and facilitating evasion of immune detection [ 87 ]. There is currently no documented evidence regarding the presence of miRNAs encoded by HHV-7. For non-cellular samples isolated from whole blood samples, such as plasma, or supernatant separated from cell culture, a positive DNA test typically indicates active infection, as HHV infectious particles containing DNA are released during active infection. However, a negative result could not rule out the possibility of a latent infection. False negatives are possible if infectious particles are present at a very low level in the tested samples. HHV-6 and HHV-7 can localize to specific tissues during active infection, potentially resulting in false-negative results in non-cellular samples [ 35 ]. Therefore, additional investigations into assay sensitivity and specificity, or supplementary tests such as RNA assays, which will be discussed in the next paragraph, may be necessary to provide a clearer picture of the infection status. RNA assays ( Figure 2 B), such as quantitative reverse transcription (qRT-PCR) and RNA sequencing (RNA-seq), are used to detect replicating HHV-6 and HHV-7 viruses in cellular samples. The extensive understanding of transcriptional dynamics in HHV-6 infection has enabled the selection of specific transcripts for investigation ( Box 2 ). A recent patent application uses single-cell sequencing to quantify HHV-6 transcription in CAR-T cell culture [ 36 ]. These assays take advantage of the fact that only actively replicating HHV-6 and HHV-7 viruses transcribe certain RNA transcripts, making them effective for indicating active infection [ 31 ]. However, a positive result cannot completely dismiss the likelihood of latent iciHHV-6 (and potentially iciHHV-7) infections, as the integrated viral DNA can utilize the host transcriptional machinery to produce RNA transcripts [ 37 ]. Conversely, a negative result, which indicates the absence of detectable RNA transcripts in the samples, could suggest either the absence of viruses, a latent infection, or a false negative due to suboptimal sensitivity and specificity. This could be a result of the selection of inappropriate viral targets, or the timing and location of sample collection. Further testing is needed to accurately interpret the true infection status. In application, DNA assays are more sensitive for identifying latent or active infections due to the stability and persistence of viral DNA. Therefore, they are more suitable for first-line testing, such as donor selection, where straightforward information is required. However, if there is any suspicion of reactivation during the manufacturing process, an RNA assay should be performed along with a DNA test to confirm the presence of active viral replication. It is important to carefully select which RNA transcripts to detect, as viral RNA transcripts are dynamically expressed at different infection stages [ 38, 39 ]. Relying on a single gene transcript may not be reliable to provide a comprehensive view of viral activity. A comparative study on advancements in metagenomic next-generation sequencing (mNGS) has demonstrated its superiority over qRT-PCR for virus detection [ 40 ]. Furthermore, mNGS possesses the capability to simultaneously detect both viral RNA and DNA within a pipeline [ 40, 41 ]. Genetic engineering as a tool for HHV mitigation Genetic engineering technologies have proven effective in disrupting both latent and active herpesvirus infections within infected cells. For instance, genetic editing tools, such as CRISPR/Cas9 and adeno-associated virus (AAV)-mediated gene editing using meganucleases, can target critical viral genes during latency to prevent replication, thereby limiting the ability of the virus to reactivate ( Figure 3 A ) [ 42., 43., 44., 45. ]. Alternatively, editing the host cell genes responsible for encoding receptors or co-receptors used by herpesviruses can reduce the ability of these viruses to infect additional cells ( Figure 3 B) [ 46 ]. Modifying host genes involved in the immune response may also enhance the host to recognize and combat infections [ 47 ]. In the case of HHV-6, transgenesis was used where human T cells are engineered to constitutively express short interfering RNAs that target U51, a positive regulator of virus replication ( Figure 3 C) [ 48 ]. Although in vivo and in vitro studies demonstrated promising results, efforts to translate these findings into clinical applications have not yet achieved the desired outcomes [ 49 ]. Despite this setback, research continues to hold promise for future advancements in applying genetic editing to intervene with HHVs. It is anticipated that similar strategies will emerge to enhance HHV-6 and HHV-7 safety in CAR-T cells in the future. Download: Download high-res image (722KB) Download: Download full-size image Figure 3. Genetic engineering and chemical intervention involve the manipulation or modification of biological systems to prevent or mitigate human herpesvirus (HHV) infection. (A) Genetic engineering can be used to modify the HHV viral DNA that integrates into human DNA. Some viral sequences critical for viral replication and reactivation can be modified to prevent latent HHV from reactivating. (B,C) Genetic modification can also target the human genome. In (B), some genes encode transmembrane receptors that serve as entry points for HHVs. Editing these genes to produce transgenic receptors can inhibit HHV binding, therefore suppressing its spread to uninfected cells. In (C), certain genes responsible for producing negative modulators of HHVs, such as certain siRNAs that post-transcriptionally suppress HHV gene expression by targeting HHV RNA, can be added to boost the host immunity against HHVs. (D–F) Chemical intervention involves the use of chemicals to affect the host or HHV immune response. (D) Chemicals can specifically interact with viral transcription machinery to directly inhibit viral DNA replication. The mechanisms include chemicals (e.g., cidofovir, acyclovir, and ganciclovir) that act as analogs to compete with nucleotides. Once these analogs are incorporated into the DNA, they stop DNA from further elongation. Or others (e.g., foscarnet) inhibit viral DNA polymerase activity. (E) IFNα can bind to host cells and induce the host immune system to produce antiviral proteins against HHVs. (F) Other chemicals, such as phosphonoacetyl-l-aspartate, can indirectly interfere with replication by inhibiting nucleoside biosynthesis in general, such as pathways supplying nucleotide precursors and enzymes involved in replication, thereby indirectly inhibiting HHV replication. Chemical interventions of HHV reactivation The application of chemical substances has been demonstrated to inhibit the proliferation of the HHV-6 and HHV-7 viruses. There is in vitro evidence that some antiviral drugs can inhibit HHV-6 replication in T cell cultures; examples include acyclovir, ganciclovir, foscarnet, and cidofovir [ 50, 51 ]. Recent research has put forth the idea of using foscarnet during CAR-T cell expansion to inhibit HHV-6 reactivation and its spread during cell therapy production [ 10 ]. Figure 3 D summarizes different strategies of these antiviral drugs used to inhibit viral DNA polymerase in action [ 52 ]. Additionally, the application of human type I interferon α (IFNα) has been shown to enhance human immunity against viruses by inducing expression of genes that encode proteins which prevent viral replication ( Figure 3 E) [ 53 ]. A patent application suggests that IFNα can prevent or inhibit HHV-6 replication in CAR-T cell culture [ 54 ]. Another mechanism by which chemicals can indirectly interfere with viral replication is by inhibiting the biosynthesis pathways that generate the essential molecules and compounds required for the process, such as phosphonoacetyl-l-aspartate ( Figure 3 F) [ 55 ]. This idea is less well understood in the context of CAR-T cell antiviral application due to its non-specific nature. For HHV-7, several antiviral agents, including foscarnet, cidofovir, vidarabine, lobucavir, phosphonoacetyl-l-aspartate, phosphonoacetyl-β-alanine, and S2242, dideoxyinosine, have shown anti-HHV-7 activity in CD4+ cells [ 56 ]. More research is needed to determine if any of these compounds can effectively suppress HHV-7 activity in CAR-T cell manufacturing while minimizing their impact on the final CAR-T cell drug products. Concluding remarks and future perspectives While allogeneic CAR-T cell therapy represents a promising advancement in cancer treatment, the risk of HHV-6 and HHV-7 reactivation requires careful consideration and management in manufacturing. This review highlights the importance of robust prevention and molecular testing strategies, as well as the potential benefits of chemical interventions and genetic engineering to address these risks. Moving forward, continued research in HHV virology is crucial to develop better testing strategies. A sensitive and specific molecular assay to detect viral nucleic acids relies on our prior knowledge of target molecules, which includes, but is not limited to, genomic sequences of HHV variants, as well as spatial and temporal viral loads or expression. Understanding these aspects will help us design better assays to inform active and latent infection. A better understanding of virology not only aids detection strategies but also enhances overall preventive management. This is particularly relevant for HHV-7, where knowledge is significantly less compared with HHV-6. Specific gaps in our understanding include the details of HHV-7 latency and reactivation, particularly the molecular mechanism of infection, such as the receptors involved in HHV-7 entry into host cells, and regulatory elements that contribute to its reactivation. Identifying the viral genes and proteins essential for HHV-7 replication can allow the design of targets for genetic modification and intervention strategies. There is a need for more extensive investigation into whether any steps of the CAR-T cell manufacturing can trigger viral reactivation. The ex vivo condition during manufacturing often differs from the in vivo environment. The conditions and formulas used for the culture, expansion, and activation of T cells may induce a state of stress in T cells, which could lead to HHV reactivation. It is imperative to identify the specific factors in the manufacturing process that can disrupt the latent state and promote the reactivation of latent viruses (see Outstanding questions ). As clinical data accumulates on the events of HHV reactivation in recipients of CAR-T cell therapy, it is evident that most studies have focused on risk factors related to patient epidemiology. We propose an interdisciplinary analysis to investigate whether there is a correlation between the latent infection status of donor-derived materials and the likelihood of viral reactivation following infusion into patients. To date, no studies have reported such an extensive analysis. Addressing this hypothesis requires a collaborative approach that investigates data from both CAR-T manufacturing processes and clinical observations. By integrating this data, we can determine if these factors are related and develop a more effective strategy for selecting donors for CAR-T cell production. Outstanding questions How does HHV-7 remain latent and what causes it to reactivate? What is the molecular mechanism behind this? Is there a correlation between the latent infection status of the donor and the likelihood of HHV reactivation following infusion into patients? What in the manufacturing process causes HHV-6/7 reactivation during CAR-T cell therapy? For the HHV-6 and HHV-7 PCR tests recommended by FDA draft guidance, what is the validated cutoff value that can be used to establish the release criteria for cel therapy products? What types of manufacturing control systems can be robustly implemented by CAR-T cell manufacturers for donor screening, in-process testing, and final product release? How might future research validate the use of intervention and mitigation strategies, such as a gene editing approach and chemical treatment, to inhibit HHV-6 and HHV-7 reactivation during CAR-T cell manufacturing, while demonstrating minimal impacts on therapeutic efficacy, stability, and patient safety? Acknowledgments The author would like to thank their colleague Dayue Chen for initiating this review idea and for the discussions during manuscript preparation. This work was funded internally by Roche/Genentech. Declaration of interests The authors declare no conflict of interest. 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Antiviral prophylaxis the use of medications as a preventive treatment aims to inhibit the onset or spread of viral infections. It is commonly used in situations where individuals are at high risk of encountering a specific virus or have already been exposed and require protection against infection. Autologous in the context of CAR-T cell therapy, cells are obtained and modified from the patients’ cells. Cell expansion the process of growing and multiplying cells in a controlled environment to increase their number for therapeutic purposes. Chimeric antigen receptor T (CAR-T) cell therapy an advanced form of immunotherapy that involves genetically modified T cells to target specific antigens present on the surface of cancerous cells. Droplet digital polymerase chain reaction (ddPCR) a molecular technique that performs absolute nucleic acid quantitation in a sample. It quantifies target DNA or RNA molecules by amplifying them in individual droplets. Each droplet undergoes PCR and generates fluorescent signals, which are then analyzed. Fluorescent in situ hybridization (FISH) a molecular assay used to detect and localize the presence of particular DNA sequences on chromosomes. This method utilizes fluorescent labeled probes that hybridize specific sequences and visualizes the signals under a fluorescent microscope. In-process detection a quality control process that continuously monitors and inspects products throughout the manufacturing process in real-time to ensure compliance with standards. Leukopaks a collection of white blood cells taken and isolated from a donor’s whole blood. Metagenomic next-generation sequencing (mNGS) the comprehensive analysis of genetic material from all organisms present in a complex sample; it sequences all the DNA or RNA directly from the sample. Plasma the liquid portion of blood that remains after the removal of blood cells. It contains water, electrolytes, antibodies, antigens, and clotting factors, etc. Polymerase chain reaction (PCR) and quantitative PCR (qPCR) molecular biology techniques used to amplify a specific DNA sequence through cycles of denaturation, annealing, and extension. Quantitative PCR (qPCR) is an advanced version of PCR that allows for the simultaneous amplification and quantification of DNA. Preemptive therapy early treatment based on initial signs of an infection to prevent its progression and minimize impact. This approach is often used in high-risk situations or for immunocompromised individuals. Quantitative reverse transcription PCR (qRT-PCR) an approach for quantifying RNA or gene expression that involves converting RNA into complementary DNA (cDNA) by reverse transcription, followed by amplification of this cDNA using standard PCR methods. RNA sequencing (RNA-seq) a high-throughput technique used to analyze the quantity and sequences of RNA in a sample. This method involves converting RNA into complementary DNA, which is subsequently sequenced using next-generation sequencing technologies. Supernatant the clear liquid that remains above and separated from the cell pellet after centrifuging cell culture. Transcription the process whereby genetic information from DNA is transcribed into messenger RNA (mRNA), which then acts as a template for guiding protein synthesis. © 2025 The Author(s). Published by Elsevier Ltd. About ScienceDirect Remote access Advertise Contact and support Terms and conditions Privacy policy Cookies are used by this site. Cookie Settings All content on this site: Copyright © 2025 Elsevier B.V., its licensors, and contributors. All rights are reserved, including those for text and data mining, AI training, and similar technologies. For all open access content, the relevant licensing terms apply. Cookie Settings
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