Search

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 ‘super-expressors’ (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5–7 and has broad implications for the design, production, and monitoring of cell therapies.

In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.

Allogeneic chimeric antigen-receptor T-cell process flows comprise large-scale primary cultures incorporating traditional biologics processing steps such as cell expansion, with novel processing steps such as electroporation. In addition, these process flows begin with donor-derived peripheral blood mononuclear cells and generate single batches of product, which may be used to treat hundreds of patients. The corresponding unit operation-process safety considerations are individually diverse, and in combination are currently unique and rapidly evolving. The use of available regulations and the principles of quality by design are useful in designing appropriate control strategies into the process during development to ensure process safety.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA).

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO− and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV. Biotechnol. Bioeng. 2012;109: 157–165. © 2011 Wiley Periodicals, Inc.

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of env in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.

The rate of disease development in simian immunodeficiency virus (SIV) infection of macaques varies considerably among individual macaques. While the majority of macaques inoculated with pathogenic SIV develop AIDS within a period of 1 to 2 years, a minority exhibit a rapid disease course characterized by absence or transience of humoral and cellular immune responses and high levels of virus replication with widespread dissemination of SIV in macrophages and multinucleated giant cells. The goal of this study was to examine viral evolution in three SIVsmE543-3-inoculated rapid progressors to determine the contribution of viral evolution to the development of rapid disease and the effect of the absence of immune pressure upon viral evolution. PCR was used to amplify and clone the entire SIV genome from tissues collected at necropsy, and the course of viral evolution was assessed byenvsequences cloned from sequential plasma samples of one rapid progressor (RP) macaque. The majority of sequence changes in RP macaques occurred in the envelope gene. Substitutions were observed in all three animals at specific conserved residues in envelope, including loss of a glycosylation site in V1/V2, a D-to-N/V substitution in a highly conserved GDPE motif, and a P-to-V/H/T substitution in the V3 loop analog. A cell-cell fusion assay revealed that representativeenvclones utilized CCR5 as a coreceptor, independent of CD4. The selection of specific substitutions in envelope in RP macaques suggests novel selection pressures on virus in such animals and suggests that viral variants that evolve in these animals may play a role in disease progression.

The role of the immunoreceptor tyrosine-based activation motif (ITAM) that is unique to the Nef protein of the acutely pathogenic simian immunodeficiency virus SIVsmPBj was studied in the context of two AIDS-inducing simian immunodeficiency virus molecular clones. NefY+variants of SIVagm9063-2 and SIVsmE543-3 replicated in and induced proliferation of unstimulated pig-tailed macaque PBMC. The pathogenesis of the NefY+and NefY−clones of SIVagm9063-2, SIVsmE543-3, and PBj6.6 were evaluated by intravenous inoculation of pig-tailed macaques (Macaca nemestrina). Introduction of the ITAM did not increase plasma viral RNA levels nor alter the kinetics of viremia compared with the NefY−versions of each clone. Clinical symptoms were not observed in animals inoculated with the NefY−variants. In contrast, characteristic PBj symptoms were observed in animals inoculated with any of the three NefY+clones. Blunting and fusion of intestinal villi and multifocal infiltration of mononuclear cells were observed in the gastrointestinal tracts of macaques inoculated with the NefY+versions. Lesions were associated with active viral replication, as demonstrated by simian immunodeficiency virus-specific in situ hybridization. However, only the macaque inoculated with wild-type NefY+SIVsmPBj developed fatal disease; lesions were more widespread and severe in this animal. A switch to macrophages as a viral reservoir and the presence of interleukin-6 in plasma was unique to the macaque infected with PBj6.6. Overall, these data suggest that the ITAM in SIV Nef alters the pathogenesis of simian immunodeficiency virus regardless of the viral background. The change in pathogenesis occurs without enhancement of viral replication. However, NefY+variants of SIVagm and SIVsm did not fully recapitulate the virulence of SIVsmPBj, implicating additional viral factors in this unique virus pathogenesis.

Previous studies with simian immunodeficiency virus (SIV) infection of rhesus macaques suggested that the intrinsic susceptibility of peripheral blood mononuclear cells (PBMC) to infection with SIV in vitro was predictive of relative viremia after SIV challenge. The present study was conducted to evaluate this parameter in a well-characterized cohort of six rhesus macaques selected for marked differences in susceptibility to SIV infection in vitro. Rank order relative susceptibility of PBMC to SIVsmE543-3-infection in vitro was maintained over a 1-year period of evaluation. Differential susceptibility of different donors was maintained in CD8+T-cell-depleted PBMC, macrophages, and CD4+T-cell lines derived by transformation of PBMC with herpesvirus saimiri, suggesting that this phenomenon is an intrinsic property of CD4+target cells. Following intravenous infection of these macaques with SIVsmE543-3, we observed a wide range in plasma viremia which followed the same rank order as the relative susceptibility established by in vitro studies. A significant correlation was observed between plasma viremia at 2 and 8 weeks postinoculation and in vitro susceptibility (P< 0.05). The observation that the two most susceptible macaques were seropositive for simian T-lymphotropic virus type 1 may suggests a role for this viral infection in enhancing susceptibility to SIV infection in vitro and in vivo. In summary, intrinsic susceptibility of CD4+target cells appears to be an important factor influencing early virus replication patterns in vivo that should be considered in the design and interpretation of vaccine studies using the SIV/macaque model.

Ensuring the safety of therapeutic modalities produced and purified from biological systems is of high concern. Regulatory authorities and the biopharmaceutical industry are continuously seeking to improve methods for the detection, identification, inactivation and removal of potentially contaminating pathogens in biotherapeutic products. Current methods for pathogen detection and identification are designed to discover adventitious as well as known microbial species in product samples. Many of these approaches require weeks or even months of observation, and the time involved is often a constraint on product release. This review focuses on current practices and technologies that have emerged in recent years, and highlights advances that have accelerated the time required for, and accuracy of, pathogen detection and identification. The biopharmaceutical industry has employed a multifaceted approach in pathogen detection, including the rigorous screening of blood/plasma donations; documented sourcing and screening of raw materials; thorough testing of production cell substrates and cell culture harvest material during processing, and at the stage of a final purified drug substance; and the evaluation of microbe clearance during purification operations. All these practices strive to ensure safety and mitigate the risk to patients undergoing biotherapeutic treatment.