Candidate Cell Substrates, Vaccine Production, and Transmissible Spongiform Encephalopathies

Candidate cell substrates neither accumulated abnormal prion protein nor propagated infectivity.

T ransmissible spongiform encephalopathies (TSEs or prion diseases) are a heterogeneous group of fatal neurodegenerative diseases that affect animals and humans. TSEs can be sporadic, transmitted iatrogenically, or expressed as familial disorders. TSEs include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in cervid ruminants, and mink encephalopathy. In humans the most common TSEs are sporadic, familial, and variant Creutzfeldt-Jakob disease (sCJD, fCJD, and vCJD, respectively). TSEs are characterized by the accumulation in the central nervous system and, less often, in lymphoid tissues of TSEassociated prion protein (PrP TSE ), a conformational variant of a normal host cellular prion protein (PrP C ). PrP C is a nonessential protein but, at least in mice and cows, must be expressed by animals susceptible to TSE infection. There is compelling evidence that the BSE agent has infected humans, causing vCJD. Most cases of vCJD are attributed to exposure to contaminated beef products (1)(2)(3). In addition, vCJD infections have been transmitted by transfusions of nonleukoreduced erythrocyte concentrates and by a humanderived coagulation factor (factor VIII) (4)(5)(6).
The conclusion that PrP TSE is central to the pathogenesis of TSE is based on the temporal and anatomic correlations between accumulation of PrP TSE and the development of pathologic changes in tissues of the central nervous system (1). However, TSEs can develop in the absence of detectable PrP TSE and, conversely, PrP TSE might accumulate without causing either clinical illness or the neuropathologic alterations typical of TSEs (i.e., a progressive fatal illness with spongiform degeneration of the brain) (7,8). In short, the molecular basis of TSE infection and the role of PrP TSE (unquestionably important in pathogenesis of TSEs) are not yet entirely clear, and both remain key issues in TSE research (9,10). The standard assay for detecting a TSE agent remains bioassay in susceptible animals.
Many investigators once believed that TSE agents infected mainly, if not exclusively, cells of neuronal and lymphoid lineages. It has become clear, however, that the susceptibility of cells to infection with TSE agents cannot be reliably predicted either from their tissue of origin or level of expression of PrP C (11,12). Studies showing that murine fi broblast cell lines are susceptible to infection with mouse-adapted scrapie agent (11,12) increased concern that nonneuronal cell substrates used to propagate viruses for vaccine production might become infected with a TSE agent contaminating some component of culture medium, especially bovine serum (13). The theoretical risk of contaminating vaccines or other biologic products prepared in culture cells with TSE agents from animal-derived materials in media has been considered low. However, 1) as noted above, the blood of asymptomatic humans has transmitted vCJD, and 2) in a variety of experimentally TSE-infected animals, TSE agent has been detected in blood, mainly in nucleated cells and plasma (4)(5)(6)(14)(15)(16)(17). Fortunately, no human vaccine has ever been implicated as a source of iatrogenic TSE. However, 2 animal tissuederived vaccines have caused outbreaks of scrapie in sheep, and 2 medical products of human origin-dura mater allograft and human cadaveric pituitary hormones (no longer marketed in the United States)-have transmitted hundreds of cases of CJD; corneal grafts have transmitted a few cases as well (2,18). Since 1993, the US Food and Drug Administration has recommended against the use in the manufacture of biological products of bovine-derived materials from countries identifi ed by the US Department of Agriculture (USDA) as having BSE or being at increased risk for BSE in native cattle (19).
The recognition of >20 BSE cases in North America since 2003 (most in Canada) has increased the need to determine whether cell substrates that might be accidentally exposed to the BSE agent are capable of acquiring and propagating the infectious agent and potentially transmitting infections to vaccine recipients (20). To address these issues, we investigated the susceptibility of cell lines used or proposed for manufacture of biologics and controls to propagate TSE agents, especially the BSE agent, under simulated worst-case conditions.
The BSE-infected bovine brain was a 10% brain suspension in 250 mmol/L sucrose. The infectivity titer of that material is being determined in squirrel monkeys (>10 2 / inoculum [22]) intracerebral 150 μL and intraperitoneally 150 μL with 10 −1 (wt/vol) through 10 −9 dilutions of low speed-clarifi ed brain extracts. Nonhuman primates were also inoculated with a 10 −2 dilution of brain extract after fi ltration through a 0.45-μm Millipore membrane to eliminate bacterial contamination. Three animals each  were inoculated with the BSE agent dilution 10 −1 to 10 −6 ; 2 animals each were inoculated with the same material dilution 10 −7 to 10 −9 . Similar bacteria-free samples were used to expose cell substrates. The ic infectivity titer of the BSE agent in transgenic mice expressing the bovine PrP gene (TgBo) was 5.0 log 10 LD 50 /30 μL (L. Cervenakova, unpub. data). Brain extracts from animals with neurologic signs contained PrP TSE by Western blot (WB) analysis, and TSE was confi rmed neuropathologically. All BSE experiments were performed under BioSafety Level 3 containment conditions in facilities inspected and approved by the USDA. Scrapie agent used as a positive control was the 22L mouse-adapted strain (11).

Inoculation of Cells with TSE Agents
Cells were grown in 25-cm 2 tissue culture fl asks and overlaid with 200 μL of a detergent-free homogenate of brain tissue diluted to 1% (wt/vol) in Opti-MEM (Gibco, Grand Island, NY, USA). Bacteria-free inocula were obtained by fi ltration of 1% brain extracts through premoistened 0.45-μm Millipore membranes (Millipore, Billerica, MA, USA). In an attempt to mimic a worst-case scenario, in some instances cells were exposed to brain extracts by gently centrifuging them in 25-cm 2 tissue culture fl asks for 5 min at ≈900 × g to ensure maximum contact with TSE agents. After incubation of the cell cultures at 37°C for 4 h, 400 μL of fetal bovine serum/Dulbecco modifi ed Eagle medium was added, and cells were incubated for 96 h before further passaging. To determine the presence of PrP TSE in cells, WB analyses were done, usually at passages 0, 5, 10, 15, 20, and 30. The same protocol was used with NIH-3T3 and L929 murine cells exposed to mouse-adapted 22L scrapie agent (as positive controls).

Animal Models
TgHu and TgBo mice were developed at the American Red Cross (L. Cervenakova, unpub. data). TgHu mice express in the brain ≈4-fold higher levels of PrP than wild-type mice. TgBo mice express in the brain wild-type levels of PrP. These Tg mice do not develop spontaneous neurologic illness. Neuropathologic studies of selected aged mice showed no abnormalities such as vacuolation of the neuropil typical of TSEs. Conventional C57/BL6 mice were used to titrate 22L scrapie agent. Squirrel monkeys were purchased from Osage Research Primates (Kaiser, MO, USA) and housed in a USDAapproved BioSafety Level 3 animal facility. In a separate series of experiments, a work in progress, we are addressing the hypothesis, postulated by some authorities (13), that a TSE agent might develop spontaneously in cell cultures expressing mutated or nonmutated PrP. To that end, we are investigating whether transfected cell lines overexpressing wild-type or mutant PrP might become spontaneously infectious. Transfected cells are being bioassayed in squirrel monkeys. Although none of those monkeys showed development of any neurologic disease (7 years after inoculation), 3 animals have died of unrelated causes (1 with pneumonia and 2 culled with unexplained nontuberculous granulomatosis) without evidence of TSE neuropathologic changes. We used tissues of those 3 monkeys as negative controls. All animal experiments were reviewed and approved by the Food and Drug Administration and the American Red Cross Institutional Animal Care and Use Committees.

Preparation of Inocula for Bioassay in Mice and Primates
Samples of selected cultures at various passage levels and the fi nal subcultures after >30 passages were examined for PrP TSE by WB by using published protocols (14). Tg mice were inoculated intracerebrally with 30 μL of cell lysates from 1 × 10 8 cells/mL to 2 × 10 9 cells/mL (depending on the cell line) by 3 cycles of freezing and thawing followed either by sonication or by forcing through hypodermic needles of increasingly small gauge. Tg mice were inspected daily for signs of illness and euthanized either after illness developed or at the end of a normal expected lifespan (Tables 2, 3). The same neuropathologist, blinded to the experimental design, interpreted all stained sections used for neuropathologic studies. The same cell lysates were also inoculated into primates, 150 μL intracerebrally and 150 μL intraperitoneally. Animals were inspected daily for signs of illness and promptly euthanized when defi nite neurologic signs or intercurrent illnesses developed. Animals without clinical illness will be maintained for their expected normal lifespans. After euthanasia, brains were removed and portions stored frozen for WB (22) or in 10% formalin solution for histopathologic and immunohistochemical studies by using published protocols (8).

TSE in TgBo Mice and Primates Inoculated with BSE agent
Neuropathologic characterization of TgBo mice inoculated with BSE agent showed spongiform degeneration in the cerebrum with variable amounts of fi ne-punctate, coarse, and, in some cases, plaque-like deposits in the cerebrum, cerebellum, and brain stem. Mice inoculated with the bacteria-free fi ltrate of 1% BSE-infected brain suspension used to expose cells also developed signs of TSE, and PrP TSE was detected in brains, demonstrating that the inoculum used to expose cell cultures contained a TSE agent transmissible to mice (Figure 1).
Squirrel monkeys were inoculated with serial dilutions of the same material used to inoculate TgBo mice. At the time this report was written, 6 monkeys have already developed neurologic signs typical of TSE. Three animals inoculated with 10 -1 (10%) unfi ltered low-speed clarifi ed BSE reference material became ill and were euthanized ≈3.2 years after inoculation; 2 primates inoculated with 10 -2 unfi ltered, low speed-clarifi ed BSE suspension were euthanized 3.7 years after inoculation; and 1 primate inoculated with the 0.45-μm fi ltered bacteria-free 10 -2 (1%) BSE-infected brain suspension used to expose cells also developed signs of TSE and was euthanized 3.3 years after inoculation. Brain extracts from each of these monkeys contained PrP TSE demonstrated by WB. Preliminary neuropathologic studies of formalin-fi xed paraffi n-embedded brain sections of each of the 6 animals showed severe spongiform degeneration of the cerebrum, cerebellum, and brainstem. Immunohistochemical studies showed widespread accumulations of PrP-immunopositive deposits throughout the brain of each monkey ( Figure 2). Control brains from 4 other squirrel monkeys dying of nonneurologic diseases, including 1 housed with monkeys used to titrate BSE agent, showed no evidence of TSE ( Figure 2). A detailed neuropathologic report of all monkeys is in press (22).

Analysis of Cell Cultures and Bioassay in Rodents
No PrP TSE was detected in cells exposed to normal brain extracts (controls) or after passage 5 in any of the cell substrates or control TSE-resistant cells exposed to brain extracts containing TSE agents. PrP TSE was detected in some samples of cells collected 96 h after inoculation (passage 0), suggesting the probable presence of residual inoculum. The consistent failure to detect PrP TSE in any cell line exposed to TSE agents after >5 passages suggests that proteinase K-resistant PrP was not generated de novo Table 2. Cell lines exposed to BSE or vCJD or normal bovine or human brain suspension and bioassayed in TgBo mice*   under these experimental conditions. WB analyses of cells collected after 30 serial passages showed no detectable PrP TSE in any cell line (Figure 3). Samples of each cell line exposed to human TSE agents (sCJD, vCJD) and BSE agent were expanded after 30 passages for bioassay in TgHu and TgBo mice. At the time of this report, no mice inoculated with cells exposed to TSE agent have evidence of TSE illness during their expected lifespan (Tables 2, 3). WB with extracts of brain tissue from all culled animals showed no PrP TSE . Neuropathologic analyses of selected mouse brains found no spongiform encephalopathy or accumulations of PrP (data not shown). The results confi rm that no cell substrate propagated infectivity detectable by mouse bioassay, even when mice were observed for their expected lifespan.

Nonhuman Primate Bioassay
Lysates of Vero, CHO, WI-38, and R9ab cells exposed to BSE agent and passaged 30 times were inoculated into squirrel monkeys in May 2006. Lysates of MDCK and HEK-293 cells exposed to BSE agent and passaged 30 times were inoculated into squirrel monkeys in August 2007. No monkey inoculated with those cells had neurologic signs. One monkey inoculated with Vero cells exposed to the BSE agent was attacked by a cage mate; the wound became infected, suppuration increased despite treatment with antimicrobial drugs, and the injured monkey was euthanized several days later without ever showing any sign of neurologic disease. The brain showed no neuropathologic changes of TSE or PrP TSE by WB (data not shown). In contrast, as noted above, primates inoculated with Swiss Reference BSE brain extracts at 10 -1 and 10 -2 dilutions developed typical TSE confi rmed by neuropathologic results (Figure 2) and by WB (not shown). These results indicate that 1) squirrel monkeys are susceptible to infection with the BSE agent, and 2) BSE infectivity was present in the bacteria-free fi ltrate used to expose cell substrates.

Murine Fibroblast Cell lines Generate PrP TSE after Exposure to Mouse-adapted Scrapie Agent
Several murine tissue cultures have been successfully infected with TSE agents, providing a promising alternative to assays of TSE agents by time-consuming and expensive bioassays in animals (9,11,12,(23)(24)(25)(26)(27)(28). Previous studies reported that several commonly used mouse fi broblast cell lines can be effi ciently infected with the scrapie agent and support formation of PrP TSE (11). We studied 2 such cell lines as a positive control to confi rm that our protocol would have detected an infected cell line and that our failures to 2266 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011 fi nd PrP TSE or infectivity by bioassay in cell substrates exposed to the BSE agent were more likely to have resulted from an intrinsic resistance of the cells to infection rather than to some technical problem. We exposed monolayers of NIH-3T3 and L929 murine fi broblast cells to the mouse-adapted 22L strain of scrapie agent and observed the formation of readily detectable PrP TSE that persisted through 30 passages (Figure 4). We are performing bioassays of the PrP TSE -positive cells in C57/BL6 mice to determine amounts of infectivity. Several mice inoculated with samples of NIH-3T3 and L929 fi broblasts collected 30 passages after exposure to 22L mouse-adapted scrapie agent have already developed spongiform encephalopathy, confi rming that the agent was successfully propagated in vitro (unpub. data).

Discussion
Candidate cell substrates used to produce biologics were not infected by a simulated worst-case exposure to BSE agent. Similar more limited studies exposing the same cultures to vCJD and sCJD agents also gave negative results.
The fi nding of PrP TSE in several cell culture samples collected at passages 0-4 probably resulted from small amounts of residual inoculum. This conclusion is reinforced by our consistent failure to detect PrP TSE in any cell sample at or after passage 5. However, we cannot rule out the possibility of a transient de novo generation of PrP TSE in the earliest passages of the cultures. Other investigators (23) have shown some immediate (acute) formation of new PrP TSE in infection-resistant cell cultures exposed to scrapie agent; the new PrP formed did not depend upon the strain of TSE agent used or cell type involved and was not associated with infectivity (23). Whether the failure of infectivity to persist after transient formation of PrP TSE resulted from death of the infected cells or the dilution of a small amount of TSE agent is unknown. We recognized no overt cytotoxicity in any cell line inoculated with a TSEinfected brain suspension. Thus, our data so far suggest that several cell substrates actually or potentially used to produce biologics were not susceptible to the propagation of TSE agents under the experimental conditions we used. In agreement with these results, others showed that MDCK cells were refractory to infection with human and mouse TSE agents (24). MRC5 human diploid cells also failed to support the replication of a TSE agent (25).
Because bioassays are time-consuming and expensive, a few lines of cells susceptible to infection with certain strains of TSE agent have been derived (9,11,12,(26)(27)(28)(29)(30). However, for unknown reasons, most cell lines have resisted TSE infections. In addition, most cell lines infectable with TSE agents have been highly heterogeneous and not stable, requiring repeated subcloning of susceptible cells, and they have been successfully infected with only a few strains of TSE agent (27). Thus, it was vital to verify that the protocol we chose as a simulated worst-case model would successfully infect previously characterized cell lines with a TSE agent to which they were known to be susceptible. We demonstrated that the protocol we used was valid by persistently infecting 2 murine fi broblast cell lines with a mouse-adapted scrapie agent. However, we must caution that even cell lines susceptible to infection have shown widely different responses when exposed to various TSE agent strains (27). Furthermore, the emergence of atypical forms of BSE raises a new concern, i.e., that cell substrates resistant to infection with the original classic BSE agent (31) might not resist infection with newer strains of BSE agent (if new strains are implicated in atypical BSE).  5) and Vero cell (lanes 6-9). Cells exposed to normal bovine brain and passaged 30 times (lanes 2, 3, 6, 7). Cells exposed to bovine spongiform encephalopathy agent and passaged 30 times (lanes 4,5,8,9). Total PrP (cell extracts without proteinase K [PK] digestion) are shown in lanes 2, 4, 6, 8; cell extracts treated with PK are shown in lanes 3, 5, 7, 9. Western blots were probed with PrP monoclonal antibody 6D11. Figure 4. Western blot of brain extract from C57/Bl mouse inoculated with 22L mouse-adapted scrapie agent (lanes 1, 2); NIH-3T3 cells exposed to normal mouse brain and passaged 30 times (lane 3); NIH-3T3 (lane 4) and L929 (lane 5) cells exposed to 22L scrapie agent and passaged 30 times. Non-proteinase K [PK]-treated samples (lane 1), PK-treated samples (lanes 2-5). Western blots were probed with prion protein monoclonal antibody 6H4.
Although BSE has been transmitted to many animal species, the effi ciency of transmission between species has been diffi cult to predict. Low transmission rates and long incubation periods are often observed when TSEs are transmitted to a new species-commonly known as a species barrier (32)-but experience with squirrel monkeys (sensitive to almost all TSE agents affecting humans and to several animal TSEs as well) suggests that they are an especially useful model species (33). To that end we initiated titration experiments of the BSE agent and found at the time of writing this report that squirrel monkeys develop TSE when inoculated with BSE brain suspensions at high concentration 10 -1 and 10 -2 dilutions (work in progress). Given the long incubation time (years) needed to elicit disease in this primate model, we will continue the observation of monkeys inoculated with cell cultures exposed to the BSE agent in an attempt to determine whether low infectivity could potentially be detected in this model.
Because few cell lines used as substrates for biologics are of bovine origin, it is tempting to speculate that the potential for contaminating biologics with the BSE agent is low. However, an observation that cells derived from a pheochromocytoma of the rat adrenal medulla (PC12 cells) could be infected with a mouse-adapted scrapie agent and murine hypothalamic cells with a human TSE agent demonstrates that cross-species transmissions of TSE agents in cells in culture are possible (29). Recent studies showed that passage of TSE agents through different animal species altered key characteristics of the agent, sometimes producing variants with increased virulence or broader host range, as happened when BSE agent was passaged through sheep (34). Thus, further experiments are needed to evaluate the potential susceptibility of various cultured cell lines to infection with new emerging TSE strains.