Oral Ingestion of Synthetically Generated Recombinant Prion Is Sufficient to Cause Prion Disease in Wild-Type Mice

Prion disease is a group of transmissible neurodegenerative disorders affecting humans and animals. The prion hypothesis postulates that PrPSc, the pathogenic conformer of host-encoded prion protein (PrP), is the unconventional proteinaceous infectious agent called prion. Supporting this hypothesis, highly infectious prion has been generated in vitro with recombinant PrP plus defined non-protein cofactors and the synthetically generated prion (recPrPSc) is capable of causing prion disease in wild-type mice through intracerebral (i.c.) or intraperitoneal (i.p.) inoculation. Given that many of the naturally occurring prion diseases are acquired through oral route, demonstrating the capability of recPrPSc to cause prion disease via oral transmission is important, but has never been proven. Here we showed for the first time that oral ingestion of recPrPSc is sufficient to cause prion disease in wild-type mice, which was supported by the development of fatal neurodegeneration in exposed mice, biochemical and histopathological analyses of diseased brains, and second round transmission. Our results demonstrate the oral transmissibility of recPrPSc and provide the missing evidence to support that the in vitro generated recPrPSc recapitulates all the important properties of naturally occurring prions.


Introduction
Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), and Kuru in humans, scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle [1,2]. Different from other late age onset neurodegenerative disorders, prion disease is a naturally occurring infectious disease that can be transmitted within and between species [2]. It is now well established that in prion disease, the host-encoded PrP converts from its normal, soluble and protease sensitive PrP C form to the disease-associated, aggregated and protease resistant PrP Sc conformer [3]. The prion hypothesis posits that PrP Sc is the infectious agent and because of its seeding capability, PrP Sc is able to seed the conversion of host-encoded PrP C to PrP Sc , resulting in prion disease [1].
Using purified recombinant PrP (recPrP) plus defined non-protein cofactors, we previously showed that recPrP Sc can be generated in vitro with serial protein misfolding cyclic amplification (sPMCA) technique [4,5]. Similar to naturally occurring prions, recPrP Sc is aggregated, highly resistant to proteinase K (PK) digestion, and able to chronically infect susceptible cell lines. Importantly, recPrP Sc has a high titer of infectivity [6,7], causing bona fide prion disease in wild-type mice through i.c. or i.p. inoculation [4][5][6]. The pathogenic process of recPrP Sc -caused disease is identical to those observed in naturally occurring prion disease [6]. Biophysical analyses revealed that the conformation of recPrP Sc is similar to that of native PrP Sc [8] and the unique recPrP Sc conformation determines its pathogenicity in animals [9,10].
The studies of recPrP Sc provide strong experimental evidence to support a causative role of PrP Sc in prion disease. But whether the in vitro generated recPrP Sc is able to cause prion disease in wild-type animal via oral route remains unclear. Oral ingestion is a well-established route for the transmission of many naturally occurring prion diseases, including the outbreaks of Kuru and variant CJD in humans, scrapie in sheep and goats, CWD in cervids and BSE in cattle [2]. Establishing the oral infectivity of recPrP Sc would provide a critical evidence to unambiguously support that prion is the infectious agent for these naturally occurring prion diseases. In this study, we determined the oral transmissibility of recPrP Sc and showed that a single oral ingestion of recPrP Sc is sufficient to cause prion disease in wild-type mice.

A Single Oral Feeding of RecPrP Sc Causes Prion Disease in Wild-Type Mice
To determine whether recPrP Sc is able to cause prion disease via oral route, we prepared recPrP Sc with sPMCA. The sPMCA substrate without going through sPMCA reaction was used as a negative control. Brain homogenates prepared from a mouse that succumbed to prion disease (DBH, representing prion diseased brain homogenates) was used as a positive control. The presence or absence of PK-resistant PrP in these samples was verified by PK digestion and immunoblot analysis ( Figure 1A).
Pathogens 2020, 9, x FOR PEER REVIEW 2 of 8 PrP Sc is the infectious agent and because of its seeding capability, PrP Sc is able to seed the conversion of host-encoded PrP C to PrP Sc , resulting in prion disease [1]. Using purified recombinant PrP (recPrP) plus defined non-protein cofactors, we previously showed that recPrP Sc can be generated in vitro with serial protein misfolding cyclic amplification (sPMCA) technique [4,5]. Similar to naturally occurring prions, recPrP Sc is aggregated, highly resistant to proteinase K (PK) digestion, and able to chronically infect susceptible cell lines. Importantly, recPrP Sc has a high titer of infectivity [6,7], causing bona fide prion disease in wild-type mice through i.c. or i.p. inoculation [4][5][6]. The pathogenic process of recPrP Sc -caused disease is identical to those observed in naturally occurring prion disease [6]. Biophysical analyses revealed that the conformation of recPrP Sc is similar to that of native PrP Sc [8] and the unique recPrP Sc conformation determines its pathogenicity in animals [9,10].
The studies of recPrP Sc provide strong experimental evidence to support a causative role of PrP Sc in prion disease. But whether the in vitro generated recPrP Sc is able to cause prion disease in wildtype animal via oral route remains unclear. Oral ingestion is a well-established route for the transmission of many naturally occurring prion diseases, including the outbreaks of Kuru and variant CJD in humans, scrapie in sheep and goats, CWD in cervids and BSE in cattle [2]. Establishing the oral infectivity of recPrP Sc would provide a critical evidence to unambiguously support that prion is the infectious agent for these naturally occurring prion diseases. In this study, we determined the oral transmissibility of recPrP Sc and showed that a single oral ingestion of recPrP Sc is sufficient to cause prion disease in wild-type mice.

A Single Oral Feeding of RecPrP Sc Causes Prion Disease in Wild-Type Mice
To determine whether recPrP Sc is able to cause prion disease via oral route, we prepared recPrP Sc with sPMCA. The sPMCA substrate without going through sPMCA reaction was used as a negative control. Brain homogenates prepared from a mouse that succumbed to prion disease (DBH, representing prion diseased brain homogenates) was used as a positive control. The presence or absence of PK-resistant PrP in these samples was verified by PK digestion and immunoblot analysis ( Figure 1A). The presence of PK-resistant PrP in the inocula was detected by immunoblot analysis with 3F10 anti-PrP antibody. C, sPMCA substrate; S, recPrP Sc ; B, prion diseased mouse brain homogenate. Samples were digested with 25 µg/mL PK at 37 °C for 30 min (for C and S) or 60 min (for B). (B) Survival curve of mice that received indicated inocula via i.c. or p.o. route. DBH, prion diseased mouse brain homogenate. (C) The presence of PK-resistant PrP in mouse brain homogenates was detected by immunoblot analysis with 3F10 anti-PrP antibody after 25 µg/mL PK digestion at 37 °C for 60 min. C1, a negative control mouse that was orally fed with sPMCA substrate; C2, a positive control mouse that received i.c inoculation of recPrP Sc . Mice that received oral feeding of recPrP Sc (mouse #6-#16) or DBH (mouse #17-#22) were indicated. The presence of PK-resistant PrP in the inocula was detected by immunoblot analysis with 3F10 anti-PrP antibody. C, sPMCA substrate; S, recPrP Sc ; B, prion diseased mouse brain homogenate. Samples were digested with 25 µg/mL PK at 37 • C for 30 min (for C and S) or 60 min (for B). (B) Survival curve of mice that received indicated inocula via i.c. or p.o. route. DBH, prion diseased mouse brain homogenate. (C) The presence of PK-resistant PrP in mouse brain homogenates was detected by immunoblot analysis with 3F10 anti-PrP antibody after 25 µg/mL PK digestion at 37 • C for 60 min. C1, a negative control mouse that was orally fed with sPMCA substrate; C2, a positive control mouse that received i.c inoculation of recPrP Sc . Mice that received oral feeding of recPrP Sc (mouse #6-#16) or DBH (mouse #17-#22) were indicated.
For oral feeding, each mouse was individually housed in a clean cage without food and bedding. A mouse food pellet doused with recPrP Sc , sPMCA substrate or DBH was placed on the floor of the cage. After complete ingestion of the food pellet, the mouse was returned to its home cage and monitored for the development of signs of neurodegeneration. Intracerebral inoculation of the same batch of recPrP Sc or DBH was performed as positive controls to demonstrate the prion infectivity in these preparations.
As expected, all mice that received i.c. inoculation of recPrP Sc or DBH developed neurological signs of prion disease, including clasping, tail plasticity, hypokinesia, kyphosis and ataxia, and reached terminal stage at 196.2 ± 3.59 and 176.4 ± 5.17 days post inoculation (dpi), respectively ( Figure 1B and Table 1). Around 250 days after oral feeding, clasping was observed in one mouse (#9) from recPrP Sc -fed group and another mouse (#19) from DBH-fed group. Both mice developed unsteady gait, tail plasticity, kyphosis and weight loss, but urinary incontinence was only observed in #19 mouse. Disease progressed quickly and both mice reached terminal stage in about 40 days ( Figure 1B and Table 1). Except for these two mice, no sign of prion disease was observed in other mice that were orally fed with recPrP Sc , DBH or sPMCA substrate. All mice were sacrificed at 650 dpi. Mouse brain homogenates were prepared and subjected to PK digestion. Immunoblot analysis revealed that the classic PK-resistant PrP Sc was detected in the positive control mice that received i.c. prion inoculation, the #9 and #19 mice that were orally fed with recPrP Sc and DBH, respectively, but not in other mice that were orally fed with recPrP Sc or DBH ( Figure 1C). Histopathological analyses revealed classical pathological changes in #9 and #19 mice, including spongiosis, astrogliosis, microgliosis and the accumulation of PK-resistant PrP ( Figure 2).

Second Round Transmission
If #9 and #19 mice indeed developed prion disease, the disease should be able to serially transmit in mice. In addition, the lower infectivity of orally fed recPrP Sc may create a subclinical state with prion infectivity generated in the brains of clinically normal mice [11][12][13]. Alternatively, the orally fed recPrP Sc may be subject to truncation or alteration in conformation due to oral digestion, which may need further adaptation in vivo to become a fully infectious prion [14]. To test these possibilities, we selected three groups of mice that (1) developed classic characteristics of prion disease (#9 and #19); (2) developed clasping, but without other signs of prion disease (#10, #14 and #21); (3) appeared normal (#8 and #18). Wild-type C57BL/6 mice were intracerebrally inoculated with 1% brain homogenates prepared from these mice.
All mice that were inoculated with #9 or #19 mouse brain homogenates developed fatal neurodegeneration with a survival time of 191 ± 4.97 dpi and 187 ± 3.38 dpi, respectively ( Figure 3A and Table 2). For mice that received the inoculation of other mouse brain homogenates, although clasping was observed in some of them, none of the mice developed typical signs of prion disease and all were sacrificed at 330 dpi ( Figure 3A and Table 2). Biochemical analysis revealed that the PK-resistant PrP Sc was only detected in mice that were inoculated with #9 or #19 mouse brain homogenates ( Figure 3B). Consistent with this finding, spongiosis and PK-resistant PrP were only detected in mice that were inoculated with #9 or #19 brain homogenate, but not in mice that received other inocula (Figure 4). orally fed with recPrP , DBH or sPMCA substrate. All mice were sacrificed at 650 dpi. Mouse brain homogenates were prepared and subjected to PK digestion. Immunoblot analysis revealed that the classic PK-resistant PrP Sc was detected in the positive control mice that received i.c. prion inoculation, the #9 and #19 mice that were orally fed with recPrP Sc and DBH, respectively, but not in other mice that were orally fed with recPrP Sc or DBH ( Figure 1C). Histopathological analyses revealed classical pathological changes in #9 and #19 mice, including spongiosis, astrogliosis, microgliosis and the accumulation of PK-resistant PrP (Figure 2).  Immunohistochemical stains were counterstained with hematoxylin. Scale bar represents 200 µm.

Second Round Transmission
If #9 and #19 mice indeed developed prion disease, the disease should be able to serially transmit in mice. In addition, the lower infectivity of orally fed recPrP Sc may create a subclinical state with prion infectivity generated in the brains of clinically normal mice [11][12][13]. Alternatively, the orally fed recPrP Sc may be subject to truncation or alteration in conformation due to oral digestion, which may need further adaptation in vivo to become a fully infectious prion [14]. To test these possibilities, we selected three groups of mice that 1) developed classic characteristics of prion disease (#9 and #19); 2) developed clasping, but without other signs of prion disease (#10, #14 and #21); 3) appeared normal (#8 and #18). Wild-type C57BL/6 mice were intracerebrally inoculated with 1% brain homogenates prepared from these mice.
All mice that were inoculated with #9 or #19 mouse brain homogenates developed fatal neurodegeneration with a survival time of 191 ± 4.97 dpi and 187 ± 3.38 dpi, respectively ( Figure 3A and Table 2). For mice that received the inoculation of other mouse brain homogenates, although clasping was observed in some of them, none of the mice developed typical signs of prion disease and all were sacrificed at 330 dpi ( Figure 3A and Table 2). Biochemical analysis revealed that the PKresistant PrP Sc was only detected in mice that were inoculated with #9 or #19 mouse brain homogenates ( Figure 3B). Consistent with this finding, spongiosis and PK-resistant PrP were only detected in mice that were inoculated with #9 or #19 brain homogenate, but not in mice that received other inocula (Figure 4). inoculation of brain homogenates (BH) prepared from indicated mice. Control, mice that received i.c. inoculation of brain homogenate prepared from mice which were orally fed with sPMCA substrate in the first round. (B) PK-resistant PrP Sc in mouse brain homogenates was detected by immunoblot analysis with 3F10 anti-PrP antibody after 25 µg/mL PK digestion at 37 °C for 60 min. C, a control mouse that was i.c. inoculated with brain homogenate prepared from a negative control mouse which was orally fed with sPMCA substrate in the first round. #9-3 and #19-4 are representative mice that were i.c. inoculated with brain homogenates prepared from #9 and #19 mice, respectively. For mouse numbering, the first letter represents the first round mouse number. inoculation of brain homogenates (BH) prepared from indicated mice. Control, mice that received i.c. inoculation of brain homogenate prepared from mice which were orally fed with sPMCA substrate in the first round. (B) PK-resistant PrP Sc in mouse brain homogenates was detected by immunoblot analysis with 3F10 anti-PrP antibody after 25 µg/mL PK digestion at 37 • C for 60 min. C, a control mouse that was i.c. inoculated with brain homogenate prepared from a negative control mouse which was orally fed with sPMCA substrate in the first round. #9-3 and #19-4 are representative mice that were i.c. inoculated with brain homogenates prepared from #9 and #19 mice, respectively. For mouse numbering, the first letter represents the first round mouse number.   Figure 4. Pathological changes in mice that received second round transmission. As indicated, mouse brains were subjected to HE stain or PET blot analysis to determine the pathological changes. Control is a negative control mouse that was i.c. inoculated with brain homogenate prepared from a negative control mouse in the first round. Mice were indicated by the first round mouse number. BH, brain homogenates. Scale bar represents 200 µm.

Discussion
Since the first success in generating recPrP Sc 10 years ago [4], recPrP Sc has been extensively studied by multiple labs. However, its ability to cause prion disease via oral route had never been proven. Our study provided the first experimental evidence that oral feeding of recPrP Sc is sufficient to cause prion disease in wild-type mice. Because orally ingested prions are exposed to the digestion process in the gastrointestinal tract that is known to degrade PrP Sc [15], our results indicate that the in vitro generated recPrP Sc is able to survive the digestion, maintain its infectious conformation, spread to the central nervous system and cause a fatal neurodegenerative disease in wild-type mice.
Although orally ingested recPrP Sc successfully causes prion disease, the attack rate is low and Sc Figure 4. Pathological changes in mice that received second round transmission. As indicated, mouse brains were subjected to HE stain or PET blot analysis to determine the pathological changes. Control is a negative control mouse that was i.c. inoculated with brain homogenate prepared from a negative control mouse in the first round. Mice were indicated by the first round mouse number. BH, brain homogenates. Scale bar represents 200 µm.

Discussion
Since the first success in generating recPrP Sc 10 years ago [4], recPrP Sc has been extensively studied by multiple labs. However, its ability to cause prion disease via oral route had never been proven. Our study provided the first experimental evidence that oral feeding of recPrP Sc is sufficient to cause prion disease in wild-type mice. Because orally ingested prions are exposed to the digestion process in the gastrointestinal tract that is known to degrade PrP Sc [15], our results indicate that the in vitro generated recPrP Sc is able to survive the digestion, maintain its infectious conformation, spread to the central nervous system and cause a fatal neurodegenerative disease in wild-type mice.
Although orally ingested recPrP Sc successfully causes prion disease, the attack rate is low and only one out of 11 mice developed disease. The i.c. inoculation of the same batch of recPrP Sc , however, resulted in 100% attack rate and a rather synchronized survival time of 196.2 ± 3.59 days ( Figure 1B and Table 1), indicating a higher infectivity. The reduced efficiency of orally fed recPrP Sc is consistent with previous reports that compared to i.c. prion inoculation, the infectivity of oral dosing reduces by a factor~10 5 in mice [16] and~10 9 in hamster [17]. With gastric gavage, Sigurdson et al. showed that 6.4 LogLD 50 infectious dose of RML prion caused prion disease in one of 11 mice [18]. Although we did not titrate the batch of recPrP Sc used in this study, our previous study showed that a batch of similarly prepared recPrP Sc contains~10 4 LD 50 (by i.c. route)/µg of PrP [6], which indicates that the infectious dose of recPrP Sc fed to a mouse in this study would be around 5 LogLD 50 (by i.c. route). Even including the consideration of potential variations in each in vitro recPrP Sc preparation, the dose used in our study would be similar or lower than that used by Sigurdson et al. [18]. Therefore, we concluded that the attack rate of orally fed recPrP Sc is comparable to that of naturally occurring prions. Notably, the attack rate of orally fed DBH is only 1 out of 6 mice in our study, which is also significantly lower than that of i.c. inoculation ( Figure 1B and Table 1).
In order to more stringently recapitulate the natural spread of prion via oral route, we chose to feed mice with recPrP Sc instead of using gastric gavage, which would increase its exposure to the digestion and may also contribute to the low attack rate. In future studies, the infectivity of the inocula should be carefully titrated by i.c. inoculation. The dosage of orally fed recPrP Sc can be increased and if necessary, the inocula can be concentrated by centrifugation. Together with increasing the size of the experimental group, these measures will allow us to accurately compare the rate of oral transmissibility of recPrP Sc to that of DBH. In addition, a second round p.o. transmission may help to determine whether the disease can be serially transmitted via oral route, and whether the particular prion strain induced by orally fed recPrP Sc has a preference for oral transmission. Despite these limitations of our study, the success of orally fed recPrP Sc to cause prion disease in wild-type mice does support the idea that PrP Sc , the pathogenic conformer of PrP, is the cause for orally transmitted prion disease.
Several naturally occurring prion diseases, such as scrapie in sheep and goats and CWD in cervids, appear to be quite efficient in oral transmission [19][20][21], which may be attributed to the particular prion strains and animal species. Oral transmission of these natural prion diseases could also be enhanced by other factors, such as the binding of prion to soil particles [22], the presence of bacterial colitis [18], the alteration of intestinal M cells density regulated by RANKL [23] and lesions to the oral mucosa [24]. It will be interesting to determine whether any of these factors are able to enhance the oral transmission of recPrP Sc in future studies.
Collectively, our study revealed that oral ingestion of in vitro generated recPrP Sc is sufficient to cause prion disease in wild-type mice, which provides the first example that an in vitro generated pathogenic conformer of a recombinant protein is able to cause a fatal neurodegenerative disease via oral route. Together with previous findings, our results support that as the prion hypothesis postulated, the misfolded PrP conformer is responsible for the transmissibility of prion disease.

Mice
Wild-type C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and 6-week-old female mice were used in this study. Mice were maintained under specific pathogen free (SPF) conditions. All mouse experiments were performed according to

Prion Exposure and Disease Monitoring
Preparation of inocula, i.c. inoculation, second-round i.c. transmission and the analyses of mouse brains were performed as previously described [5,25]. Oral exposure was performed according a previously reported protocol [23,26]. Briefly, a single food pellet was doused with 50 µL of sPMCA substrate, recPrP Sc or 1% (w/v) DBH, and allowed to dry at room temperature. Mice were individually housed in bedding-and food-free cages during the oral feeding. A single food pellet was placed on the floor of the cage. Once the food pellet was completely ingested, the mouse was returned to its home cage. For i.c. inoculation, 30 µL of recPrP Sc or 1% (w/v) DBH was injected into an anesthetized mouse. After oral feeding or i.c. inoculation, all mice were monitored three times a week. Once neurological signs were clearly identified, mice were monitored daily and euthanized at terminal disease stage. Efforts were made to minimize pain and suffering of animals.