Unswitched immunoglobulin M response prolongs mouse survival in ...

Journal of General Virology (2009), 90, 777–782

Short Communication

DOI 10.1099/vir.0.005041-0

Unswitched immunoglobulin M response prolongs mouse survival in prion disease Mourad Tayebi,1,2 John Collinge3 and Simon Hawke2 1

Correspondence

Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK

Simon Hawke [email protected]

2

Brain & Mind Research Institute, University of Sydney, 100 Mallett Street, Camperdown, NSW 2050, Australia

3

MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK

Received 6 July 2008 Accepted 14 October 2008

Several studies have failed to demonstrate the presence of immune responses to infectious prions during the course of prion disease, reflecting the identical primary structure of normal and disease-associated isoforms and the widespread expression of the normal cellular form of prion protein, PrPC, leading to B- and/or T-cell tolerance of disease-associated isoforms and also possibly because antigen-presenting cells are unable to process the highly aggregated, detergent-insoluble, protease-resistant form, PrPSc. Under certain circumstances, PrPSc can be revealed to the immune system in immunogenic form, and it has been shown previously that antiPrP antibodies can be induced to prions immunoadsorbed to Dynabeads using specific anti-PrP monoclonal antibodies, even in PrP-sufficient mice. This study demonstrated in a murine scrapie model that PrP–Dynabeads effectively stimulated the immune system to produce anti-PrP IgM antibodies over prolonged periods after repeated immunization. It was also shown that these immune responses prolonged incubation times in murine scrapie.

Prions diseases, or transmissible spongiform encephalopathies, which include Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy and scrapie in animals, are a group of invariably fatal diseases with a pathogenesis that involves the transformation of the mainly a-helical normal cellular prion protein, PrPC, into a disease-associated isoform, PrPSc, that acquires increased b-sheet content, detergent insolubility and resistance to proteases. How the formation of prions induces neurotoxicity is still poorly understood, but there is an absolute requirement for the expression of PrPC (Bueler et al., 1993). Although prions potentially adopt non-native conformations that might be recognized by the immune system during the course of natural infection, no antibody responses to prions have yet been identified, undoubtedly because of the widespread systemic expression of PrPC, rendering the adaptive immune system tolerant of it (McBride et al., 1992). Furthermore, native PrPSc has not been considered immunogenic (Peretz et al., 1997), possibly because antigen-presenting cells are incapable of processing the highly aggregated, protease-resistant protein. Native prions have generally failed to stimulate an immune response in experimental animal models, and few antibodies have been produced that recognize infectious prions in native form (Tayebi & Hawke, 2006). 005041 G 2009 SGM

Initially, scrapie-associated fibrils (SAFs) were used as the immunogen to generate an immune response in mice and rabbits. Although purified intact infectious SAFs in native form did not elicit detectable natural or experimental immune responses (Kascsak et al., 1987), a weakly measurable immunoreactive antibody reaction was evoked if SAFs were exposed to formic acid or SDS. So-called prion rods were also purified and then used to produce anti-PrP antibodies in rabbits (Prusiner et al., 1993) and mice (Williamson et al., 1996). Similar to the SAF preparations, prion rods also underwent substantial and stringent purification steps (Prusiner et al., 1993), which may have led to their denaturation and increased immunogenicity The immune responses induced by both the SAF preparations and the prion rods produced antibodies that bound weakly or not at all to diseaseassociated isoforms. Generally, the induction of anti-PrP antibodies using native or recombinant PrP has required using PrPC-null mice (Bueler et al., 1993), unless species-specific sequence variations are present in the immunogen. Nakamura et al. (2003) used native PrP to immunize PrPnull mice to generate anti-PrP antibodies. In their study, scrapie-infected cells were used as the immunogen to generate anti-PrP antibodies, demonstrating de facto that native PrP could be used as a potent immunogen without

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recourse to intensive and stringent protocols for preparing the native immunogen, as has been shown with SAFs (Rubenstein et al., 1999). The monoclonal antibodies (mAbs) raised against scrapie-infected cells did not discriminate between PrP proteins, indicating that PrPC, PrPSc or both lead to antibody stimulation in PrP-null mice (Nakamura et al., 2003). Finally, Spinner et al. (2007) immunized PrP-null and wild-type mice with native PrPSc, with coincident stimulation of Toll-like receptor 9 using CpG oligodeoxynucleotides. Stimulation of the humoral response following prion immunization was found to greatly enhance anti-PrP antibody production. In general, immunizing rodents with syngeneic native PrP does not elicit responses in wild-type mice. We were therefore surprised to find that PrP-sufficient mice immunized with partially purified, native PrPSc immunoadsorbed to Dynabeads using anti-PrP mAbs raised in our laboratory (Tayebi et al., 2004) produced polyclonal anti-PrP antibodies predominantly of the IgM subclass. Of note, our immunization strategy revealed an immunodominant region that lies between aa 101 and 120, a strong indication of its immunogenic influence in native PrPSc (Tayebi et al., 2004; Jones et al., 2009). In more recent work, Jones et al. (2009) successfully used this unravelled motif to produce antibodies that were apparently PrPSc-specific. Antibody-based therapy appears to be the best approach and has been effective in scrapie-susceptible neuroblastoma (N2a) cells (Enari et al., 2001; Peretz et al., 2001; Gilch et al., 2003). Furthermore, its in vivo application and, more specifically, in transgenic mice harbouring a PrP antibody m-chain led to resistance to peripheral infection (Heppner et al., 2001). Vaccination with recombinant PrP (rPrP) (Sigurdsson et al., 2002) and prion peptides (Schwarz et al., 2003) and mucosal vaccination (Goni et al., 2005, 2008) were shown to have some efficacy in prolonging the disease incubation period. Goni and colleagues used a live attenuated strain of Salmonella typhimurium expressing the mouse PrP gene (Prnp) and achieved full protection against oral prion infection only in mice with high antibody titres (Goni et al., 2005, 2008). Most importantly, anti-PrP mAbs were found to be effective in preventing prion replication in vivo and animals remained free of detectable prion infection for over 500 days after equivalent untreated animals had succumbed to the disease (White et al., 2003). The purpose of the current study was to determine whether long-lived IgM anti-PrP antibodies can influence the course of experimental murine scrapie. All procedures involving animals were carried out under a project and personal licence authority issued in accordance with The Animals (Scientific Procedures) Act 1986 and approval by the Institutional Ethics Committee. FVB/N mice (Harlan-Olac, UK) (10 per group) were immunized via the intraperitoneal route with 100 ml PrP– 778

Dynabeads emulsified with complete Freund’s adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) prepared as described previously (Tayebi et al., 2004). Animals were monitored daily for clinical symptoms of scrapie (White et al., 2003). PrP–Dynabeads were shown to be highly effective at stimulating strong polyclonal anti-PrP responses in Prnp0/0 mice by rPrP ELISA (Tayebi et al., 2004). Briefly, medium-binding, 96-well plates (Greiner) were coated with 50 ml rPrP solution (10 mg ml21) in coating buffer. The plates were incubated for 1 h at 37 uC, washed three times with PBS/0.05 % Tween 20 (PBST), and then blocked with 10 % fetal calf serum for 1 h at room temperature. After decanting the RF10, 50 ml of the relevant mAb was added and incubated for 1 h at 37 uC. The plates were then washed three times with PBST, a 1 : 1000 dilution of horseradish peroxidase-conjugated anti-mouse IgM (Sigma) was added for 25 min at 37 uC and the plates were washed four times with PBST. Finally, the plates were developed with OPD buffer (Sigma) until optimum development occurred; the reaction was stopped with 3 M H2SO4 prior to spectrophotometric reading at 490 nm. We next used Dynabead-adsorbed antigens and appropriate controls to immunize mice inoculated with scrapie in order to demonstrate long-term persistence of anti-PrP antibodies and to look for prolongation of the incubation period. Groups of 6-week-old female PrP-sufficient FVB/N mice were inoculated by the intracerebral route with 30 ml 1 % brain homogenate derived from terminally ill mice infected with the RML mouse-adapted scrapie strain. The anti-PrP mAbs ICSM 35 and ICSM 18 (D-Gen), which detect both PrPC and PrPSc in Western blots, were adsorbed to Dynabeads. The experimental groups of mice were immunized with ICSM 35–Dynabeads to which PrPSc had been adsorbed from proteinase K-digested, scrapieinfected brain homogenate (ICSM35/Dynabeads/RML) and ICSM 18–Dynabeads to which PrPSc had been adsorbed from PK-digested, heat-treated, scrapie-infected brain homogenate (ICSM18/Dynabeads/RML). Control groups comprised non-immunized mice (prion inoculated only), a group immunized with Dynabeads that were adsorbed with scrapie-infected brain homogenate lacking the capture antibody (Dynabeads/RML) and a group immunized with CFA followed by IFA (plus Dynabeads) only (Table 1). The amount and concentration of scrapieinfected brain homogenate adsorbed to the Dynabeads had been optimized previously (Tayebi et al., 2004), and 100 ml of 1 % homogenates was incubated with Dynabeads saturated (or not) with either ICSM 18 or ICSM 35 (White et al., 2003). After washing with PBS/0.1 % Tween 20, the immunocomplex was emulsified with CFA and IFA prior to the subcutaneous immunizations. All groups of mice were immunized 30 days after intracranial or intraperitoneal inoculation with strain RML.


Journal of General Virology 90

IgM anti-PrP responses prolong murine scrapie

Table 1. Effect of active immunization with PrPSc–Dynabeads on survival of FVB/N mice inoculated with RML scrapie IC or IP inoculation* IC IC IC IC IC IP IP IP IP IP

ImmunogenD

ICSM35/Dynabeads/RML ICSM18/Dynabeads/RML Dynabeads/RML CFA+IFA None ICSM35/Dynabeads/RML ICSM18/Dynabeads/RML Dynabeads/RML CFA+IFA None

Start of active immunization (days p.i.)

FVB/N mice succumbing to scrapied

Mean survival time (days p.i.)

Increase in survival compared with untreated mice (%)

30 30 30 30 – 15 15 15 15 –

10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10

149±4 153±3 146±2 148±1 151±2 224±16 224±22 198±2 198±2 202±2

0 3 3 0 – 20 23 0 0 –

*Pooled RML scrapie brain homogenate was inoculated intracerebrally or intraperitoneally. The infectious titre of the pooled homogenate was determined as 8.1 log LD50 (g brain)21 by infectivity assay with tga20 indicator mice. FVB/N mice were inoculated intracerebrally (IC) with 30 ml or intraperitoneally (IP) with 100 ml 1 % homogenate. DOnce every 2 weeks. dNo. of animals succumbing to scrapie/no. of animals inoculated.

Although the level of antiserum induced was consistently high (Fig. 1) as shown by ELISA analysis throughout the incubation period, all mice that were challenged with prions via the intracerebral route succumbed to disease at the same time as unimmunized, prion-challenged controls (Figs 1a and 2a), namely around 144–156 days post-inoculation (p.i.) (Table 1). We have shown previously (White et al., 2003) that passive immunization of anti-PrP mAbs was ineffective if begun at the onset of clinical scrapie or after intracerebral challenge, probably reflecting the inadequacy of IgG translocation across the blood–brain barrier, which limits molecular translocation depending on charge and molecular size unless specific transporters are utilized. PrP–Dynabeads have been shown to induce a predominantly IgM response in vivo (Tayebi et al., 2004). IgM is a large molecule. Once the infectious agent, PrPSc, has established itself in the brain parenchyma, it will ultimately lead to PrPSc accumulation in the brain (Kimberlin & Walker, 1988), even in splenectomized mice (Kimberlin & Walker, 1989). It is therefore probable that, as occurred with our anti-PrP IgG mAbs, the IgM anti-PrP antibodies, being considerably larger molecules, were unable to cross the blood–brain barrier to neutralize the infectious agent. Of note, the group of mice that received CFA and IFA only showed no increase in incubation period when compared with untreated mice. These results are not in agreement with the findings of Tal et al. (2003), who demonstrated an increase in the incubation period and survival of scrapieinfected mice after injection with CFA, although equivalent amounts of PrPSc were immunodetected in control and CFA-immunized mice. Prions accumulate in the lymphoreticular system, including the spleen, long before neuroinvasion occurs in most http://vir.sgmjournals.org

Fig. 1. Repeated immunization with PrPSc–Dynabeads induces prolonged IgM secretion in wild-type mice challenged intracerebrally and intraperitoneally with RML scrapie. Mice were immunized once every 2 weeks with ICSM35/Dynabeads/RML (&), ICSM18/ Dynabeads/RML (m), Dynabeads/RML (.) or CFA/IFA (with Dynabeads, $) following intracranial (a) or intraperitoneal (b) inoculation with RML-infected brain homogenates. Anti-PrP responses were assayed in mice (n510 for each group) using a standard recombinant protein ELISA. Mice were bled once a month and a 50 ml volume of a 1 : 100 dilution of the IgM-containing serum (non-purified) was assayed for anti-PrP antibody by ELISA.


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(Table 1). The various immunogens were emulsified with CFA prior to subcutaneous immunizations. It has been established that when an animal is immunized with an antigen, it produces a primary antibody response of low magnitude and short duration, peaking at about 10– 17 days. A second immunization with the same antigen results in a secondary response that is greater in magnitude, peaks in less time (2–7 days) and lasts longer than the primary response. We decided to immunize mice at 15 days p.i. so that significant amounts of anti-PrP antibodies had been induced at the time of maximal accumulation of prions in the spleen (the so-called plateau phase occurring at 30 days p.i.).

Fig. 2. Effect of active immunization with PrPSc–Dynabeads on prion disease progression in mice. Mice were immunized once every 2 weeks and anti-PrP responses were measured using a standard recombinant protein ELISA. (a) Mice were inoculated intracranially with RML scrapie prions and immunized by active immunization with ICSM35/Dynabeads/RML, ICSM18/ Dynabeads/RML, Dynabeads/RML and CFA/IFA (with Dynabeads). (b) Mice were inoculated intraperitoneally with RML scrapie prions and immunized by active immunization with the immunogens described in (a). The mean survival times of mice immunized with ICSM35/Dynabeads/RML was 224±16 days (P52¾10”8) and with ICSM18/Dynabeads/RML was 224±22 days (P52¾10”5) compared with a survival time of 202±2 days for untreated intraperitoneally inoculated mice.

experimental scrapie mouse models (Eklund & Hadlow, 1973; Kimberlin & Walker, 1979). Time-course analyses of peripheral PrPSc accumulation in mice confirmed that PrPSc was detectable in the spleens at 7 days and reached a plateau by 30–40 days after peripheral challenge (Beringue et al., 2000). Similar to the intracerebral inoculation, groups of FVB/N female wild-type mice, at 6 weeks of age, were challenged via the intraperitoneal route with prions (100 ml 1 % brain homogenate derived from terminally ill RML-challenged mice). Each group was immunized 15 days after the intraperitoneal RML challenge with various immunogens, including ICSM35/Dynabeads/RML and ICSM18/ Dynabeads/RML. Control groups included non-immunized mice (prion inoculated only), a group immunized with Dynabeads adsorbed with scrapie-infected brain homogenate lacking the capture antibody (Dynabeads/ RML) and a group immunized with CFA and IFA only 780

Mice that were inoculated intraperitoneally with RML scrapie (Fig. 2b) and subjected to immunization with ICSM35/Dynabeads/RML and ICSM18/Dynabeads/RML once every 2 weeks, starting at 15 days p.i., survived for much longer than untreated mice or mice immunized with CFA and IFA only (Table 1 and Fig. 2b). The mean survival time for untreated mice inoculated by the intraperitoneal route was 202±2 days. Mice immunized with ICSM35/ Dynabeads/RML or ICSM18/Dynabeads/RML from 15 days p.i. had a mean survival time of 224±16 days [P5261028; analysis of variance (ANOVA)] and 224±22 days (P5261025; ANOVA), respectively. Therefore, the increase in survival of both groups compared with untreated mice was significant and in the order of 24 days (Table 1), an extension of the incubation period of at least 11 %. Clinical signs were observed in all mice after the delay of disease onset. Furthermore, as already shown with mice challenged through the intracerebral route, mice challenged intraperitoneally and then immunized with CFA followed by IFA at 15 days p.i. did not show any increase in the incubation period when compared with untreated intraperitoneally challenged mice (Fig. 2b). The mean survival time of CFA-immunized mice was 198±2 days p.i. and 202±2 days p.i. for the untreated group. Antibody titres were determined by serial dilutions of sera in 96-well microtitre ELISA plates coated with b-sheet rPrP (data not shown). To determine the correlation between antibody levels and duration of disease incubation time (Fig. 1), the mice were bled once a month and a 50 ml volume of a 1 : 100 dilution of the serum was assayed for anti-PrP antibody by ELISA. Interestingly, antibody levels were not dramatically reduced prior to the onset of clinical signs of disease, indicating that, although antisera increased the incubation period, probably through a peripheral reduction in PrPSc accumulation, they did not completely alter or prevent neuroinvasion or subsequent fatal encephalopathy. Sigurdsson et al. (2002) also used rPrP in active immunization and showed no apparent differences in the degree of spongiform change or PrPSc levels at the time of sacrifice using histological and Western blot evaluations, although immunized groups of mice had a



IgM anti-PrP responses prolong murine scrapie

significant delay in the onset of scrapie symptoms. In another study, rPrP did not have any effect on disease incubation (Schwarz et al., 2003), emphasizing the importance of the specificity of the antibodies raised in vivo. In this report, we have shown that active immunization with PrPSc adsorbed to Dynabeads elicited an immune response that was effective in delaying the onset of prion disease in rodents. Support for immunotherapy for prion diseases was initially provided by in vitro studies in which anti-PrP antibodies suppressed prion replication in cell lines (Enari et al., 2001; Peretz et al., 2001; Beringue et al., 2004). Translating this strategy to the in vivo situation, Heppner et al. (2001) produced B-cell transgenic mice expressing an anti-PrP antibody m-chain and found that these animals were resistant to peripheral infection. To date, the most efficient suppression of active prion replication in vivo has come from our work showing that anti-PrP mAbs, administered systemically, efficiently suppress prion replication in peripheral infection. Passively transferred antibodies obviously had no effect after prions had gained access to the central nervous system (White et al., 2003). Because of the large amounts of anti-PrP mAbs required for these experiments, the approach is not likely to gain utility in treating animal prion diseases, and providing sufficient quantities of inhibitory antibodies in the brain will be a major challenge for treating humans with Creutzfeldt– Jakob disease, but the experiments suggest that humoral immunity might have an effect in preventing disease prior to or after exposure. Certainly, there is a pressing need to develop strategies to increase the resistance of animals to prion diseases. Active vaccination would be the logical approach. Indeed, vaccination with mouse rPrP has been shown to delay the onset of prion disease in mice (Sigurdsson et al., 2002). In these experiments, murine rPrP was used to immunize rodents before or after peripheral inoculation with infectious prions. A delay in disease onset was observed in both groups, but was more prolonged in animals immunized before exposure. Antibody titres were measured and were found to correlate closely with an increase in incubation time. Interestingly, we have been unable to induce antibody to murine PrP in PrP-sufficient mice using highly purified murine rPrP either in full-length or truncated (aa 91–231) form as the immunogen (S. Hawke and Z. Sattar, unpublished data). The active immunization approach was also found to be effective through the use of synthetic PrP-derived peptide (aa 105–125) in mice that were infected by dietary exposure to the scrapie infectious agent (Schwarz et al., 2003), with prolonged survival time. In our study, active immunization using PrP–Dynabeads as the immunogen modestly delayed the onset of prion disease. This is the first report of using native PrP as a vaccine, and also the first to report on the efficacy of IgM anti-PrP antibodies to inhibit prion replication in wildhttp://vir.sgmjournals.org

type mice (Tayebi et al., 2004; Jones et al., 2009). Of course, vaccinating naı¨ve animals with prions would not be reasonable, but other means of inducing antibodies to repetitive protein units without resorting to using prions might be possible (Kayed et al., 2003). Paramithiotis et al. (2003) have provided data indicating that IgM anti-PrP antibodies are inducible with synthetic peptides, although information regarding the efficacy of these antibodies at inhibiting prion replication has not been published. Adsorption of PrPSc to Dynabeads using either ICSM 18 or ICSM 35 led to a significant increase in the incubation period of RML-challenged mice (Fig. 2b). The use of ICSM 18 and ICSM 35, which recognize distinct epitopes, in the passive immunization process (White et al., 2003) has shown differences in reducing peripheral PrPSc accumulation, as ICSM 18 was shown to be more efficient in PrPSc clearance. ICSM 18 recognizes a region in helix 1, thought to play a crucial role in prion replication (Heppner et al., 2001; Westaway & Carlson, 2002). The various studies using antibodies as a basis for the treatment of prion diseases in mice have shown great promise, but it remains to be established whether the findings in rodents are transferable to humans. Furthermore, the reported problems with amyloid b1–42 vaccination in Alzheimer’s disease (Dodart et al., 2003) and the neuronal-mediated apoptosis with anti-PrP antibodies (Solforosi et al., 2004) suggest that caution is required when targeting self antigens expressed in the central nervous system, even if they are also expressed in the periphery. Nevertheless, vaccination would be the only economical way to rid animal populations of prion disease, and in this study we have shown that there are epitopes on PrPSc to which the IgM anti-PrP antibodies that can be induced can bind, presumably reflecting repeated peptide motifs exposed on the ordered prion aggregate. These epitopes may not be exposed in PrPC, thus avoiding the potential problem of autoimmunity. Finally, it remains an open question as to whether small quantities of anti-PrP IgM antibodies are induced during natural infection with prions and whether such antibodies have a role in susceptibility or resistance to disease.

Acknowledgements This work was supported by the Jessie and Isabel Alberti Program grant from the University of Sydney Medical Foundation and the Medical Research Council (UK).

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