Mucosal vaccination delays or prevents prion infection via an oral route

Neuroscience 133 (2005) 413– 421

MUCOSAL VACCINATION DELAYS OR PREVENTS PRION INFECTION VIA AN ORAL ROUTE F. GOÑI,a,i E. KNUDSEN,b F. SCHREIBER,h H. SCHOLTZOVA,a J. PANKIEWICZ,a R. CARP,d H. C. MEEKER,d R. RUBENSTEIN,e D. R. BROWN,f M.-S. SY,g J. A. CHABALGOITY,h E. M. SIGURDSSONb,c AND T. WISNIEWSKIa,b,c*

Key words: scrapie, immunization, Salmonella vaccine strain, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, chronic wasting disease.

Prion diseases are a unique category of illness where the pathogenesis is related to a conformational change of a normal protein, PrPC (prion protein cellular), to a form with a high ␤-sheet content, PrPSc (prion protein scrapie), that is infectious (Sadowski et al., 2004). Disease may be related to exogenous exposure, sporadic conformational change or to mutations that increase the propensity of PrPC to take up the PrPSc state. A major outbreak of a new prionosis, bovine spongiform encephalopathy (BSE), devastated the British beef industry, with new isolated cases being reported in many countries throughout the world, including the USA. BSE is related to the oral exposure of cattle to meat and bone dietary supplements that were contaminated with scrapie-infected sheep and/or other cattle with BSE. Subsequently, BSE was transmitted to humans with the emergence of new variant Creutzfeldt-Jakob disease (vCJD). By some estimates at least several thousand Britons are asymptomatic carriers of prion infection and will develop vCJD in the future (Hilton et al., 2002; Spinney, 2003). The emergence of human cases of vCJD many years after the near elimination of BSE in the UK is related to the very long incubation times of prion diseases, that range from months to decades (Sadowski and Wisniewski, 2004). However, once clinical symptoms begin the disease course progresses to death typically in a few months. Prion diseases are also found among wild animals. The disease, termed chronic wasting disease (CWD), has been affecting increasing numbers of wild and captive deer and elk populations in Western USA (Miller et al., 2004), raising the possibility of another source of animal prions which could overcome the species barrier and spread to humans (Belay et al., 2004). Currently no effective form of therapy exists for prion disease (Aguzzi et al., 2004; Sadowski and Wisniewski, 2004). Immune modulation is a promising therapeutic approach for neurodegenerative diseases which are characterized by the accumulation of a normal host protein in an abnormal conformation, such as prion and Alzheimer’s disease (AD). It has been shown in many studies that active immunization of AD model transgenic mice can reduce the amyloid plaque burden and produce behavioral improvement (Sigurdsson et al., 2004; Solomon, 2004). A number of reports suggest that such an approach may also be feasible for prion disease. It has been shown that mice, which transgenically express an anti-PrP monoclonal antibody, are almost completely resistant to prion infection

a

Department of Neurology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA b Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA c

Department of Psychiatry, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

d New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA e Department of Biochemistry, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA f

Departments of Biology and Biochemistry, University of Bath, UK

g

Institute of Pathology and Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA h Laboratory for Vaccine Research, Department of Biotechnology, Instituto de Higiene, Facultad de Medicina, University of Uruguay i

Department of Immunology, School of Chemistry, University of Uruguay

Abstract—In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrPC (prion protein cellular), to a toxic and infectious form, PrPSc (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. *Correspondence to: T. Wisniewski, New York University School of Medicine, Millhauser Laboratory, Room HN419, 550 First Avenue, New York, NY 10016, USA. Tel: ⫹1-212-263-7993; fax: ⫹1-212-263-7528. E-mail address: [email protected] (T. Wisniewski). Abbreviations: A␤, amyloid ␤; AD, Alzheimer’s disease; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; ELISA, enzyme-linked immunosorbent assay; GI, gastro-intestinal; Ig, immunoglobulin; LPS, lipopolysaccharide; PBST, phosphate-buffered saline, 0.1% Tween-20; PrPC, prion protein cellular; PrPSc, prion protein scrapie; recPrP, recombinant prion protein; TetC, fragment C of tetanus toxin; vCJD, variant Creutzfeldt-Jakob disease.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.02.031

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from a peripheral source (Heppner et al., 2001). A number of studies in tissue culture have shown that anti-PrP antibodies can suppress prion replication (Enari et al., 2001; Peretz et al., 2001). However, for prion disease active immunization is hampered by auto-tolerance against PrP. Our own studies have shown that active immunization with recombinant PrP (recPrP) given with Freund’s adjuvant can induce anti-PrP antibodies in wild-type mice and prolongs the incubation period of prion infection (Sigurdsson et al., 2002a). Nonetheless the increases in the incubation period in our past study and in a recent report of active immunization with synthetic PrP peptides (Schwarz et al., 2003), are relatively modest. In the present study we have focused on inducing mucosal immunity, because oral infection is the major route of prion transmission for many prionoses including: BSE, CWD (Belay et al., 2004), transmissible mink encephalopathy (Bartz et al., 2003), scrapie (Andreoletti et al., 2000; van Keulen et al., 2000) and in vCJD (Bruce et al., 1997). Induction of gut immunity holds the possibility of preventing entry of PrPSc before it can make significant contact with host PrPC. Furthermore, the possibility of toxicity related to autoimmunity is much lower, since the primary response is gut immunoglobulin (Ig)A production with a more limited systemic immune response. We have used a live attenuated S. typhimurium vaccine strain (LVR01) as a vector expressing the mouse PrP cDNA. Salmonella vaccine strains have been extensively used in mice to deliver foreign antigens and elicit a mucosal immune response (Lillard et al., 2001; Mastroeni et al., 2001; Pasetti et al., 1999, 2003), inducing mucosal IgA and systemic IgG production. This approach has also been successfully used in humans (Nardelli-Haefliger et al., 1996). We report that such an approach is successful for induction of mucosal anti-PrP immunity, which can significantly prolong the incubation period or prevent infection following oral exposure to the prion agent.

EXPERIMENTAL PROCEDURES Construction of a recombinant Salmonella vaccine strain expressing tandem copies of PrP The construction of the S. typhimurium aroC LVR01 has been previously described (Chabalgoity et al., 2000). The construction of the PrP expression vector was as follows: plasmid pTECH2 (kindly provided by Dr. C. M. Anjam Khan, Institute for Cell and Molecular Biosciences & School of Biomedical Sciences, University of Newcastle, UK) allows the expression of multiple tandem copies of a foreign antigen as a C-terminal fusion to the non-toxic fragment C of tetanus toxin (TetC) (Khan et al., 1994). The full length coding sequence of mouse PrP was amplified by PCR from a plasmid (Brown, 2003) using primers especially designed according to the published sequence to amplify the full cDNA, beginning with the start codon and finishing at the stop codon. Forward and reverse primers were tailored with BamHI and SpeI respectively to allow directional cloning into pTECH2. The construction of TetC fusions comprising two tandem copies of mouse PrP was done as previously described (Chabalgoity et al., 1996). Briefly, aliquots of the recombinant fusion vector were simultaneously digested with either XbaI and PstI, or with SpeI and PstI. Each digest generated two restriction fragments; from each, the fragment containing the PrP sequence was purified. The over-

hangs generated by XbaI and SpeI are compatible, but the recognition sites for both of these enzymes are destroyed upon ligation. Thus, the XbaI and SpeI sites flanking the PrP sequence remain unique and the procedure can be serially repeated, doubling the copy number of the peptide with each cycle. The plasmid constructs encoding either one or two copies of PrP insert were introduced into Salmonella LVR01 by electroporation. The expression of rPrP by the Salmonella LVR01 strain was assessed by SDS-PAGE and Western blotting using anti-PrP 7F9 (Liu et al., 2001), as previously described (Chabalgoity et al., 1996).

Animal and vaccination protocols Prior to inoculation into mice, the bacteria were cultured overnight on Luria broth at 37 °C with continuous shaking. The bacterial suspensions were centrifuged at 1200⫻g for 20 min at 15 °C, washed once with sterile PBS, centrifuged again and diluted to 1⫻1011 colony forming units/ml. Groups of 20 female CD-1 mice, 6 weeks of age, were orally immunized with S. typhimurium LVR01 expressing one or two copies of mouse PrP (PrPx1 or PrPx2, respectively). A control group of 20 mice were orally immunized with S. typhimurium LVR01 carrying pTECH without the PrP insert. The mice were subject to a 3 h food fast and each exposed to 2⫻1010 viable cells of the vaccine strain in 0.36 M NaHCO3, pH 8.3 in a 0.5 ml volume mixed in a 4:1 ratio with alum ([Al(OH)3], Alhydrogel, Superfos Biosector, Denmark), via gavage (direct placement of a feeding tube orally into the stomach and delivering the bacteria over approximately 10 s). The use of the sodium bicarbonate and alum was to neutralize gastric secretions, in order to increase survival of the LVR01 within the stomach and provide additional adjuvanticity in the intestine, as previously reported (Hajishengallis et al., 1995). The number of viable cells in every inoculum was determined by motility, using a hemocytometer on light microscopy at 400⫻ magnification. The oral vaccinations were repeated 7 days and 4 weeks after the original inoculation. Seven weeks after the original mucosal vaccination the mice were orally challenged via gavage with 100 ␮l of a 1:5 dilution of a brain homogenate of the mouseadapted scrapie strain 139A. An additional control group of 10, age-matched CD-1 mice received the oral challenge of 139A scrapie strain, without first receiving the vaccine nor the Salmonella vaccine strain. Mice were bled prior to the first vaccination (T⫽0), immediately prior to oral challenge with scrapie strain 139A (T⫽1) and at the time of kill (T Final). In addition, all animals which did not show signs of clinical infection were bled 400 days following scrapie strain 139A challenge (T Final). In addition to being bled, at the same time points feces were collected from the mice in PBS pH 7.2, 0.25% SDS/1 mM PMSF in order to quantitate gut IgA titers, essentially as has been described previously (Dounce et al., 1997). At least six fresh pellets per animal were collected and immediately placed in 1.2 ml of the above buffer. The pellets were immediately homogenized with a spatula, vortexed at high speed for 15 s and centrifuged at 14,000⫻g for 20 min; the clear supernatant was separated and stored at ⫺20 °C until used. Kill was performed when the mice scored positive for clinical signs of prion using a test of motor co-ordination for 3 consecutive weeks by observers blinded to the experimental status of the animals, using an established protocol (Aucouturier et al., 2001; Carp et al., 1984; Sigurdsson et al., 2002a). In addition, mice clinically healthy 500 days following inoculation, were killed to assess for the presence of sub-clinical prion infection. Mice were clinically assessed once a week starting 100 days following the 139A scrapie strain challenge. The analysis of clinical symptoms consists of observing the activity level and competency of the mice on an apparatus containing a series of parallel bars (3 mm in diameter) placed 7 mm apart. The initial clinical findings are

F. Goñi et al. / Neuroscience 133 (2005) 413– 421 a reduction in activity and/or the ability of the mice to traverse the parallel bars. This clinical endpoint correlates with the pathological development of CNS scrapie infection (Aucouturier et al., 2001; Carp et al., 1984; Kimberlin and Walker, 1989; Sigurdsson et al., 2002a). All experiments conformed to guidelines on the ethical use of animals. The number of animals used was minimized, as was the animals’ suffering. The plasma was tested for reactivity against recPrP by enzyme-linked immunosorbent assay (ELISA). The brains from ketamine/xylazine-anesthetized mice were removed, and the right hemisphere was immersion-fixed overnight in periodate–lysine–paraformaldehyde, whereas the left hemisphere was snap frozen for Western blots. The fixed brain hemispheres were subsequently transferred to a solution containing 20% glycerol and 2% dimethylsulfoxide in 0.1 M sodium phosphate buffer, and stored at 4 °C until sectioned. Serial coronal sections (40 ␮m) were cut and series of sections at 0.2 mm intervals were obtained for histological analysis. The diagnosis of prion disease was confirmed by staining brain sections with Cresyl Violet, immunostaining with a monoclonal anti-PrP antibody 7H9 (Liu et al., 2001) and by the detection of proteinase K resistant PrP on Western blots as previously described (Kascsak et al., 1997; Sigurdsson et al., 2003a).

Antibody levels IgG and IgA antibody levels to recPrP were determined on a 1:125 dilution of plasma in duplicate, in which mouse recPrP in 50 mM ammonium bicarbonate pH 9.6 at 50 ng/well was coated overnight onto microtiter wells. Antibodies were detected by a goat antimouse IgG or IgA linked to horseradish peroxidase (Sigma-Aldrich Co.) and tetramethyl benzene (Pierce, Rockford, IL, USA) was the substrate. In the feces supernatant extract, IgA levels to recPrP were determined in a 1:5 and 1:20 dilution of the extract in PBST, using the above protocol. The titers of specific anti-PrP IgA were normalized, correlated to the total IgA in each sample to account for the differing levels of collected feces and extracted IgA from these feces homogenates. The total IgA levels in each feces extract (␮g/ml) were determined using a mouse IgA quantitation kit and following the manufacturer’s instructions (Bethyl Laboratory. Inc., Montgomery, TX, USA). To test the efficacy of the delivery and adjuvanticity system, IgA antibody production against Salmonella LVR01 was assessed by measuring antibodies specific to Salmonella lipopolysaccharide (LPS) in the feces homogenates, of vaccinated and nonvaccinated mice at the time of oral challenge. Plates were coated with a solution consisting of 5 ␮g/ml LPS from S. typhi (SigmaAldrich Co.) in Reggiardo’s buffer, 0.1% deoxycholate (50 ␮l per well) and incubated overnight in a moist chamber at 37 °C. The plates were washed three times with PBS, 0.1% Tween-20 (PBST) and then feces supernatants were added diluted 1:3, 1:10 and 1:20 in 1%BSA PBST to wells (50 ␮l) in duplicate and incubated for 3 h at room temperature, followed by the same procedure as above.

Data analysis The Kaplan and Meier survival curve of the vaccinated and control animals was analyzed by the logrank test (GraphPad Prism, version 4; GraphPad Inc., San Diego CA, USA). The feces anti-LPS IgA level results were analyzed by one way ANOVA followed by a Neuman-Keuls test for post hoc analysis (GraphPad Prism). The anti-PrP IgA and IgG level results were analyzed by two-way repeated measures ANOVA followed by Bonferroni post hoc test (GraphPad Prism). Linear regression analyses were done using GraphPad Prism.

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Fig. 1. Shows the expression of recPrP by S. typhimurium LVR01 vaccine strain assessed by SDS-PAGE and Western blotting using anti-PrP antibody 7F9 (Liu et al., 2001). Five micrograms of bacterial suspension was run in each lane. In lane 1 only S. typhimurium without the PrP insert was run, while in lanes 2 and 3 S. typhi expressing either one or two copies of mouse PrP, respectively, were electrophoresed. The arrow indicates the PrP band. As has been previously reported when using this system to express tandem copies of other antigens in Salmonella (Chabalgoity et al., 1996), PrPx2 (lane 3) do not express the full fusion and instead express a fusion corresponding with the size of the fusion of tetC with a single copy of PrP, but at a higher level than the PrPx1 (lane 2). S. typhimurium LVR01 without the mouse PrP insert (lane 1) expresses no PrP.

RESULTS Expression of PrP by mouse-adapted Salmonella typhimurium LVR01 The expression of recPrP by S. typhimurium LVR01 vaccine strain was assessed by SDS-PAGE and Western blotting using anti-PrP antibody 7F9 (Liu et al., 2001). Fig. 1 shows the Western blot, in which 5 ␮g of bacterial suspension was run in each lane. In lane 1 only S. typhimurium without the PrP insert was run, while in lanes 2 and 3 LVR01 expressing either one or two copies of mouse PrP, respectively, was electrophoresed. The arrow indicates the PrP band. As can be seen, the strain encoding the construct with two tandem copies of PrP, did not express the full length fusion, and instead expressed a band of similar size to that corresponding with the fusion of TetC with a single copy of PrP, but at a higher level than the monomer construct (Fig. 1, lanes 2 and 3). Similar results have been previously observed when using this system to express tandem copies of other antigens in Salmonella vaccine strains (Chabalgoity et al., 1996). S. typhimurium LVR01 without the mouse PrP insert (lane 1) expressed no PrP. Survival of vaccinated mice Fig. 2 shows the Kaplan and Meier survival curve of the different groups. The two control groups that either received the S. typhimurium LVR01 without PrP expression or where not exposed to Salmonella were combined in a single control group, as there was no difference in survival between these control groups. By 300 days post-oral chal-

F. Goñi et al. / Neuroscience 133 (2005) 413– 421

0.0 Pr Px 1

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Feces IgA production to S. typhimurium LVR01 LPS was measured in all animals prior to the oral challenge with scrapie strain 139A. As can be seen in Fig. 3 there was no significant difference in the anti-LPS levels in the LVR01exposed groups, regardless of whether the LVR01 was expressing one or two copies of PrP or the vector alone. This verified that all vaccinated animals received an adequate oral challenge, indicating that gastric inoculation was successful and the bacteria were able to survive in the GI tract and induce an immune response. As expected, the control group of mice that was not exposed to LVR01 (but was then challenged with the scrapie strain 139A in the same manner as the other mouse groups) had no detectable anti-LPS antibodies. Since the control group that was exposed to LVR01 and the group not exposed did not differ in their survival time, these control group results were pooled for clarity of the data presentation (anti-PrP antibody level and survival time results). Linear regression analysis of the feces IgA anti-LPS compared with the feces anti-recPrP shows positive correlation (data not shown; r2⫽0.345, P⫽0.0005). This correlation is as expected, since mice would need to be exposed to the LVR01 in order to develop anti-PrP antibodies. Fig. 4 shows the anti-PrP IgA levels extracted from feces at the three collection time points. ANOVA analysis

LV R 01

indicates the differences between the treatment groups are significant (P⫽0.01). Post hoc Bonferroni tests show that at T⫽1 (immediately prior to scrapie 139A challenge) the IgA levels in the PrPx2 and PrPx1 groups are significantly different from their initial levels (P⬍0.01 and P⬍0.001,

PrPx1 Final

lenge with scrapie strain 139A all the control animals had shown clinical signs of prion infection which were confirmed by Western blotting. Five hundred days following the oral challenge with 139A prion strain 30% of each treatment group were alive and without clinical signs of infection. This represents a statistically significant difference between vaccinated and control groups (PrPx2 group versus controls, P⫽0.0004; PrPx1 group versus controls, P⫽0.0015) using the logrank test (GraphPad Prism, version 4; GraphPad Inc.).

Fig. 3. Shows the mean feces IgA antibody levels to S. typhi LVR01 LPS 7 weeks following the first inoculation and prior to the oral challenge with scrapie strain 139A (error bars indicate the S.E.M.). There was no significant difference in the anti-LPS levels in the LVR01 exposed groups, regardless of whether the LVR01 was expressing one or two copies of PrP or the vector alone. As expected the control group which was not exposed to LVR01 (last column) did not have detectable anti-LPS antibodies.

PrPx1 T0

Fig. 2. Shows the Kaplan and Meier survival curve of the different groups. By 300 days post-oral challenge with scrapie strain 139A all the control animals had shown clinical signs of prion infection which was confirmed by Western blotting. Five hundred days following the oral challenge with 139A prion strain, 30% of each treatment group were alive and without clinical signs of infection. This represents a statistically significant difference between vaccinated and control groups (PrPx2 group versus controls, P⫽0.0004; PrPx1 group versus controls, P⫽0.0015) using the logrank test (GraphPad Prism, version 4; GraphPad Inc.).

LV R 01 Pr P

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Fig. 4. Shows the mean anti-PrP IgA levels (⫾S.E.M.) extracted from feces supernatants at the three collection time points. ANOVA analysis indicates the differences between the treatment groups is significant (P⫽0.01). Post hoc Bonferroni tests show that at T⫽1 (immediately prior to scrapie 139A challenge) the IgA levels in the PrPx2 and PrPx1 groups are significantly different from the initial levels (* P⬍0.01, # P⬍0.001). The control group at T⫽1 is not statistically different from T⫽0. At the final collection time, there is a clear trend for the IgA levels to be higher then at the T⫽0 level. However, these differences just missed statistical significance at the P⬍0.05 level, by post hoc test.

Control Final

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control T1

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Histological and Western blot evaluations

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Fig. 5. Illustrates the mean anti-PrP IgG plasma levels (⫾S.E.M.) at the three time points. ANOVA analysis indicates the differences between the treatment groups is significant (P⫽0.0016). Post hoc Bonferroni tests show that the IgG levels in the PrPx2 and PrPx1 groups at T⫽1 are significantly different from the initial levels (* P⬍0.001) and the final collection time is also significantly different from the initial levels(# P⬍0.01, ⫹ P⬍0.05). The control mice do not differ significantly at either T⫽1 or at the final collection time from the T⫽0 levels (although at final collection there is a trend for an increase compared with T⫽0, which just missed significance by post hoc test).

respectively). The control group at T⫽1 is not statistically different from T⫽0. At the final collection time, although there is a clear trend for the IgA levels to be higher then at the T⫽0 level, these differences are not statistically significant. Fig. 5 illustrates the IgG plasma levels at the three time points. ANOVA analysis indicates the differences between the treatment groups are significant (P⫽0.0016). Post hoc Bonferroni test shows that the IgG levels in the PrPx2 and PrPx1 groups are significantly different from the initial levels at T⫽1 (P⬍0.001 for both groups) and at the final collection time (P⬍0.01 and P⬍0.05, respectively). The control mice do not differ significantly at either T⫽1 or at the final collection time from the T⫽0 levels. IgA production in the plasma was not detected above background by ELISA. Fig. 6 shows the correlation between the gastrointestinal (GI) IgA levels at T⫽1 and the incubation period. Animals which are still alive and asymptomatic currently are included as day 500 of incubation. There is significant correlation (r2⫽0.6974, P⬍0.0001), suggesting that GI IgA production may be protective against oral prion exposure, preventing penetration of the infectious agent through the mucosa. There is also positive correlation between the plasma IgG titer at T⫽1 and the incubation period, which is somewhat less strong (data not shown, r2⫽0.6291, P⫽0.001). Plasma IgG may also contribute to protection against the reduced dosage of scrapie agent which is able to penetrate the GI mucosa, but may also in part reflect the expected positive correlation between gut IgA and plasma IgG production (data not shown, r2⫽0.3598, P⫽0.001).

Histological and Western blot evaluations of all the brains of treated and control animals which were clinically ill, did not reveal any apparent differences in the degree of spongiform change or PrPSc levels at the time of kill (data not shown). Hence, mucosal immunization with LVR01 expressing PrP in animals which ultimately showed clinical signs of infection reduced the dosage of PrPSc entry and/or PrPSc propagation, but ultimately similar pathology and PrPSc levels were obtained. Clinically healthy animals which were killed 500 days after 139A infection, did not show evidence of PrPSc in their brains or spleens by Western blot evaluation.

DISCUSSION Historically, vaccination has been one of the most successful medical interventions. Recent studies have expanded the conditions where vaccination may be used, including neurodegenerative conformational disorders. These conditions include prion disease and AD. They are characterized by the accumulation of a constitutively expressed protein in an abnormal, pathology associated conformation. Numerous recent reports, including from our laboratories, as well as from many others, (reviewed in Solomon, 2004) have documented that active immunization with amyloid ␤ (A␤) peptides (a major constituent of the amyloid deposits in AD) or A␤ homologous peptides can significantly reduce the amyloid burden in AD transgenic mice, and also lead to cognitive improvements (Solomon, 2004). In prion disease, a difficulty with active immunization is overcoming tolerance against PrP. We and others have shown that vaccination with recPrP or PrP peptides can induce anti-PrP antibodies in wild-type mice, which are associated with a prolongation of the incubation period; although, the increase in the incubation period of the treatment groups was relatively modest (Schwarz et al., 2003; Sigurdsson et al., 2002a) i.e. about 2 weeks in these studies. However, these responses were achieved 1.25

Feces IgA Levels

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1.00 0.75 0.50 0.25 0.00 100

200

300

400

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Days of Incubation Fig. 6. Shows the correlation between the gut IgA levels at T⫽1 and the scrapie incubation period. Animals which are still alive and asymptomatic currently are included as day 500 of incubation. There is significant correlation (r2⫽0.6974, P⬍0.0001), suggesting that gut IgA production may be protective against oral prion exposure, preventing penetration of the infectious agent through the mucosa.

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by using parenteral routes of inoculation and conventional polyclonal activating and pro-inflammatory adjuvants. In the present report, the effect of mucosal vaccination is more striking. Our control group has a 185 day median survival time with all controls being dead by 300 days (see Fig. 2). In our treatment groups about 30% of the animals were still alive and without symptoms at 500 days post-oral scrapie exposure. These clinically healthy animals did not show evidence of PrPSc in their brains or spleens by Western blot evaluation (data not shown), suggesting these animals did not have sub-clinical infection, as has been reported to occur following some prion oral infections (Baier et al., 2003). However, we cannot completely rule out the possibility of low levels of PrPSc, below the level of our detection in these asymptomatic animals. Our treatment groups received LVR01 expressing either one or two copies of PrP. Although expression of two copies of PrP led to increased production of PrP (Fig. 1), this was not sufficient to affect the level of protection in the treated animals. For a mucosal anti-prion vaccine to be effective, the challenge is to break both general oral tolerance, as well as tolerance to a self-antigen (Polymenidou et al., 2004). In our mucosal vaccine, we succeeded in consistently obtaining a humoral immune response against PrP by using Salmonella as a delivery and adjuvant system. Of additional interest was the concomitant use of alum to enhance adjuvanticity at the mucosal level. The rationale for the use of alum is that it acts as a strong alkali that speeds passage from the stomach to the intestine and it can interact with antigen presenting cells in the Peyer’s patches, helping to produce a response against PrP in the same manner that alum promotes a response against a foreign antigen (Hajishengallis et al., 1995). Although we could not differentiate the influence of each adjuvant, our results of a modest but neutralizing response in the gut and an appreciable systemic IgG response, are consistent with the expected influence of these two adjuvants. Mucosal administration of auto-antigens typically results in the development of peripheral immunological tolerance (Mayer and Shao, 2004). Numerous studies have shown that mucosal tolerance induced by oral or nasal antigen inoculation effectively prevents several experimental autoimmune diseases (Mayer and Shao, 2004). However, oral antigen exposure can also lead to an immune response (Blanas et al., 1995; Mayer and Shao, 2004; Xiao and Link, 1997). The balance of tolerance to response is dependent on many factors including the dose of antigen, use of adjuvants and the age of the animal (Mayer and Shao, 2004). In our experiment we were able to overcome mucosal tolerance with the use of attenuated Salmonella as an adjuvant. Interestingly, mucosal immunization and induction of auto-antibodies has already been used in model animals to successfully treat AD, another conformational disorder similar to prion disease (Sadowski and Wisniewski, 2004). AD is characterized by the accumulation of a normal peptide, A␤, in an abnormal conformation (Wisniewski et al., 2002). Both nasal and oral administration of A␤ peptides in transgenic AD model mice has been shown to lead to the production of anti-A␤ auto-

antibodies associated with a reduction of amyloid deposits without the use of an adjuvant (Weiner et al., 2000). Intranasal co-administration of A␤ peptides with Escherichia coli heat-labile enterotoxin as an adjuvant increased anti-A␤ autoantibody titers (Lemere et al., 2002). Use of a recombinant adeno-associated virus vaccine expressing the A␤ peptide via a nasal or oral route has also been shown to reduce the amyloid burden and prevent behavioral impairment in model AD mice, due to anti-A␤ antibody production in the absence of a T cell proliferative response to A␤ (Hara et al., 2004; Zhang et al., 2003). These past studies and our study suggest that mucosal immunization may be a potential therapeutic approach for many conformational diseases (Sadowski and Wisniewski, 2004). It is interesting to note that in our control groups of mice at the final plasma and feces collection point a low anti-PrP IgG and feces IgA titer (which did not reach statistical significance on post hoc testing) was detectable in some animals (see Figs. 4 and 5). These control animals were orally exposed to brain homogenates of scrapie infection animals as their prion inoculum. We speculate that these low, but significant levels of gut IgA and plasma IgG antiPrP titers are a result of oral exposure to PrPSc, with the brain homogenate acting as an adjuvant. These low levels of anti-PrP antibodies did not provide protection against prion infection as they were not present prior to the time of exposure (T⫽1, see Figs. 4 and 5), and as a result could not prevent the entry of the scrapie agent systemically. It is not surprising that orally presented brain homogenate could act as a weak adjuvant, since the immune system normally has limited exposure to CNS tissue. In fact autoantibodies to a variety of CNS antigens, such as neurofilament as well as the prion protein, have been reported in prion disease where exposure to the prion agent is thought to occur following oral exposure to CNS tissue contaminated with the scrapie agent (Sotelo et al., 1980; Wilson et al., 2003; Wilson et al., 2004). These include the human prion disease kuru and in BSE (Sotelo et al., 1980; Wilson et al., 2003, 2004). The presence of these auto-antibodies in BSE has been suggested to play a role in the pathogenesis of the disease and also to have utility as a potential antemortem diagnostic test (Axelrad, 1998; Wilson et al., 2003, 2004). In another ongoing set of experiments we have orally inoculated mice with scrapie-infected brain homogenates or normal brain homogenate mixed with recombinant mouse PrP and are sampling plasma and feces at multiple times points. Our preliminary results (data not shown) indicate that a subset of both these groups of animals develops low level anti-PrP antibodies. The scrapie brain-exposed mice are not protected against infection as it takes at least 3 weeks for the anti-PrP antibodies to start developing, a time point where systemic dissemination of the prion agent has already occurred. In many prion diseases infection occurs from a peripheral source such as oral exposure. In these diseases, there is propagation and transport of prions outside the CNS, which is asymptomatic and occurs long before neuroinvasion. These include vCJD, BSE, scrapie, iatrogenic CJD and CWD (Sadowski et al., 2004). Our oral vaccination

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approach may be ideally suited for these prionoses. The prolongation of the incubation period in our experiments correlated significantly with the gut IgA production (see Fig. 6); although, there was some variability in the gut IgA among individual treated mice. This correlation suggests that our mucosal vaccination could in part prevent entry of the prion agent through the mucosal barrier. An important unanswered question is for how long mucosal immunization protects against prion infection? This question is being addressed in ongoing, additional experiments. In prion disease following oral challenge, there is first an early rise in prion infectivity in the distal ileum (Aguzzi et al., 2004). A number of studies have shown that Peyer’s patches accumulate prions and that mature follicular dendritic cells, located in the patches are critical for the transmission of scrapie out from the GI tract (Aguzzi et al., 2004). An additional advantage of a mucosal vaccination approach is that toxicity related to autoimmunity is less likely to occur. In the human AD vaccination trial, toxicity in the form of meningoencephalitis occurred in 6% of patients (Solomon, 2004), leading to the premature closure of this trial. Current evidence suggests that this was related to an excessive Th1 immune response, while the beneficial effects of immunization have been linked to the humoral Th2 response (Solomon, 2004). Mucosal immunization can lead to a predominantly Th2 response of local IgA and systemic IgG production. The cell-mediated immunity which has been linked to toxicity in the AD vaccine studies does not occur with many mucosal vaccines. In our study we did not see any evidence of toxicity in our vaccinated mice. Histological examination of the killed, vaccinated animals did not show any indication of autoimmune disease. Salmonella can potentially elicit a strong Th1 immune response, even when administered via an oral route (Xu and Ulmer, 2003). However, in the case of our Salmonella vaccine we specifically chose LVR01, which is not able to produce a systemic infection when delivered by an oral route (our unpublished results), reducing the possibility of producing a Th1 immune response. This is likely a significant reason why we did not observe toxicity. Whether toxicity could be a problem in other species remains to be determined. In addition to inducing a gut anti-PrP, IgA humoral response, our vaccine approach leads to low-level systemic anti-PrP IgG production. Several studies have shown that anti-PrP antibodies can inhibit the PrPC to PrPSc conversion (Enari et al., 2001; Peretz et al., 2001). Furthermore mice expressing both PrPC and a transgene for a defined anti-PrP antibody, were shown to be virtually resistant to prion infection. Our own studies and that of others, using passive immunization with anti-PrP antibodies in wild-type mice have also shown that antibodies can increase the resistance to prion infection (Sigurdsson et al., 2003b; White et al., 2003). Hence, the prolongation of the incubation period or prevention of disease in our vaccinated animals may result from a combination of a reduced infective inoculum via IgA neutralization in the gut and the presence of systemic anti-PrP IgG. However in our experimental system, we believe that it is more likely that IgA production is playing the critical role for the following reason: our previous active immunization exper-

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iment using recPrP injected with Freund’s adjuvant s.c., induced higher systemic anti-PrP IgG levels but was associated with a much lower degree of protection against prion infection (Sigurdsson et al., 2002a). In this prior experiment we used the same 139A prion inoculum and the same strain of mice; although, the route of infection was via intra-peritoneal injection. This active immunization protocol led to a prolongation of the incubation period of about 2 weeks, but all treated mice became clinically sick. In the present mucosal vaccination about 1/3 of the animals are without clinical signs of infection, consistent with our hypothesis that the mucosal neutralizing IgA is playing a significant role. Salmonella vaccine strains have been extensively used in mice and also in humans to deliver foreign antigens and elicit a mucosal immune response (Lillard et al., 2001; Mastroeni et al., 2001; Nardelli-Haefliger et al., 1996; Pasetti et al., 1999, 2003; Tacket et al., 1997). As indicated by our results, mucosal vaccination can be a highly effective method for inducing a Th2 non-inflammatory response, which we have suggested is an optimal immune modulating approach for neurodegenerative disorders (Sadowski and Wisniewski, 2004; Sigurdsson et al., 2001). Our findings in AD (Sigurdsson et al., 2001, 2002b, 2004) and prion animal models (Sigurdsson et al., 2002a, 2003b) reinforce the hypothesis that immunization is an important potential therapeutic approach for conformational disorders. Acknowledgments—This work was supported by NIH grants: NS47433, AG20197, AR2594 and the Alzheimer’s Disease Association.

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(Accepted 20 February 2005) (Available online 4 May 2005)