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Originally published online as doi:10.1189/jlb.1106665 on March 30, 2007 Originally published online as doi:10.1189/jlb.1106665 on March 22, 2007

Published online before print March 22, 2007
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(Journal of Leukocyte Biology. 2007;81:1374-1385.)
© 2007 by Society for Leukocyte Biology

CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils

Daryl S. Spinner*,1, Regina B. Kascsak*, Giuseppe LaFauci*, Harry C. Meeker*, Xuemin Ye*,2, Michael J. Flory*, Jae Il Kim*,3, Georgia B. Schuller-Levis*, William R. Levis{dagger}, Thomas Wisniewski{dagger}, Richard I. Carp* and Richard J. Kascsak*,1

* New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, USA; and
{dagger} New York University School of Medicine, New York, New York, USA

1 Correspondence: New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314, USA. E-mail: darylspinner{at}aol.com; rrkascsak{at}msn.com

ABSTRACT

Prion diseases are characterized by conversion of the cellular prion protein (PrPC) to a protease-resistant conformer, the srapie form of PrP (PrPSc). Humoral immune responses to nondenatured forms of PrPSc have never been fully characterized. We investigated whether production of antibodies to PrPSc could occur in PrP null (Prnp–/–) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrPSc antibody levels in wild-type (Prnp+/+) mice was also investigated. Prnp–/– and Prnp+/+ mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp–/– mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrPSc and recombinant PrP and exhibits a Kd in the 10–11 M range. In Prnp+/+ mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrPSc in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrPSc and suggest methods for optimizing the generation of mAbs to PrPSc, many of which could be used for diagnosis and treatment of prion diseases.

Key Words: transmissible spongiform encephalopathy (TSE) • monoclonal antibodies • innate immunity • Toll-like receptor 9 (TLR9 or TLR-9)

INTRODUCTION

The transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of infectious, invariably fatal, neurodegenerative diseases that affect the CNS. These diseases, which include scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids, and Creutzfeldt-Jakob disease in humans, are characterized by the development of spongiform degeneration in the brain and the conversion of the normal cellular prion protein (PrPC) to a pathologic, ß-rich conformer, which is designated the scrapie form of PrP (PrPSc; reviewed in ref. [1 ]). As PrPSc is highly resistant to proteolytic degradation, its clearance from the body is slow. Deposition of PrPSc occurs throughout the lymphoreticular tissues and the brain in patterns that depend on the TSE strain, the host genotype, and the route of infection (reviewed in ref. [2 ]).

The abnormal PrP, PrPSc, is a notoriously poor immunogen. This may partly be a result of its high degree of protease resistance, rendering it resistant to proteolytic degradation within phagocytic APCs, such as dendritic cells (DCs), macrophages, and microglia. As presentation of PrPSc fragments by APCs to T cells is so impaired, initiation of adaptive immune responses is blocked. While this may be true in PrPC-expressing and nonexpressing hosts, those hosts that express PrPC are further prevented from developing humoral immunity to PrPSc by immune tolerance [3 4 5 6 7 ]. Immune tolerance to these agents, which are composed largely or entirely of PrPSc, is avoided in PrP null (Prnp–/–) mice that support the production of anti-PrP antibodies [8 9 10 ].

Stimulation of innate immune signaling through members of the TLR family (TLR1–13) potentiates immune activation and the destruction of foreign pathogens such as bacteria, viruses, and fungi, and a variety of cancers [11 12 13 14 ]. The function of TLRs is to recognize pathogen-associated macromolecules and subsequently, to activate immune cells on which the TLRs reside [11 12 13 14 ]. TLR ligation also plays a role in adaptive immunity to certain pathogen-associated proteins by leading to a strong antibody response [15 ]. Bacterial DNA, which contains unmethylated CpG dinucleotides, or similar nucleic acid sequences, e.g., synthetic CpG oligodeoxynucleotides (ODNs), signal through TLR9 [16 , 17 ]. CpG ODNs have been shown to heighten humoral immunity to proteins such as the hepatitis B virus (HBV) surface antigen [18 , 19 ] and Leishmania major antigens [20 ]. This effect is attributable to several factors, including enhanced activation and mobilization of immune system components such as macrophages, DCs, and B cells [16 , 17 ], as well as reduction of CD4+CD25+ regulatory T cell (Treg)-mediated immune suppression [21 22 23 24 ].

In the present study, we investigated whether humoral immune responses to nondenatured, infectious murine (m)PrPSc could be developed in Prnp–/– mice and whether stimulating innate immunity through TLR9 signaling increases this response. These studies were prompted in part by results showing effective prevention of disease in scrapie-infected mice treated with the CpG ODN 1826 [25 ]. Therefore, we also investigated the effect of TLR9 stimulation on humoral immunity to mPrPSc in wild-type (WT; Prnp+/+) mice. To address these issues, mice were immunized with nondenatured, infectious 139A scrapie-associated fibrils (SAF) in a standard adjuvant (TiterMax), with or without CpG ODN 1826 (also called ODN 1826). 139A SAF were chosen as the immunogen, as relative to other mouse-adapted scrapie strains, 139A PrPSc is one of the more protease-sensitive PrPSc conformers [26 ] and is therefore more amenable to proteolytic processing and antigen presentation in vivo. Our data indicate that immunized Prnp–/– and Prnp+/+ mice develop measurable titers of antibodies to 139A SAF, and the levels of humoral immunity are heightened in the mice treated with ODN 1826. These results represent the first characterization of humoral immune responses to a nondenatured, infectious PrPSc immunogen in Prnp–/– or Prnp+/+ mice and further indicate how such responses are enhanced by CpG ODNs.

MATERIALS AND METHODS

Antibodies
mAb 3F4 and rabbit polyclonal antibody (pAb) 78295 have been described previously [5 , 6 , 27 ]. mAb 4B4 was derived from a BALB/c mouse immunized with formic acid-denatured, proteinase K (PK)-treated sheep SAF in TiterMax adjuvant (CytRx, Norcross, GA, USA); the corresponding hybridoma line was produced by standard splenocyte fusion protocols using the P3/X63-Ag8.653 myeloma line [American Type Culture Collection (ATCC), Manassas, VA, USA]. mAbs 3F4 and 4B4 are available from Signet Laboratories (Dedham, MA, USA). mAb 7A12 [28 , 29 ] was a gift from Man-Sun Sy (Case Western Reserve University, Cleveland, OH, USA).

mAbs were purified from concentrated tissue-culture supernatant produced in Integra CL bioreactor flasks (Integra Biosciences, Chur, Switzerland) or from ascites, using Pierce UltraLink Immobilized Protein A/G Plus affinity resin (Pierce, Rockford, IL, USA). Concentrations of antibodies were determined by mouse IgG radial immunodiffusion assay kits (MP Biomedicals, Irvine, CA, USA). Isotypes of antibodies were determined with ImmunoPure mAb isotyping kits (Pierce). Determination of Kd values for antibody-antigen binding was performed by ELISA as described previously [30 , 31 ] with modifications. Briefly, PolySorp immunomodule (Nalge Nunc, Rochester, NY, USA) wells were coated with varying concentrations of recombinant mPrP (rmPrp; 5, 2.5, 1.25, 0.63, 0.31, or 0.16 µg/ml in PBS), and each concentration of PrP was titrated with varying concentrations of mAb representing a titration series. Apparent Kd (Kdapp) values were obtained from each titration of mAb against a given PrP concentration. Finally, data were plotted as graphs of Kdapp for a given PrP concentration (ordinate) versus PrP concentration (abscissa), and the extrapolated y-intercept of the resulting linear regression represented the true Kd value for the mAb-PrP interaction. Pepscan epitope analyses were conducted by Pepscan Systems (Lelystad, The Netherlands) with overlapping 15- or 30-mer PrP peptides, which were shifted by one residue at a time from the N-terminal to the C-terminal, and together spanned the 1–254 or the 61–240 region of mPrP, respectively. Plasma was analyzed at dilutions ranging from 125- to 1000-fold. Peaks representing antibody epitopes were defined by extended regions of OD values exceeding twice the background for a given antibody.

Preparation of proteins
SAF
Isolation and purification of 139A SAF were performed from brains of clinically positive, 139A scrapie-infected C57BL/6J or CD-1 mice, according to the protocol of Hilmert and Diringer [32 ], with modifications as described previously [6 ]. The final isolated pellets were resuspended in water (~0.5 ml/25 g starting brain material final concentration) and dialyzed into water. The SAF preparations were sonicated prior to analyses or immunizations, as this treatment was shown to disaggregate and increase the number of infectious particles present [33 ]. For formic acid denaturation of SAF, preparations were treated for 2 h at 4°C with 3 vol 1.0 M formic acid, lyophilized, resuspended in water, relyophilized, and finally resuspended in a volume of PBS equivalent to the volume of the starting SAF preparation.

r-PrPs
Full-length rmPrP23–230 (recPrP) unmodified with affinity tags, including Residues 23–230, was prepared in Escherichia coli as described previously [34 ]. Syrian hamster recPrP was a gift from Ilia V. Baskakov (University of Maryland, Baltimore, MD, USA) and Robert G. Rohwer and Luisa Gregori (Department of Veterans Affairs Medical Center, Baltimore, MD, USA).

Protein concentrations
Protein concentrations were determined by bicinchoninic acid protein assay (Pierce).

Mice
Prnp–/– mice, crossed into the FVB/N genetic background, were originally obtained from Charles Weissmann (Scripps Research Institute, Jupiter, FL, USA) [35 ].

Transgenic sheep (TgShp), Tg666 [Tg human (TgHu)], TgElk, and Tg mule deer (TgMuD) mice, exclusively expressing sheep (Q171 genotype), human (M129 genotype), elk (eGMSE allele) [36 ], or mule deer PrPC, respectively, were developed at the New York State Institute for Basic Research in Developmental Disabilities (NYS IBRDD; Staten Island, NY, USA; G. LaFauci, personal communication). All TgPrP mice were homozygous with respect to the transgene, except the TgMuD mice, which were heterozygous. The TgElk mice have been described elsewhere [37 ].

All of the strains of mice above were bred and maintained at the NYS IBRDD Animal Facility. Female BALB/cJ and C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice expressing bovine PrPC (BoTg 3204), of which only brains were used, were developed by Larisa Cervenakova at the American Red Cross (Rockville, MD, USA) and were not bred or maintained on site.

All protocols involving animals were approved by the NYS IBRDD or the New York University (New York, NY, USA) Institutional Animal Care and Use Committee prior to beginning experiments.

ELISA analyses
For ELISA analyses against PrP, PolySorp immunomodule wells were coated with ~5 µg/ml protein. For determinations of plasma antibody titers, each plasma sample was run in triplicate at three sequential tenfold dilutions. The secondary reagent was an alkaline phosphatase conjugate of goat antimouse IgG + M (Biosource, Camarillo, CA, USA) used at 0.4 µg/ml. Plasma Ig isotype titer determination was done similarly to the IgG + M determinations described above but using ImmunoPure mAb isotyping kits (Pierce), according to the manufacturer’s protocol. The ELISA buffer used for blocking and for antibody binding steps, when appropriate, contained 0.2% Tween-20 and 1% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS, and wash buffer contained 0.05% Tween-20 in PBS. p-Nitrophenyl phosphate was used for ELISA colorimetric development. All ELISA analyses were done on an ELx800 microplate reader from BioTek Instruments (Winooski, VT, USA). In determining endpoint dilution (E.D.), a threshold value for considering a measurable response was set at 0.200 OD405 units. For determining antibody titers in plasma, OD405 was plotted against plasma dilution for each dilution in the series on a log-log transform, and the plasma dilution, at which the resulting regression line crossed OD405 = 0.200, was taken as the E.D., which for each mouse plasma sample, was derived by averaging the results of three separate assays of each sample. For statistical analyses, two-tailed Student’s t tests were used. Differences between groups of data were considered statistically significant for P values ≤0.05.

SDS-PAGE/Western blotting analyses
Protein samples in sample loading buffer containing SDS and 2-ME were heated at 100°C for 5 min and loaded to and run on SDS-PAGE gels with 12% polyacrylamide (Bio-Rad, Hercules, CA, USA), blotted onto nitrocellulose membranes of 0.45 µm pore size (Bio-Rad) using premade buffers (Bio-Rad), blocked with 5% nonfat dry milk in 0.2% Tween-20/TBS, and stained with antibodies in 1% normal goat serum/0.2% Tween-20/TBS, according to standard methods. Secondary antibodies were antimouse IgG + M or antirabbit IgG alkaline phosphatase conjugates at 0.1 µg/ml (Biosource). The Mr values of protein bands were identified by running gels with Kaleidoscope prestained standards (Bio-Rad). Brain homogenates were 10% or 20% (w/v) in 1% sarcosyl/TBS. PK treatment of brain homogenates and purified proteins was done with 50 µg/ml PK at 37°C for 1 h, followed by treatment with Pefabloc SC (Roche, Indianapolis, IN, USA) and Complete protease inhibitor cocktail (Roche). Protein concentrations in brain homogenates were quantified as described above under Preparation of proteins.

Immunohistochemistry
As described previously [38 , 39 ], clinical, 87V mouse-adapted, scrapie-infected MB/Dk mice [40 ] were lethally anesthetized with sodium pentobarbital i.p. and perfused transaortically with PBS, followed by 4% paraformaldehyde/PBS. Brains were subsequently removed, incubated for 2 days in 4% paraformaldehyde/PBS at 4°C, and then transferred to a preservation solution containing 30% glycerol/10% DMSO in PBS, also at 4°C until sectioning. Serial coronal brain sections of 40 µm thickness were prepared by microtome (Leica, Nussloch, Germany) and subsequently stained with mAb 6D11 at 3 µg/ml, with or without prior PK treatment (50 µg/ml for 1 h). Tissue staining, including peroxidase quenching and nonspecific signal blocking, was completed with a Vector Elite kit (Vector Laboratories), according to the manufacturer’s directions.

Mouse immunizations
Six-week-old female mice were immunized i.p. with semipurified, nondenatured, PK-treated 139A SAF (25 µg) in PBS (25 µl), emulsified with 10% AlCl3 (50 µl) in TiterMax (150 µl) with 25 µl water or ODN 1826 (63 µg, 10 nmol). After the first immunization only, mice were administered daily doses of ODN 1826 (63 µg) or vehicle (PBS) for the subsequent 4 (Prnp–/– and BALB/cJ) or 2 (C57BL/6J) days. C57BL/6J mice were administered ODN 1826 for only 2 days, as further dosing with these levels of ODN 1826 was found to be lethal for these mice. Mice were retro-orbitally bled under Avertin-induced anesthesia 3 days prior to the first immunization (for preimmune controls) and at 2-week intervals postimmunization.

ODN 1826 [5'-TCC ATG ACG TTC CTG ACG TT-3' (CpG motifs underlined)], with a complete phosphorothioate backbone, was purchased from Integrated DNA Technologies (Coralville, IA, USA).

RESULTS

ODN 1826 greatly potentiates the humoral immune response to PrPSc in PrPC null mice
Preliminary studies had been conducted to examine the immunogenicity of nondenatured PrPSc in Prnp–/– mice. Plasma from Prnp–/– mice, which were immunized four times with 139A SAF, displayed moderate immunoreactivity to the immunogen (E.D., ~20x103; pepscan results, see Fig. 5B ). This finding indicated that antibodies to nondenatured PrPSc could be generated in Prnp–/– mice and suggested that stimulation of the innate immune response using CpG ODNs may result in a significantly heightened response to PrPSc after fewer immunizations.


Figure 5
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  		Figure 5. ODN 1826 induces antibodies to a unique N-terminal epitope in 139A SAF-immunized Prnp–/– mice. Pepscan epitope analyses of plasma from immunized Prnp–/– mice. Number ranges above pepscan peaks represent PrP epitope residues in the corresponding peak. The shade of the numbers (black or gray) corresponds to that of the plot that they describe. (A, upper panel) Analyses with 30-mer peptides spanning mPrP 61–240 from mice immunized twice with 139A SAF alone. (A, lower panel) Analyses with 30-mer peptides (as described for A) from mice immunized twice with 139A + 1826. (B) Analyses with 15-mer peptides spanning PrP 1–254 from mice immunized four times with 139A alone.

 
Prnp–/– mice were immunized with inocula containing 139A SAF alone (identical to that described for the mice immunized four times) or 139A SAF and ODN 1826 to stimulate innate immunity, henceforth referred to as Inocula A (139A only) and B (139A+1826), respectively. Plasma PrPSc immunoreactivity (specific IgG+M) was then monitored over time. After an initial spike in plasma anti-PrPSc levels following immunization (i.e., antibodies capable of reacting to PrPSc), titers in all mice decreased to relatively low levels by Week 10. Mice were reimmunized at Week 11 and monitored for anti-PrPSc levels for an additional 11 weeks. As reported in Table 1 (see also Fig. 1 ), immunization with Inoculum B resulted in antigen-specific antibody titers, which were 18.9-fold greater than Inoculum A when analyzed at Week 12 postimmunization (the time-point of highest mean peak anti-PrP antibody levels for the Inoculum B mice; Fig. 1A and Table 1 ) or 13.2-fold greater when analyzed at peak time-points of response for each mouse (Fig. 1B and Table 1 ).


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  		Table 1. Ratios for Analyzing ODN 1826 Effect in 139A SAF-Immunized Prnp–/– Mice

 

Figure 1
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  		Figure 1. ODN 1826 increases anti-PrP antibody levels in Prnp–/– mice immunized with 139A SAF. ELISA analyses of plasma anti-PrP IgG + M from Prnp–/– mice immunized with 139A SAF with (139A+1826) or without (139A) ODN 1826. ELISA plate wells coated with nondenatured 139A SAF or recPrP at 5 µg/ml. Data are mean E.D. ± SEM of n = 3 mice per group and three determinations per plasma sample. (A) Time-course analysis of anti-139A PrPSc titers in mouse plasma. Mice were immunized at time = 0 and reimmunized at 11 weeks postimmunization. E.D. at time = 0 reflects preimmune plasma taken 3 days prior to immunization. ND, Not detected. *, Difference between groups is 18.9-fold (P<0.05). (B) Analysis of pooled group data at the peak time-point of humoral immune response for each mouse from A. The difference is 13.2-fold (P<0.001). (C) Analysis of plasma from each mouse at the peak of response (from B) against recPrP. The difference between groups is 43.1-fold.

 
The same mice were analyzed next at the peak time-points of their responses for plasma immunoreactivity to purified recPrP. Mice given Inoculum B had titers of antibody recognizing recPrP, which were 43.1-fold higher than mice given Inoculum A {E.D.±SEM: 448.0±220.9x103 vs. 10.4±2.0x103 [not significant (ns; Fig. 1C )]}. This indicated that the bulk of the immune response in the two groups of mice was mounted to PrPSc and not to other copurifying, proteinaceous contaminants present in the semipurified 139A SAF immunogen, such as ferritin [41 , 42 ]. Plasma from all immunized Prnp–/– mice also displayed immunoreactivity by Western blot to the bacterially expressed recPrP, which is nonglycosylated [43 ], as well as to all three glycoforms of PrPSc (di-, mono-, and nonglycosylated), indicating that the immune response to PrPSc in these mice was not glycoform-specific (Fig. 2 ).


Figure 2
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  		Figure 2. Polyclonal response in 139A SAF-immunized mice recognizes all three PrP glycoforms. Western blot analysis of plasma from Prnp–/– mice immunized with (139A+1826) or without (139A) ODN 1826, against 2 µg recPrP and semipurified, PK-treated 139A SAF. Blots from two representative mice from each immunization group are shown. Mouse plasma samples were tested at 1000-fold (139A) or 10,000-fold (139A+1826) dilutions, and mAb 3F4 ascites (negative control for mPrP) was run at 1000-fold dilution. Positions of Mr standards are noted by bands to the left of the figure.

 
ODN 1826 induces antigen-specific Ig isotype switching indicative of a conversion from TH2 to TH1 immune response in Prnp–/– mice
To examine isotypes of antigen-specific Igs present in the immune responses of Prnp–/– mice immunized with Inoculum A or B, plasma was analyzed at the time-point of the highest response for each mouse by IgG-specific ELISA for reactivity to nondenatured 139A PrPSc. Antigen-specific IgG1, 2a, and 2b were elevated greatly in mice given Inoculum B relative to mice given Inoculum A (Fig. 3 and Table 2 ). A 32.6-fold shift in the ratio of specific IgG2a + 2b:IgG1 was observed with Inoculum B as compared with Inoculum A, and a 42.4-fold shift was seen when considering the ratio of specific IgG2a:IgG1 (as derived from data in Table 2 ). The results indicate that although there is an increase in overall humoral immunity across TH1 and TH2 profiles in the mice treated with ODN 1826, the shift from TH2 to TH1 immune response dominates the seroconversion. These data also confirm results of previous IgG + M analyses (Fig. 1 and Table 1 ) and demonstrate a 22.2-fold increase in total IgG levels (IgGtotal, IgG1+2a+2b) in the Inoculum B mice [as derived from data in Table 2 ; E.D.: IgGtotal, 2879.2x103 vs. 129.5x103 (ns)]. The large discrepancy observed between measured plasma antibody titers (IgG+M, Fig. 1 ) and specific IgG isotype titers (Fig. 3) is a result of the additional antibody-dependent step in the ELISA used to detect the specific antibody isotypes.


Figure 3
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  		Figure 3. ODN 1826 alters the isotype distribution of antigen-specific IgG in 139A SAF-immunized mice. ELISA analyses of plasma from Prnp–/– mice immunized with 139A SAF with (139A+1826) or without ODN 1826 (139A). ELISA plate wells coated with nondenatured 139A SAF at 5 µg/ml. Analysis of pooled group data at peak time-point of humoral immune response for each mouse from Figure 1B measuring levels of specific IgG1, 2a, and 2b. Data are mean E.D. ± SEM for n = 3 mice per group and three determinations per plasma sample. *, P < 0.05; #, P < 0.07.

 

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  		Table 2. Ig Isotype Levels and Ratios in 139A SAF-Immunized Prnp–/– Mice

 
Levels of antigen-specific IgM were also examined by ELISA, as described above, at the peak of humoral immunity for each mouse; relatively low levels of antigen-specific IgM were found in all mice (Table 2 ). When compared against mean antigen-specific IgGtotal in the same mice, mean antigen-specific IgM levels were 113.4-fold lower in Inoculum B-immunized mice [25.4 vs. 2879.2x103 (ns)] and 11.9-fold lower in Inoculum A-immunized mice [10.9 vs. 129.5x103 (P<0.08)].

ODN 1826 increases the humoral immune response to PrPSc in mice expressing PrPC
Next, we studied the effects of innate immune stimulation with ODN 1826 on humoral immune responses to PrPSc in Prnp+/+ mice. BALB/cJ and C57BL/6J mice were immunized with Inoculum A or B, and plasma was collected at 2-week intervals and analyzed for specific IgG + M. PrP-specific IgG + M was undetectable in mouse plasma prior to immunization. After a single immunization, BALB/cJ mice developed a measurable PrP-specific humoral immune response, which was 59% greater in the mice given Inoculum B than those given Inoculum A [E.D., 364.5±60.8 vs. 229.7±10.2 (ns; Fig. 4 )]. Although the humoral immune response to PrPSc in C57BL/6J mice was weaker than in BALB/cJ mice after a single immunization, the former displayed a greater relative increase (84%) in anti-PrPSc antibody levels following immunization with Inoculum B versus Inoculum A [E.D., 200.8±9.3 vs. 109.4±10.5 (Fig. 4) ]. Thus, the humoral immune response to PrPSc in WT mice is enhanced by the administration of ODN 1826; however, the response is 100- to 1000-fold lower than that observed in Prnp–/– mice (Fig. 1 and Table 1 ).


Figure 4
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  		Figure 4. ODN 1826 increases anti-PrPSc antibody levels in 139A SAF-immunized Prnp+/+ mice. ELISA analyses of plasma from Prnp+/+ mice immunized with 139A SAF with (139A+1826) or without ODN 1826 (139A). ELISA plate wells coated with nondenatured 139A SAF at 5 µg/ml. Analysis of pooled group data (n=3 mice per group and three determinations per plasma sample) at the peak time-point of humoral immune response for each mouse. Data are mean E.D. ± SEM. The difference between immunized BALB/cJ groups is 1.59-fold and between C57BL/6J groups is 1.84-fold (*, P<0.005).

 
The humoral immune response of Prnp–/– mice immunized with PrPSc and ODN 1826 yields reactivity to a previously unrecognized PrP N-terminal epitope
Plasma from the Prnp–/– mice immunized with Inoculum A or B were analyzed by pepscan using mPrP peptides to detect linear (and restricted, nonlinear) epitopes within the 88–231 PK-resistant region of PrPSc. Plasma from mice that received Inoculum A (139A#1, #2, and #3) showed no linear epitopes (Fig. 5A ); one of the mice (139A#1), however, displayed a possible low-level response to the most C-terminal region of PrP (Residues 206–230; Fig. 5A and Table 3 ). In contrast, all mice given Inoculum B (139A+1826#1 and #2 and M003-3) generated responses to various epitopes along PrP (Fig. 5A and Table 3 ); plasma from these mice consistently displayed robust responses to the N- and C-terminal regions (Residues 88–103 and 207–230, respectively).


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  		Table 3. mPrP Epitopes Represented in Pepscan Analyses of Plasma from 139A SAF-Immunized Prnp–/– Mice

 
To better characterize the epitope specificity of the humoral immune response induced by Inoculum A, two mice that had been immunized four times with this inoculum (described previously in Results; M001-9 and M002-1) were analyzed. Mice immunized four times with Inoculum A generated detectable responses to epitopes at the C-terminal region of PrP between Residues 205 and 229 (Fig. 5B and Table 3 ). Although one of these mice exhibited an antibody response with epitopes as close to the N-terminal as Residues 120–126, none exhibited any in the 88–103 region, as found in the Inoculum B-immunized mice. Thus, the absence of detectable epitopes in the plasma of mice immunized twice with Inoculum A was probably a reflection of the low level of humoral immunity in these mice. In addition, these results indicate that the presence of ODN 1826 in Inoculum B allowed for the generation of antibodies to the N-terminal region of PrP, which were notably absent from the mice treated with Inoculum A (Fig. 5A and Table 3 ). Although pepscan-identifiable epitopes in the N-terminal domain of PrP were displayed only in Inoculum B-treated mice, it cannot be excluded that nonlinear epitopes to this region of PrP were present but not detected in mice given Inoculum A.

Characterization of mAb 6D11 derived from a Prnp–/– mouse immunized with PrPSc and ODN 1826
The significantly elevated antibody response and unique N-terminal epitope observed in Inoculum B-treated mice prompted us to use these mice to generate mAbs. The Inoculum B-immunized Prnp–/– mouse with the greatest level of humoral immunity to PrPSc (M003-3; see Fig. 5A ) was used to generate hybridomas (fusion with myeloma line P3/NSI/1-Ag4-1 from ATCC), and an array of hybridoma clones was generated; the anti-PrP mAb secreted by the hybridoma line 6D11 was fully characterized in this study.

Binding and reactivity
mAb 6D11 is of the IgG2a{kappa} isotype, which is the subclass (IgG2a) that exhibited the greatest increase (>560-fold; see Table 2 ) in the Prnp–/– mice treated with ODN 1826. mAb 6D11 exhibits a broad species specificity by Western blot, which includes PrP of bovine, cervid (elk and mule deer), human, ovine, and rodent (hamster and mouse; Fig. 6 ). Reactivity of 6D11 was also tested by ELISA against nondenatured forms of 139A and hamster-adapted scrapie 263K PrPSc, semidenatured (formic acid-treated) 139A PrPSc, and recPrP and was found to bind very well to these antigens (data not shown). The binding affinity/avidity of 6D11 for recPrP, as measured by Kd, was determined by ELISA to be 40 ± 28 pM (Kd±SD, n=2). This Kd is comparable with that of the mAb 3F4 for Syrian hamster rPrP and that of 7A12 for recPrP (Kd values of ~80 and ~190 pM, respectively; D. S. Spinner and R. J. Kascsak, unpublished data). In addition, 6D11 reacted strongly with nondenatured PrPSc deposits in PK- and non-PK-treated brain sections taken during the clinical phase from mice infected with the plaque-forming, 87V mouse-adapted scrapie strain (Fig. 7 ); immunostaining of intraneuronal and extracellular PrPSc, but not PrPC, was observed. The recognition of PrPC and nondenatured and denatured forms of PrPSc in a variety of formats indicates that mAb 6D11 recognizes an epitope whose structure is conserved in PrPC and PrPSc and which is readily accessible in the two PrP isoforms.


Figure 6
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  		Figure 6. mAb 6D11 recognizes PrP isoforms from multiple species. Analysis of mAb 6D11 reactivity to PrP by Western blotting. SDS-PAGE/Western blot of 6D11 (0.4 µg/ml), pAb 78295 (positive control for homogenate PrP levels; 5000-fold dilution), and secondary antibodies only (0.1 µg/ml) tested against 8 µg protein (measured prior to PK treatment) loaded to gel from brain homogenates of a normal mouse (C57BL/6J), 139A scrapie-infected mouse (139A in C57BL/6J), Prnp–/– mouse [knockout (KO)], Syrian hamster (SHa), or TgPrP mice expressing sheep, human, cow, elk, or mule deer PrP.

 

Figure 7
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  		Figure 7. mAb 6D11 reacts to PrPSc deposits in PK- and non-PK-treated brain sections from plaque-forming, scrapie-infected mice. Immunohistochemistry on fixed brain sections from clinical, 87V scrapie-infected MB/Dk mice. Fixed tissue sections were treated with (+) or without (–) PK prior to immunostaining with mAb 6D11 (3 µg/ml). Original magnifications, 200x. Closed and open arrowheads mark select mAb 6D11-stained extracellular and intraneuronal PrPSc deposits, respectively. (A) Coronal brain section of cortical region overlying the hippocampus. (B) PK-treated coronal brain section.

 
Epitope determination
The epitope of 6D11 was mapped by pepscan analysis to within the N-terminal region of PrP, Residues 89–100 (GQGGGTHNQWNK; Fig. 8A ). This region of PrP also displayed the strongest immunoreactivity in the polyclonal response of the mouse, from which 6D11 was derived (M003-3; Figs. 5A and 8A ). On the basis of the species specificity exhibited in Western blot, peptide reactivity determined previously in dot-blot [44 ], and knowledge of the PrP sequences in the various species, the 6D11 epitope was further narrowed to Residues 97–100 (QWNK; Fig. 8B ). The asparagine of QWNK (murine Position 99) is replaced by a glycine in the rabbit and mink PrP sequences, which we had not examined previously by Western blotting; it is notable that in the region of 89–100, the PrP sequences of rabbit and mouse differ only at Residue 99 (Fig. 8B) . To confirm the epitope of 6D11 to be QWNK, Western blotting was performed against normal rabbit and mink brain homogenates (Fig. 8C) . As expected, the reactivity of 6D11 in the Western blot excludes PrPC of rabbit and mink, whereas mAbs 4B4 and 7A12 stain these proteins, confirming the sequence QWNK as the epitope for 6D11. In addition, more extensive, higher resolution pepscan analyses on 6D11 using truncated peptides further confirmed QWNK as the epitope (data not shown). This epitope imparts upon 6D11 unique and highly desirable diagnostic and therapeutic properties (see Discussion).


Figure 8
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  		Figure 8. The epitope of mAb 6D11 is the amino acid sequence QWNK. Determination of epitope for mAb 6D11. (A) Comparison of pepscan analyses of mAb 6D11 and plasma from mouse M003-3, from which hybridoma line 6D11 was derived; 30-mer peptides spanning mPrP 61–240 were used. The epitope of 6D11 resides within PrP Residues 89–100, a major region of polyclonal response in mouse M003-3. (B) Alignment of PrP sequences from various species within the region homologous to mPrP Residues 89–100. Epitope for 6D11 is underlined. Note that the only point of difference in this region between the mouse and human PrP sequences is at mAsn 96 and between the mouse and rabbit PrP sequences is at mAsn 99 (bold). (C) SDS-PAGE/Western blot comparing reactivity of mAb 6D11 (0.1 µg/ml), 4B4 (0.6 µg/ml; positive control for rabbit and mink PrP), 7A12 (0.4 µg/ml; positive control for all tested species), and 3F4 (ascites 1000-fold dilution; negative control for all tested species) against 35 µg protein from normal rabbit and mink brain homogenates and 18 µg normal mouse and Prnp–/– mouse brain homogenates.

 
DISCUSSION

We have shown that Prnp–/– mice are capable of generating a humoral immune response to nondenatured 139A PrPSc, which is increased significantly by the TLR9 agonist ODN 1826. Several other studies have demonstrated humoral immunity to PrP in Prnp–/– mice and the ability to develop anti-PrP mAb-secreting hybridomas from them [6 , 7 , 10 , 27 , 28 , 45 46 47 48 49 50 51 52 53 54 ]. Of these studies, one reported the generation of mAbs following immunization with nondenatured PrPSc [51 ]; however, in that study, the nature of the humoral immune response was not described, and the mAbs produced failed to interact with the nondenatured antigen.

Following ODN 1826 administration, IgG subclasses present in the PrP-specific humoral immune response of Prnp–/– mice reflect a shift from IgG1 in favor of IgG2a and 2b. This switch to a TH1 Ig profile was anticipated on the basis of results obtained from previous studies with other antigens [18 19 20 ] and confirms that CpG ODNs act with similar mechanisms for PrPSc in Prnp–/– mice. Previously reported results [55 ] also indicated that elevated IgM titers were produced in Prnp–/– mice immunized with murine Rocky Mountain Laboratory scrapie PrPSc-coated affinity beads. By contrast, our results showed that levels of specific IgM are low, at least at the point of maximal humoral immune response, regardless of whether ODN 1826 was administered.

In Prnp–/– mice, administration of ODN 1826 with 139A PrPSc immunization induced the production of anti-PrP antibodies, a subset of which was specific to an N-terminal region (Residues 89–103) not represented in the repertoire of mice immunized with 139A alone. The mAb 6D11 is specific to an epitope within this region of PrP (Residues 97–100, QWNK). The mouse from which this mAb was derived exhibited an immune response to regions of PrPSc (89–103, 132–149, and 211–230), which overlap with regions 89–112 and 136–158 identified as key domains in the PrPC-to-PrPSc conversion [56 , 57 ]. mAbs generated by others to the N-terminal region have been found to bind PrPSc selectively [56 , 57 ]. mAb 6D11 also binds to nondenatured PrPSc in a variety of assay formats and to PrPSc in scrapie brain homogenates [44 ] but is not selective for the PrPSc isoform. Although we found that plasma from the immunized mice reacted to PrPSc, as we used pepscan and other methods incapable of discerning PrPSc-selective antibodies in plasma, we were unable to verify whether such selective antibodies were produced in the immunized mice. Nonetheless, the results collectively suggest that our method of immunizing Prnp–/– mice with nondenatured PrPSc and CpG ODNs may generate antibodies that can inhibit formation of PrPSc; in fact, mAb 6D11 has been used to prevent and reverse PrPSc accumulation in prion-infected neuronal cells in culture [44 ] and to significantly prolong the scrapie incubation period in scrapie-infected mice [58 ].

BALB/cJ and C57BL/6J mice, which express PrPC, were immunized with 139A PrPSc and exhibited low-level, specific humoral immune responses. The mice given ODN 1826 developed higher levels of anti-PrPSc antibodies in their plasma. Although the anti-PrP titers in the WT mice were much lower than those seen in their Prnp–/– counterparts, the concentrations achieved may be effective for prion disease vaccination, particularly as even low levels of specific antibodies are sufficient to delay onset and attenuate disease symptoms [44 , 59 60 61 62 63 64 65 66 ]. Furthermore, the immunized BALB/cJ mice mentioned above were monitored for the scrapie incubation period as described previously [37 ], and similar to the results of Sethi et al. [25 ], incubation time was found to be prolonged significantly (by 42%) in the mice given ODN 1826 [mean±SD; 284.0±27.7 vs. 200.0±17.3 days (P<0.05)]; the immunized C57BL/6J mice were not monitored for the scrapie incubation period. Development of humoral immunity to PrP in Prnp+/+ mice has been reported previously [5 , 27 , 60 , 61 , 64 , 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 ]. However, as none of the studies used nondenatured PrPSc, ours represents the first characterization of such a response.

Our results demonstrate that innate immune stimulation with the TLR9 agonist ODN 1826 enhances humoral immunity to PrPSc in mice. This effect resulted most likely from a combination of increased uptake, processing, and presentation of antigen [82 83 84 ] and down-regulated Treg function [21 22 23 24 , 85 ], which have been described for a number of TLR ligands. Previous studies have shown that non-CpG-containing ODNs [19 , 86 ] or those that contain methylated CpG elements [87 ] do not enhance humoral immune responses. As our study did not include a non-CpG ODN control group, it cannot be stated with certainty that the effects on humoral immunity to PrPSc were attributable exclusively to TLR9 signaling and not to general stimulatory properties of phosphorothioate backbone-containing ODNs [88 , 89 ]. It is interesting that recent studies indicate that certain non-CpG-containing ODNs could activate TLR9 [90 ]. Nonetheless, it is clear that the strategy to include ODN/1826 as part of the immunization protocol was highly successful. Similar immune enhancement resulting from CpG ODN administration has been observed for numerous non-PrP antigens such as the HBV surface antigen [18 , 19 ] and L. major antigens [20 ]. Obviously, mice lacking the gene encoding PrPC possess a greater capacity to respond to PrP than do their Prnp+/+ counterparts. Primarily, this effect is believed to be from tolerance induced by endogenous PrPC, which does not occur in Prnp–/– mice. However, some of the differences observed between Prnp–/– and Prnp+/+ mice may be attributable to a lower capacity of Prnp+/+ immune cells for phagocytosis [91 ], a process crucial for antigen presentation and hence, initiation of adaptive immunity. It is also possible that antibody titers in WT mice may appear artificially low as a result of circulating blood cells and other cell types that express high levels of surface PrP [92 , 93 ], which can adsorb anti-PrP antibodies and mask their presence. Similar antibody adsorption effects have been seen in amyloid-ß-immunized mice [94 ].

A recent study by Heikenwalder and colleagues [95 ] reported ODN 1826-dependent induction of lymphoreticular damage in WT mice after just 2 consecutive days of administration in doses similar to those used in the present study. They demonstrated that severe damage to the lymphoreticular system occurred by Day 7 of treatment and grew progressively worse with increasing numbers of successive daily doses. One would expect increasing lymphoreticular destruction to severely diminish the capacity of mice to initiate adaptive immune responses. Results obtained in the present study (and those of others including Sethi and colleagues [96 ]) differ from those of Heikenwalder et al. [95 ] in that we found repeated treatments with ODN 1826 to increase humoral immune responses in Prnp+/+ and Prnp–/– mice. In our study, after 3 or 5 consecutive daily doses of ODN 1826, significant potentiation of anti-PrP antibody production occurred. In addition, total plasma IgG levels in ODN 1826-treated Prnp–/– mice were in the normal range after the first and second immunizations at the peaks of humoral immune response for each mouse when measured by radial immunodiffusion assay. After one immunization, Prnp–/– mice inoculated with 139A + 1826 had levels of total plasma IgG 1.8-fold higher (P<0.05) than mice immunized with 139A alone (mean±SD; 27.1±0.9 vs. 15.4±1.5 mg/ml; n=3 mice per group, with two determinations per plasma sample). Levels of total plasma IgG in mice of both immunization groups were identical after the second immunization (139A 14.0±2.0 vs. 139+1826 14.1±1.8 mg/ml).

Heikenwalder et al. [95 ] reported mouse strain-dependent differences in toxicity, which we also observed. In the current study, C57BL/6J mice were administered ODN 1826 for only 3 consecutive days (including the immunization day; vs. 5), as the levels of ODN 1826 used were found to be lethal for some of these mice when administered over a longer period. The Prnp–/– (FVB/N strain) and BALB/cJ mice received five doses of ODN 1826, which may be expected to result in toxicity and diminished immune response, yet we observed no overt symptoms. It may be noted that one difference between our study and that reported by others [95 ] was the use of the TiterMax adjuvant in the present study.

Our data for Prnp+/+ mice suggest that CpG ODN-induced protection against prion disease in mice [25 ] may be partly attributable to therapeutic antibody production, along with increased clearance of the scrapie agent by activated phagocytes. The antigen-specific IgGs present and particularly those of the IgG2a or 2b isotype, which we found to be increased substantially following ODN 1826 treatment in the Prnp–/– mice, are likely to be highly effective opsonins for PrPSc [97 ]. In addition, T cell-based mechanisms, including down-regulation of Treg function [21 22 23 24 , 85 ], enhanced levels of antigen cross-presentation [98 99 100 101 ], and increased activation of CD8+ T cells [86 , 102 103 104 ] are likely to play important roles in the therapeutic effect of CpG ODNs on scrapie; these effects may be especially pertinent, as T cell effector functions are diminished in scrapie-infected mice despite their ability to bind major histocompatibility complex:PrP peptide tetramers [105 ]. Recently, additional mechanisms have been proposed for phosphorothioate backbone-containing ODNs in scrapie prophylaxis involving the binding of these molecules to PrPC and preventing the conversion to PrPSc [106 ].

Our studies demonstrate that the ODN 1826 enhances adaptive/humoral immune responses to PrPSc in mice and therefore, also may greatly improve the efficacy of vaccination [60 , 61 , 64 65 66 ] and passive immunization [44 , 59 , 62 , 63 ] against TSE infection. mAbs 6D11, induced in the presence of ODN 1826, and similar mAb possessing high affinities and unique epitope specificities derived from PrPSc appear to be ideal candidates that offer considerable diagnostic [27 , 107 , 108 ] and therapeutic [44 ] promise. As improved therapy at later stages of disease and diagnosis at earlier stages of prion disease remain major goals of this field, it is clear that further studies pertinent to the development of such antibodies to PrP are important.

ACKNOWLEDGEMENTS

This work was supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, National Institutes of Health, contract N01-NS-0-2327 (R. J. K., Subcontract Principal Investigator; Robert G. Rohwer, Contract Principal Investigator) and Grant NS047433 (to T. W.), and Alzheimer’s Association/Stranahan Foundation Grant NIRG-04-1162 (to D. S. S.). The authors gratefully acknowledge Larisa Cervenakova (American Red Cross, Rockville, MD, USA) for the gift of bovine PrP Tg mouse (BoTg 3204) brains, Man-Sun Sy (Case Western Reserve University, Cleveland, OH, USA) for providing mAb 7A12, and Ilia V. Baskakov (University of Maryland, Baltimore, MD, USA), Robert G. Rohwer, and Luisa Gregori (Department of Veterans Affairs Medical Center, Baltimore, MD, USA) for Syrian hamster rPrP used for mAb Kd determination. We also thank Jerry Slootstra (Pepscan Systems, Lelystad, The Netherlands) for help in interpreting pepscan analyses; Michael Natelli and Marisol Cedeno for maintaining Tg mouse lines; Victor Sapienza, Heni Hong, and Chengmo James Chen for technical assistance; Patricia A. Merz for helpful discussions; Mary Ellen Cafaro and Robert L. Freedland for graphical assistance; and Elaine J. Marchi for editorial assistance. Conflict of Interest Disclosure: mAb 6D11 is currently marketed through Covance/Signet Laboraties (Dedham, MA, USA) and royalties may be received in the future as a result of these sales.

FOOTNOTES

2 Current address: Department of Ophthalmology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Back

3 Current address: Department of Physiology and Biophysics, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA. Back

Received October 23, 2006; revised December 8, 2006; accepted February 23, 2007.

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