Prevention of Scrapie Pathogenesis by Transgenic Expression of Anti-Prion Protein Antibodies

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Science  05 Oct 2001:
Vol. 294, Issue 5540, pp. 178-182
DOI: 10.1126/science.1063093


Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) μ chain in Prnp o/o mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction ofPrnp + alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrPC) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.

Prion diseases are slow, lethal transmissible neurodegenerative illnesses that affect humans and many animal species. Although human prion diseases are rare, the incidence of variant Creutzfeldt-Jakob disease (vCJD) in the United Kingdom appears to be increasing exponentially (1), probably as the result of exposure to bovine spongiform encephalopathy (BSE) prions (2, 3). Given the large amount of BSE-infected material that may have entered the human food chain, and the many people who may eventually develop vCJD (4), it will be important to introduce strategies to prevent the development of symptoms in these individuals. For many conventional infectious agents, vaccination is an effective method of infection control. However, there has been little evidence that vaccines might be effective for protection against prion diseases. The endogenous cellular prion protein (PrPC) is expressed by most tissues of the body and is therefore known to be a poor immunogen, probably because of host tolerance. However, ablation of the Prnp gene (5), which encodes PrPC, makes it possible to immunize mice with prions (6). We reasoned that genes encoding high-affinity anti-PrP antibodies generated inPrnp o/o mice might be used to redirect B cell responses of prion-susceptible mice that express PrPC.

Epitope-binding variable regions of the immunoglobulin heavy (VH) and light chains (VL) from cDNA of hybridoma 6H4, which expresses an immunoglobulin G1 (IgG1) monoclonal antibody (mAb) recognizing murine PrPC, were amplified by polymerase chain reaction (7). The same method was used to amplify the VH and VL chains of 15B3, which expresses an IgM antibody recognizing PrPSc(7, 8). These sequences were then expressed as single-chain variable fragment (scFv), tested for their anti-PrP binding capacity, and were finally expressed as transgenes (7). To avoid autoimmunity, we used fertilized eggs from Prnp o/o Sv129 × C57BL/6 mice (5), which express IgM/D of the a or b allotype. The resulting mice expressed transgenic IgMa μ chain combined with a repertoire of endogenous λ and κ light chains, but no detectable endogenous IgM/D, indicating that the transgene induced allelic exclusion of endogenous heavy chains (Fig. 1A) (9). When compared with nontransgenic Prnp o/o orPrnp +/o mice, the B cell compartments of 6H4μ and 15B3μ transgenic mice were reduced (7), whereas T cell compartments (7) were normal (Fig. 1B). Transgenic Prnp o/o-6H4μ mice had consistently high spontaneous anti-PrP titers at the age of 4 weeks, whereas 15B3μ transgenic mice did not display signals above background (Fig. 1C) (10). In addition, 15B3μ sera showed no titer when tested in an enzyme-linked immunosorbent assay with a target peptide recognized by mAb 15B3 (11). Because 15B3μ mice do not contain the original 15B3 light chain, their combinatorial repertoire may differ in specificity from that of mAb 15B3. Therefore, 15B3μ was operationally regarded as of irrelevant specificity. Sera from Prnp o/o-6H4μ, but notPrnp o/o-15B3μ mice, were able to decorate Western blot membranes loaded with brain extracts of wild-type mice and with PrPREC (12) (Fig. 1D). In addition,Prnp o/o-6H4μ, but notPrnp o/o-15B3μ sera, bound to splenocytes fromtg94 transgenic mice, which overexpress PrPC on lymphocytes (13) (Fig. 1E). The above data indicate that the 6H4μ transgene, but not 15B3μ, conferred anti-PrP titers toPrnp o/o mice.

Figure 1

Expression of anti-PrP μ in transgenic mice. (A) FACS analysis of surface immunoglobulins on B220+ peripheral blood B cells. Transgenic 6H4μ and 15B3μ mice expressed only immunoglobulins of the IgMa, but not of the IgM/D, class, indicating allelic exclusion of endogenous heavy chains. Instead, nontransgenic littermates expressed IgMa/D and/or IgMb/D. Relative B cell numbers were reduced in both 6H4μ and 15B3μ mice. (B) FACS analysis of peripheral blood revealed no alteration in CD4+and CD8+ T cell subsets in transgenic mice. (C) ELISA showing anti-PrPREC titers in sera of 6H4μ mice (red circles), but not of 15B3μ mice (green triangles), nor of nontransgenic littermates (black crosses). Each data point represents the average of four mice. Immunoreactivity of 6H4μ mice was only detectable with anti-IgM and anti-IgM+A+G, but not with anti-IgG secondary antibodies (11). Based on the concentration of anti-PrPREC immunoglobulins (6H4μ mice: IgMa; mAb 6H4 control: IgG1), the anti-PrP titer of transgenic 6H4μ immunoglobulins is ∼500-fold lower than that of mAb 6H4 (blue diamonds). Because IgM is about fives times heavier than IgG, the total anti-PrP avidity of IgMa in 6H4μ serum (M−1) is ∼100-fold lower than that of mAb 6H4. (D) Transgenic IgMa immunoglobulins of 6H4μ mice, in contrast to 15B3μ mice or nontransgenic littermates, recognized PrPC in immunoblotted wild-type brain homogenates (left column) and full-length PrPREC, but yielded no signal withPrnp o/o brain homogenate. mAb 6H4 produced a pattern similar to that of the serum of 6H4μ mice exhibiting the un-, mono- and diglycosylated band of PrPC. Markers on the right indicate molecular size at 35 and 28 kD. (E) FACS analysis. (Left) Twenty-five–fold diluted serum of 15B3μ mice (red line, corresponding to 8 μg/ml IgMa) or of nontransgenicPrnp o/o littermates (gray dotted line) did not bind to native PrPC of tg94 splenocytes that overexpress PrPC. (Middle) In contrast, 25-fold–diluted serum of 6H4μ transgenic mice recognized native PrPC of tg94 splenocytes (red line, 8 μg/ml IgMa) and produced a shift of >1 logarithmic unit of fluorescence intensity. (Right) Twenty-five–fold diluted 6H4μ serum (red line, corresponding to 8 μg/ml IgMa) yielded only a faint signal when wild-type (wt) splenocytes expressing low levels of PrPC were used as targets. Gray dotted lines: 25-fold–diluted serum of nontransgenicPrnp o/o mice.

Because prion replication requires PrPC(14), we reintroduced one or two Prnpalleles by crossbreeding transgenic mice with wild-type C57BL/6 mice. The number of transgenic IgMa+IgD B cells (7), as well as IgMa surface expression levels in Prnp +/o-6H4μ mice, were slightly higher than those of Prnp o/o-6H4μ mice (Fig. 2A).Prnp +/o-6H4μ andPrnp +/+-6H4μ mice developed substantial spontaneous anti-PrP serum levels, albeit more slowly thanPrnp o/o-6H4μ mice (Fig. 2B). Because unresponsiveness of B cells may correlate with the level of self-antigen (15) and B cell receptor affinity/avidity (16), the delayed build-up of anti-PrP serum titers in Prnp +/o-6H4μ mice may have reflected negative selection of B cells expressing combinations of 6H4μ with light chains yielding high-avidity binding to PrPC that was not permissive for B cell survival. If this were the case, it would be expected that (i) the B cell repertoires ofPrnp o/o-6H4μ,Prnp +/o-6H4μ, andPrnp +/+-6H4μ mice should differ; (ii) the unexpected expansion of B cells inPrnp +/o-6H4μ andPrnp +/+-6H4μ mice may consist of clones expressing lower affinities; and (iii) peripheral 6H4μ-expressing B cells may be susceptible to tolerization in PrP-overexpressing mice.

Figure 2

Coexpression of PrPC and anti-PrP μ chain: (A) FACS analysis of peripheral blood gated on B220+ B cells. Transgenic IgMa surface expression in Prnp +/o-6H4μ (black line) was slightly higher than in Prnp o/o-6H4μ mice (dotted red line), whereas tg94-6H4μ mice (gray line) displayed drastically reduced IgMa expression. (B) Anti-PrPREC titers determined by ELISA.Prnp o/o-6H4μ mice (red circles),Prnp +/o-6H4μ mice (black squares), andPrnp +/+-6H4μ mice (green triangles) showed similar titers at the age of 4 to 5 months. However,Prnp o/o-6H4μ mice already had substantial titers at the age of 1 month, whereasPrnp +/o-6H4μ mice andPrnp +/+-6H4μ mice displayed delayed kinetics in reaching considerable anti-PrP serum levels. Each data point represents the average of four mice. (C) FACS analysis of peripheral blood. Tg94-6H4μ mice displayed only a small residual population of B220+ B cells expressing IgMa (lower left) but not IgMb (lower right), suggesting partial clonal deletion of self-reactive B cells.Tg94 mice without the 6H4μ transgene displayed normal numbers of IgMb-positive B cells (upper right). Numbers indicate the percentage of cells. (D) The residual population of B cells in tg94-6H4μ mice (red line) appeared to be B220dull, whereasPrnp +/o-6H4μ mice (black line) orPrnp +/+-6H4μ mice (green dotted line) with physiological PrPC levels or tg94 controls (gray dotted line) displayed B cells with a normal B220 expression pattern.

To test the latter hypothesis, 6H4μ transgenic mice were bred ontotg94 transgenic mice that overexpress PrP in B and T cells ∼1000-fold (13). WhereasPrnp +/o-6H4μ andPrnp +/+-6H4μ mice were indistinguishable from each other (7), tg94-6H4μ mice exhibited almost complete loss of B cells in peripheral blood (Fig. 2C) (7). In the blood of tg94-6H4μ mice, we found a residual population of B220dullIgMa-positive B cells (Fig. 2D) that was CD11bCD5CD62L+(11) and may represent immature/transitional B cells that recently emigrated from the bone marrow (17, 18). Therefore, PrP can be coexpressed with PrP-specific antibodies inPrnp +/o-6H4μ orPrnp +/+ -6H4μ transgenic mice without inducing autoimmune disease or hematological disorders. Only when PrPC was expressed at nonphysiologically high levels were 6H4μ-expressing B cells strongly reduced.

Transgenic mice (Prnp o/o andPrnp +/o) were next inoculated intraperitoneally with scrapie prions (RML strain, passage 5) (19). Spleens were harvested at 35 and 50 days after inoculation (dpi), and prion titers were determined by bioassay with tga20mice (20, 19). Although nontransgenicPrnp +/o mice developed measurable prion titers, no infectivity could be detected inPrnp +/o-6H4μ or inPrnp o/o mice at any time (Table 1).

Table 1

Intraperitoneal prion challenge of transgenic mice. Spleen infectivity titers were reduced by >4 logarithmic units in the presence of the 6H4μ transgene. dpi, days after inoculation; LD50, median lethal dose.

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We next quantitated the level of deposition of PrPScin the spleens of scrapie-infected 6H4μ and 15B3μ transgenic mice (12). PrPSc accumulated inPrnp +/o andPrnp +/o-15B3μ spleens at various time points investigated, but was undetectable inPrnp +/o-6H4μ spleens (Fig. 3, A and B). The sensitivity of Western blot analyses may be inadequate if PrPSc is distributed inhomogeneously in tissue, because PrPSc-free material may dilute the PrPSc-positive regions. We controlled for this possibility with histoblots of spleen and brain sections (21, 22). Protease-resistant PrPScwas distributed inhomogenously in prion-inoculatedPrnp +/o andPrnp +/o-15B3μ spleens (Fig. 3C), reflecting accumulation within splenic germinal centers (23). Again, no signal was detectable in Prnp +/o -6H4μ mice (Fig. 3C), confirming that 6H4μ effectively prevented splenic accumulation of protease-resistant PrP.

Figure 3

PrPSc in transgenic mice after prion inoculation. (A) Western blot of spleen samples after sodium phosphotungstic acid (NaPTA) precipitation. PrPScwas not detected in the spleens ofPrnp +/o-6H4μ mice at any time point investigated (35 to 234 dpi), whereas nontransgenic littermates and Prnp +/o-15B3μ showed substantial PrPSc deposition. All samples were digested with proteinase K (PK). All mice were Prnp +/o. (B) Determination of the sensitivity of Western blots after NaPTA precipitation. In the spleens of Prnp +/omice 170 dpi, the PrPSc-specific signal was detected in 32-fold–diluted samples. Therefore, PrPScaccumulation in the spleens of Prnp +/o-6H4μ mice was ≤3% that of nontransgenic Prnp +/omice. (C) Histoblot of spleens (lanes 1 to 6) and brains (lanes 7 and 8) 50 to 234 dpi. Discrete PrPSc deposits were detected in the spleens of Prnp +/o-15B3μ and nontransgenic Prnp +/o mice, whereas there was no PrPSc accumulation in Prnp +/o-6H4μ mice. The brains of Prnp +/o-6H4μ displayed no PrPSc deposits, whereasPrnp +/o-15B3μ controls exhibited strong PrPSc accumulation at 234 dpi. (D) PrPSc deposition in the brains of nontransgenic littermates and Prnp +/o-15B3μ was detected as early as 170 dpi and, to a larger extent, at 234 dpi. In contrast, no signal was detected in the brains of 6H4μ mice, even when 1 mg of protein was used and the blots were overexposed. (+) indicates proteinase K (PK) digestion.

We next tested whether anti-PrP humoral immunity in 6H4μ mice could inhibit prion transport from peripheral sites to the central nervous system. The brains of scrapie-inoculatedPrnp o/o, Prnp +/o,Prnp +/o-15B3μ, andPrnp +/o-6H4μ mice were analyzed by Western blot. PrPSc was not detected in any transgenicPrnp +/o-6H4μ brain, whereas there was a strong signal in the brains of all nontransgenicPrnp +/o andPrnp +/o-15B3μ mice as early as 170 dpi (Fig. 3D). Histoblots confirmed PrPSc accumulation in the brains of Prnp +/o-15B3μ mice, whereas there was no signal in Prnp +/o-6H4μ mice (Fig. 3C).

It has been observed that ablation of B-lymphocytes prevents neuropathogenesis of prion disease after intraperitoneal inoculation (24, 25). This is probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs) (26), which are a major extracerebral prion reservoir (23). Neuropathogenesis is also impaired by ablation of complement factors and receptors that mediate opsonization and capture by FDCs (27). We therefore tested whether antiprion protection of 6H4μ may result from altered B cell physiology, development, or histoarchitecture and cellular composition of germinal centers. Such alterations may impair antiviral responses. However, 6H4μ and 15B3μ (Prnp o/o orPrnp +/o) mounted IgM and IgG responses to vesicular stomatitis virus or lymphocytic choriomeningitis virus with similar kinetics (7). Therefore, expression of transgenic μ chains did not alter the immune system (7) in a way that would suppress B cell responses, although a slight reduction of B cell counts per volume unit was seen in peripheral blood and spleen (7).Prnp +/o-6H4μ andPrnp +/o- 15B3μ mice displayed germinal centers of slightly smaller size than nontransgenicPrnp +/o mice, but had normal FDC clusters that were found to colocalize with PrPC (Fig. 4A), implying that mature B cells were present (28). Splenic B and T cell subsets, dendritic cells, and macrophages were unaltered in all transgenic mice (7). All of the above data indicate that 6H4μ prevents, or drastically delays, scrapie pathogenesis.

Figure 4

Histological and biochemical characteristics associated with scrapie protection. (A) Histological examination of age-matched transgenic and nontransgenic littermates (genotype as indicated above) revealed normal networks of follicular dendritic cells (FDCs; detected with mAb FDC-M1, middle row) forming networklike structures within germinal centers and colocalized with PrPC expression (detected with polyclonal XN antibody, upper row). (Lower row) Overlay of upper and middle rows. (B) Western blot. PrPC expression in the brain (left) and spleen (right) was similar inPrnp +/o-6H4μ and nontransgenicPrnp +/o mice. This was confirmed by quantitative chemiluminescent analysis (11).Prnp +/+ and tga20 mice served as positive controls, Prnp o/o mice as negative controls. Equal amounts of protein were loaded, as assessed by β-actin quantification (lower bands). (C) FACS analysis of peripheral blood of tg94-6H4μ mice revealed transgenic IgMa immunoglobulins adhering to Thy1.2+ T cells, suggesting masking of PrPC (solid black line). Because of the low percentage of B cells in the peripheral blood oftg94-6H4μ mice, there was a relative increase in the number of T cells compared with that in tg94 control mice (gray dotted line) within the sample population (10,000 lymphocytes).

Genetic ablation of Prnp abrogates susceptibility of mice to scrapie (14) and prevents subclinical prion replication in the brain and spleen (29). Therefore, we investigated whether 6H4μ repressed PrPC expression inPrnp +/o mice. However, Western blot analysis showed that the content of PrPC in the spleens and brains of Prnp +/o-6H4μ mice before (Fig. 4B) (7) and after prion inoculation (11) was similar to that of nontransgenic Prnp +/o mice (12). We conclude that prion resistance ofPrnp +/o-6H4μ mice is not mediated by down-regulation of PrPC expression. Masking of PrPC at critical sites of prion replication may be involved in protection, as suggested by the finding that intg94-6H4μ mice, T cells overexpressing PrPCwere highly decorated with transgenic IgMa antibodies (Fig. 4C). This may hinder the interaction of prions with PrPC. Other mechanisms, such as capturing and immune-mediated degradation of the incoming PrPSc inoculum, steric competition with template-directed refolding, or interference with a seeded PrPSc nucleation reaction, may also be involved.

Although in vitro preincubation with anti-PrP antisera was reported to reduce the prion titer of infectious microsomes from hamster brain homogenates (30) and an anti-PrP antibody was found to inhibit formation of PrPSc in a cell-free system (31), prevention of neuroinvasive scrapie in vivo by specific anti-PrP antibodies has not been previously reported. Because PrPC is broadly expressed, it is possible that induction of anti-PrP immune responses may induce an autoimmune disease and defeat prospects for prion vaccination. However, we observed no overt symptoms of autoimmune disease as a result of anti-prion immunization unless PrPC was expressed at extremely high levels. At the same time, there appears to be no appreciable clonal deletion of autoreactive immune cells, suggesting that B cells are not tolerant to PrPC, similar to other examples of B cells specific for transgenic antigens (32). If B cells of wild-type and transgenic 6H4μ mice are not tolerant to PrPC, lack of immunity to prions may be due to T helper cell tolerance—a condition that may be overcome by presenting PrPC in an appropriate, adjuvant context.

From an applied viewpoint, transgenesis is too elaborate as a defensive strategy against prions. However, the findings described here encourage reassessment of the value of active and passive immunization, and perhaps of reprogramming B cell repertoires by μ chain transfer, in prophylaxis or therapy of prion diseases.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: adriano{at}


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