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Rapid induction of autoantibodies against Nogo-A and MOG in the absence of an encephalitogenic T cell response: implication for immunotherapeutic approaches in neurological diseases ¹

Brain Research Institute, University of Zurich and Department of Biology, Swiss Federal Institute of Technology Zurich, CH-8057 Zurich, Switzerland; and
^* Institute of Experimental Immunology, University Hospital, Zurich, Switzerland

²Correspondence: Institute of Neuropathology, Georg-August-University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany. E-mail: merkler{at}med.uni-goettingen.de

SPECIFIC AIMS

Vaccination against central nervous system (CNS) proteins has been developed as a therapeutic approach for several neurological diseases including Alzheimer’s disease, stroke, and spinal cord injury. Use of these vaccination approaches, however, has so far been limited by the inherent risk of autoimmune side effects. To overcome these limitations, we have designed a conjugate vaccine approach with particular attention to the safety and the kinetics of the antibody response. As antigen targets, we used the neurite outgrowth inhibitory protein Nogo-A and the highly encephalitogenic myelin-oligodendrocyte glycoprotein MOG.

PRINCIPAL FINDINGS

1. Limited time window of blood–brain barrier breakdown allowing antibody influx after spinal cord injury in adult rats
Intravenous injection and subsequent immunohistochemical detection of Nogo-A-specific mouse antibodies in the rat spinal cord revealed the window of time allowing circulating antibodies to cross the damaged blood–brain barrier after spinal cord injury. Antibody influx was massive at and around the lesion site for up to 3 days after injury, followed by a substantial reduction of antibody influx on days 6 to 9. Antibody concentrations in the cerebrospinal fluid measured by ELISA 1 day after spinal cord injury were greater than three orders of magnitude below serum levels, demonstrating the rapid antibody clearance from the cerebrospinal fluid even after considerable damage of the blood–brain barrier. For a postlesion vaccine approach, these data emphasize the necessity to induce a very rapid therapeutic antibody response.

2. Priming and route of antigen administration determine the kinetics of antibody response against Nogo-A
To develop a vaccine that fulfills the demands of a rapid immune response, we took advantage of the fact that intrasplenic injection of a high antigen dose can induce a T cell-independent IgM response that arises faster than in a T cell-dependent manner. In our present study the intrasplenic immunization with the Nogo-A-specific region NiG (amino acids 174-979) fused to the C fragment of tetanus toxin (TTC) elicited a rapid IgM response against NiG within 4 days (Fig. 1 ). This rapid IgM response was not elicited when the same antigen dose was applied subcutaneously in incomplete Freund’s adjuvant. We further analyzed whether TTC, which contains strong and unrelated T cell helper epitopes, can facilitate an immunoglobulin class switch to IgG against NiG after intrasplenic immunization with the NiG-TTC fusion protein. Animals were subcutaneously immunized with TTC prior to spinal cord injury for T cell priming. Although intrasplenic immunization with the NiG-TTC fusion protein elicited a comparable primary IgM response in primed and unprimed rats, a rapid immunoglobulin class switch to IgG (after 4–7 days) was observed only in TTC primed animals (Fig. 1) . Furthermore, the induction of the anti-NIG IgG response in these animals was considerably faster than in animals that received a conventional subcutaneous immunization of NiG-TTC in incomplete Freund’s adjuvant.

View larger version (27K): [in this window] [in a new window]   Figure 1. IgM and IgG response against Nogo-A after intrasplenic or subcutaneous (s.c.) immunization, respectively. A) Fast IgM response against the Nogo-A-specific region NiG (amino acids 174-979) was already detectable 4 days after injection of 100 µg NiG-TTC in PBS into the spleen independent of prior TTC priming (open squares and filled diamonds). In contrast, s.c. immunization of 100 µg NiG-TTC in adjuvant did not elicit a detectable IgM response in TTC primed animals (open triangles). Intrasplenic immunization of PBS (open squares) served as negative control. B) TTC primed rats elicited a rapid immunoglobulin class switch to IgG within 4 to 7 days (filled diamonds) in contrast to unprimed rats (open triangles) after intrasplenic immunization with NiG-TTC. Immunoglobulin class switch after s.c. immunization with NiG-TTC in IFA was delayed compared with intrasplenic NiG-TTC immunization in TTC primed rats. Priming against TTC was performed by s.c. immunization of 40 µg TTC in adjuvant. Antibody titers were determined by ELISA. Results are given as the mean ± SE of 4–8 rats per group. Difference statistically significant with **P < 0.01.

3. Specificity of the immune sera against Nogo-A
Antigen specificity of the immune sera was analyzed with Western blots of rat spinal cord extract and by immunocytochemistry. Immune sera recognized Nogo-A as soon as 6 days after intrasplenic immunization with NiG-TTC in TTC primed animals. Nogo-A transfected COS cells showed a reticular staining typical of the main subcellular localization of Nogo-A to membranes of the endoplasmic reticulum. These findings demonstrate that a specific antibody response can be induced against Nogo-A within few days after spinal cord injury.

4. Absence of in vitro T cell activation and absence of EAE symptoms and inflammation in vivo after intrasplenic MOG-TTC and NIG-TTC immunization
The development of an autoimmune disease after immunization against a CNS antigen is strongly dependent on the activation of encephalitogenic T cells. As Nogo A is not a strong encephalitogen we used the highly encephalitogenic myelin-oligodendrocyte glycoprotein (MOG) to address specifically the autoreactive T cell activation after intrasplenic immunization. We injected MOG as a fusion protein with TTC at a concentration known to induce EAE after subcutaneous immunization in incomplete Freund’s adjuvant. Although a rapid antibody response could be measured after single intrasplenic or subcutaneous immunization, only animals that were subcutaneously immunized developed clinical signs of inflammatory CNS disease (Fig. 2 ). Measurement of IL-2, IL-10, and IFN- secretion upon stimulation with MOG in vitro confirmed the lack of autoreactive T cell activation after intrasplenic immunization. These findings suggest that our conjugate vaccine approach leads to the induction of a robust B cell response against a self-antigen in the absence of measurable T cell activation.

View larger version (25K): [in this window] [in a new window]   Figure 2. Induction of EAE after s.c. immunization with MOG but not after intrasplenic immunization with MOG-TTC in TTC primed animals. Rats immunized subcutaneously with 100 µg MOG in IFA (open squares, n=6) developed classical signs of EAE starting 13 days after vaccination. In contrast, rats primed against TTC and injected with 100 µg MOG-TTC into the spleen (filled triangles, n=6) showed no clinical symptoms during the observed period, although an IgG response against MOG was measured after both intrasplenic MOG-TTC and s.c. MOG immunization (box top left). No clinical symptoms were observed after s.c. in incomplete Freund’s adjuvant (100 µg/animal, open circles, n=4) or intrasplenic immunization with NiG-TTC (not shown). #Rats were killed because of progressive disease or bad general state.

CONCLUSIONS AND SIGNIFICANCE

In the present study, the demands for a therapeutic vaccine are investigated with respect to the kinetics of the antibody response against CNS self proteins and to safety after spinal cord injury. Intrasplenic immunization with the Nogo-A fragment NiG as a fusion protein with TTC induced a rapid IgM and IgG response in TTC primed animals. These findings are best explained by the concept that autoreactive Nogo-A-specific B cells receive cross-linked help from preactivated T helper cells which are directed against epitopes of the covalently linked non-self protein TTC (Fig. 3 ). For potential application in humans, it is interesting to note that TTC-specific immunity is already present in a large portion of the population due to tetanus vaccination programs in most countries.

View larger version (67K): [in this window] [in a new window]   Figure 3. TTC-directed T cell help for IgG switch in NiG-specific B cells. Tetanus toxin C fragment (TTC, red) in adjuvant is taken up by antigen-presenting cells (APC) in a subcutaneous depot (1). Processing and presentation on MHC class II in secondary lymphoid organs leads to activation and expansion of TTC-specific T helper cells (T -TTC) (2). Intrasplenic immunization with high-dose NiG-TTC (3) activates NiG-specific B cells (B -NiG) via their NiG-specific IgM surface receptors, resulting in a rapid T cell-independent IgM response (4). At the same time, the NiG-TTC conjugate is internalized via receptor binding, and NiG-derived (green) as well as TTC-derived (red) peptides are processed and presented on MHC class II (5). Primed TTC-specific T helper cells interact with NiG-specific B cells presenting TTC peptides and provide the necessary signals (6) for isotype switch to IgG (7). Self-reactive NiG-specific T helper cells (T -TTC) apparently are not induced to a measurable degree or are centrally deleted (8).

For the therapeutic purpose of specifically neutralizing a CNS antigen, it is crucial to induce an autoimmune B cell response in the absence of activation of self-reactive T cells. Uncontrolled T cell activation can lead to autoimmune side effects as recently highlighted by postvaccination syndromes that developed in some patients of the AN-1792 Alzheimer trial. The conjugate vaccine approach provides the basis for the design of safer antibody-based immunotherapeutics for neurological diseases such as stroke, Alzheimer’s disease, or spinal cord injury.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1203fje; doi: 10.1096/fj.02-1203fje

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