Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury promotes recovery via a T-cell-mediated neuroprotective response: comparison with other myelin antigens

Abstract

The myelin-associated protein Nogo-A has received more research attention than any other inhibitor of axonal regeneration in the injured central nervous system (CNS). Circumvention of its inhibitory effect, by using antibodies specific to Nogo-A, has been shown to promote axonal regrowth. Studies in our laboratory have demonstrated that active or passive immunization of CNS-injured rats or mice with myelin-associated peptides induces a T-cell-mediated protective autoimmune response, which promotes recovery by reducing posttraumatic degeneration. Here, we show that neuronal degeneration after incomplete spinal-cord contusion in rats was substantially reduced, and hence recovery was significantly promoted, by posttraumatic immunization with p472, a peptide derived from Nogo-A. The observed effect seemed to be mediated by T cells and could be reproduced by passive transfer of a T cell line directed against the Nogo-A peptide. Thus, it seems that after incomplete spinal-cord injury, immunization with a variety of myelin-associated peptides, including those derived from Nogo-A, can be used to evoke a T cell-mediated response that promotes recovery. The choice of peptide(s) for clinical treatment of spinal-cord injuries should be based on safety considerations; in particular, the likelihood that the chosen peptide will not cause an autoimmune disease or interfere with essential functions of this peptide or other proteins. From a therapeutic point of view, the fact that the active cellular agents are T cells rather than antibodies is an advantage, as T cell production commences within the time window required for a protective effect after spinal-cord injury, whereas antibody production takes longer.

Figure 1

Active posttraumatic vaccination with the Nogo-derived peptide p472 reduces paralysis after incomplete spinal-cord injury in SPD rats. Female SPD rats were subjected to spinal contusion at T8 inflicted by the N.Y.U. Impactor with a 10-g weight dropped from a height of 50 mm. Immediately after contusion, rats in one group (n = 6) were vaccinated with p472 emulsified in CFA and rats in another group (n = 6) were injected with PBS only. Motor behavior was assessed weekly in an open field by observers blinded to the treatment received by the rat. Recovery was best in rats vaccinated with p472. Results are mean values of the motor score ± SEM. Significance of the difference between the groups was determined by using a two-tailed Student's t test (*, P < 0.05; **, P < 0.01).

Figure 2

Comparison between vaccination with the Nogo-derived peptide p472 and the MBP-derived altered peptide Ala-91 in male SPD rats. (A) Male SPD rats were subjected to spinal contusion as described for Fig. 1. Immediately after the contusion, the animals were randomly divided into two groups (n = 6 in each group). Rats in the first group were immunized with p472 in CFA, and rats in the second group were injected with PBS in CFA. Motor behavior was assessed as described for Fig. 1. Results are mean values of the motor score ± SEM. (B) Male SPD rats were injured and grouped as in A and were immunized with Ala-91 in CFA or injected with PBS in CFA. Motor recovery in the Ala-91-treated group, assessed as in A, was at least as good as with p472 treatment. Results are mean values of the motor score ± SEM. (C) Assessment of recovery from a milder spinal contusion inflicted by dropping the 10-g weight from a height of 25 mm (*, P < 0.05; **, P < 0.01).

Figure 3

Presence of proliferating T cells specific to p472 and absence of anti-p472 antibodies 1 week after vaccination with p472. (A) The proliferative response of splenocytes prepared from rats injected with p472 in CFA or with PBS in CFA to their specific antigen was compared with their proliferative response to the nonspecific antigen (OVA) in CFA or in the absence of antigen. Results are expressed as the mean cpm values of quadruplicate samples obtained from three different animals ± SEM. (B) Antibodies in the treated rats were assayed by ELISA. The amount of antibodies detected in the rats immunized with p472 in CFA was not significantly greater than in those injected with PBS in CFA. Results are expressed by mean values (expressed in arbitrary units) ± SEM obtained from three rats, each tested in triplicate at each of the indicated dilutions.

Figure 4

Passive transfer of p472-specific T cells into spinally contused rats promotes recovery. Male SPD rats were subjected to spinal contusion as described for Fig. 1. Immediately after contusion, the rats were divided into two groups (n = 5 in each group). Rats in one group were injected intraperitoneally with p472-specific T cells (1 × 10⁷ cells) in PBS, and rats in other groups were injected with PBS only. Motor behavior was assessed in an open field by observers blinded to the treatment received. Results are mean values of the motor score ± SEM. Significance of the differences between the groups at each time point was determined by using a two-tailed Student's t test (*, P < 0.05).

Figure 5

Immunization with p472 reduces RGC loss after partial crush injury of the rat optic nerve. (A) Rats were subjected to a calibrated crush injury of the optic nerve immediately before or 5 days after injection with p472 in CFA, PBS in CFA, or T cells directed to p472. (B) Rats were immunized with Ala-91 in CFA or PBS in CFA immediately after optic-nerve crush. In both A and B, the optic nerves were re-exposed 2 weeks later, and a dye was injected distally to the primary lesion. After 5 days, the retinas were excised and whole-mounted, and the RGCs were counted by observers blinded to the treatment received by the rats. Results are expressed as the mean numbers of RGCs per mm² ± SEM.

Figure 6

Retrograde labeling of cell bodies in the red nucleus. Three months after spinal contusion, rats from each group were reanesthetized, and the dye rhodamine dextran amine (Fluoro-ruby) was applied below the site of contusion. Five days later, the rats were killed, and their brains were excised, processed, and cryosectioned. Sections taken through the red nucleus were inspected and analyzed by fluorescence confocal microscopy. The photographs are of p472-immunized rats and of the control rats shown in Fig 2C.