Vaccination with CNS antigens can induce autoreactive T cell responses that home to sites of injury in the CNS and can inhibit neuronal degeneration in different models of neurological disease and injury, including spinal cord and head injury, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, glutamate toxicity, and glaucoma . It is thought that immunization activates CNS-reactive T cells that enter the CNS, secrete neurosupportive factors and shift the phenotype of resident microglia to one that is more neurosupportive. The initial studies of this therapeutic strategy immunized animals with oligodendrocyte antigens such as myelin basic protein . Since autoimmunity to oligodendrocyte antigens can lead to a multiple sclerosis -like disease in experimental animals, subsequent studies of neuroprotective vaccines have focused on vaccinating with CopaxoneH . CopaxoneH is a mixture of synthetic polypeptides composed of four amino acids in a random sequence dissolved in an aqueous solution. Frequent CopaxoneH injection induces regulatory T cell responses that have partial cross-reactivity with myelin antigens and this treatment has been approved as a therapy for relapsing-type MS. The vast majority of neuroprotective vaccine studies in animal models of neuropathological disorders have, however, administered myelin antigens or CopaxoneH in complete Freund’s adjuvant , an adjuvant that is unsuited for human use. There have been few reports of adjuvant-free CopaxoneH having beneficial effects in animal models of neuropathological disorders other than MS. Our previous studies of antigen-based vaccine therapies for inhibiting autoimmune disease have shown that the ability of a vaccine to induce protective T cell responses depends critically on which self-antigen is administered. This is because each self-antigen has a unique expression pattern and impact on T cell selftolerance induction. Accordingly,vertical tower for strawberries self-antigens have different immunogenicities and should vary in their ability to induce neuroprotective T cell responses.
Random copolymers as CopaxoneH may not be optimal immunogens for inducing neuroprotective T cell responses since only a small portion of the induced T cell response may be capable of cross-reacting with CNS antigens. Hence, further studies are needed to examine how the nature of the antigen used in neuroprotective vaccines affects the efficacy of the treatment. Current treatments for Parkinson’s disease temporarily ameliorate its symptoms but do not slow progressive loss of dopaminergic neurons. Accordingly, new approaches to slow the degeneration of the nigrostriatal dopaminergic system are urgently needed. It is thought that oxidative stress, protein nitration andactivated microglia contribute to the loss of dopaminergic function in human PD. Additionally, there is a growing appreciation that CD8+ and CD4+ T cells significantly infiltrate the SN of patients with PD . All of these potentially pathogenic factors are elicited by treatment with the neurotoxin MPTP. The MPTP mouse model of PD has therefore been extensively used to assess neuroprotective strategies. Several studies have shown that vaccination with oligodendrocyte antigens or CopaxoneH in CFA preserves dopaminergic neurons in MPTP treated mice. These studies, however, did not determine whether the vaccine-induced immune responses limited the initial nigrostriatal dopamine system damage and/or promoted long-term neurorestoration. We began our studies asking whether vaccination with tyrosine hydroxylase , a neuronal protein involved in dopamine synthesis, could protect striatal dopaminergic neurons to a greater extent than CopaxoneH in the MPTP model of PD in mice. Contrary to our expectations, we observed that immune stimulation by the CFA adjuvant, regardless of the emulsified antigen, appeared to be the major neuroprotective factor.The BCG vaccine developed against childhood tuberculosis contains live attenuated Mycobacterium bovis that is closely related to Mycobacterium tuberculosis, and has been administered safely to billions of individuals since the 1920s [29,30]. We describe the neuroprotective effects of BCG vaccination in the MPTP mouse model and discuss possible underlying mechanisms.
Our results suggest that general immune stimulation in the periphery may provide a new strategy to help slow disease progression in some neurodegenerative diseases.Studies of neuroprotective vaccines have focused on using CopaxoneH since it induces protective immune responses that cross-react with myelin antigens and because it is in clinical use for treating MS. We wanted to test whether immunization with a dopaminergic neuron antigen might have a more beneficial effect in the MPTP mouse model of PD, since this should direct vaccine induced T cells to the brain areas that were damaged by MPTP treatment and that slowly degenerate in human PD. We chose tyrosine hydroxylase as a test antigen because it is involved in dopamine synthesis and is predominantly expressed in striatal dopaminergic neurons. We isolated TH from recombinant E. coli inclusion bodies, and purified it using affinity chromatography and preparative SDS-PAGE as described in Materials and Methods. Gel analysis of the purified TH is shown in Supplement Figure S1. Since it takes 10–14 days for vaccine-induced immune responses to peak, and MPTP has a very immediate toxic effect, we immunized mice with TH or CopaxoneH in CFA 10 days before MPTP treatment. A group of control mice received only saline. The animals were sacrificed 21 days after the last MPTP treatment, which is a relatively long time point for such studies, because we wanted to test for potential neurorestorative effects of vaccination. As an initial read-out of the vaccine’s ability to preserve dopaminergic system integrity, we measured [3 H]WIN 35,428 binding to DAT in mouse striatal homogenates. We found that the mean DAT WIN binding levels were higher in striata from MPTP-treated mice that received CFA, regardless of whether they received CFA alone, TH/CFA, or CopaxoneH/ CFA, compared to that in unvaccinated MPTP-treated mice . Specifically, compared to unvaccinated MPTP-treated mice, the levels of striatal WIN binding were 43% higher in MPTP-treated mice that received CFA alone and 34% higher in mice that received CopaxoneH/CFA . The level of striatal WIN binding was 17% higher in MPTP-treated mice that received TH/CFA, but this was not statistically significant. These results argue against our initial hypothesis that a neuronal self-antigen may provide a more efficacious neuroprotective vaccine. Rather, the results suggest that peripheral immunostimulation by CFA was the major beneficial factor.It is possible that immune responses elicited by CFA vaccination limited MPTP’s direct effects or promoted the subsequent restoration of dopaminergic neuron integrity. We therefore performed a more detailed study of the effects of CFA immunization on DAT levels 4 and 21 days after the last MPTP treatment. Groups of mice were vaccinated with CFA or saline, and 10 days later were given MPTP for 5 consecutive days.
Four days after the last MPTP treatment, the mean levels of striatal WIN binding were 18% higher in the CFA treated group than in unvaccinated MPTP-treated mice, but this increase was not statistically significant . This suggests that CFA vaccination did not differentially affect the uptake, distribution or metabolism of MPTP and that CFA-induced immune responses have little or no ability to limit the acute toxicity of MPTP. We also examined similarly treated mice 21 days after the last MPTP treatment. We found that the mean levels of striatal DAT WIN binding was 29% higher in CFA vaccinated mice that received MPTP,vertical growing compared to unvaccinated MPTP-treated mice . These data again demonstrate the beneficial effects of CFA treatment in our model. Additionally, the increase in striatal WIN binding observed in vaccinated vs. unvaccinated MPTP-treated mice from 4 to 21 days post treatment , suggests that CFA treatment promoted a greater rate of neurorestoration. Indeed, 21 days after MPTP treatment, only CFA-treated mice displayed a significant increase in striatal DAT WIN binding compared to levels 4 days post-treatment, suggestive of a neurorestorative effect.CFA is unsuitable for human use, but its main immunogenic component, inactivated Mycobacterium tuberculosis, is closely related to the live attenuated Mycobacterium bovis used in the BCG vaccine against childhood tuberculosis. We hypothesized that the peripheral immune responses induced by BCG immunization may also be neuroprotective. C57Bl/6 mice were immunized with BCG and 10 days later they, and a control group of unvaccinated mice, received MPTP. Twenty one days later, their striatal WIN binding levels were measured. Mice vaccinated with BCG had significantly higher levels of WIN binding than MPTP controls . In addition, striatum from mice vaccinated with BCG also had significantly higher DA content . Thus, BCG vaccination had a significant beneficial effect on both striatal DA content and DAT ligand binding levels.Previous studies have shown that the number of microglia increases rapidly in the striatum after MPTP treatment and play an active role in MPTP-induced nigro-striatal system damage. Inflammatory type microglia are considered detrimental to neuron survival after a neuro-toxin insult and blockade of microglia activation was neuroprotective in the MPTP mouse model of PD. To examine whether BCG vaccination also affected the microglial reaction to MPTP toxicity, we treated other groups of mice with BCG or saline prior to MPTP treatment and counted the number of microglia in their midbrains three days post-MPTP treatment. We found that the number of Iba1+ microglia cells was significantly greater in animals that received MPTP compared to that in mice that received only saline , as also reported by others . In contrast, the Iba1+ cell number in SNc of BCG-vaccinated mice that received MPTP was similar to that in mice that only received saline . We also observed that the Iba1+ cells in the unvaccinated MPTP-treated mice had large cell bodies with only a few short thick processes, a morphology associated with microglia activation. In contrast, the Iba1+ cells in mice that received BCG before MPTP treatment had small cell bodies with long-fine processes similar to those in saline-treated control mice, suggesting a resting state. Thus, BCG vaccination prevented the MPTP induced increase in the number of activated microglia in the SNc, suggesting that general immune stimulation in the periphery can limit CNS microglia responses to a neuronal insult. At 21 days post-MPTP, stereological analysis revealed that the number of TH+ cells in the SNc of animals that received BCG was on average 6% greater than that in mice that received only MPTP , although this was not statistically significant .Vaccination with CNS antigens has beneficial effects in a number of different animal models of neurological disease and injury. This strategy is based on inducing CNS-reactive T cells which home to areas of damage and exert beneficial effects locally in a process termed ‘‘protective autoimmunity’’ . Early studies of neuroprotective vaccines administered myelin antigens, which raised safety concerns because of their potential for inducing a MS-like disease. Subsequent studies used CopaxoneH which has some resemblance with MBP, and in its aqueous form is approved for MS treatment. Almost all these studies, however, used CFA as an adjuvant and often did not report on the effects of CFA alone. The studies that did examine CFA often reported that these treatments had some beneficial effect, although of lower magnitude than the myelin antigen or CopaxoneH in CFA. Contrary to our initial expectations, we found that immunization with a dopaminergic neuron antigen did not provide a greater beneficial effect. Rather, CFA itself appeared to be main factor associated with higher levels of striatal WIN binding in vaccinated MPTP-treated mice. CFA treatment did not significantly alter the level of striatal DAT WIN binding 4 days after MPTP treatment, suggesting that CFA-induced immune responses cannot limit the acute toxicity of MPTP. Twenty one days post-MPTP treatment, however, the average levels of striatal DAT WIN binding in CFA treated MPTP-treated mice was significantly greater than that in unvaccinated MPTP-treated mice. The ratio of striatal WIN binding in vaccinated mice versus unvaccinated MPTP-treated mice increased from 4 to 21 days, suggesting that the CFAinduced responses promoted a greater rate of neurorestoration. CFA-treated mice, but not unvaccinated mice, had significantly higher striatal WIN binding 21 days vs. 4 days after MPTP treatment, indicating a neurorestorative effect. Based on the neuroprotective effects of CFA, we turned to testing BCG vaccination. Potential advantages of BCG vaccination include not only its established safety record over many decades of worldwide use in humans, but also that the attenuated BCG slowly replicates in the vaccinated individual, inducing immune responses over many months. Accordingly, BCG vaccination could provide a long-term source of neurosupportive immune responses.We observed that BCG vaccination significantly preserved striatal DAT WIN binding and DA content compared to that in unvaccinated MPTP-treated mice.