Conceptually the best way to treat multiple sclerosis is to specifically inhibit the immune response against the myelin sheath, while leaving the immune response against other proteins intact.  Additionally, the best way to treat it, is also to induce regeneration of neurons and central nervous system components that have already been damaged, perhaps through the use of stem cells.

Successful use of stem cell therapy in multiple sclerosis is believed to occur because the fat contains numerous cell populations that inhibit pathological immune responses, such as T regulatory cells (whose function is suppressed in multiple sclerosis), while at the same time containing stem cells, especially mesenchymal stem cells, which can repair damaged tissue.

The reprogramming of the immune system to selectively stop attacking one protein, or a series of specific proteins is called “immunological tolerance”.  Originally the concept of tolerance came from experiments by the scientists Billingham-Brent-Medawar decades ago who demonstrated that if two genetically distinct animals had shared circulation during embryonic development, when the animals reached adulthood they would readily accept tissue grafts from each other but reject grafts from others.  In other words the animals were made “tolerant” to each other. 

This concept of selectively “teaching” the immune system that it should not attack a specific antigen is how approaches such as the myelin basic protein DNA vaccine developed by Bayhill Therapeutics seems to work.  This vaccine, called BHT-3009, was demonstrated to induce antigen-specific immune modulation in multiple sclerosis patients in a Phase I/II study, and was subsequently demonstrated to be capable of causing a 50-61% reduction in new lesion formation as detected by MRI and profound reduction of anti-myelin antibodies in a subsequent Phase II study in multiple sclerosis patients.  DNA vaccines seem to work in part through inducing interferon beta, which seems to be involved in shifting of the T cell cytokine production profile away from Th1 and Th17, although this is controversial.

Another way to “trick” the immune system into selectively not attacking an antigen is to provide the antigen orally.  A published study fed multiple sclerosis patients cow myelin (which contains both myelin basic protein and proteolipid protein) and examined whether this affected immune response to myelin (Hafler, DA et al. Oral administration of myelin induces antigen-specific TGF-beta 1 secreting T cells in patients with multiple sclerosis. Ann NY Acad Sci 1997 Dec 19;835:120-31).

The investigators took blood from 34 patients with relapse remitting multiple sclerosis that were either fed cow myelin (17 patients) or not fed it (17 control patients) and generated T cell lines that recognized either myelin basic protein (MBP), proteolipid protein (PLP), or tetanus toxin (TT). 

A profound increase in the number of cells secreting the antiinflammatory cytokine TGF-b on stimulation with MBP or PLP was seen in patients who ate the cow myelin as opposed to controls.  Interestingly, there was no increase in production of the inflammatory cytokine interferon gamma, nor were there alterations in response to tetanus toxin. 

These data suggest that at least at an immunological level administration of oral cow myelin is helpful in patients with multiple sclerosis.  The question now because, can one increase therapeutic effects by combining the cow myelin with something like lithium, or with fat stem cells?  Furthermore, clinically used drugs such as metformin, which may conceptually increase Treg generation by suppressing IL-17 may be useful to expand the overall tolerogenic profile of oral tolerance induction.

We previously discussed the concept of DNA vaccination for antigen-specific modulation of the immune system in conditions of autoimmunity such as multiple sclerosis.  In essence, it appears that DNA-based administration of the same proteins that are targets of the immune system during autoimmunity seem to suppress the immune response in a specific manner.  This is different than other immune modulators such as anti-CD25 antibody or Tysabri which suppresses in a non-specific manner.

A published Phase I/II trial of BHT-3009, Bayhill Therapeutics DNA vaccine for myelin basic protein in 30 patients with either secondary progressive or relapse-remitting multiple sclerosis was performed in which antigen-specific immune modulation was seen, and safety of the vaccine was demonstrated.  Here we will discuss a larger trial by the same group that was recently published (Garren et al. Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis. Ann Neurol 2008 May;63(5):611-20).

This trial was much larger than the previous trial (267 patients), and was limited to patients with relapse-remitting multiple sclerosis.  The patients were randomized to receive either placebo, or two doses (0.5 mg and 1.5 mg) of the DNA vaccine (BHT-3009).  DNA vaccination was performed intramuscularly at the timepoints of initiation, after 2 weeks, after 4 weeks and subsequently given for every month until the 44th week. 

The data demonstrated that in comparison to patients receiving placebo there were no positive effects of the 1.5 mg dose.  In contrast, the 0.5 mg dose caused a 50-61% reduction in new lesion formation as detected by MRI and profound reduction of anti-myelin antibodies.

These data support further expansion of the DNA vaccine approach into phase III clinical trials.  These data, as well as other antigen-specific tolerogenic vaccines may be one of the reasons why entered a $350 million deal with Genetech.

We previously discussed that interferon beta therapy is associated with upregulation of T regulatory cell (Treg) activity in patients with multiple sclerosis, and that this may be one of the mechanisms by which it mediates its therapeutic effect.  We also discussed the point that current methods of measuring Treg activity are somewhat limited in that they do not measure the specific Treg that protect the body against the specific autoreactive T cells that are attacking the myelin, but measure activity of all the Tregs as a whole.

Here we are going to discuss a publication (Andersson et al. Impaired autoimmune T helper 17 cell responses following DNA vaccination against rat experimental autoimmune encephalomyelitis. PLoS One 2008;3(11):e3682) that seems to demonstrate, at least in the rat model, that protection from multiple sclerosis (well actually EAE) by DNA immunization with autoantigen is dependent on production of interferon beta by the immunized animal.

To induce EAE (experimental allergic encephalomyelitis) the investigators vaccinated the rats with peptide 91-108 of the myelin oligodendrocyte glycoprotein in Complete Freund’s Adjuvant.  This stimulates an autoimmune reaction against the rat myelin, and rats develop a progressive disease that resembles multiple sclerosis. 

When rats were administered a DNA vaccine encoding the same peptide that the animals were immunized with to induce EAE, the progress of the disease was potently inhibited.  This is similar to what we discussed about in multiple sclerosis patients who were administered the DNA vaccine made by Bayhill Therapeutics against myelin basis protein.  In that situation, specific inhibition of the autoimmune responses was also observed.

What is interesting about the current study is that experiments were performed to determine through what molecular mechanism the DNA vaccine was mediating protection against disease.  It was found that T cells isolated from animals at the peak of disease had a much higher expression of the cytokine IL-17 when restimulated in vitro with the antigen myelin oligodendrocyte glycoprotein 91-108.  Animals that were treated with the DNA vaccine had a lower amount of IL-17 production.  Since IL-17 is associated with multiple sclerosis in both mice and human, it would make sense that a protective vaccine would reduce IL-17 production.  As a quick aside, the anti-diabetes drug metformin, which inhibits EAE, also suppresses production of IL-17 by autoreactive T cells.  Additionally, vitamin D has been demonstrated to reduce IL-17.

With the knowledge that the DNA vaccine seems to reduce ability of autoreactive T cells to produce the “bad” cytokine IL-17, the investigators next wanted to see what exactly is it that is responsible for this reduction in IL-17 producing cells.  So the DNA microarray technique was used to analyze 6240 genes at once to randomly see expression of which genes goes and which goes down after DNA vaccination. 

What was found was that the gene for interferon beta, as well as other genes that are associated with interferon beta were upregulated after immunization.  We know that interferon beta is part of the current-day treatment of multiple sclerosis, but also that it has several systemic side effects.  Therefore this study may be suggesting that DNA vaccination seems to be stimulating the body to make its own interferon beta.  This would be a much more attractive approach as compared to what is currently being performed for two reasons.  Firstly, the DNA vaccination encodes a specific antigen, therefore in addition to receiving interferon beta, the vaccination may offer the possibility of inducing tolerance to the autoantigen selectively.  Secondly, the production of interferon beta stimulating by the DNA vaccine would hypothetically be released at smaller and more localized concentrations, thus not having to “flood” the body with interferon beta from the outside and hopefully reducing the side effects.

But here is the catch…how do we know that the interferon beta being produced as a result of the DNA vaccination is actually responsible for suppression of disease progression?  In order to address this, the investigators created a DNA plasmid for vaccination that not only induced expression of the myelin oligodendrocyte glycoprotein peptide, but also short interferon RNA to target gene expression of interferon beta. 

In other words, the investigators repeated their experiments using two sets of DNA plasmids, one that has previously been shown to protect from disease by inducing expression of the myelin oligodendrocyte glycoprotein peptide, and another one in which the peptide is expressed but interferon beta gene is silenced (actually they used more controls, but these are the main ones to make the point).  It was found that suppression of the interferon beta gene expression resulted in loss of the therapeutic effect !  Also, recall response to peptide in terms of IL-17 secretion was augmented 20-fold if interferon beta was silenced.

What does all this mean?

Firstly, why would interferon beta be made by the simple procedure of DNA vaccination?  After all, DNA vaccination is performed as an alternative delivery of antigen.  Instead of injecting protein or peptides of the protein with adjuvant (which in the case of myelin oligodendrocyte glycoprotein results in disease) one injects DNA encoding the protein or peptide so that the cell generates the protein or peptide internally.  So if its the same protein or peptide being made, then why would one cause disease (direct injection of protein or peptide) but the other (DNA vaccination of the same thing) cause protection from disease and interferon beta production?

The authors state that one likely explanation is that the DNA vaccine backbone (the other parts of the DNA plasmid that do not encode the protein or peptide) has CpG motifs.  CpG motifs are parts of DNA that are recognized by human cells as being foreign and induce production of interferons, including interferon beta.  So it may be the interact effect of the CpG motifs that is responsible for protection.  In fact, the authors cite several papers, such as (Lobell et al. J Immunol 2003 Feb 15;170(4):1806-13) that demonstrate removal of CpG motifs from plasmid backbone used to “immunize” animals from autoimmunity, results in loss of effect.

In conclusion, the study discussed seems to provide a very interesting concept…that DNA vaccination can concurrently induce a cytokine that is therapeutic for multiple sclerosis, while at the same time antigen-specifically modulating an immune response.  It will be very interesting to see how the trials of Bayhill Therapeutics progress using a similar antigen-specific DNA-”vaccine” based approach.

As we discussed here on StemNow.com, the possibility of antigen-specific tolerance for multiple sclerosis is, along with regenerative medicine, the most promising area of research in this field. 

Clinical trials have previously tried to induce tolerance in multiple sclerosis by selectively inducing immune response against the autoreactive T cells, with some degree of effect. 

The current clinical trial that we will discuss here uses immunization with a DNA vaccine encoding myelin basic protein to induce antigen-specific tolerance, or inhibition of immunity.   It was published as a collaboration between the company Bayhill Therapeutics, and several academic centers (Bar-Or et al. Induction of antigen-specific tolerance in multiple sclerosis after immunization with DNA encoding myelin basic protein in a randomized, placebo-controlled phase 1/2 trial. Arch Neurol 2007 Oct;64(10):1407-15). 

Since myelin basic protein is what the immune system is attacking in multiple sclerosis, or at least one of the main targets, the idea was that if myelin basic protein could be administered intramuscularly, or better said, expressed intramuscularly, then through the immune system may actually be suppressed in response to it.  Below is a picture of the plasmid that was used for injection. 

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This study is interesting because it is, to our knowledge, the first time that DNA vaccination was used for induction of immunological tolerance in patients in an antigen-specific manner.  Unfortunately, the lack of actual efficacy in MRI lesion size, or even lack of symptomological improvement (it was not discussed), suggests that numerous other modifications, or at least large sample sizes, need to be performed to see an effect.

On a positive note, one of the interesting features of this study was that even though the antigen myelin basic protein was administered as a “tolerogen”, inhibition of immune responses against other autoantigens, such as PLP.  This suggests that the immune system may be somehow “re-educating itself”.  We proposed the concept of “epitope spreading” in the context of immune regulation many years ago, and this may be an excellent example.

Given that autologous stromal vascular fraction contains antiinflammatory cells in addition to regenerative mesenchymal stem cells, it may be worthwhile to see whether adipose non-expanded stem cell therapy affects epitope spreading both from the pathogenic and regulatory perspective.

Multiple sclerosis is associated with T cell attacks against components of the central nervous system, specifically the myelin sheath.  This is why immunizing mice with components of the myelin sheath induces an MS-like disease called experimental allergic encephalomyelitis (EAE), which interestingly enough can be induced to resolve by stem cell therapy.

An interesting idea would be to take the autoreactive T cells (T cells causing the damage) and “immunize” against them.  This would theoretically induce the immune system to fight the part of the immune system that is pathological.  Additionally, such an approach would have advantages over non-specific immune suppressants which non-specifically shut down all T cell responses.

A recent publication (Loftus et al. Autologous attenuated T-cell vaccine (Tovaxin) dose escalation in multiple sclerosis relapsing-remitting and secondary progressive patients nonresponsive to approved immunomodulatory therapies. Clin Immunol 2009 May;131(2):202-15. Epub 2009 Feb 18) took exactly this approach in a clinical trial.

Myelin-reactive T cells were generated by stimulation with peptides from myelin basic protein, proteolipid protein, and myelin oligodendrocyte protein and subsequently inactivated by irradiation so as to not proliferate.  This would prevent the autoreactive cells from causing damage when used for immunization.  The authors used the term “attenuated myelin reactive T-cells” (MRTC).

Vaccination was performed subcutaneously and patients were assessed for over 52 weeks. 

Reduction in autoreactive T cells in the patients as well as suppression of relapses was reported.  MRI studies did not reveal resolution of plaques, nor progression in patients that responded by clinical parameters such as EDSS. 

This study demonstrates the potential of inducing an immune response against pathology-causing T cells. It may be that future studies will seek to amplify the therapeutic response by combining this approach with mesenchymal stem cells.