Pertussis Toxin Stokes Autoimmune Reaction in Aβ-Vaccinated Mice

In this month’s Brain, researchers at the Universities of Milan and Genoa, Italy, report that immunizing C57/Bl6 mice with Aβ42 generated an inflammatory response similar to an autoimmune disease in the brains of these mice. None of the prior studies of Aβ42 immunotherapy of various mouse AD models, which were conducted in preparation for clinical trials of this approach, had reported this side effect (see Schenk et al., 1999; Janus et al., 2000; and Morgan et al., 2000). Roberto Furlan, working with Gianvito Martino and colleagues, injected human Aβ42 into six- to eight-week-old female mice, following the dosing regime and schedule as described previously. In a second group of mice, they added pertussis toxin (PT), an immunostimulant known to boost T cell responses and to predispose to autoimmune reactions. Sixteen out of 18 mice in this second group developed a chronic CNS inflammation reminiscent of experimental allergic encephalitis (EAE), a condition in mice widely used to model multiple sclerosis.

The mice showed inflammatory aggregates of macrophages and T cells surrounding small venules in the leptomeningeal space and the brain and spinal cord parenchyma. The authors saw occasional areas of demyelination, though much less widespread than in EAE mice. Mice treated with adjuvant plus PT, but no Aβ, had macrophages but no T cells in these areas. Some of the Aβ- and PT-injected mice had small areas of necrosis in the spleen and gastrointestinal tract. T cells isolated from the lymph nodes of these animals showed a dose-dependent response to stimulation with Aβ and produced cytokines typical of the Th-1 responses seen in autoimmune diseases. Aβ antibodies were present in serum irrespective of pertussis toxin injection. The authors write that the complement-fixing IgG2a antibodies seen in this study were probably pathogenic in the PT-treated mice, but can be beneficial under other circumstances, as in prior studies, where such antibodies triggered microglia into clearing amyloid.

The authors write that the difference between this study and prior work in transgenics hinges on the presence of the pertussis toxin, but they do not discuss how this factor relates to humans. They do conclude, however, that the demonstration of a quasi-autoimmune response against AβPP/Aβ in mice poses a challenge to the idea of Aβ vaccination as a therapy for Alzheimer’s disease.—Gabrielle Strobel

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Comments

    • User Profile Image Howard Weiner
      Brigham and Women's Hospital Harvard Medical School
    • Posted:

    The authors show inflammation in the CNS following use of pertussis and immunization with Aβ. Their ability to induce an EAE-type response in C57/Bl6 mice when it wasn’t seen in AβPP-transgenic mice may relate not only to the use of pertussis, but to the fact that AβPP-transgenic mice may have a form of immunologic tolerance to Aβ due to the overexpression of Aβ (Monsonego et al., 2001). One would like to have seen the adoptive transfer of an EAE-like picture with Aβ-reactive T cells in the absence of pertussis.

    It does seem a likely hypothesis that the adverse events observed in the Elan trial were related to a Th1-type response against Aβ induced by the adjuvant (Weiner and Selkoe, 2002). Of note, T cell reactivity to Aβ has not been documented in patients with AD. However, in extensive ongoing studies in our laboratories, we have found such T cell reactivity and are in the process of characterizing it—something that will be required should further vaccination trials be attempted in AD. The human result does not mean that Aβ vaccination cannot be attempted if modifications of the immunization protocol are made. This includes immunizing with fragments of Aβ to obtain only antibody responses, or immunizing in a fashion that does not induce Th1 type responses. We are planning the latter approach in humans by using the mucosal route to administer Aβ, inducing both antibodies and Th2/Th3 responses rather than Th1 responses (Monsonego et al., 2000). We also postulate that, in addition to antibodies, T cells which secrete cytokines such as TGF-β may also be of benefit in AD.

    References:

    Monsonego A, Maron R, Zota V, Selkoe DJ, Weiner HL. Immune hyporesponsiveness to amyloid beta-peptide in amyloid precursor protein transgenic mice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10273-8. PubMed.

    Weiner HL, Selkoe DJ. Inflammation and therapeutic vaccination in CNS diseases. Nature. 2002 Dec 19-26;420(6917):879-84. PubMed.

    Weiner HL, Lemere CA, Maron R, Spooner ET, Grenfell TJ, Mori C, Issazadeh S, Hancock WW, Selkoe DJ. Nasal administration of amyloid-beta peptide decreases cerebral amyloid burden in a mouse model of Alzheimer's disease. Ann Neurol. 2000 Oct;48(4):567-79. PubMed.

    View all comments by Howard Weiner

  2. The key point of the Furlan et al. paper is that they used pertussis toxin as a necessary adjuvant to produce the autoimmune encephalitis; that is not the protocol of others who carried out Aβ vaccination experiments in transgenic mice. The results are nevertheless of considerable interest to EAE and possibly multiple sclerosis. In their discussion, the authors state that "this experimental evidence may explain the unexpected appearance of clinical signs consistent with CNS inflammation occurring in 15 patients with Alzheimer's disease undergoing the Aβ vaccination trial." Many physicians would disagree that the encephalitis cases were unexpected, and would contend that the surprising result was that only 15 came down with symptoms.

    References:

    McGeer PL, McGeer E. Is there a future for vaccination as a treatment for Alzheimer's disease?. Neurobiol Aging. 2003 May-Jun;24(3):391-5. PubMed.

    View all comments by Pat McGeer

  3. This paper is interesting. Pertussis toxin mimics certain aspects of bacterial inflammation and enhances traffic of T cells into the CNS, thereby favoring EAE-type inflammatory responses. One could speculate that some of the trial subjects may have harbored subclinical inflammatory responses, perhaps associated with AD, that contributed to vaccine sequelae.

    It is good to see that more attention is being given to the immunogenicity of the peptide/HL-A complexes. A better outcome would be wonderful, but active immunization of AD patients at this stage of our understanding of the disease remains risky.

    View all comments by Harvey Cantor

  4. The adverse reactions of vaccines can have various and complex origins related to both the antigen and/or the adjuvant. We have previously addressed concerns about the use of full-length Aβ in vaccines, but adjuvants can also add to the toxicity of the preparation. Humoral and cell-mediated immune responses have side effects, and are enhanced by adjuvants to a different degree. These immunostimulatory additives may also activate a latent infection in the patient, and have the potential to promote amyloidosis, particularly when administered with an amyloidogenic peptide (Sigurdsson et al., 2002). Their selection depends on what the vaccine is designed to accomplish. An enhanced cellular immune response is important to combat various microorganisms, whereas antibody-mediated effects may be more appropriate to promote clearance of self-antigens, such as the Aβ peptide.

    The article by Roberto Furlan and colleagues indicates that vaccination with Aβ in Freund’s adjuvant may induce encephalomyelitis in mice when coadministered with pertussis toxin (PT). Based on the known properties of PT, this vaccine combination could be expected to generate a T-1-type immune response which, among other things, results in the activation of macrophages, cytotoxic T-lymphocytes (CTLs), and high levels of IgG2a antibodies. The authors point out that the mouse encephalitis observed in their studies might resemble the cerebral inflammation seen in some of the AD patients receiving the Elan vaccine AN-1792, in which QS-21 served as an adjuvant. Although the cause of the adverse effects in the clinical trial remains to be determined, it is interesting to note similarities in the properties of these two adjuvants. Like PT, QS-21 promotes a T-1 type response and, in mice, both are associated with an increase in complement-fixing antibodies, such as IgG2a (Kensil et al., 1995). Related to this type of response, PT induces CD4+ T-cells as shown by Furlan and colleagues, whereas QS-21 elicits both a CD4+ and a CD8+ effect in various species (Kensil et al., 1995). Interestingly, most CTLs are CD8+, suggesting that QS-21 may have a more pronounced CTL effect than PT. In designing vaccines for promoting Aβ clearance, it is obviously important to avoid a CTL induction, and any detrimental complement activation. As we discussed previously, adjuvants stimulating a T-2 type response such as alum may be more appropriate for human vaccination with Aβ derivatives (Sigurdsson et al., 2001). These adjuvants promote predominantly a humoral immune response, which is more likely to lead to Aβ clearance without the potentially destructive effects of CTLs.

    References:

    Sigurdsson EM, Wisniewski T, Frangione B. Infectivity of amyloid diseases. Trends Mol Med. 2002 Sep;8(9):411-3. PubMed.

    Kensil CR, Wu JY, Soltysik S. Structural and immunological characterization of the vaccine adjuvant QS-21. Pharm Biotechnol. 1995;6:525-41. PubMed.

    Sigurdsson EM, Scholtzova H, Mehta PD, Frangione B, Wisniewski T. Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice. Am J Pathol. 2001 Aug;159(2):439-47. PubMed.

    View all comments by Blas Frangione

  5. I’d like to note two additional things that might have relevance here. First, there is a severe neurological response when another protein is coadminstered with PT (myelin oligodendrocyte glycoprotein, or MOG25-35) that seems much greater than that observed with Aβ. Second, the sequence for Aβ in mice differs from the human sequence. I am a little concerned whether the response the authors are observing is actually against endogenous murine Aβ (unlikely, as murine Aβ is mostly intracellular in nontransgenic mice) or AβPP (not very common in white matter or the leptomeninges). It would have been interesting to evaluate other proteins that provoke a strong immune reaction in this model, for example, the nonmammalian keyhole limpet hemocyanin, with PT to see if this is a nonspecific T cell response against myelin and CNS vessels whenever a strong immunogenic protein is used. In other words, the PT plus adjuvant may not be the most relevant control group to imply specificity of the response for Aβ as opposed to other immunogenic peptides.

    Conversely, an excessive T cell response is precisely what Elan claims is responsible for the problem they observed in the five percent of vaccinated patients who exhibited signs of CNS inflammation. For this reason they are planning further study with vaccines that have less of a T cell response.

    Another option would be to evaluate passive antibody immunotherapy, as this should totally lack T cell activation. Until Elan releases more information about the cases that had adverse reactions, it will be difficult to know if the model described here is relevant to human AD cases.

    View all comments by Dave Morgan

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References

Paper Citations

  1. Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 1999 Jul 8;400(6740):173-7. PubMed.
  2. Janus C, Pearson J, McLaurin J, Mathews PM, Jiang Y, Schmidt SD, Chishti MA, Horne P, Heslin D, French J, Mount HT, Nixon RA, Mercken M, Bergeron C, Fraser PE, St George-Hyslop P, Westaway D. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease. Nature. 2000 Dec 21-28;408(6815):979-82. PubMed.
  3. Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, Duff K, Jantzen P, DiCarlo G, Wilcock D, Connor K, Hatcher J, Hope C, Gordon M, Arendash GW. A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease. Nature. 2000 Dec 21-28;408(6815):982-5. PubMed.

Other Citations

  1. ARF Live Discussion

Further Reading

Papers

  1. Petrushina I, Tran M, Sadzikava N, Ghochikyan A, Vasilevko V, Agadjanyan MG, Cribbs DH. Importance of IgG2c isotype in the immune response to beta-amyloid in amyloid precursor protein/transgenic mice. Neurosci Lett. 2003 Feb 20;338(1):5-8. PubMed.

Primary Papers

  1. Furlan R, Brambilla E, Sanvito F, Roccatagliata L, Olivieri S, Bergami A, Pluchino S, Uccelli A, Comi G, Martino G. Vaccination with amyloid-beta peptide induces autoimmune encephalomyelitis in C57/BL6 mice. Brain. 2003 Feb;126(Pt 2):285-91. PubMed.

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