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Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice.

Petrushina I, Ghochikyan A, Mkrtichyan M, Mamikonyan G, Movsesyan N, Ajdari R, Vasilevko V, Karapetyan A, Lees A, Agadjanyan MG, Cribbs DH - J Neuroinflammation (2008)

Bottom Line: Mannan was purified, activated and chemically conjugated to Abeta28 peptide.This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice.Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Abeta antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Institute for Brain Aging and Dementia, University of California Irvine, Irvine, CA 92697-4540, USA. ipetrush@uci.edu

ABSTRACT

Background: New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Abeta28 conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576).

Methods: Mannan was purified, activated and chemically conjugated to Abeta28 peptide. Humoral immune responses induced by the immunization of mice with mannan-Abeta28 conjugate were analyzed using a standard ELISA. Abeta42 and Abeta40 amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry.

Results: Immunizations with low doses of mannan-Abeta28 induced potent and long-lasting anti-Abeta humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Abeta antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Abeta28 prevented Abeta plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Abeta28 showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups.

Conclusion: Mannan conjugated to Abeta28 provided sufficient adjuvant activity to induce potent anti-Abeta antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Abeta self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Abeta28 immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Abeta antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

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Immunostaining of neocortex for C1q and C3b components of complement, and concomitant staining of meninges for C1q and ThS. A. Representative images of cortex areas of coronal brain sections of mice immunized with mannan-Aβ28 or mannan-BSA, immunostained with antibodies specific for C1q and C3b components of complement. Original magnification, 20×, scale bar = 50 μm. B. Representative images of colocalization of vascular fibrillar Aβ detected with ThS (green) with C1q component of complement (red) in the meninges of coronal brain sections of mice immunized with mannan-Aβ28, mannan-BSA or non-immunized animals. Original magnification, 70×.
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Figure 6: Immunostaining of neocortex for C1q and C3b components of complement, and concomitant staining of meninges for C1q and ThS. A. Representative images of cortex areas of coronal brain sections of mice immunized with mannan-Aβ28 or mannan-BSA, immunostained with antibodies specific for C1q and C3b components of complement. Original magnification, 20×, scale bar = 50 μm. B. Representative images of colocalization of vascular fibrillar Aβ detected with ThS (green) with C1q component of complement (red) in the meninges of coronal brain sections of mice immunized with mannan-Aβ28, mannan-BSA or non-immunized animals. Original magnification, 70×.

Mentions: Since the activation of the complement cascade by antibody-Aβ immune complexes at sites of cerebral amyloid angiopathy (CAA) may be one possible cause of microhemorrhage, it was of interest to analyze the opsonization of sites of microhemorrhage with C1q and C3 proteolytic fragments, C3b, complement components in mice immunized with mannan-Aβ28. We detected the accumulation of these complement proteins in the cerebral vasculature of immunized, but not control mice immunized with mannan-BSA (Figure 6A). In the meningeal vessels, in the group of mannan-Aβ28 immunized animals we have demonstrated partially cleared ThS-stained vascular fibrillar Aβ deposits abundantly decorated with C1q component of complement, while only trace amount of C1q was co-localized with compact Aβ load in the meninges of mannan-BSA immunized group, and practically none of the C1q was detected in the Aβ-laden meninges of non-immunized animals (Figure 6B). The similar co-localization pattern was shown for C3b component of complement (data not shown).


Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice.

Petrushina I, Ghochikyan A, Mkrtichyan M, Mamikonyan G, Movsesyan N, Ajdari R, Vasilevko V, Karapetyan A, Lees A, Agadjanyan MG, Cribbs DH - J Neuroinflammation (2008)

Immunostaining of neocortex for C1q and C3b components of complement, and concomitant staining of meninges for C1q and ThS. A. Representative images of cortex areas of coronal brain sections of mice immunized with mannan-Aβ28 or mannan-BSA, immunostained with antibodies specific for C1q and C3b components of complement. Original magnification, 20×, scale bar = 50 μm. B. Representative images of colocalization of vascular fibrillar Aβ detected with ThS (green) with C1q component of complement (red) in the meninges of coronal brain sections of mice immunized with mannan-Aβ28, mannan-BSA or non-immunized animals. Original magnification, 70×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2567310&req=5

Figure 6: Immunostaining of neocortex for C1q and C3b components of complement, and concomitant staining of meninges for C1q and ThS. A. Representative images of cortex areas of coronal brain sections of mice immunized with mannan-Aβ28 or mannan-BSA, immunostained with antibodies specific for C1q and C3b components of complement. Original magnification, 20×, scale bar = 50 μm. B. Representative images of colocalization of vascular fibrillar Aβ detected with ThS (green) with C1q component of complement (red) in the meninges of coronal brain sections of mice immunized with mannan-Aβ28, mannan-BSA or non-immunized animals. Original magnification, 70×.
Mentions: Since the activation of the complement cascade by antibody-Aβ immune complexes at sites of cerebral amyloid angiopathy (CAA) may be one possible cause of microhemorrhage, it was of interest to analyze the opsonization of sites of microhemorrhage with C1q and C3 proteolytic fragments, C3b, complement components in mice immunized with mannan-Aβ28. We detected the accumulation of these complement proteins in the cerebral vasculature of immunized, but not control mice immunized with mannan-BSA (Figure 6A). In the meningeal vessels, in the group of mannan-Aβ28 immunized animals we have demonstrated partially cleared ThS-stained vascular fibrillar Aβ deposits abundantly decorated with C1q component of complement, while only trace amount of C1q was co-localized with compact Aβ load in the meninges of mannan-BSA immunized group, and practically none of the C1q was detected in the Aβ-laden meninges of non-immunized animals (Figure 6B). The similar co-localization pattern was shown for C3b component of complement (data not shown).

Bottom Line: Mannan was purified, activated and chemically conjugated to Abeta28 peptide.This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice.Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Abeta antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Institute for Brain Aging and Dementia, University of California Irvine, Irvine, CA 92697-4540, USA. ipetrush@uci.edu

ABSTRACT

Background: New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Abeta28 conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576).

Methods: Mannan was purified, activated and chemically conjugated to Abeta28 peptide. Humoral immune responses induced by the immunization of mice with mannan-Abeta28 conjugate were analyzed using a standard ELISA. Abeta42 and Abeta40 amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry.

Results: Immunizations with low doses of mannan-Abeta28 induced potent and long-lasting anti-Abeta humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Abeta antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Abeta28 prevented Abeta plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Abeta28 showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups.

Conclusion: Mannan conjugated to Abeta28 provided sufficient adjuvant activity to induce potent anti-Abeta antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Abeta self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Abeta28 immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Abeta antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

Show MeSH
Related in: MedlinePlus