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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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Vaccination with mannan-Aβ28 attenuates amyloid deposition in the cortical and hippocampal regions of brains. A. Representative images of hippocampal region from non-immunized controls and mice immunized with mannan-Aβ28 showing reduction of Aβ42 and Aβ40 amyloid burden or cored amyloid plaques. Aβ deposits were stained with anti-Aβ42, anti-Aβ40 antibodies, and with ThS. Original magnifications, 5× for anti-Aβ42/40 staining and 4× for ThS (scale bars = 200 μm). B, C, D. Image analysis of Aβ42 (B), Aβ40 (C) staining, and number of ThS-positive cored plaques (D) in cortex of 18–18.5 mo old Tg2576 non-immunized mice and mice immunized with mannan-Aβ28. Vaccinated mice showed significant reduction in Aβ42 (P = 0.0075), Aβ40 (P = 0.0103) burden, and in number of ThS-positive cored plaques (P = 0.0019) compared to that in non-immunized control group. Bars represent mean ± SE from n = 5 in the group of non-immunized mice, and n = 8 in the group of mice immunized with mannan-Aβ28.
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Figure 2: Vaccination with mannan-Aβ28 attenuates amyloid deposition in the cortical and hippocampal regions of brains. A. Representative images of hippocampal region from non-immunized controls and mice immunized with mannan-Aβ28 showing reduction of Aβ42 and Aβ40 amyloid burden or cored amyloid plaques. Aβ deposits were stained with anti-Aβ42, anti-Aβ40 antibodies, and with ThS. Original magnifications, 5× for anti-Aβ42/40 staining and 4× for ThS (scale bars = 200 μm). B, C, D. Image analysis of Aβ42 (B), Aβ40 (C) staining, and number of ThS-positive cored plaques (D) in cortex of 18–18.5 mo old Tg2576 non-immunized mice and mice immunized with mannan-Aβ28. Vaccinated mice showed significant reduction in Aβ42 (P = 0.0075), Aβ40 (P = 0.0103) burden, and in number of ThS-positive cored plaques (P = 0.0019) compared to that in non-immunized control group. Bars represent mean ± SE from n = 5 in the group of non-immunized mice, and n = 8 in the group of mice immunized with mannan-Aβ28.

Mentions: Next, we analyzed the extent of AD-like pathology in brains of vaccinated Tg2576 mice (18–18.5 months old). As controls, we used Tg2576 mice of the same age injected with mannan-BSA, or non-immunized group of animals. Image analysis of anti-Aβ40 and anti-Aβ42 antibody-stained parietal cortical sections demonstrated a significant reduction of amyloid plaques in brains of mice vaccinated with mannan-Aβ28 compared with similar brain staining of non-immunized mice (Figure 2) or animals injected with mannan-BSA (data not shown). While staining with anti-Aβ40 and anti-Aβ42 antibodies detected both cored and diffuse plaques, ThS binds only to cored amyloid deposits. Therefore, we compared image analyses with ThS staining of parietal cortical brain sections from vaccinated and control Tg2576 mice. We found that mice actively immunized 11 months earlier with the mannan-Aβ28 conjugate had significantly reduced depositions of cored amyloid plaques (Figure 2D). A similar reduction in Aβ40 and Aβ42 amyloid burden, as well as cored plaques was observed in the hippocampus of vaccinated mice compared with control non-immunized animals (Figure 2A). Of note, we observed no changes in Aβ depositions by immunohistochemistry or histochemistry in the brains of mice immunized 6 times with mannan-BSA compared to non-immunized control (data not shown). Collectively, these results demonstrate that the mannan-Aβ28 immunogen induced therapeutically potent anti-Aβ antibodies, predominantly of the IgG1 isotype, that effectively reduced the deposition AD-like plaque pathology in 18–18.5 months old Tg2576 mice.


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)

Vaccination with mannan-Aβ28 attenuates amyloid deposition in the cortical and hippocampal regions of brains. A. Representative images of hippocampal region from non-immunized controls and mice immunized with mannan-Aβ28 showing reduction of Aβ42 and Aβ40 amyloid burden or cored amyloid plaques. Aβ deposits were stained with anti-Aβ42, anti-Aβ40 antibodies, and with ThS. Original magnifications, 5× for anti-Aβ42/40 staining and 4× for ThS (scale bars = 200 μm). B, C, D. Image analysis of Aβ42 (B), Aβ40 (C) staining, and number of ThS-positive cored plaques (D) in cortex of 18–18.5 mo old Tg2576 non-immunized mice and mice immunized with mannan-Aβ28. Vaccinated mice showed significant reduction in Aβ42 (P = 0.0075), Aβ40 (P = 0.0103) burden, and in number of ThS-positive cored plaques (P = 0.0019) compared to that in non-immunized control group. Bars represent mean ± SE from n = 5 in the group of non-immunized mice, and n = 8 in the group of mice immunized with mannan-Aβ28.
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Related In: Results  -  Collection

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Figure 2: Vaccination with mannan-Aβ28 attenuates amyloid deposition in the cortical and hippocampal regions of brains. A. Representative images of hippocampal region from non-immunized controls and mice immunized with mannan-Aβ28 showing reduction of Aβ42 and Aβ40 amyloid burden or cored amyloid plaques. Aβ deposits were stained with anti-Aβ42, anti-Aβ40 antibodies, and with ThS. Original magnifications, 5× for anti-Aβ42/40 staining and 4× for ThS (scale bars = 200 μm). B, C, D. Image analysis of Aβ42 (B), Aβ40 (C) staining, and number of ThS-positive cored plaques (D) in cortex of 18–18.5 mo old Tg2576 non-immunized mice and mice immunized with mannan-Aβ28. Vaccinated mice showed significant reduction in Aβ42 (P = 0.0075), Aβ40 (P = 0.0103) burden, and in number of ThS-positive cored plaques (P = 0.0019) compared to that in non-immunized control group. Bars represent mean ± SE from n = 5 in the group of non-immunized mice, and n = 8 in the group of mice immunized with mannan-Aβ28.
Mentions: Next, we analyzed the extent of AD-like pathology in brains of vaccinated Tg2576 mice (18–18.5 months old). As controls, we used Tg2576 mice of the same age injected with mannan-BSA, or non-immunized group of animals. Image analysis of anti-Aβ40 and anti-Aβ42 antibody-stained parietal cortical sections demonstrated a significant reduction of amyloid plaques in brains of mice vaccinated with mannan-Aβ28 compared with similar brain staining of non-immunized mice (Figure 2) or animals injected with mannan-BSA (data not shown). While staining with anti-Aβ40 and anti-Aβ42 antibodies detected both cored and diffuse plaques, ThS binds only to cored amyloid deposits. Therefore, we compared image analyses with ThS staining of parietal cortical brain sections from vaccinated and control Tg2576 mice. We found that mice actively immunized 11 months earlier with the mannan-Aβ28 conjugate had significantly reduced depositions of cored amyloid plaques (Figure 2D). A similar reduction in Aβ40 and Aβ42 amyloid burden, as well as cored plaques was observed in the hippocampus of vaccinated mice compared with control non-immunized animals (Figure 2A). Of note, we observed no changes in Aβ depositions by immunohistochemistry or histochemistry in the brains of mice immunized 6 times with mannan-BSA compared to non-immunized control (data not shown). Collectively, these results demonstrate that the mannan-Aβ28 immunogen induced therapeutically potent anti-Aβ antibodies, predominantly of the IgG1 isotype, that effectively reduced the deposition AD-like plaque pathology in 18–18.5 months old Tg2576 mice.

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