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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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Semi-quantitative analysis of CAA in the neocortex areas of animals immunized with mannan-Aβ28, mannan-BSA, or non-immunized mice. A, B, C. Examples of CAA severity grades: grade 1, Aβ immunoreactivity confined strictly to the vessel wall (A); grade 2, Aβ immunoreactivity in and around vessel wall with focal infiltration of the amyloid in the neuropil (B); grade 3, extensive infiltration of amyloid into the neuropil with a complete amyloid coat around the vessel (C). Original magnification 20×, scale bar = 50 μm. D. The CAA score detected in the neocortex of mice vaccinated with mannan-Aβ28, was similar to that detected in neocortex of animals injected with mannan-BSA or non-immunized mice (P > 0.05). Bars represent mean ± SE from n = 5 in the group of non-immunized mice, n = 6 in the group of mannan-BSA immunized group, and n = 8 in the group of mice immunized with mannan-Aβ28.
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Figure 5: Semi-quantitative analysis of CAA in the neocortex areas of animals immunized with mannan-Aβ28, mannan-BSA, or non-immunized mice. A, B, C. Examples of CAA severity grades: grade 1, Aβ immunoreactivity confined strictly to the vessel wall (A); grade 2, Aβ immunoreactivity in and around vessel wall with focal infiltration of the amyloid in the neuropil (B); grade 3, extensive infiltration of amyloid into the neuropil with a complete amyloid coat around the vessel (C). Original magnification 20×, scale bar = 50 μm. D. The CAA score detected in the neocortex of mice vaccinated with mannan-Aβ28, was similar to that detected in neocortex of animals injected with mannan-BSA or non-immunized mice (P > 0.05). Bars represent mean ± SE from n = 5 in the group of non-immunized mice, n = 6 in the group of mannan-BSA immunized group, and n = 8 in the group of mice immunized with mannan-Aβ28.

Mentions: Previous studies have shown that passive immunotherapy with high doses of anti-Aβ antibodies may induce microhemorrhage in aged APP/Tg mice [18,21]. More recently, it was reported that multiple (10 times) immunizations of APP+PS1 mice with high doses of fibrillar Aβ42 formulated in conventional adjuvant (FCA/FIA) could also increase microhemorrhage in 20 months old mice [22]. Interestingly, in PDAPP mice passive vaccination with anti-Aβ antibodies cleared vascular amyloid, but high doses of antibodies induced microhemorrhages that are limited to focal perivascular sites [62]. Here, we analyzed microhemorrhage in 18–18.5 months-old Tg2576 mice, vaccinated with low doses of mannan-Aβ28, for 11 months prior to sacrifice, by hemosiderin staining with Prussian blue. Numbers of positive profiles per section of the brains of mannan-Aβ28 vaccinated animals were compared with that in the brains of control age-matched animals immunized with mannan-BSA, or non-immunized mice. We observed low numbers of microhemorrhage in both non-immunized and mannan-BSA injected control aged mice. In contrast, following vaccination with mannan-Aβ28, we detected significantly (P < 0.001) higher numbers of microhemorrhage (Figure 4A). Additionally, we observed larger microhemorrhage in mice vaccinated with mannan-Aβ28 (Figure 4B, C, D), than in mannan-BSA (Figure 4E, F), or non-immunized control animals (Figure 4G). Previously, it was demonstrated that passive Aβ-immunizations resulted in reduction of amyloid deposition, but also increased incidence of microhemorrhage associated with increased CAA in humans [14] and APP/Tg mice [20]. Thus, we undertook a semi-quantitative analysis of the CAA in the cortical brain sections of mice injected with mannan-Aβ28, mannan-BSA, or non-immunized animals. CAA score was calculated based on the frequency and severity of the Aβ immunoreactivity confined strictly to the vessel wall, around vessel wall with focal infiltration of the amyloid in the neuropil, or extensively infiltrated into the neuropil with a complete amyloid coat around the vessel as it is shown in Figure 5A, B, C. Using this approach we calculated CAA in neocortical areas of experimental and control animals and demonstrated that the CAA score was almost equal in mice vaccinated with mannan-Aβ28, mannan-BSA or non-immunized control (P > 0.05, Figure 5D). Of note, we did not quantitate CAA in the meninges in this study, because the above mentioned standard Aβ burden measurements made in the parenchyma and normalized to brain region were not applicable to the meningeal location of vascular Aβ deposits.


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)

Semi-quantitative analysis of CAA in the neocortex areas of animals immunized with mannan-Aβ28, mannan-BSA, or non-immunized mice. A, B, C. Examples of CAA severity grades: grade 1, Aβ immunoreactivity confined strictly to the vessel wall (A); grade 2, Aβ immunoreactivity in and around vessel wall with focal infiltration of the amyloid in the neuropil (B); grade 3, extensive infiltration of amyloid into the neuropil with a complete amyloid coat around the vessel (C). Original magnification 20×, scale bar = 50 μm. D. The CAA score detected in the neocortex of mice vaccinated with mannan-Aβ28, was similar to that detected in neocortex of animals injected with mannan-BSA or non-immunized mice (P > 0.05). Bars represent mean ± SE from n = 5 in the group of non-immunized mice, n = 6 in the group of mannan-BSA immunized group, and n = 8 in the group of mice immunized with mannan-Aβ28.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Semi-quantitative analysis of CAA in the neocortex areas of animals immunized with mannan-Aβ28, mannan-BSA, or non-immunized mice. A, B, C. Examples of CAA severity grades: grade 1, Aβ immunoreactivity confined strictly to the vessel wall (A); grade 2, Aβ immunoreactivity in and around vessel wall with focal infiltration of the amyloid in the neuropil (B); grade 3, extensive infiltration of amyloid into the neuropil with a complete amyloid coat around the vessel (C). Original magnification 20×, scale bar = 50 μm. D. The CAA score detected in the neocortex of mice vaccinated with mannan-Aβ28, was similar to that detected in neocortex of animals injected with mannan-BSA or non-immunized mice (P > 0.05). Bars represent mean ± SE from n = 5 in the group of non-immunized mice, n = 6 in the group of mannan-BSA immunized group, and n = 8 in the group of mice immunized with mannan-Aβ28.
Mentions: Previous studies have shown that passive immunotherapy with high doses of anti-Aβ antibodies may induce microhemorrhage in aged APP/Tg mice [18,21]. More recently, it was reported that multiple (10 times) immunizations of APP+PS1 mice with high doses of fibrillar Aβ42 formulated in conventional adjuvant (FCA/FIA) could also increase microhemorrhage in 20 months old mice [22]. Interestingly, in PDAPP mice passive vaccination with anti-Aβ antibodies cleared vascular amyloid, but high doses of antibodies induced microhemorrhages that are limited to focal perivascular sites [62]. Here, we analyzed microhemorrhage in 18–18.5 months-old Tg2576 mice, vaccinated with low doses of mannan-Aβ28, for 11 months prior to sacrifice, by hemosiderin staining with Prussian blue. Numbers of positive profiles per section of the brains of mannan-Aβ28 vaccinated animals were compared with that in the brains of control age-matched animals immunized with mannan-BSA, or non-immunized mice. We observed low numbers of microhemorrhage in both non-immunized and mannan-BSA injected control aged mice. In contrast, following vaccination with mannan-Aβ28, we detected significantly (P < 0.001) higher numbers of microhemorrhage (Figure 4A). Additionally, we observed larger microhemorrhage in mice vaccinated with mannan-Aβ28 (Figure 4B, C, D), than in mannan-BSA (Figure 4E, F), or non-immunized control animals (Figure 4G). Previously, it was demonstrated that passive Aβ-immunizations resulted in reduction of amyloid deposition, but also increased incidence of microhemorrhage associated with increased CAA in humans [14] and APP/Tg mice [20]. Thus, we undertook a semi-quantitative analysis of the CAA in the cortical brain sections of mice injected with mannan-Aβ28, mannan-BSA, or non-immunized animals. CAA score was calculated based on the frequency and severity of the Aβ immunoreactivity confined strictly to the vessel wall, around vessel wall with focal infiltration of the amyloid in the neuropil, or extensively infiltrated into the neuropil with a complete amyloid coat around the vessel as it is shown in Figure 5A, B, C. Using this approach we calculated CAA in neocortical areas of experimental and control animals and demonstrated that the CAA score was almost equal in mice vaccinated with mannan-Aβ28, mannan-BSA or non-immunized control (P > 0.05, Figure 5D). Of note, we did not quantitate CAA in the meninges in this study, because the above mentioned standard Aβ burden measurements made in the parenchyma and normalized to brain region were not applicable to the meningeal location of vascular Aβ deposits.

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