The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 57:B355-B358 (2002)
© 2002 The Gerontological Society of America
Age-Related Differences in the Immune Response to Immunization With Human Aß42 Peptide
Jeannette Pifera,
Jason L. Hennesa,
John M. Leeb,c and
Pamela L. Wittea
a Departments of Cell Biology, Neurobiology, & Anatomy,
b Pathology, Loyola University Medical Center, Maywood, Illinois
c Pharmacology, Loyola University Medical Center, Maywood, Illinois
Pamela L. Witte, Department of Cell Biology, Neurobiology, & Anatomy, Loyola University Chicago, 2160 S. First Avenue, Maywood, IL 60153 E-mail: pwitte{at}lumc.edu.
Decision Editor: James R. Smith, PhD
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Abstract
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Several studies show that plaque burden is resolved in young to middle-aged amyloid precursor protein transgenic mice after rigorous immunization with Aß42 peptide. We determined if wild-type 20-month-old and 3-month-old animals could produce high-titer antibody against Aß42 with a less strenuous immunization protocol. All treated young animals mounted a high-titer (20,000–50,000) response after two immunizations and sustained a strong response for 6 months following the initial treatment with Aß42. However, 6 of 8 immunized aged animals did not respond after three immunizations. The 2 responding aged mice produced low-titer antibody (5,000–10,000), which rapidly declined to control levels within 5 weeks after the third immunization. Aged animals may require alternate strategies for successful vaccination, such as inclusion of stimulatory cytokines or better adjuvants. If tolerance to Aß42 underlies the poor response observed in aged animals, then a mechanism to overcome this response will have to be investigated.
ALZHEIMER'S disease (AD) is the most common form of dementia that increases in incidence with age. Incidence of AD increases progressively past the age of 60 years, and 47% of individuals are affected by age 85 (1). The pathology of AD is associated with the presence of neuritic and diffuse plaques. The main component of plaques, Aß42, is a 42 amino acid peptide that results from abnormal cleavage of membrane-bound amyloid precursor protein. New treatments are currently under investigation to reverse symptoms or halt progression of AD. One remarkable and promising strategy is based on studies that demonstrated that humoral immune response against Aß42 may be a potential treatment for AD and may provide a vaccine to prevent onset of disease (2)(3)(4)(5). These studies used a transgenic mouse model of AD in which young and middle-aged animals were vigorously immunized with human Aß42 for periods up to 11 months, by means of immunization by nasal or subcutaneous administration. These prolonged immunization regimens resulted in the formation of high-titer anti-Aß42 antibodies (2)(3)(4).
Although a vaccine-based treatment may be effective in treating AD, these previous studies did not address that efficiency in a truly aged population. This is important because AD patients are often quite elderly. Additionally, the prior studies were performed with stringent courses of immunization. A lengthy protocol may not be suitable for aged individuals or necessary as a vaccine in a young population. These points led us to test the following hypotheses. First, a short course of immunization may be sufficient to yield high-titer antibodies against Aß42. Second, aged animals may be deficient in antibody production as a result of functional alterations in immune response with age.
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Methods
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Animals
Female C57BL/6 mice, 3 or 20 months of age, were obtained from the National Institute of Aging colony at Harlan (Indianapolis, IN). Animals were housed in the Animal Research facility at Loyola University.
Immunization
Four groups (8 per group) of female C57BL/6 mice, 3 or 20 months of age, were injected subcutaneously with either 100 µg aggregated synthetic human Aß42 (American Peptide, Sunnyvale, CA) or phosphate-buffered saline (PBS) in Titremax Gold adjuvant (Sigma, St. Louis, MO). Secondary immunizations were given at 2 and 8 weeks after initial injection.
Detection of Serum Antibodies Against Aß42
Serum samples were collected by tail bleed at the indicated times. Sera were diluted 1:16 to 1:262,000 and analyzed by enzyme-linked immunosorbent assay (ELISA) as previously described (5). The titer of antigen-specific antibody was determined by using a best-fit line analysis of the absorbance at 405 nm versus serum dilution and is presented as the reciprocal of the dilution that yielded 50% maximum optical density. Positive samples were analyzed by ELISA to determine isotypes of Aß42-specific antibodies (BD Pharmingen, San Diego, CA).
Immunocytochemistry
Immunocytochemistry was performed as previously described (4). The highest-titer serum sample from 1 young and 1 aged animal was used at 1:500 dilution in immunoperoxidase staining to determine if the antibodies bound specifically to human temporal lobe amyloid plaques. Sera taken from randomly chosen, age-matched PBS-treated animals were used as controls.
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Results
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Serum samples from young Aß42 and PBS-immunized animals were analyzed by ELISA for the presence of antibodies against Aß42. No response was detected in serum samples from young animals 2 weeks following the primary injection (Fig. 1). However, moderately high-titer antibodies were detected in 5 of 8 young Aß42-treated animals 2 weeks after the first boost injection (Fig. 1). All Aß42-immunized young animals had detectable serum antibody 1 week following the second boost injection. Most animals produced moderate-titer (20,000) to high-titer (50,000) antibodies. By the third immunization, only 1 young animal had a low-titer (10,000) response. Antibody production was sustained at 5 and 9 weeks, and as long as 6 months, following the third immunization of most young animals.
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Figure 1. Detection of anti-Aß42 antibody in A, young and B, aged animals immunized with Aß42 peptide ( ) or phosphate-buffered saline ( ). At start, young animals were 3 months old and aged animals were 20 months old. Serum samples were taken by tail bleed biweekly following the first and second injections, at 1 week following the third injection, and then at indicated time points for the course of the experiment. Samples were analyzed by enzyme-linked immunosorbent assay. Each data point represents 1 individual animal (n = 8 animals per experimental group at start of experiment). Arrows indicate injections; n.d. = not determined. Note: # is the same young animal at 9, 14, 19, and 45 weeks and * is the same young animal at 9, 14, 19, and 45 weeks. Young animals with lower responses tended to continue producing lower responses, whereas young animals with higher responses tended to continue exhibiting higher responses. All aged animals were sacrificed at 19 weeks; hence titer was not determined at 45 weeks.
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In contrast, aged animals treated with Aß42 demonstrated a profound deficiency in ability to mount a humoral response against Aß42 (Fig. 1). No response to immunization was observed in aged animals treated with Aß42 following both the initial and second immunizations. A low-titer response was observed in 2 of 8 aged animals 1 week following the third injection with Aß42, but this response had disappeared by 5 weeks following the third injection. Of 8 aged mice, 6 had no detectable antibody to Aß42 after three injections.
The predominant isotype of anti-Aß42 antibody in young mice was immunoglobulin G2b (IgG2b), shown in Fig. 2. This finding is consistent with previous studies using the amyloid precursor protein transgenic (APP-Tg) mouse model of human AD (2)(4)(6). IgG2b was also the predominant isotype of anti-Aß42 antibody detected in the 2 responding aged animals, although IgG1 was also detected.
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Figure 2. Isotypes of anti-Aß42 antibody detected in serum from young (solid bars) and aged (striped bars) animals. Serum samples from immunopositive animals, collected by tail bleed at 9 weeks (1 week following the third injection) were analyzed by enzyme-linked immunosorbent assay for isotype of Aß42-specific antibody. In this experiment, serum samples from analyzed animals were diluted to 1:500 because of low titer of detectable antibody in aged animals. Shown is the average of 2 randomly analyzed young animals, and the average of the 2 responding aged animals. The data for the 2 young animals shown are representative of all data obtained for young immunopositive animals. OD = optical density.
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Immunoperoxidase staining of sequential sections taken from the temporal lobe of an AD brain determined that immune serum antibody recognized primarily diffuse AD plaques, but not core plaques (Fig. 3). The highest-titer serum from an aged animal showed similar but weak staining of diffuse plaques (Fig. 3).
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Figure 3. Anti-Aß42 antibodies in serum from immune mice stain amyloid plaque in temporal lobe tissue. Samples taken at 9 weeks (1 week following the third injection) from the highest responding young (A) and aged (C) Aß42-treated animals stain diffuse plaques (arrowheads) in sections from a patient with midstage Alzheimer's disease. Phosphate-buffered saline control treated young (B) and aged (D) animals yielded no staining. All photos were treated equally and are at 40x magnification.
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Discussion
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Our findings demonstrate that an immunization schedule of only three subcutaneous injections is sufficient to achieve a strong, sustained antibody response against Aß42 in young, wild-type animals. Notably, half of the mice housed for an additional 6 months after the final immunization continued to have strong antibody titers. This may be evidence that long-term immune memory can be established by a relatively short course of immunization with Aß42. Alternatively, the response to human Aß42 may cross-react with an epitope of mouse amyloid protein such that continuous antigen stimulation results. Further investigation is required to resolve this issue.
In distinct contrast to the response of young mice, aged animals exhibited a negligible immune response to aggregated Aß42. Although we anticipated that aged animals would give a less robust antibody response, the almost complete absence of a response was unexpected. The magnitude of antibody responses by aged mice has been reported to vary among strains and with antigen. Nicoletti and Cerny (7) have demonstrated that aged C57BL/6 mice respond as well as young mice to some antigens but are deficient in response to others. For example, aged C57BL/6 mice responded poorly to phosphorylcholine compared with young mice. However, the aged mice responded well to TNP, and NP has been frequently used as an antigen in aging studies that use the C57BL/6 strain (7)(8)(9). We used C57BL/6 mice in this study because the APP-Tg mice have been backcrossed on this background.
The mechanism that yields such a profoundly deficient response to Aß42 in aged animals will be important to resolve, because the most urgent candidates for AD vaccine therapy would be elderly patients. Among the first consideration is the progressive loss of T-helper cell function that may impinge on humoral immune function because of insufficient production of necessary cytokines. T-helper cells and the ability to generate T-memory cells are affected by age. The failure is primarily mediated by a deficiency in interleukin-2 production (10). Therefore, a vaccine strategy that supplements the interleukin-2 deficiency may enhance the ability to produce anti-Aß42 antibodies, as reported for influenza vaccines (11). Another possibility is a deficiency or alteration in the B-cell antibody repertoire (12)(13). This could be partially due to deficits in affinity maturation resulting from a decreased germinal center response as reported by Miller and Kelsoe (8), who observed that germinal centers in aged mice are smaller with underdeveloped marginal zones. Improvements in adjuvants, which are currently under investigation to immunize the elderly population successfully against influenza and pneumonia, may improve response to immunization with Aß42.
Aged animals do respond to many antigens. The responses are not as immediate or profound as those observed in younger animals; nonetheless, there is usually some response. The almost complete lack of Aß42-specific antibody in aged mice may indicate that the animals are tolerant to Aß42 (although these wild-type mice had not been previously exposed to this antigen). If the human peptide cross-reacts with mouse amyloid protein, the ability of these animals to mount an immune response may be impaired through deletion or anergy of lymphocytes. Some degree of overcoming tolerance by means of repeated boost injections may be necessary and may explain the lag observed in the initial response of young C57BL/6 mice or reported in APP-Tg mice (14). If tolerance underlies the poor response observed in aged mice, this difficulty becomes an even greater consideration in humans, and novel vaccine strategies may be necessary in the elderly population to break tolerance. Taken together, our results suggest that the development of an effective and practical vaccine protocol for the treatment and/or prevention of AD must consider the age and immune status of candidates for immunization.
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Acknowledgments
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This work was made possible by National Institutes of Health Grants KO7 AG00997 and RO1 AG13874 to P. Witte and the Immunology and Aging Program at Loyola University Medical Center.
We thank Debra Magnuson, Zheng Yu, and Matthew Ewert for expert technical assistance.
Received April 16, 2002
Accepted July 22, 2002
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Abstract
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