Article Figures & Data
Figures
- FIGURE 1.
Proliferative responses to human IFN-β peptides by BALB/cByJ mice. Three mice were immunized with 25 μg of human IFN-β i.p. in alum on days 1, 3, and 10. Splenocytes were tested for proliferation to 5 μg/ml peptides in vitro on day 15. All 52 peptides describing the sequence of human IFN-β were tested in triplicate. The peptide numbers are listed on the x-axis. The first amino acid in each peptide can be calculated using the following formula: ((3n) − 2), where n is the peptide number listed. SIs were calculated for each peptide. The three mice were tested independently. The average SI for the three mice ± SD is shown on the y-axis. Average background proliferation ranged from 4100 to 6450 cpm.
- FIGURE 2.
Proliferative response to IFN 118–132 amino acid variants. Three BALB/cByJ mice were immunized with 25 μg of human IFN-β i.p. in alum on days 1, 3 and 10. Splenocytes were tested for proliferation in response to peptides in vitro on day 15. The sequence of the 118–132 peptide, SSLHLKRYYGRILHY, was modified by substituting an alanine at each position, except the lysine at position 130. Each peptide was tested in triplicate. The three mice were tested independently, and SIs were calculated. The average SI for the three mice ± SD is shown on the y-axis. Average background cpm ranged from 3984 to 4513.
- FIGURE 3.
Immunization with the variant IFN-β molecule results in reduced T cell responses to both IFN-β T cell epitope regions. BALB/cByJ mice were immunized with 25 μg of either human IFN-β (A and B) or the I129V IFN-β variant (C and D) i.p. in alum on days 1, 3, and 10. Proliferation in vitro was tested on day 15. Results are shown as average cpm from duplicate wells (A and C) or as SIs (B and D). Average background cpm were 1499 for the I129V-immunized mouse splenocytes and 1517 for the IFN-β-immunized mouse splenocytes.
- FIGURE 4.
Proliferative responses to IFN-β and the I129V IFN-β variant. Groups of four BALB/cByJ mice were immunized with human IFN-β (A, C, E, and G) or the I129V IFN-β variant (B, D, F, and H) on days 1, 3, 10, 17, 21, and 26. On day 31 pooled splenocytes were tested for proliferative responses in vitro to human IFN-β (♦) or I129V IFN-β variant (□). Mice were immunized i.p. using alum as an adjuvant. Mice received 5 μg (A and B), 10 μg (C and D), 25 μg (E and F) or 50 μg (G and H) per immunization.
- FIGURE 5.
Ab responses to the I129V IFN-β variant do not increase either over time or with an increasing dose of protein. Four BALB/cByJ mice per group were immunized i.p. with 5, 10, 25, or 50 μg of human IFN-β (♦) or the I129V IFN-β variant (□) in alum on days 1, 3, 10, 17, and 21. Serum samples were obtained and tested by ELISA for IFN-β-specific IgG1 on day 15 (A), day 21 (B), and day 26 (C). A time course is presented in D for the 50-μg dose.
- FIGURE 6.
The I129V mutation does not disrupt Ab-binding epitopes in IFN-β and does not create a new specificity. BALB/c mice were immunized with 50 μg of either IFN-β (♦ and ⋄) or the I129V-modified variant (• and ○) i.p. in alum on days 1, 3, and 10. Serum samples were taken on d = 15. ELISA plates were coated with 2 μg/ml of either IFN-β (♦ and •) or the I129V-modified variant (⋄ and ○), and the assay was performed as described in Materials and Methods.
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