Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies

Abstract

High avidity and long-lasting autoantibodies to a self-polypeptide (TNF-alpha) were generated after parenteral vaccination of mice with low doses of virus-like particle-based (VLP-based) vaccines that were constructed by linking mouse TNF-alpha peptides to the surface of papillomavirus VLPs. High-titer autoantibodies were induced with or without coadministration of potent conventional adjuvants, but were enhanced by coadministration of CFA. Compared with immunization with the fusion protein alone, attachment to VLPs increased autoantibody titers 1,000-fold. A comparison of Ab responses against the self (TNF-alpha) and foreign components of the fusion protein showed that VLP conjugation abrogated the ability of the humoral immune system to distinguish between self and foreign. Similar levels of IgM were detected to self and foreign epitopes regardless of the assembly state of the antigen, suggesting that conjugation of self-peptides to VLPs promotes survival or expansion of mature autoreactive B cells. In a mouse model, vaccination with conjugated particles inhibited development of type II collagen-induced arthritis. Together, these results suggest a potentially flexible method to efficiently generate autoantibodies against specific self-proteins that mediate arthritis and other diseases.

Figure 1

Binding of SA–TNF-α fusion protein to biotinylated VLPs. (a) Drawing summarizing conjugated vaccine construction. BPV L1 VLPs were coated with activated biotin and then purified on a linear sucrose gradient. Biotinylated particles were incubated with purified SA–TNF-α fusion protein to generate conjugated particles. (b) Binding of SA–TNF-α fusion protein to VLPs. Binding of recombinant SA or purified SA–TNF-α(3–22) to wild-type or biotinylated VLPs was measured by ELISA. (c) Cosedimentation of biotinylated VLPs with SA–TNF-α(3–22). Biotinylated VLPs were incubated with SA–TNF-α(3–22) and then separated on a 24–54% sucrose gradient. Gradient fractions were analyzed by Western blot analysis, using an anti-L1 mAb (mAB837) (top panel) or an anti-SA Ab (bottom panel). Fraction 1 represents the bottom and fraction 10 represents the top of the gradient.

Figure 2

Serum IgG Ab titers in immunized C57Bl/6 mice. Anti–TNF-α (open squares) and anti–SA (filled circles) geometric mean titer (GMT) in sera from three mice immunized with VLPs conjugated to SA–TNF-α(3–22) and followed for over 1 year. Mice were immunized at weeks 0, 2, 4, and 58 (indicated by arrows).

Figure 3

Immunization with conjugated VLPs ameliorates the symptoms of collagen-induced arthritis. DBA/1 mice were immunized four times with either VLPs conjugated to SA–TNF-α(3–22) or wild-type VLPs plus Titermax and then injected intradermally with bovine type II collagen plus Freund’s adjuvant 1 and 3 weeks after the final vaccination. (a) Percentage of arthritic mice; groups immunized with wild-type VLPs (solid line) or VLPs conjugated to SA–TNF-α(3–22) (dashed line). (b) Clinical score: VLP-immunized mice (filled circles; VLP:SA–TNF-α(3–22)–immunized mice (open squares). Clinical score data is for arthritic mice only. Error bars reflect SEM. (c) Representative joints taken from naive or collagen-injected mice immunized with either wild-type VLPs or VLPs conjugated to SA–TNF-α(3–22). Joints were taken 4 weeks after the second collagen injection. The first column of panels shows hematoxylin and eosin (H&E) staining of synovial joints. ×10. B, bone; S, synovial membrane. The second column of panels shows representative immunohistochemical analysis of TNF-α expression in each set of mice. ×40. The percentage of TNF-α–positive cells in joints from protected vaccinated mice was similar to that in naive mice.