Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide

Abstract

Filamentous bacteriophage are widely used as immunogenic carriers for "phage-displayed" recombinant peptides. Here we report that they are an effective immunogenic carrier for synthetic peptides. The f1.K phage was engineered to have an additional Lys residue near the N-terminus of the major coat protein, pVIII, so as to enhance access to chemical cross-linking agents. The dimeric synthetic peptide, B2.1, was conjugated to f1.K (f1.K/B2.1) in high copy number and compared as an immunogen to B2.1 conjugated to ovalbumin (OVA/B2.1) and to phage-displayed, recombinant B2.1 peptide. All immunogens were administered without adjuvant. The serum antibody titers were measured against: the peptide, the carrier, and, if appropriate, the cross-linker. All immunogens elicited anti-peptide antibody titers, with those elicited by OVA/B2.1 exceeding those by f1.K/B2.1; both titers were greater than that elicited by recombinant B2.1 phage. Comparison of the anti-peptide and anti-carrier antibody responses showed that f1.K/B2.1 elicited a more focused anti-peptide antibody response than OVA/B2.1. The anti-peptide antibody response against f1.K/B2.1 was optimized for the injection route, dose and adjuvant. Dose and adjuvant did not have a significant effect on anti-peptide antibody titers, but a change in injection route from intraperitoneal (IP) to subcutaneous (SC) enhanced anti-peptide antibody titers after seven immunizations. The optimized anti-peptide antibody response exceeded the anti-carrier one by 21-fold, compared to 0.07-fold elicited by OVA/B2.1. This indicates that phage as a carrier can focus the antibody response against the peptide. The results are discussed with respect to the advantages of phage as an alternative to traditional carrier proteins for synthetic peptides, carbohydrates and haptens, and to further improvements in phage as immunogenic carriers.

Fig. 1

Western blots of f1.K/B2.1 peptide conjugate. (A) Detected with MAb b12. Lanes: (1) f1.K phage, negative control; (2) f1.K/B2.1 peptide conjugate, 5 × 10¹⁰ phage particles. (B) Detected with rabbit anti-phage polyclonal Ab. Lanes: (1) f1.K/B2.1 peptide conjugate, 5 × 10¹⁰ phage particles; (2) OVA/B2.1 conjugate-negative control; (3) f1.K phage-positive control, 2 × 10¹⁰ phage particles. The positions of the pIII (MW 42.5 kDa) and pVIII (MW 5.5 kDa) bands are indicated by arrows.

Fig. 2

Study 1 Ab titers: shown are the mean antibody titer±standard error after third, fifth and seventh immunizations against: (A) B2.1 peptide; (B) f1.K, f88-4; and OVA carriers (C) peptide-to-carrier ratios; (D) BS³ cross-linker for f1.K/B2.1 and OVA/B2.1; and (E) peptide-to-cross-linker ratio. Also shown are ANOVA p-values that indicate the existence of significant differences between groups that received different antigens at the third, fifth and seventh immunizations.

Fig. 3

Study 2 Ab titers: shown are the mean antibody titer±standard error after third, fifth and seventh immunizations against: (A) B2.1 peptide; (B) f1.K carrier; (C) peptide-to-carrier ratios; (D) BS³ cross-linker; and (E) peptide-to-cross-linker ratio. Also shown are ANOVA p-values that indicate the existence of significant differences between groups that received different immunization conditions at the third, fifth and seventh immunizations.