Humoral immune response recognizes a complex set of epitopes on human papillomavirus type 6 l1 capsomers

Abstract

Although epitope mapping has identified residues on the human papillomavirus (HPV) major capsid protein (L1) that are important for binding mouse monoclonal antibodies, epitopes recognized by human antibodies are not known. To map epitopes on HPV type 6 (HPV6) L1, surface-exposed loops were mutated to the corresponding sequence of HPV11 L1. HPV6 L1 capsomers had one to six regions mutated, including the BC, DE, EF, FG, and HI loops and the 139 C-terminal residues. After verifying proper conformation, hybrid capsomers were used in enzyme-linked immunosorbent assays with 36 HPV6-seropositive sera from women enrolled in a study of incident HPV infection. Twelve sera were HPV6 specific, while the remainder reacted with both HPV6 and HPV11 L1. By preadsorption studies, 6/11 of these sera were shown to be cross-reactive. Among the HPV6-specific sera there was no immunodominant epitope recognized by all sera. Six of the 12 sera recognized epitopes that contained residues from combinations of the BC, DE, and FG loops, one serum recognized an epitope that consisted partially of the C-terminal arm, and three sera recognized complex epitopes to which reactivity was eliminated by switching all five loops. Reactivity in two sera was not eliminated even with all six regions swapped. The patterns of epitope recognition did not change over time in women whose sera were examined 9 years after their first-seropositive visit.

FIG. 1.

Capsomers with mutagenized loops are efficiently produced and purified from bacterial cultures. Linear model of HPV6 L1 loops mutated, showing amino acid residues targeted and changed (A). These regions are color coded in the molecular model of HPV6 L1 (B), after aligning HPV6 and HPV16 L1 sequences and modeling the crystal structure of HPV16 L1. Capsomers with different loop changes can be purified from bacterial cultures, as seen in the Coomassie gel that shows the capsomer purification scheme (C) where the high-salt elution contains a relatively pure band of 55 kDa corresponding to L1 lane 1: insoluble fraction; 2: NH₄⁺ supernatant; postdialysis 3: pellet and 4: supernatant; column load flowthrough for 5: DE52 and 6: P11; resin residue on 7: DE52 and 8: P11; 9: 250 mM, 10: 500 mM, and 11: 1 M (L1 fraction) elutions. Immunoblotting for L1 with Camvir (D) following the purification of L1 shows that fainter lower-molecular-weight bands seen on coomassie staining are L1 breakdown products lane 3: insoluble fraction; 4: NH₄⁺ supernatant; postdialysis 5: pellet and 6: supernatant; column load flowthrough for 7: DE52 and 8: P11; resin residue on 9: DE52 and 10: P11; 11: 250 mM, 12: 500 mM, and 13: 1 M (L1 fraction) elutions.

FIG. 2.

Capsomers with mutagenized loops fold properly. Capsomer preparations with different loop changes yielded a substantial fraction of the capsomer preparation resistant to trypsin because of proper folding, as evidenced by the 42-kDa L1 fragment (A). When assayed by ELISA with type-specific, conformation-dependent mouse monoclonal antibodies H11.A3, H11.B2, H6.N8, and H6.M48, as indicated, capsomers with different loop changes bound MAbs, as expected (B). Capsomer loop changes are shown on the x axis, and optical density is shown on the y axis.

FIG. 3.

Overview of HPV6 and HPV11 seroreactivity of sera used. Overview of serum reactivity by capture ELISA against HPV6 L1 (solid bars) and HPV11 (open bars) for all HPV6-specific sera (A) and some sera that are reactive to both HPV6 and HPV11 (B). Optical density is on the y axis and serum samples are along the x axis.

FIG. 4.

Defined epitopes are different for different individuals. Four of the HPV6-specific sera had epitopes defined as indicated (A to D), where altering the HPV6 L1 loop(s), shown on the x axis, diminished OD reactivities, shown on the y axis. Altering loop DE obliterates binding in serum I (A), while altering either loop DE or FG does away with binding in serum II (B). Serum IV (C) targets both loops BC and DE, while serum VI targeted an epitope involving the C terminus (D). Three sera required altering all five loops to obliterate antibody binding (E). The majority of the HPV6-seropositive sera were cross-reactive (F), binding HPV11 L1 to significant levels such that they could not be used to define epitopes.

FIG. 5.

Preadsorption studies validated by MAbs and HPV6-specific human serum. Either HPV6 L1, HPV11 L1, HPV6-FRM, or HPV6L1:Cterm capsomers were covalently coupled to agarose beads and incubated with MAb H6 M.48 (A) or H11.B2 (B) after blocking. Unbound supernatants were subsequently reacted against HPV6 L1 (A) or HPV11 (B) capsomers in direct ELISAs, and competition of reactivity by preadsorbing measured by OD (y axis). HPV6-specificsera were also used to validate covalently linked capsomers (C). The serum sample for which the epitope was elucidated via capture ELISAs was preadsorbed onto HPV6 L1, HPV11 L1, HPV6-FRM:Ctrm, or blank beads and subsequently reacted against capsomers with different loop mutations (x axis) and residual antibody binding was measured by optical density (y axis). A human serum sample for which the epitope could not be defined by capture ELISAs was preadsorbed onto HPV6 L1, HPV11 L1, HPV6-FRM:Ctrm, or blank beads and subsequently reacted against HPV6 L1M capsomers or HPV6:BC+DC capsomers.

FIG. 6.

HPV6 and HPV11 reactivity is due to cross-reactive antibodies according to preadsorption studies. Human sera reactive to both HPV6 and HPV11 were preadsorbed onto HPV6 L1, HPV11 L1, or empty beads and subsequently reacted against HPV6 L1M or HPV11 L1M capsomers (x axis) and residual antibody binding was measured by optical density (y axis) after subtracting readings from blank wells. Equal reactivity to HPV6 and HPV11 capsomers after preadsorption to either HPV6 or HPV11 capsomer beads suggests that cross-reactivity is due to cross-reactive antibodies (A). One sample had a small amount of differential binding after preadsorption (B).

FIG. 7.

HPV6 L1 targets do not change over time. Sera from individuals who had samples either 4 months apart (A and B) or 9 years apart (C) were tested against capsomers with different loop changes via capture ELISA, and optical density (y axis) was normalized to that of HPV6 L1.