Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein

Abstract

The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.

FIG. 1.

Sequences of L2-derived peptides used for vaccination

FIG. 2.

Immunoblots of serum reactivity for purified L2 proteins. Immune rabbit serum at a 1:250 dilution was used to probe blots of His₆-tagged, nickel-purified fusion proteins of insect cell-derived ROPV L2 (lanes 3), bacterially derived CRPV L2 (lanes 4), and HPV 16 L2 (lanes 5); included are bacterial cell lysates (lanes 1) and insect cell lysates (lanes 2). Sera from rabbits immunized with ROPV L2.1 (A), ROPV L2.2 (B), CRPV L2.1 (C), CRPV L2.2 (D), and HPV 16 L2 (E) are shown. An anti-His₆ antibody was used as a control (F). Arrows, 66-kDa marker. L2 is ∼70 to 75 kDa.

FIG. 3.

Immunohistochemical staining of rabbit tissue infected with CRPV (A to F) and ROPV (G to L). A positive antibody response is indicated by dark red nuclear staining in virus-infected cells. Hematoxylin and eosin staining reveals tissue morphology (A and G). Sera from immunization with HPV 16 (C and I), CRPV L2.1 (D), CRPV L2.2 (E and L), ROPV L2.1 (F and J), and ROPV L2.2 (K) are shown. The rabbit polyclonal HPV L1 control antibody recognizes CRPV (B). ROPV L2-specific control monoclonal antibody RL2.5D11 is also shown (H). Magnifications: ×40 (C, F, I, and L) and ×100 (A, B, D, E, G, H, J, and K).

FIG. 4.

RT-PCR analysis of CRPV neutralization by rabbit CRPV L2 peptide immune serum. (Top) Amplimers of the E1^E4 transcript. Lanes 1 and 15, virus only; lanes 2 and 16, uninfected cells; lanes 3 to 8 and 17 to 19, CRPV L2.2 serum diluted 1:80 (lane 3), 1:40 (lane 4), 1:20 (lanes 5 and 18), 1:10 (lanes 6 and 19), and 1:5 (lane 7) and undiluted preimmune serum (lanes 8 and 17); lanes 9 to 11 and 20 and 21, CRPV L2.1 serum diluted 1:20 (lanes 9 and 21) and 1:10 (lane 10) and undiluted preimmune serum (lanes 11 and 20); lane 12, 1:5 dilution of ROPV L2.1 serum; lane 13, ROPV L2.2 serum; lane 14, HPV 16 serum. (Bottom) β actin controls for each lane.

FIG. 5.

Cutaneous papilloma sizes on peptide-immunized rabbits following infection with CRPV. Sizes are the average geometric mean diameters (GMD) of papillomas at sites (two sites per rabbit) infected with a 10⁻² dilution of viral stock 11 weeks postinfection. Each bar corresponds to one animal. ∗, rabbit died on day 28.

FIG. 6.

Physical appearance of cutaneous and oral papillomas on vaccinated rabbits. (Top) CRPV infections at week 10; (bottom) ROPV infections on day 28.