Protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of L2

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   Fig. 1.

   All components of the P25-P2C-HPV vaccine are required to generate a potent L2-specific antibody response. (a) Schematic representation of the three components of the lipopeptide construct P25-P2C-HPV. (b) Mice vaccinated with P25-P2C-HPV were bled 2 weeks after the third immunization (week 10). The titer for HPV16 L2-specific antibody was determined by HPV16 L2 17–36 peptide-based ELISA. HPV, HPV16 minor capsid protein L2 amino acids 17–36; P25, T_h epitope derived from the fusion protein of the morbillivirus canine distemper virus; Lys, lysine; Ser, serine; Pam₂Cys, lipid component of macrophage-activating lipopeptide 2; s.c., subcutaneous; i.n., intranasal; OD₄₀₅, optical density at a wavelength of 405 nm.

   
   
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   Fig. 2.

   Class II MHC and MyD88 signaling are critical for an L2-specific antibody response to P25-P2C-HPV. BALB/c or C57BL/6 wild-type mice as well as MyD88^−/−, class II MHC-deficient (def) or CD40^−/− mice were vaccinated with P25-P2C-HPV and were subsequently bled 2 weeks after the third immunization (week 10). The titer of HPV16 L2-specific antibody was determined by HPV16 L2 17–36 peptide ELISA. L2-specific antibody was not detected in sera diluted 200 and derived from MyD88^−/−, class II MHC def, or CD40^−/− mice that were vaccinated with P25-P2C-HPV.

   
   
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   Fig. 3.

   Vaccination with P25-P2C-HPV via s.c. or intranasal routes induces high titers of L2-specific HPV16-neutralizing serum antibodies. (a–d) BALB/c mice vaccinated with P25-P2C-HPV via the s.c. (a and c) or i.n. (b and d) routes were bled 2 weeks after the second immunization (week 6) or 2 weeks after the third immunization (week 10). The titer for the HPV16 L2-specific antibody was determined by HPV16 L2 17–36 peptide-based ELISA (a and b). In vitro HPV16 neutralization titers also were determined (c and d). s.c., subcutaneous; i.n., intranasal. Statistical significance is indicated by an asterisk and P value.

   
   
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   Fig. 4.

   Antibodies elicited by vaccination with P25-P2C-HPV cross-neutralizes multiple heterologous HPV pseudovirions. The ability of antiserum generated by immunization of mice with P25P2CHPV vaccine to neutralize heterologous HPV5, HPV18, HPV45, and BPV1 pseudovirions was tested. Using serial dilutions of antiserum, mean titers of 5320, 2845, 360, 110, and 180 were generated when reacted with HPV16, HPV5, HPV18, HPV45, and BPV1, respectively.

   
   
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   Fig. 5.

   P25-P2C-HPV vaccination protects mice from cutaneous challenge with HPV16 and HPV45. (a–c) BALB/c mice were vaccinated s.c. three times with saline, HPV16 L2 17–36 peptide, P25 peptide, HPV45 L1 VLP, HPV16 L1 VLP, or P25-P2C-HPV and challenged on their belly with HPV16 (a and b) or HPV45 (c) pseudovirions carrying a luciferase reporter 2 weeks after the third immunization (week 10). To detect pseudoinfection, the mice were injected with luciferin 3 days after viral challenge and imaged for bioluminescence by using the IVIS 200 imaging system [see a for HPV16 challenge and supporting information (SI) Fig. S1 for HPV45 challenge]. The bioluminescence was quantified in relative light units by using Living Image 2.20 software for HPV16 challenge (b) or HPV45 challenge (c). rlu, relative light units.

   
   
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   Fig. 6.

   P25-P2C-HPV vaccination protects mice from vaginal challenge with HPV16. BALB/c mice were vaccinated s.c. three times with P25-P2C-HPV or not (controls) and challenged in their genital tracts with HPV16 pseudovirions carrying an RFP reporter gene 2 weeks after the third immunization (except for negative control). To detect pseudoinfection, the mice were killed 3 days after viral challenge, and their genital tracts were dissected and isolated. (a) The lumen of each genital tract was imaged for red fluorescence by using a Maestro instrument. (b) The red fluorescence was quantified by using Image J software.