Figure 1.
Peptide display sites on MS2 and PP7 VLPs.
(A) RasMol-generated structures of MS2 VLPs (left panel) and PP7 VLPs (right panel). Red and purple colors depict the locations of the N-terminus of MS2 VLPs and the AB-loop of PP7 VLPs, respectively. In our recombinant VLPs, only 90 of the 180 coat proteins/VLP display the L2 peptide. B) Amino acid sequences of 16L2 peptides (capital bold characters; amino acids 20–29, 17–31, 14–40, and 14–65) inserted at the N-terminus of the single-chain dimer of MS2 coat protein. The start codon in capital italicized characters is underlined. Linker sequences are shown in small characters.
Figure 2.
Characterization of recombinant 16L2-MS2 coat proteins.
(A) An ethidium bromide stained-agarose gel showing encapsidated RNAs (left panel) and the same gel (right panel) stained with coomassie blue, showing the coat proteins at the same position of encapsidated RNAs. (B) Representative transmission electron micrographs of purified wild-type MS2 VLPs, 16L2(20–29) Nterm MS2 VLPs, 16L2(17–31) Nterm MS2 VLPs, and 16L2(14–40) Nterm MS2 VLPs. Images are magnified 70,000x. (C) Western blot analysis of recombinant L2-VLPs. Blots were probed with either mouse anti-16L2 sera (left panel) or rabbit anti-MS2 sera (right panel).
Figure 3.
Immunogenicity of L2-MS2 and L2-PP7 VLPs in mice.
(A) Groups of Balb/c mice were immunized twice intramuscularly at two-weeks interval with 5 µg each of the three 16L2-MS2 VLPs (displaying epitopes 20–29, 17–31, and 14–40) or control MS2 or with 16L2(17–31) AB-loop PP7 VLPs or control PP7 VLPs with IFA. Sera were collected after two weeks following each immunization. (B) Balb/c mice were immunized i.m. with 250 ng of 16L2(17–31) Nterm MS2 VLPs or control MS2 VLPs (without IFA) and four weeks later, both groups of mice were boosted with 500 ng of their respective VLPs without IFA. Sera was collected at weeks one, three, and five. In both cases, IgG titers were determined by end-point titration ELISA using 16L2 peptide (amino acid 14–40) as target antigen. Open red and blue circles represent titers after one immunization; broken red and blue lines represent geometric mean IgG titers after one immunization. Red- & Blue-filled circles represent titers after two immunizations and the solid lines represent geometric mean IgG titers after two immunizations. White-filled black circles represent both one and two immunizations.
Figure 4.
Analysis of cross-reactivity of antiserum to L2 peptides from diverse HPV types.
(A) Cross-reactivity of 16L2 Nterm MS2 VLP sera with diverse HPV L2 peptides. ELISA plates were coated with 500 ng of streptavidin-conjugated L2 peptides (amino acid 14–40) from HPV 1, 5, 6, 16, and 18. Diluted sera (1∶640) from mice immunized with 16L2(20–29) Nterm MS2 VLPs or 16L2(17–31) Nterm MS2 VLPs or 16L2(14–40) Nterm MS2 VLPs was reacted with the peptides in ELISA plates. Shown are the average optical densities (ODs) at 405 nm for 3 mice. Error bars represent standard error of the mean (SEM). (B) Comparison of cross-reactivity levels of 16L2(17–31) Nterm MS2 VLP sera and 16L2(17–31) AB-loop PP7 VLP sera with diverse HPV L2 peptides. ELISA plates were coated with peptides as described above and then reacted with sera from mice immunized with 16L2(17–31) Nterm MS2 VLPs or 16L2(17–31) AB-loop PP7 VLPs or MS2/PP7 VLPs at a 1∶2,560 dilution. Average ODs (405 nm) from 3 mice are shown. Error bars represent SEM.
Figure 5.
Reactivity of sera with short, linear HPV16 L2 peptides.
ELISA plates were coated with 500 ng of streptavidin-conjugated 16L2 peptides (amino acids 17–24, 20–27, 22–29, 24–31, and 14–40) and reacted with a 1∶40 dilution of sera from mice immunized with either 16L2(17–31) Nterm MS2 VLPs or 16L2(17–31) AB-loop PP7 VLPs or MS2/PP7 VLPs. Average ODs (405 nm) from a group of five mice are shown in solid black horizontal lines.
Figure 6.
L2 cysteines displayed on PP7 and MS2 VLPs are not reactive with BMCC-biotin.
Purified VLPs were mock-treated, DTT-treated and/or denatured prior to incubation with thiol-reactive BMCC-biotin. Proteins were separated by SDS-PAGE, transferred to membranes, and biotinylation was detected using streptavidin-HRP.
Table 1.
Summary of in vivo HPV PsV challenge experiments.
Figure 7.
Mice immunized with 16L2(17–31) Nterm MS2 VLPs show broader protection from infection with diverse HPV PsV types compared to those immunized with 16L2(17–31) AB-loop PP7 VLPs.
Balb/c mice were immunized (intramuscularly) twice at two weeks intervals with 5 µg of 16L2(17–31) Nterm MS2 VLPs or 16L2(17–31) AB-loop PP7 VLPs or HPV16 L1L2 VLPs or a mixture of MS2/PP7 VLPs. Three to five weeks after the last immunization, mice were challenged with: A) PsV16 and B) PsV5, 6, 31, 33, 35, 39, 45, 51, 53, and PsV58. All challenges were done vaginally except PsV5, which was done intradermally. Forty-eight hours post-challenge, 0.4 mg of luciferin was administered via the same route used for PsV challenge and average radiance (p/s/cm2/sr) values for each mouse was determined using Living Image 3.2 software. White-filled black circles represent mice immunized with MS2/PP7 VLPs, black-filled circles represent mice immunized with HPV16 L1L2 VLPs, blue-filled circles represent mice immunized with 16L2(17–31) AB-loop PP7 VLPs, and red-filled circles represent mice immunized with 16L2(17–31) Nterm MS2 VLPs. Black solid lines represent the geometric mean of average radiance.