Model for the induction of neutralizing antibodies by high-density versus low-density epitope display.

<p>Black arrows represent antibody species during lineage evolution that are not reactive with self; red arrows represent species that are reactive with self. Inverted blue Ys denote B cell lineages that result in production of a virion binding antibody; red Xs denote deletion or tolerization of a self-reactive B cell lineage.</p

What's different about HIV virions?

<p>Surface projections of bovine papillomavirus (BPV), HIV, dengue virus, and influenza virus are shown (not to scale). The images of papillomavirus (PV) and dengue virus were obtained from the Viper database (PMID: 18981051). The image of influenza virus is courtesy of cdc.gov. The HIV image was generously provided by Sriram Subramaniam, National Cancer Institute.</p

Amino acid sequence alignment of L2 (residue 17–31, or equivalent) from selected carcinogenic, genital, and/or cutaneous HPV types.

<p>The consensus sequence was derived from an alignment of 15 carcinogenic HPVs, 2 genital HPVs, 4 cutaneous HPVs plus 2 animal papillomaviruses using ClustalW2. Recombinant L2 PP7 VLPs displaying these peptide sequences were constructed. Amino acids are shown in single-letter code, dots indicate that the amino acid is identical to consensus sequence, and amino acid differences from the consensus are shown for each HPV type. A plus (+) symbol indicates that there is no consensus amino acid at this position.</p

Reactivity of the RG-1 monoclonal antibody with recombinant L2 PP7 VLPs.

<p>500 ng of wild-type PP7 VLPs or L2 PP7 VLPs were used to coat ELISA plates. Binding of a 1∶5,000 dilution of RG-1 was detected using a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody followed by development with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Reactivity was determined by measuring the mean optical density (OD) values at 405 nm. Error bars indicate standard error of the mean (SEM) of duplicate wells.</p

Immunization with a mixture of L2-PP7 VLPs induces broad anti-L2 IgG responses.

<p>Shown is the reactivity of sera from mice immunized with mixed L2 PP7 VLPs to L2 peptides. Mice were immunized i.m. three times with 10 µg of wild-type PP7 VLPs or 10 µg of mixed L2 PP7 VLPs. Sera were collected 2 weeks after the last immunization and serum anti-L2 IgG titer was determined by end-point dilution ELISA against streptavidin-conjugated HPV L2 peptides (amino acid 14–40) from HPV1, HPV5, HPV6, HPV16, and HPV18. Black-filled circles denote mice immunized with PP7 VLPs and diamonds denote mice immunized with mixed L2 PP7 VLPs.</p

Mice immunized with mixed L2 PP7 VLPs are protected from vaginal challenge with diverse PsVs.

<p>Groups of 5 Balb/c mice were immunized i.m. three times with 10 µg of PP7 VLPs or mixed L2 PP7 VLPs. Three weeks after the last immunization, mice were vaginally challenged with 1.3×10⁵– 6.5×10⁶ IU of PsV5, PsV6, PsV16, PsV18, PsV31, PsV45, PsV52, or PsV58. Forty-eight hours later, luciferin was instilled vaginally and images were taken 3 minutes post-luciferin instillation. Average radiance (p/s/cm²/sr) values for each animal were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023310#pone-0023310-g006" target="_blank">Figure 6</a>. Black-filled circles denote mice immunized with wild-type PP7 VLPs and white-filled circles denote mice immunized with mixed L2 PP7 VLPs.</p

Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs are protected from vaginal challenge with HPV18 and HPV16 PsV.

<p>Groups of 5 BALB/c mice were immunized i.m. twice with 5 µg of PP7 VLPs, 16L2 PP7 VLPs or 18L2 PP7 VLPs with IFA. Three weeks after the second immunization, mice were vaginally challenged with 1.3×10⁵ (PsV18) or 3.0×10⁶ (PsV16) IU of PsV. Forty-eight hours later, luciferin was instilled vaginally and images were taken 3 minutes post-luciferin instillation. Images showing the magnitude of vaginal infection with PsV18 or PsV16 are shown in panels A and B, respectively. The colors reflect the intensity of luciferase expression. Colors are scaled for each image and shown to the right of the image. Quantitative data (shown in each panel) was extracted by drawing equally sized regions of interests surrounding the site of PsV instillation and determining average radiance (p/s/cm²/sr) by using Living Image 3.2 software. Background radiance (determined by gating on another region of the mouse) was subtracted from this value. Black-filled circles denote mice immunized with control wild-type PP7 VLPs and white-filled circles denote mice immunized with either 16L2 PP7 VLPs or 18L2 PP7 VLPs. Lines reflect the geometric mean radiance for each group.</p

Mice immunized with mixed L2 PP7 VLPs are protected from cutaneous challenge with PsV5.

<p>Balb/c mice were immunized i.m. as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023310#pone-0023310-g008" target="_blank">Figure 8</a>. Three weeks after the last immunization, mice were subcutaneously challenged in the belly with 6.0×10⁵ IU of PsV5. Three days post-challenge, mice were anesthetized and 0.7 mg luciferin was injected subcutaneously. Images of mice immunized with control PP7 VLPs and mixed L2 PP7 VLPs are shown in panels A and B, respectively. Average radiance values for the two groups are shown in panel C. Black-filled circles denote mice immunized with PP7 VLPs and white-filled circles denote mice immunized with mixed L2 PP7 VLPs.</p

Epitope-Specific Anti-hCG Vaccines on a Virus Like Particle Platform

<div><p>The possibility of a contraceptive vaccine targeting human chorionic gonadotropin has long been recognized, but never fully realized. Here we describe an epitope-specific approach based on immunogenic display of hCG-derived peptides on virus-like particles of RNA bacteriophage. A number of recombinant VLPs were constructed, each displaying a different hCG-derived peptide. Some were taken from the disordered C-terminal tail of the hormone, another came from an internal loop, and yet another was an epitope mimic produced by affinity-selection on an hCG-neutralizing antibody target. Immunization of mice with some VLPs yielded antisera that bound the hormone and neutralized hCG biological activity.</p></div

Immunogenicity of L2-MS2 and L2-PP7 VLPs in mice.

<p>(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.</p