Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Sequence alignment of MS, PP7, and Qβ coat proteins. The coat protein sequences of MS2, Qβ, and PP7, aligned using the Clustal Omega35 multiple sequence alignment tool. Dashed lines mark alignment gaps, asterisks (green highlight) indicate identical residues, and single (red) and double (blue) dots indicate similar residues.

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Sequencing

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Hybrid and Homogeneous PP7 Virus-like Particles

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Virus

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Successful C-Terminal Extension Modifications of the PP7 Virus-like Particle

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Virus

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Successful Loop Insertion Modifications of the PP7-PP7 Dimer Particle

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Sequencing

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Representative analyses of thermal stability of the indicated PP7-PP7-based particles (0.1 mg/mL in PBS buffer, heated at 10 °C per minute) in the absence (top) and presence (bottom) of 1 mM DTT. Additional traces for other particles are provided in Supporting Information (Figure S1).

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques:

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Melting ( T m ) and Onset-of-Aggregation ( T agg ) Temperatures of PP7 Particles a

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques:

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Particle Sizes Measured from TEM Images

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques:

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Selected cryo-EM 2D class averages corresponding to the projection along the twofold axis of monomeric PP7-ZZ and ZZ-PP7 particles and dimeric PP7-PP7-ZZ particle. The apparent coronas around the first two particles are artifacts of sample preparation (scale bar = 20 nm).

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Cryo-EM Sample Prep, Sample Prep

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Cryo-EM structure of the PP7-PP7 virus-like particle [top, T = 4 (h = 2; k = 0) structure, PDB ID 6N4V] compared to the previously published structure of the PP7 particle [bottom, T = 3 (h = 1; k = 1) subunits colored the same way, PDB ID 1DWN]. (left) View down the fivefold symmetry axes; (middle) view down the twofold symmetry axis; (right) a cartoon representation showing the organization of the coat-protein dimers in the T = 4 structure. The linker loop is labeled “L”, and the termini are labeled “NC”.

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Cryo-EM Sample Prep, Virus, Labeling

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Cryo-EM structural details. (A, left) PP7-PP7 dimer threefold organization, (A, right) expanded view of one single-chain dimer, with AYGG linker (green), N-terminal Ser (cyan), and C-terminal Arg (purple) residues shown in stick representation. (B) Comparison of particles bearing the a-loop insertion and C-terminal ZZ-domain extension. Arrows mark regions of poorly resolved amino acid density in the reconstruction of the PP7-a-loop-PP7 particle following the initial Ala residue of the a-loop (left) and following the last Arg residue at the C-terminus (Arg127) (right). Circles delineate well-resolved linker loop density for the PP7-PP7-ZZ particle (left) and the Gly residue that starts the linkage to the ZZ-domain but not the rest of that domain (right).

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Cryo-EM Sample Prep, Comparison, Residue

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Mapping of extra density on the surface of PP7-a-loop-PP7, PP7-PP7-ZZ, and PP7-a-loop-PP7–150-loop particles. Overall surface density maps: (A) PP7-PP7-ZZ, (B) PP7-a-loop-PP7, and (C) PP7-a-loop-PP7–150-loop. Extra densities highlighted: (D) PP7-PP7-ZZ, (E) PP7-a-loop-PP7, and (F) PP7-a-loop-PP7–150-loop, comparing to PP7-PP7. (G) PP7-PP7-ZZ, (H) PP7-a-loop-PP7, and (I) PP7-a-loop-PP7–150-loop, showing the same loop insertions and C-terminal extensions as in (D–F). Subunit organization in (G–I) is indicated as in Figure 5.

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques:

Journal: ACS nano

Article Title: Engineering the PP7 Virus Capsid as a Peptide Display Platform

doi: 10.1021/acsnano.8b09683

Figure Lengend Snippet: Characterization of Rev-CD encapsulated PP7-PP7-OVA particles. (A) Electrophoretic analysis: purified particles showing PP7-PP7-OVA1, PP7-PP7-OVA2, and Rev-CD bands. (B) TEM; images are indistinguishable from those of PP7-PP7-OVA1 and PP7-PP7-OVA2 without enzyme packaged. (C) Kinetic analysis, plotted on a per-enzyme basis, of the conversion of 5-FC to 5-FU catalyzed by the indicated forms of cytosine deaminase. Solid curves show the best fit using the Michaelis–Menten equation. The @ symbol designates the encapsidated enzyme.

Article Snippet: The PP7 VLPs were expressed and assembled in BL21(DE3) E. coli cells (Biogen) as previously described for Q β .

Techniques: Purification

Journal: Frontiers in Microbiology

Article Title: Cutaneous Papillomaviruses and Non-melanoma Skin Cancer: Causal Agents or Innocent Bystanders?

doi: 10.3389/fmicb.2018.00874

Figure Lengend Snippet: Preclinical studies showing reactivity of HPV vaccines to cutaneous types/challenge.

Article Snippet: PP7/MS2 bacteriophage VLPs , HPV16 L2(20–29), (17–31), (14–40) and (14–65) , ND , HPV5 , Being developed by the company Agilvax with DMID/NIAID/NIH support ( ). , .

Techniques: Vaccines, In Vitro, Bioprocessing, Produced, Clinical Proteomics

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: 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.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Sequencing, Derivative Assay, Recombinant, Construct

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: Shown are A) wild-type PP7 VLPs, B) 16L2 PP7 VLPs, and C) 18L2 PP7 VLPs. VLPs were visualized at a magnification of 40,000×.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques:

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: 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.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: Mice were immunized i.m. twice with 5 µg of PP7 or L2 PP7 VLPs with IFA and their sera collected 2 weeks after the last immunization. Serum anti-L2 IgG titer was determined by end-point dilution ELISA against streptavidin-conjugated HPV L2 peptides (amino acid 14–40) from HPV1 (squares), HPV5 (inverted triangles), HPV6 (upright triangles), HPV16 (white-filled circles), and HPV18 (diamonds). Black-filled circles denote reactivity of PP7 sera with the different five L2 peptides. Each datum point shows antibody titer from each mouse and lines represent the geometric mean titers for each group.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: (A) ELISA plates were coated with the streptavidin-conjugated L2 peptides described in and reacted with a 1∶160 dilution of serum from mice immunized with wild-type PP7 VLPs or L2 PP7 VLPs followed by secondary antibody. Shown are the averages of the optical density (OD 405 ) values of sera from three individual mice in each group. Error bars represent SEM. (B) A summary of these results. “++++” indicates a mean OD 405 >1.2, “+++” indicates a mean OD 405 between 0.8 and 1.2, “++” indicates a mean OD 405 between 0.4 and 0.8, “+” indicates a mean OD 405 between 0.2 and 0.4, and “−” indicates a mean OD 405 below 0.2.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: 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 1.3×10 5 (PsV18) or 3.0×10 6 (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/cm 2 /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.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Infection, Luciferase, Expressing, Software, Control

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: 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.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: 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 1.3×10 5 – 6.5×10 6 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/cm 2 /sr) values for each animal were determined as described in . Black-filled circles denote mice immunized with wild-type PP7 VLPs and white-filled circles denote mice immunized with mixed L2 PP7 VLPs.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques:

Journal: PLoS ONE

Article Title: A Pan-HPV Vaccine Based on Bacteriophage PP7 VLPs Displaying Broadly Cross-Neutralizing Epitopes from the HPV Minor Capsid Protein, L2

doi: 10.1371/journal.pone.0023310

Figure Lengend Snippet: Balb/c mice were immunized i.m. as described in . Three weeks after the last immunization, mice were subcutaneously challenged in the belly with 6.0×10 5 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.

Article Snippet: L2 PP7 VLPs were made by transforming CSH41F or C41 cells (Lucigen) with plasmids p2P7K32 and pET2P7K32, respectively, containing L2 peptides from different HPV types.

Techniques: Injection, Control