Additional file 1: Figure S1. of Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi

B. burgdorferi plasmid PFam32 protein tree. Figure S2. Comparative maps of linear plasmids in 14 B. burgdorferi isolates. Maps of the following linear plasmids are shown in the following panels: A, lp5; B, lp17; C, lp21; D, lp25; E, lp28-1; F, lp28-2, lp28-6, lp28-7 and lp28-9; G, lp28-3; H, lp28-4; I, lp28-5; J, lp28-6; K, lp32-3; L, lp36; M, lp38; N, lp54; O, lp56. Figure S3. Deletions in cp32 circular plasmids. Figure S4. Comparative cp9 plasmid maps. Figure S5. All possible recombination products. Figure S6. Ancestral transfer of genetic material from the B. burgdorferi chromosome right end to a linear plasmid. Table S1. Linear plasmid locations of selected genes. (PDF 3869 kb

Immunogenicity of pIX-CS_short display is higher than that of CS protein and comparable levels cannot be achieved by mixing AdV vectors and protein.

<p>(A) Total CS-specific IgG titers in the serum of Balb/C mice 4 weeks post-immunization with 1x10¹⁰ VP/animal of HAdV35.empty.pIX-CS_short, HAdV35.CS.pIX-CS_short, with 5μg of CS protein in PBS, or with 1x10¹⁰ VP/animal HAdV35.empty. n = 7 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. (B) Total CS-specific IgG titers in the serum of Balb/C mice 4 weeks after immunization with 5μg CS protein in PBS, or mixed with 1x10¹⁰ VP/animal of empty AdV-vectors (HAdV35, HAdV26, or HAdV5), animals injected with Matrix-M only serve as control. n = 6 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. Ratio of CS-specific serum IgG2a to IgG1 levels, 4 weeks after immunization with 5μg CS protein in PBS only, or mixed with Matrix-M, or 1x10¹⁰ VP/animal of empty Ad-vectors (HAdV35, HAdV26, or HAdV5). Horizontal bars depict mean ratios (n = 5 animals/ group).</p

Characterization of the pIX-CS_short display- and display-/ expression vectors.

<p>(A) pIX-CS_short capsid incorporation in purified HAdV35 vector preparations. To confirm capsid incorporation of the pIX-CS_short (~50 kDa variants) 5 x10⁹ VP/well of each purified HAdV35 vector preparation was analyzed by Western blot using anti-CS antibody. To ensure equal loading, the blots were stained with anti-fiber 4D2 antibody (~35 kDa). The pIX-fusion proteins migrate higher than their predicted size in kDa due to the NANP-repeat in the CS protein. Marker (M) is indicated with the corresponding kDa band size. An additional band (~100 kDa) is indicated with an asterisk (*). (B) pIX-CS_short capsid display by electron microscopy (EM) anti-CS staining and gold-label staining. Representative EM images of HAdV35.empty.pIX-CS_short, HAdV35.CS.pIX-CS_short (upper row), HAdV35.empty.pIX-45-CS_short, HAdV35.CS.pIX-45-CS_short (middle row), HAdV35empty, and HAdV35 (bottom row) vectors. The bars represent 100 nm unless stated otherwise. (C) <i>In vitro</i> expression of CS transgene under non-replicating conditions in A549 cells. Western Blot analysis of cell lysates from A549 cells infected with 5000 VP/cell, using an anti-CS antibody. From left to right, the two controls HAdV35.empty and HAdV35.CS followed by the pIX-display vector variants with a CS transgene in E1 and without the transgene. Marker (M) is indicated with the corresponding kDa band size. (D) Heat Stability Assay showing percent (%) variation in luminescence in lysate of A549 cells infected with HAdV35.luc vector preparations subjected to 45°C temperature stress for 0 to 20 minutes. The percent variation was determined relative to the respective baseline time point (0 minutes).</p

pIX-CS_short display on CS-transgene expression-vectors induces strong humoral and cellular responses.

<p>(A) Total CS-specific IgG titers in the serum of Balb/C mice 2 and 8 weeks post-immunization with 1x10⁸, 1x10⁹, or 1x10¹⁰ VP/animal of HAdV35.empty.pIX-45-CS_short, HAdV35.CS.pIX-45-CS_short, a mix of HAdV35.empty.pIX-45-CS_short and HAdV35.CS, HAdV35.CS alone, or HAdV35.empty (10⁹ VP/ animal only). n = 5 per group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data by time point and dose. Black horizontal bars indicate statistical significance (p ≤ 0.05). (B) Ratio of CS-specific serum IgG2a to IgG1 levels, 4 weeks after immunization with 1x10¹⁰ VP of the HAdV35 vectors indicated in the legend. Horizontal bars depict mean ratios (n = 5 animals/ group). (C) IFNγ ELISPOT responses to stimulation of splenocytes of Balb/C mice 8 weeks post-immunization with <i>P</i>. <i>falciparum</i> CS peptide pool (left panel; “CS”) or the H-2Kd restricted immunodominant HAdV35 hexon epitope KYTPSNVTL (right panel; “HAdV35 hexon”). n = 5 animals/ group. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. ns = not significant.</p

Design of HAdV35 pIX-display-vectors

<p>Schematic representation of pIX-display HAdV35 Advac vectors containing a human CMV promoter and SV40 poly-A expression cassette in E1 and the native pIX promoter (P). The vectors either express a 376 amino acid CS protein (lacking the GPI anchor) or no transgene (empty) in the E1 region. The pIX-display-vectors, with and without the CS-transgene in E1, are genetically modified to display the 151 amino acid CSshort with or without a 3 amino acid glycine-linker (Gly) and/or 45Å-spacer.</p

N40 DNA is not cut by restriction endonuclease <i>Sfo</i>I.

<p>DNAs were prepared in agarose blocks, cleaved with the indicated restriction endonuclease, and subjected to agarose gel pulsed-field agarose electrophoresis and stained with ethidium bromide as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033280#pone.0033280-Casjens3" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033280#pone.0033280-Casjens10" target="_blank">[147]</a>. Strain M is described in the text. Identical results to those with strain M were obtained with strains B31, JD1 and 297 (data not shown).</p

Length variation of <i>B. burgdorferi</i> chromosomes.

<p>The relationships among the right end, plasmid-like, chromosomal extensions relative to known plasmids are indicated by gray shading; plasmid sizes are not drawn exactly to scale. There is a 1053 bp deletion and a 17 bp insertion in the 297 lp28-1 plasmid relative to the B31 extension. It is assumed that the 297 chromosome is essentially identical to that strain Sh-2-82 (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033280#pone.0033280-Huang1" target="_blank">[51]</a>).</p

<i>B. burgdorferi</i> plasmids present in four isolates.

a<p>Plasmids known to be in some cultures of the indicated strain, but which were not present in the sequenced culture. Their sequences remain undetermined.</p>b<p>Structural differences from otherwise organizationally similar plasmids are indicated as follows: trunc, truncated compared to other homologous plasmids; int, B31 cp32-10 is integrated into plasmid lp56; fused, JD1 cp32-1 and cp32-5 are fused into one large circular “cp32-1+5” plasmid.</p>c<p>In some B31 cultures the plasmid cp32-7 is replaced by cp32-2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033280#pone.0033280-Casjens2" target="_blank">[12]</a>. These two plasmids have the same apparent compatibility and appear to be prophage DNAs. Since it seems unlikely that they can exist in the same cell, and they are expected to be able to move between strains, one of them may have been inadvertently introduced in the laboratory. We use cp32-7 for this compatibility type since it is the one that is present in the completely sequenced B31 genome.</p

Organizational and open reading frame relationships among four lp38 plasmids.

<p>Maps of the four lp38s are labeled as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033280#pone-0033280-g008" target="_blank">Figure 8</a>. Yellow shading between maps marks regions of high nucleotide sequence similarity (percent identity values in black text). The pink horizontal bar indicates the region of 63 bp repeats in JD1 lp38; and blue arrows represent predicted transporter genes.</p

Comparison of three lp25 plasmids.

<p>Matrix plots with a 19 identities/23 bp window were created by DNA Strider <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033280#pone.0033280-Douglas1" target="_blank">[135]</a>. Percent identities of nucleotide sequences are indicated near the diagonal identity line for most orthologous regions. The predicted genes for B31 lp25 are shown between the two plots (open arrows with “X”s are putative pseudogenes), and regions of high similarity to other plasmids are noted on the right.</p