Adjuvants and their proposed mode of action.

<p>Adjuvants and their proposed mode of action.</p

Boost by adjuvanted RSV F DS-Cav1 can focus the RSV immune response to antigenic site Ø and II in elderly mice.

<p>Pre-immunized elderly mice (~85 weeks wait) were administered <b>(A)</b> DS-Cav1 formulated with SAS + Carbopol or <b>(B)</b> DS-Cav1 formulated with Alum and recognition of DS-Cav1, DS-Cav1 site Ø KO, DS-Cav1 site II KO, Post F and Post F site II KO probes by sera for all immunized mice assessed. Scatter plots show the geometric mean. <i>p</i> values were determined by two-tailed Wilcoxon matched-pairs signed rank test, <i>p</i> = >0.05 (ns); <i>p</i> < 0.05 (*). Associated raw data is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s005" target="_blank">S5 Table</a>.</p

Prime-boost vaccination with different adjuvant formulations induced a balanced Th1/Th2 IgG response in elderly mice.

<p><b>(A</b>) Pre-immunized elderly mice (~85 weeks wait) were administered DS-Cav1 formulated with SAS + Carbopol or Alum. Sera were assayed by ELISA for their DS-Cav1 specific IgG1 and IgG2a antibody responses. Closed circles and open triangles represent IgG1 and IgG2a antibody titers, respectively. <b>(B</b>) The ratios of IgG1 to IgG2a for each animal in the groups were calculated from the upper panel. <i>p</i> values were determined by two-tailed Wilcoxon matched-pairs signed rank test, <i>p =</i> >0.05 (ns). Associated raw data is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s006" target="_blank">S6 Table</a>.</p

Adjuvants augment DS-Cav1 response in mice.

<p><b>(A)</b> RSV F (pre-F) DS-Cav1 formulated with nine different adjuvants was used to immunize mice at an interval of 3 weeks and <b>(B)</b> neutralization titers for all nine adjuvanted plus the control unadjuvanted groups are shown. Scatter plots show the geometric mean (numerical value below), each group included 10 mice. The palivizumab protective threshold is indicated by a dotted line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.ref010" target="_blank">10</a>] and <i>p</i> values for SAS + Carbopol/DS-Cav1 versus the other eight adjuvant formulations as assessed by two-tailed Mann-Whitney test and adjusted for multiple comparisons using the Holm-Bonferroni method; <i>p =</i> > 0.05 (ns); <i>p</i> < 0.05 (*); <i>p</i> < 0.01 (**); <i>p</i> < 0.001 (***) are shown. All adjuvanted groups show <i>p</i> < 0.0001 when compared to the no adjuvant group. Associated raw data is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s001" target="_blank">S1 Table</a>.</p

Adjuvanted DS-Cav1 can augment RSV-neutralization titers in elderly mice.

<p><b>(A)</b> Pre-immunized elderly mice (DS-Cav1 adjuvanted with Poly (I:C), ~85 weeks wait) were administered DS-Cav1 formulated with Alum or SAS + Carbopol and the immune response for all mice assessed. <b>(B)</b> Neutralization titers for each group of seven elderly mice are shown. Scatter plots show the geometric mean (numerical value below), the palivizumab protective threshold is indicated by a dotted line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.ref010" target="_blank">10</a>]. <i>p</i> values were determined by two-tailed Wilcoxon matched-pairs signed rank test, <i>p =</i> >0.05 (ns); <i>p</i> < 0.05 (*). Associated raw data is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s004" target="_blank">S4 Table</a>.</p

DS-Cav1-adjuvant formulations elicit antibodies of different IgG subclass in immunized mice.

<p><b>(A)</b> Sera from naïve mice primed and boosted with DS-Cav1 adjuvant mixtures were assayed by ELISA for their RSV F DS-Cav1 specific IgG1 and IgG2a antibody responses. Scatter plots show the geometric mean, closed circles and open triangles represent IgG1 and IgG2a antibody titers, respectively. (B) The ratios of IgG1 to IgG2a for each animal in the adjuvanted groups were calculated from the upper panel. <i>p</i>-values were determined using a two-tailed Mann-Whitney test and were adjusted for multiple comparisons using the Holm-Bonferroni method, <i>p</i> < 0.001 (***). Associated raw data is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s003" target="_blank">S3 Table</a>.</p

Pairing of heavy and light 10E8 intermediates.

<p>(<b>A</b>) Heavy (top) and light (bottom) phylogenetic trees with pairing of inferred intermediates indicated by dashed lines. Intermediate pairing based on tree structure and considerations from polyreactivity (see text and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.ref018" target="_blank">18</a>]). (<b>B</b>) Heavy chain UCA, intermediates and mature sequences. (<b>C</b>) Light chain UCA, intermediates and mature sequences.</p

10E8 light chain ontogeny: lineage members and calculated intermediates.

<p>(<b>A</b>) Selection of 10E8 clonal variants by computational sieving. Identity/divergence plots showing the results of sieving on V gene (left), V, and J genes (panel 2), CDR L3 length between 9 and 12 residues (IMGT numbering) (panel 3), and CDR L3 signature (panel 4). (<b>B</b>) Phylogenetic tree based on CDR L3 length-selected sequences. To construct a phylogenetic tree from >10⁵ sequences, representative sequences were selected randomly from bins of 0.5 divergence units (as shown in left inset). The numbers in parenthesis at the end of each branch correspond to the total number of clones that share >99% nucleotide sequence identity and 99% alignment coverage. Branches terminating without numbers indicate that the sequence has more than 10 clones with >99% nucleotide sequence identity and 99% alignment coverage. The coloring scheme for the ML tree was adapted from Zhu et al., [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.ref018" target="_blank">18</a>]. Blue circles indicate nodal points on the tree. (<b>C</b>) Sequences of phylogeneticaly inferred developmental light chain intermediates compared to mature 10E8 and constituent germline genes. Numbers appearing in parenthesis on the left side of the alignment indicate the number of identical residues to the germline V gene.</p

Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus

<div><p>Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein—stabilized in the pre-fusion (pre-F) conformation by “DS-Cav1” mutations—elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These “head-only” immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines.</p></div

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p