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

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

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

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

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

A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

<div><p>Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.</p></div

Negative-stain electron microscopy of head-only immunogens.

<p>Panels <b>(A-P)</b> show typical 2D averaged classes of i-273,i-693, i-210 and i-447 alone <b>(A, E, I</b> and <b>M)</b>, in complex with D25 (in blue; <b>B, F, J</b> and <b>N</b>), in complex with motavizumab (in green; <b>C, G, K</b> and <b>O</b>) and in complex with both D25 and motavizumab (in blue, green; <b>D, H, L</b> and <b>P</b>). Yellow indicates Fabs of ambiguous identity. Two separate averages are shown for each panel. White scale bars are 50 Å long.</p

Head-only RSV F immunogen boost elicits neutralization titers surpassing DS-Cav1.

<p><b>(A)</b> All four immunogens were used to immunize mice two times, resulting in high neutralization titers for i-693 and i-447 above the protective threshold of 100 (dotted line). <i>P</i> values between i-447 and i-210 or i-273 were 0.001 and <0.0001 respectively and those between i-693 and i-210 or i-273 were 0.0014 and <0.0001 respectively. <b>(B)</b> Immunogens i-693 and i-447 were used to boost DS-Cav1 seven weeks later resulting in titers higher than a DS-Cav1 homologous boost. Scatter plots show the geometric mean (numerical value below) with error bars representing the 95% confidence level. <i>P</i> values were determined by two-tailed Mann-Whitney tests. Each group included 10 mice.</p