An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

<p>Abstract</p> <p>Background</p> <p>Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable.</p> <p>Methods</p> <p>Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described.</p> <p>Results</p> <p>The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed.</p> <p>Conclusions</p> <p>The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.</p

Immunomodulatory and Anti-Inflammatory Activities of Chicken Cathelicidin-2 Derived Peptides

Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that full-length CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. In addition, CATH-2 efficiently inhibited IL-1β and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives

Amino acid sequences and minimal inhibitory concentrations of CATH-2 analogs.

<p>Amino acid sequences and minimal inhibitory concentrations of CATH-2 analogs.</p

Primers used for quantitative real time PCR.

<p>Primers used for quantitative real time PCR.</p

Peptide-induced LDH release from HD11 cells.

<p>The percentage of lactate dehydrogenase (LDH) released from HD11 cells during 24 h exposure to medium containing CATH2 derived peptides (1–20 μM). Shown are means ± SEM for at least 3 independent experiments.</p

LPS binding affinity of CATH-2 analogs.

<p>LPS binding affinity of CATH-2 analogs.</p

CATH-2 analog neutralization of LPS-induced macrophage nitric oxide production.

<p>Nitric oxide production by HD11 cells stimulated with LPS (50 to 100 ng/ml) pre-incubated with CATH-2 derived peptides (20 μM). Nitric oxide (NO) production was measured in supernatants after 24 h incubation using the Griess assay. Amino acid substitutions: W3, [F2W, F5W, F12W]; W2, [F5W, F12W]; W, [F12W]; Y3, [F2Y, F5Y, F12Y]; Y2, [F5Y, F12Y]; Y, [F12Y]. <b>a</b> Dose-dependent production of NO by HD11 cells exposed to different sources of LPS. <b>b</b> CATH-2 significantly inhibited NO production induced by all tested LPS (100 ng/ml) sources, except wild-type <i>Neisseria meningitidis</i> LPS; bars indicate LPS-induced NO production in the absence (closed) and presence (open) of CATH-2 peptide. <b>c</b> Inhibition of <i>S</i>. <i>minnesota</i> LPS-induced (50 ng/ml) NO production by truncated CATH-2 analogs. Production of LPS-induced NO by HD11 cells was significantly reduced in the presence of C(1–21) and N-terminally truncated C(1–21) analogs up to 17 amino acid residues. <b>d, e</b> Phe/Trp substitution enhanced inhibition of active peptides C(1–21) and C(4–21), whereas Phe/Tyr substitution abrogated inhibition of LPS-induced NO production by these peptides. Data from 3 to 4 independent experiments; means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Metabolic activity of CATH-2 derived peptide treated HD11 cells.

<p>Metabolic activity of HD11 determined by WST-1 assays after 24 h stimulation with CATH-2 derived peptides (2.5–20 μM). Shown are means ± SEM for at least 3 independent experiments.</p

Induction of chemokine transcription in HD11 cells by truncated CATH-2 analogs.

<p>CXCLi2, MCP-3, CCLi4 and IL-1β transcription in HD11 cells treated with CATH-2 derived peptides (1 to 20 μM). Transcription was measured by real time QPCR analysis after 4 h (open bars) or 24 h (closed bars) of stimulation. <b>a</b> CATH-2 dose-dependently induced transcription of chemokines chCXCLi2/IL-8, MCP-3 and chCCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. <b>b</b> The capacity to induce chemokine transcription was maintained for N-terminally truncated C(1–21) analogs up to 14 amino acid residues during 24 h exposure. <b>c</b> No chemokine induction was observed when cells were exposed (4 h) to peptide C(1–15). Data from 3 to 4 independent experiments; means ± SEM * p<0.05, ** p<0.01, *** p<0.001.</p

Neutralization of LPS-induced pro-inflammatory cytokine production by CATH-2 analogs.

<p>IL-1β transcription in HD11 cells stimulated with <i>S</i>. <i>minnesota</i> LPS (50 ng/ml) and CATH-2 derived peptides (20 μM). Transcription was measured by real time QPCR analysis after 4 h of stimulation. Amino acid substitutions: W3, [F2W, F5W, F12W]; W2, [F5W, F12W]; W, [F12W]; Y3, [F2Y, F5Y, F12Y]; Y2, [F5Y, F12Y]; Y, [F12Y]. <b>a</b> LPS-induced IL-1β transcription in HD11 cells was blocked by full-length peptide and N-terminally truncated C(1–21) analogs up to 18 amino acid residues. <b>b</b> Phe/Trp substitution improved the capacity to neutralize LPS-induced IL-1β transcription of peptides C(1–21) and C(4–21) and introduced LPS-neutralizing capacity in formerly inactive peptides C(7–21) and C(10–21). <b>c</b> Phe/Tyr substitution of active peptides C(1–21) and C(4–21) abrogated neutralization of LPS-induced IL-1β expression. Data from 3 to 4 independent experiments; means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p