Detection of biomarkers for filoviral infection with a silicon photonic resonator platform

This protocol describes the use of silicon photonic microring resonator sensors for detection of Ebola virus (EBOV) and Sudan virus (SUDV) soluble glycoprotein (sGP). This protocol encompasses biosensor functionalization of silicon microring resonator chips, detection of protein biomarkers in sera, preparing calibration standards for analytical validation, and quantification of the results from these experiments. This protocol is readily adaptable toward other analytes, including cytokines, chemokines, nucleic acids, and viruses. For complete details on the use and execution of this protocol, please refer to Qavi et al. (2022)

Antibodies to <i>S</i>. <i>aureus</i> LukS-PV Attenuated Subunit Vaccine Neutralize a Broad Spectrum of Canonical and Non-Canonical Bicomponent Leukotoxin Pairs

<div><p><i>S</i>. <i>aureus</i> vaccine development has proven particularly difficult. The conventional approach to achieve sterile immunity through opsonophagocytic killing has been largely unsuccessful. <i>S</i>. <i>aureus</i> is highly toxigenic and a great body of evidence suggests that a successful future vaccine for this organism should target extracellular toxins which are responsible for host tissue destruction and immunosuppression. Major staphylococcal toxins are alpha toxin (a single subunit hemolysin) along with a group of bicomponent pore-forming toxins (BCPFT), namely Panton-Valentine leukocidin (PVL), gamma hemolysins (HlgCB and AB), LukAB and LukED. In our previous report, an attenuated mutant of LukS-PV (PVL- S subunit) named as “LukS-mut9” elicited high immunogenic response as well as provided a significant protection in a mouse sepsis model. Recent discovery of PVL receptors shows that mice lack receptors for this toxin, thus the reported protection of mice with the PVL vaccine may relate to cross protective responses against other homologous toxins. This manuscript addresses this issue by demonstrating that polyclonal antibody generated by LukS-mut9 can neutralize other canonical and non-canonical leukotoxin pairs. In this report, we also demonstrated that several potent toxins can be created by non-canonical pairing of subunits. Out of 5 pairs of canonical and 8 pairs of non-canonical toxins tested, anti-LukS-mut9 polyclonal antibodies neutralized all except for LukAB. We also studied the potential hemolytic activities of canonical and noncanonical pairs among biocomponent toxins and discovered that a novel non-canonical pair consisting of HlgA and LukD is a highly toxic combination. This pair can lyse RBC from different species including human blood far better than alpha hemolysin. Moreover, to follow-up our last report, we explored the correlation between the levels of pre-existing antibodies to new sets of leukotoxins subunits and clinical outcomes in adult patients with <i>S</i>. <i>aureus</i> bacteremia. We found that there is an inversed correlation between the antibody titer to sepsis for leukotoxins LukS-mut9, LukF-PV, HlgC, LukE and LukAB, suggesting the risk of sepsis was significantly lower in the patients with higher antibody titer against those toxins.</p></div

ELISA of clinical samples classified into sepsis and nonsepsis based on clinical outcome.

<p>Individual toxin subunits as indicated above the each panel were coated over night at 4°C. Human serum samples were diluted to 1:10,000 in PBS with 1% milk. Goat anti human HRP conjugate diluted to 1:2,000 was used as secondary antibody.</p

Noncanonical sub units: HlgA and LukD hemolysis studies.

<p>Lysis was measured at 416 nm. <b><i>A</i></b>) Hemolytic dose response plot in 2% RBC (final concentration) from different species of blood. <b><i>B</i></b>) Dose response plot in 2% rabbit RBC with all possible combination of HlgA and LukD showing that only HlgA + LukD makes a potent hemolytic pair. <b><i>C</i></b>) Comparative dose response plot between alpha toxin and HlgA + LukD in 2% rabbit RBC. <b><i>D</i></b>) Timed hemolysis between alpha toxin and HlgA + LukD in 2% rabbit RBC.</p

PMN cytotoxicity of canonical and noncanonical lukotoxin subunit EC₅₀ (nM).

<p>PMN cytotoxicity of canonical and noncanonical lukotoxin subunit EC₅₀ (nM).</p

PMN lytic activity of different bicomponent toxins.

<p>Percentage (%) survival of HL-60 derived neutrophils treated with increasing concentrations of S-and F- component from different leukotoxin groups. <b><i>A)</i></b> Canonical pair <b><i>B)</i></b> Noncanonical pair with LukF-PV as F subunit <b><i>C)</i></b> Noncanonical pair with LukD as F subunit <b><i>D)</i></b> Noncanonical pair with HlgB as F subunit.</p