Development of Fast and Portable Frequency Magnetic Mixing-Based Serological SARS-CoV-2-Specific Antibody Detection Assay

A novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in China in December 2019, causing an ongoing, rapidly spreading global pandemic. Worldwide, vaccination is now expected to provide containment of the novel virus, resulting in an antibody-mediated immunity. To verify this, serological antibody assays qualitatively as well as quantitatively depicting the amount of generated antibodies are of great importance. Currently available test methods are either laboratory based or do not have the ability to indicate an estimation about the immune response. To overcome this, a novel and rapid serological magnetic immunodetection (MID) point-of-care (PoC) assay was developed, with sensitivity and specificity comparable to laboratory-based DiaSorin Liaison SARS-CoV-2 S1/S2 IgG assay. To specifically enrich human antibodies against SARS-CoV-2 in immunofiltration columns (IFCs) from patient sera, a SARS-CoV-2 S1 antigen was transiently produced in plants, purified and immobilized on the IFC. Then, an IgG-specific secondary antibody could bind to the retained antibodies, which was finally labeled using superparamagnetic nanoparticles. Based on frequency magnetic mixing technology (FMMD), the magnetic particles enriched in IFC were detected using a portable FMMD device. The obtained measurement signal correlates with the amount of SARS-CoV-2-specific antibodies in the sera, which could be demonstrated by titer determination. In this study, a MID-based assay could be developed, giving qualitative as well as semiquantitative results of SARS-CoV-2-specific antibody levels in patient’s sera within 21 min of assay time with a sensitivity of 97% and a specificity of 92%, based on the analysis of 170 sera from hospitalized patients that were tested using an Food and Drug Administration (FDA)-certified chemiluminescence assay

Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants

Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80 degrees C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP TSR, PfTRAP TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails

Kombinierte Trochleaplastik und Rekonstruktion des MPFL bei patellofemoraler Instabilität

Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml), the blood stage (40-60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy

The purified antibody preparations used for all <i>in vitro</i> assays.

<p>The total IgG concentrations and CFCA results are listed for each component. Nomenclature is based on the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). The PlasmoMix-specific antibody concentration is the sum of the specific antibody response against the four components. The antigen-specific antibody concentration is given in mg/ml and % of total IgG.</p

Antigen-specific antibody titers of rabbit immune sera determined by ELISA.

<p>Three rabbits (R1, R2 and R3) were immunized six times with PlasmoMix and serum samples were collected on days 0 (pre-immune), 35, 63 and 91. (A) The antibody titer against the immunization mixture (PlasmoMix) as well as against the four protein-based components (CCT, E3, gAMA1 and F0) were determined. (B) To further dissect the immune response, the specific antibody response against each individual domain was analyzed. Therefore, the domains were C-terminaly fused to DsRed (fluorescent reporter protein), and expressed and purified as described in the methods section. Antigen domains comprising a fusion protein are connected by a bracket. Antibody titers are shown for each rabbit (R1: open black square, R2: open red circle, R3: open blue triangle) as well as the geometric mean (black line) of three rabbits.</p

<i>In vitro</i> growth inhibition assay (GIA) of asexual <i>P</i>. <i>falciparum</i> 3D7 parasites with purified PlasmoMix-specific rabbit IgG.

<p>The rabbit IgGs were purified from the serum of three rabbits (R1, R2 and R3) collected on days 35, 63 and 91 post-immunization with PlasmoMix. Nomenclature of the sample first features the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). (A) Four serial 1/1 dilutions from 6–0.75 mg/ml of total IgGs were used to demonstrate the concentration dependency of the assay and to calculate the IC_<b>50</b> values (the total IgG concentrations needed for 50% inhibition). (B) The same GIA but instead of total IgG, the gAMA1-specifc antibody concentration (based on CFCA and calculated from total IgG, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131456#sec002" target="_blank">methods</a> section) was used and the IC_<b>50</b> values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates.</p

Plant expression cassette and construct design, amino acid sequences, SDS-PAGE and immunoblot blot analysis.

<p>(A) Schematic presentation of the expression cassettes of the plant binary expression vector pTRAk. SAR: scaffold attachment region; CaMV 35S promoter and terminator: promoter with duplicated enhancer and terminator of the <i>Cauliflower mosaic virus</i> (CaMV) 35S gene; 5' untranslated region: 5'-UTR of the chalcone synthase gene from <i>Petroselinum crispum</i>; signal peptide sequence: transit peptide sequence of murine antibody heavy chain; GOI: Gene of interest, CCT (1), gAMA1 (2), E3 (3) and F0 (4). The restriction sites used to insert the GOI into the plant expression vector are indicated; His₆ tag: six histidine affinity purification tag; ER-retention signal: SEKDEL ER-retention signal. <b>(</b>B) Table containing all the information for the selected antigens. For each antigen the main stage of expression, the name, the plasmoDB number and the amino acid sequence are depicted. SDS-PAGE (C) and immunoblot analysis (D) under reducing conditions of the four recombinant and purified proteins. Proteins were detected using rabbit anti-His₆ antiserum and alkaline phosphatase-labeled goat anti-rabbit antiserum. M: PageRuler pre-stained protein ladder (Fermentas), lane 1: CCT, lane 2: gAMA1, lane 3: E3 and lane 4: F0.</p

<i>In vitro</i> inhibition of (A) sporozoite gliding motility, (B) hepatocyte cell traversal, (C) sporozoite invasion and liver stage development with PlasmoMix-specific rabbit IgG and (D) Dose response of sporozoite invasion and liver stage development.

<p>All three assays were performed with <i>P</i>. <i>falciparum</i> NF54 parasites and purified rabbit IgGs (R1, R2 and R3) at a total IgG concentration of 3 mg/ml from serum samples collected on days 63 and 91. For the dose response, purified rabbit IgGs from rabbit R3 (day 63 and day 91) was used at the following total IgG concentrations: 9, 3, 0.9 and 0.09 mg/ml. The CCT-specific antibody concentration was calculated based on CFCA from total IgG. Inhibitions are expressed as the mean of duplicate measurements with standard deviations.</p

Correlation of transmission blocking activity and F0-specific antibody concentration.

<p>The rabbit IgGs were purified from serum samples collected on day 91 post-immunization with PlasmoMix. To demonstrate the concentration dependency of the transmission blocking activity, the TBA was performed with purified antibodies from each rabbit (R1: black, R2: open and R3: hatched) at total IgG concentrations of 1 mg/ml (circles), 0.1 mg/ml (squares) and 0.01 mg/ml (triangles). Based on the CFCA results, the F0-specific antibody concentration was calculated, plotted and used to determine the IC₅₀ value (the antibody concentration needed to obtain 50% inhibition of transmission). For detailed results of the transmission blocking assay, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131456#pone.0131456.s005" target="_blank">S2 Table</a>.</p