Impact of Aerosol Dust on xMAP Multiplex Detection of Different Class Pathogens

Environmental or city-scale bioaerosol surveillance can provide additional value for biodefense and public health. Efficient bioaerosol monitoring should rely on multiplex systems capable of detecting a wide range of biologically hazardous components potentially present in air (bacteria, viruses, toxins and allergens). xMAP technology from LuminexTM allows multiplex bead-based detection of antigens or nucleic acids, but its use for simultaneous detection of different classes of pathogens (bacteria, virus, toxin) is questionable. Another problem is the detection of pathogens in complex matrices, e.g., in the presence of dust. In the this research, we developed the model xMAP multiplex test-system aiRDeTeX 1.0, which enables detection of influenza A virus, Adenovirus type 6 Salmonella typhimurium, and cholera toxin B subunit representing RNA virus, DNA virus, gram-negative bacteria and toxin respectively as model organisms of biologically hazardous components potentially present in or spreadable through the air. We have extensively studied the effect of matrix solution (PBS, distilled water), environmental dust and ultrasound treatment for monoplex and multiplex detection efficiency of individual targets. All targets were efficiently detectable in PBS and in the presence of dust. Ultrasound does not improve the detection except for bacterial LPS

Revaccination in Age-Risk Groups with Sputnik V Is Immunologically Effective and Depends on the Initial Neutralizing SARS-CoV-2 IgG Antibodies Level

Vaccination against COVID-19 has occurred in Russia for more than two years. According to the Russian official clinical guidelines to maintain tense immunity in the conditions of the ongoing COVID-19 pandemic, it is necessary to use booster immunization six months after primary vaccination or a previous COVID-19 contraction. It is especially important to ensure the maintenance of protective immunity in the elderly, who are at risk of severe courses of COVID-19. Meanwhile, the immunological effectiveness of the booster doses has not been sufficiently substantiated. To investigate the immunogenicity of Sputnik V within the recommended revaccination regimen and evaluate the effectiveness of booster doses, we conducted this study on 3983 samples obtained from individuals previously vaccinated with Sputnik V in Moscow. We analyzed the level of antibodies in BAU/mL three times: (i) six months after primary immunization immediately before the booster (RV), (ii) 3 weeks after the introduction of the first component of the booster (RV1), and (iii) 3 weeks after the introduction of the second component of the booster (RV2). Six months after the primary vaccination with Sputnik V, 95.5% of patients maintained a positive level of IgG antibodies to the receptor-binding domain (RBD) of SARS-CoV-2. The degree of increase in the specific virus-neutralizing antibodies level after revaccination increased with a decrease in their initial level just before the booster dose application. In the group of people with the level of antibodies up to 100 BAU/mL six months after the vaccination, a more than eightfold increase (p p p p < 0.05), regardless of the previous COVID-19 infection. Thus, revaccination is most effective in individuals with an antibody level below 500 BAU/mL, regardless of the vaccinee age and COVID-19 contraction. For the first time, it has been shown that a single booster dose of the Sputnik vaccine is sufficient to form a protective immunity in most vaccinees regardless of age and preexisting antibody level

Safety and Immunogenicity of the GamTBvac, the Recombinant Subunit Tuberculosis Vaccine Candidate: A Phase II, Multi-Center, Double-Blind, Randomized, Placebo-Controlled Study

GamTBvac is a candidate tuberculosis vaccine with two fusion proteins, containing Ag85a, ESAT6, CFP10, and a dextran-binding domain (DBD). Phase II of a double-blind, randomized, multicenter, placebo-controlled study in parallel groups in healthy adults to evaluate the safety and immunogenicity of GamTBvac in 180 previously-vaccinated with Bacillus Calmette&ndash;Gu&eacute;rin vaccine (BCG) healthy volunteers without Mycobacterium tuberculosis (MTB) infection was conducted. The dose (0.5 mL) of either the study drug or a placebo was administered subcutaneously twice with an 8-week interval. At eight timepoints from 14 to 150 days, whole blood and sera were assayed. Antigen-specific T-cell responses were measured by an in-house interferon-gamma release assay (IGRA-test), the QuantiFERON (QTF) test, and intracellular cytokine staining (ICS). For antibody response detection, the bead-based multiplex immunoassay (MIA) was applied. The vaccine confirmed an acceptable safety profile previously shown in a first-in-human clinical study. After stimulation with both fusions, the highest median level of INF-&gamma; was detected on day 21. The GamTBvac vaccine induced antigen-specific interferon-gamma release, Th1 cytokine-expressing CD4+ T-cells, and IgG responses and results support further clinical testing of GamTBvac

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

<div><p>Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (<i>R</i>_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (<i>R</i>_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.</p></div

Fractograms of BSA (1 mg/ml) preheated at 65°C.

<p>The heating times were the following: (1) 0, (2) 5, (3) 15, (4) 90 and (5) 600 min. AF4 conditions: 23°C, axial (detector) flow 1 ml/min, focus flow 5 ml/min, cross flow 5 ml/min for 10 min and then linear decay to 0.1 ml/min within 20 min plus 8 min at 0 ml/min.</p

Dependences of the hydrodynamic radius (<i>R</i>_h) on the portion of the denatured protein (γ_den) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The values of γ_den were calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.e007" target="_blank">Eq 4</a>. For each temperature parameters <i>B</i>, <i>k</i>_1,den and <i>k</i>_2,den indicated in corresponding panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.g002" target="_blank">Fig 2</a> were used. The <i>R</i>_h <i>vs</i> γ_den plots at 65°C and 70°C (panels B and C) were used for determination of the hydrodynamic radius of the primary aggregates (<i>R</i>_h,1).</p

Dependences of the light scattering intensity on time for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The dotted horizontal lines on the panels A, B and C correspond to <i>I</i>₂ values calculated from the dependences of the light scattering intensity on the portion of aggregated BSA.</p

Dependences of the hydrodynamic radius (<i>R</i>_h) on time for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The dotted horizontal lines on panels A, B, and C correspond to <i>R</i>_h,1 or <i>R</i>_h,2 values calculated from the dependences of <i>R</i>_h on the portions of denatured and aggregated BSA, respectively. The solid curve on panel D was calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.e004" target="_blank">Eq 3</a> at <i>d</i>_f = 1.76.</p

Dependences of the light scattering intensity (<i>I</i>) on the portion of the aggregated protein (γ_agg) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The vertical dotted lines correspond to γ_agg = (γ_Uhr + γ_Ulr,agg). The <i>I vs</i> γ_agg plots at 60°C, 65°C and 70°C (panels A–C) were used for determination of parameter <i>I</i>₂ corresponding to the value of the light scattering intensity after completion of the secondary BSA aggregates formation. The solid lines were calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.e013" target="_blank">Eq 5</a>.</p

Fluorescence emission spectra of ThT (20 μM; curve 1) and ThT solution incubated with heated BSA (0.4 mg/ml) for 30 min at 25°C.

<p>BSA (1 mg/ml) was preincubated for 12 h at 60°C, 65°C, 70°C and 80°C (curves 2–5, respectively). Excitation wavelength was 450 nm.</p