Efficiency and sensitivity optimization of a protocol to quantify indoor airborne SARS-CoV-2 levels.

BACKGROUND: Development of methodologies to quantify airborne micro-organisms is needed for the prevention and control of infections. It is difficult to conclude which is the most efficient and sensitive strategy to assess airborne SARS-CoV-2 RNA levels due to the disparity of results reported in clinical settings. AIM: To improve our previously reported protocol of measuring SARS-CoV-2 RNA levels, which was based on bioaerosol collection with a liquid impinger and RNA quantification with droplet digital polymerase chain reaction (ddPCR). METHODS: Air samples were collected in COVID-19 patient rooms to assess efficiency and/or sensitivity of different air samplers, liquid collection media, and reverse transcriptases (RT). FINDINGS: Mineral oil retains airborne RNA better than does hydrophilic media without impairing integrity. SARS-CoV-2 ORF1ab target was detected in 80% of the air samples using BioSampler with mineral oil. No significant differences in effectiveness were obtained with MD8 sampler equipped with gelatine membrane filters, but the SARS-CoV-2 copies/m3 air obtained with the latter were lower (28.4 ± 6.1 vs 9 ± 1.7). SuperScript II RT allows the detection of a single SARS-CoV-2 genome RNA molecule by ddPCR with high efficiency. This was the only RT that allowed the detection of SARS-CoV-2 N1 target in air samples. CONCLUSION: The collection efficiency and detection sensivity of a protocol to quantify SARS-CoV-2 RNA levels in indoor air has been improved in the present study. Such optimization is important to improve our understanding of the microbiological safety of indoor air.

Preclinical study of formulated recombinant nucleocapsid protein, the receptor binding domain of the spike protein, and truncated spike (S1) protein as vaccine candidates against COVID-19 in animal models.

BACKGROUND: In this pre-clinical study, we designed a candidate vaccine based on severe acute respiratory syndrome-related -coronavirus 2 (SARS-CoV-2) antigens and evaluated its safety and immunogenicity. METHODS: SARS-CoV-2 recombinant protein antigens, including truncated spike protein (SS1, lacking the N-terminal domain of S1), receptor-binding domain (RBD), and nucleoprotein (N) were used. Immunization program was performed via injection of RBD, SS1 +RBD, and SS1 +N along with different adjuvants, Alum, AS03, and Montanide at doses of 0, 40, 80, and 120 µg at three-time points in mice, rabbits, and primates. The humoral and cellular immunity were analyzed by ELISA, VNT, splenocyte cytokine assay, and flow cytometry. RESULTS: The candidate vaccine produced strong IgG antibody titers at doses of 80 and 120 µg on days 35 and 42. Even though AS03 and Montanide produced high-titer antibodies compared to Alum adjuvant, these sera did not neutralize the virus. Strong virus neutralization was recorded during immunization with SS1 +RBD and RBD with Alum. AS03 and Montanide showed a strong humoral and cellular immunity; however, Alum showed mild to moderate cellular responses. Ultimately, no cytotoxicity and pathologic change were observed. CONCLUSION: These findings strongly suggest that RBD with Alum adjuvant is highly immunogenic as a potential vaccine.

Immunological investigation of a multiepitope peptide vaccine candidate based on main proteins of SARS-CoV-2 pathogen.

Multiepitope vaccines could induce multiantigenic immunity against large complex pathogens with different strain variants. Herein, the in silico, in vitro and in vivo studies were used to design and develop a novel candidate antigenic multiepitope vaccine against SARS-CoV-2 pathogen. The designed multiepitope construct targets the spike glycoprotein (S), membrane protein (M), and nucleocapsid phosphoprotein (N) of SARS-CoV-2 (i.e., the S-N-M construct). This construct contains the cytotoxic T lymphocyte (CTL)-, helper T lymphocyte (HTL)-, and linear B lymphocyte (LBL)-inducing epitopes. The multiepitope s-n-m fusion gene was subcloned in prokaryotic (pET24a) and eukaryotic (pcDNA3.1) expression vectors. Its expression was evaluated in mammalian cell line using LL37 cell penetrating peptide. Moreover, the recombinant multiepitope S-N-M peptide was produced in E. coli strain. Finally, mice were immunized using homologous and heterologous regimens for evaluation of immune responses. Our data indicated that the multiepitope S-N-M peptide construct combined with Montanide 720 in homologous regimen significantly stimulated total IgG, IgG2a, IFN-γ, TNF-α, IL-15, IL-21 and IL-6, and Granzyme B secretion as compared to other groups. Moreover, the pcDNA-s-n-m/ LL37 nanoparticles significantly induced higher immune responses than the naked DNA in both homologous and heterologous regimens. In general, our designed multiepitope vaccine construct can be considered as a vaccine candidate in SARS-CoV-2 infection model.

Wax-oil lubricants to reduce the shear between skin and PPE.

Prolonged use of tight-fitting PPE, e.g., by COVID-19 healthcare workers leads to skin injuries. An important contributor is the shear exerted on the skin due to static friction at the skin-PPE interface. This study aims to develop an optimised wax-oil lubricant that reduces the friction, or shear, in the skin-PPE contact for up to four hours. Lubricants with different wax-oil combinations were prepared using beeswax, paraffin wax, olive oil, and mineral oil. In-vivo friction measurements involving seven participants were conducted by sliding a polydimethylsiloxane ball against the volar forearms to simulate the skin-PPE interface. The maximum static coefficient of friction was measured immediately and four hours after lubricant application. It was found that the coefficient of friction of wax-oil lubricants is mainly governed by the ratio of wax to oil and the thermal stability and morphology of the wax. To maintain long-term lubricity, it is crucial to consider the absorption of oil into the PPE material. The best performing lubricant is a mixture of 20 wt% beeswax, 40 wt% olive oil, and 40 wt% mineral oil, which compared to unlubricated skin, provides 87% (P = 0.0006) and 59% (P = 0.0015) reduction in instantaneous and 4-h coefficient of friction, respectively.