Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: Assessment of Lateral flow assays for detection of SARS-CoV-2 N-antigen and anti-SARS-CoV-2 spike RBD domain targeted antibodies ( A ) Expression of myc-tagged N-antigen in NOK cells by immunoblot. Vector lane indictates cells transfected with the empty plasmid, pCMV-myc. N-antigen detected with anti-myc antibody is indicated by the arrow. ( B ) LFA detected N-antigen in the lysates of NOKs transfected with myc-tagged N expression plasmid. Both, control and N-antigen bands, are indicated. Only the control band is detected in lysates from cells transfected with empty vector. ( C ) Expression and purification of His-tagged SARS-CoV-2 N-antigen in E.coli . A band consistent with the size of N-antigen (55 kD) can be seen in crude lysates from cells transformed with His-tagged, N-antigen expression plasmid. This band is eluted from Ni-beads during purification. His-tagged protein expressed in bacteria transformed with empty vector (pET30) served as a negative control. ( D ) Dilution series of His-tagged N-antigen in preCOVID-19 saliva and detection using Biomedomics N-antigen LFA. ( E ) Detection of N-antigen in saliva of preCOVID-19 and symptomatic SARS-CoV-2 NP+ participants at baseline and post baseline 14D and 28D by LFA. The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges. Control, anti-S-RBD IgM and S-RBD IgG are indicated. The 0 lane indicates unspiked, preCOVID-19 saliva only. ( G ) Detection of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 or a symptomatic SARS-CoV-2 NP+ subject at baseline and post baseline 14D saliva using Biomedomics IgG/IgM lateral flow assay cartridges.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Purification, Transformation Assay, Bacteria, Negative Control, Lateral Flow Assay

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: Study schema and asymptomatic/symptomatic participant demographics. ( A ) Enrollment inclusion criteria for participants who were COVID-19 negative (n = 15), had asymptomatic COVID-19 (n = 15), or had symptomatic COVID-19 infection (n = 17) are listed within the figure. Subsequent to enrollment, participants answered surveys and provided biospecimens (saliva, TW, serum) that were assessed for SARS-CoV-2 detection and antibody responses. ( B ) Symptomatic SARS-CoV-2 NP+ and asymptomatic participant demographics are indicated.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Infection

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: Oral virologic characterization of study participants at baseline. All participants were assayed by qPCR for detection of viral RNA in the nasopharynx. Saliva/TW was assayed by qPCR for viral RNA and for LFA-based detection of N-antigen, anti–spike IgG and anti-spike IgM. ( A ) Oligonucleotide sequences for primers used for RT-qPCR and production of cDNA for construction of expression plasmids. E_Sarbeco_F and E_Sarbeco_R have been described. The sequences of 2019-nCoV_N1-F and 2019-nCoV_N1-R, were obtained from the CDC. The SARS-CoV-2 reference sequence GenBank:MN908947 was used to determine oligonucleotide sequences and map coordinates. SfiI (ggccatggaggcc) and SalI (gtcgac) restriction enzyme sites used for cloning into the expression vector, pCMV-myc, are underlined in expression primers. The start codon and stop codons in expression primers are in bold. ( B ) Standard curves were generated by RT-qPCR using know copy numbers of SARS-CoV-2 RNA and primers targeting the nucleocapsid (N) coding region to quantitate viral RNA isolated from patient saliva and TW. ( C ) Immunoblot was used to detect SARS-CoV-2 structural proteins in cell culture media obtained from NOKs transfected with individual expression vectors encoding myc-tagged, SARS-CoV-2 structural proteins (S, E, M and N) or cotranfected with all myc-tagged expression vectors. Medium from cells transfected with empty vector (pCMV-myc) was used as a negative control. Proteins were detected using myc-specific antibody (mouse anti-myc, sc-40, Santa Cruz Biotechnology). ( D, E ) . Protein from subset of saliva and TW samples (respectively) was used for the detection of viral proteins. Immunoblot detection of SARS-CoV-2 N-antigen was assessed in a subset of symptomatic individuals (n = 10) at baseline (top panel) using N-specific antibody (PA5-114,448, LifeTechnologies, Inc). Detection of salivary alpha-amylase (anti-amylase, A8273, Sigma-Aldrich) served as a loading control (bottom panel). Saliva or TW from archived preCOVID-19 patients A and B were included as negative controls. Cell culture medium from NOK cells transfected with myc-tagged SARS-CoV-2 N-antigen expression construct was used as a positive control. Cell culture medium from NOKs transfected with empty vector was included as a negative control. N-antigen was detected at 55 KD and alpha amylase, the loading control, was detected at 58.4 KD. ( F, G) Table and graph of symptomatic participants (n = 17) who were all nasopharynx RT-qPCR positive. RT-qPCR and LFA was also performed on saliva and TW of these participants, only RNA was available for participant 0517. Spike ELISA was performed on serum (SER). Viral RNA copy number (molecules of virus RNA) in 25 µl of saliva or TW was determined using N-specific primers. Green highlight indicates a positive result. Red highlight indicates a negative result. ND indicates not determined. ( H ) Asymptomatic participants (n = 15) were all anti spike IgG positive in their serum. ( I ) COVID-19 negative participants (n = 15) were anti-spike negative and nasopharynx negative.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Quantitative RT-PCR, Expressing, Sequencing, Clone Assay, Plasmid Preparation, Generated, Isolation, Western Blot, Cell Culture, Transfection, Negative Control, Control, Construct, Positive Control, Enzyme-linked Immunosorbent Assay, Virus

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: The SARS-CoV-2 nucleocapsid mimics host RNA binding proteins that are expressed within the salivary proteome and may be responsible for cross reactivity in LFA assays. VAST+ was used to generate RNA binding protein structures demonstrating 3D similarity to the SARS-CoV-2 N-antigen. Simultaneous alignment generated molecular protein pairs between N-antigen (first column) and host RNA binding proteins (second column). Non-aligned N-antigen amino acids are rendered in green, conserved aligned amino acids are rendered in red and aligned, non-conserved amino acids are rendered in blue. Non-aligned host protein amino acids proteins are rendered in olive green, conserved aligned amino acids are rendered in pink and aligned, non-conserved amino acids are rendered in light blue. The spatial arrangement of overlapping amino acids can be seen in the merged sequence alignment (third column). The number of structurally aligned amino acids (conserved and non-conserved) is shown. The root mean square deviation (RMSD) is used as a measurement between atoms in the backbone of the molecular structures and is listed in angstroms above each merged image and detection of the host protein within the salivary proteome is displayed as yes in the fourth column while detection of a related isoform within the salivary proteome is displayed as ISO.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: RNA Binding Assay, Generated, Sequencing

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: SARS-CoV-2 subgenomic RNAs (sgRNA) are detected the saliva of symptomatic COCID patients. ( A ) Schematic diagram showing the RNA genome of SARS-CoV-2 (~ 30 kb) and the subgenomic RNAs encoding major viral structural proteins (S, E, M and N) as well as orf3a. Detection of SARS-CoV-2 RNA by RT-qPCR was performed using primer pairs corresponding to orf1 (blue arrows), E (peach arrows) and N (red arrows) coding regions. SgRNA was detected using a forward primer corresponding to the 5’UTR (black arrow) and either S-specific (yellow), orf3a-specific (green) or N-specific (red arrow) reverse primers. The RT-qPCR signal generated with N1 primers (red arrows) were used to calibrate levels of sgRNA. ( B, C, D ) The percent levels of S, orf3a or N sgRNA determined using the 5’UTR primer and the specific sgRNA reverse primer are shown. Percent sgRNA levels relative to total viral RNA levels are determined as follows: 2^-(sgRNA Ct/N Ct) × 100. Direct sequencing of RT-qPCR amplified products was performed to determine the junction sequence of the 5’UTR and the coding region of each sgRNA. Sequencing information obtained from samples is indicated with an asterisk. The overlap sequence between the 5’UTR and each specific sgRNA is indicated.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Quantitative RT-PCR, Generated, Sequencing, Amplification

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: Longitudinal analysis of SARS-CoV-2 N-antigen and anti-SARS-CoV-2 specific IgG/IgM antibodies in oral fluids of infected individuals demonstrates persistence and sex differences ( A ) Detection of N-antigen in Saliva (Sal), anti-Spike IgG and anti-Spike IgM in Saliva (Sal) and Serum of symptomatic participants, at baseline (BL), 14 days (14D), and 28 days (28D) post baseline compared to preCOVID-19, archived (Pre) TW. ( B ) LFA detection of N-antigen in TW of symptomatic participants at baseline (BL) and 28 days (28D) post baseline compared to preCOVID-19, archived (Pre) TW. The Mann–Whitney U test assessed difference between preCOVID-19 and baseline and the Wilcoxon Signed-Rank Test assessed differences between paired baseline and 28-day samples. ( C ) Saliva and TW N-antigen, anti-Spike IgG and anti-Spike IgM LFA assay sensitivity was determined relative to NP-RT-qPCR+ results in symptomatic participants. ( D ) Detection of N-antigen in TW, anti-Spike IgG and anti-Spike IgM in TW and Serum of female vs male symptomatic participants at baseline.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Infection, MANN-WHITNEY, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: Salivary IgM predicted a more favorable disease course. The relationship between cough and fatigue symptom severity and SARS-CoV-2 protein and anti-spike antibody levels in saliva (Sal) and TW of symptomatic infected individuals was assessed at baseline using LFA. The Kendall Rank Correlation test detected a decreasing relationship between salivary IgM levels and the severity of fatigue and cough.

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Infection

Journal: Scientific Reports

Article Title: Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

doi: 10.1038/s41598-024-67504-w

Figure Lengend Snippet: Salivary IgM was associated with the absence of most symptoms. ( A ) The relationship between symptom presence (weakness, muscle ache, nausea, loss of taste and smell and upper respiratory symptoms [including cough, shortness of breath, sore throat, nasal obstruction (stuffy nose), nasal discharge [runny nose]) and semi-quantitative SARS-CoV-2 N-antigen in saliva (abbreviated Sal) and TW, and anti-spike antibody levels in saliva (abbreviated Sal), TW, and Serum of symptomatic infected individuals was assessed at baseline as determined by LFA. The Mann–Whitney U test detected a relationship between the absence of symptoms and higher oral IgM levels in saliva. ( B ) Longitudinal oral virologic characterization of symptomatic participants detects viral persistence with concurrent antibody responses suggesting presentation post-acute infection. Saliva (triangle), TW (square), and Serum (circle) were assayed by LFA for detection of N-antigen (brown), anti –spike IgG (green) and anti-spike IgM (blue) at baseline, 14d and 28d (n = 15).

Article Snippet: The detection of N-antigen band is indicated. ( F ) Two-fold dilution series of anti-SARS-CoV-2 Spike RBD (S-RBD) domain IgG/IgM antibodies in pre COVID-19 saliva and detection using Biomedomics IgG/IgM lateral flow assay cartridges.

Techniques: Infection, MANN-WHITNEY