Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection).

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2-4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.

Combining recombinase polymerase amplification and DNA-templated reaction for SARS-CoV-2 sensing with dual fluorescence and lateral flow assay output.

The early phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was exacerbated by a diagnostic challenge of unprecedented magnitude. In the absence of effective therapeutics or vaccines, breaking the chain of transmission through early disease detection and patient isolation was the only means to control the growing pandemic. While polymerase chain reaction (PCR)-based methods and rapid-antigen tests rose to the occasion, the analytical challenge of rapid and sequence-specific nucleic acid-sensing at a point-of-care or home setting stimulated intense developments. Herein we report a method that combines recombinase polymerase amplification and a DNA-templated reaction to achieve a dual readout with either fluorescence (microtiter plate) or naked eye (lateral flow assay: LFA) detection. The nucleic acid templated reaction is based on an SN Ar that simultaneously transfers biotin from one Peptide Nucleic Acid (PNA) strand to another PNA strand, enabling LFA detection while uncaging a coumarin for fluorescence readout. This methodology has been applied to the detection of a DNA or RNA sequence uniquely attributed to the SARS-CoV-2.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Experimental Identification of Immuno- dominant B-cell Epitopes from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current public health crisis with devastating consequences to our societies. This COVID-19 pandemic has become the most serious threat to global public health in recent history. Given the unprecedented economic and social impact that it is causing, identification of immunodominant epitopes from SARS-CoV-2 is of great interest, not only to gain better insight into the adaptive immune response, but also for the development of vaccines, treatments and diagnostic tools. In this review, we summarize the already published or preprinted reports on the experimental identification of B-cell linear epitopes of SARS-CoV-2 proteins. Six different epitopes leading to neutralizing antibodies have been identified. Moreover, a summary of peptide candidates to be used for diagnostic tools is also included.

Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma.

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.