Biology subjects

Perreault, J.

Publications and source records attributed to Perreault, J..

5 recordsLinked to original sources

bioRxiv2020

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

BackgroundThe SARS-CoV-2 virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than a million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. Study Design and MethodsTo extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike specific antibodies in the plasma of convalescent donors. Results and ConclusionWe found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations, but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

microbiology↗

bioRxiv2020

Measurement of Kinetics of Hammerhead Ribozyme Cleavage Reactions using Toehold Mediated Strand Displacement

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of hammerhead ribozyme cleavage reactions, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5-end, while the other strand is labelled with a quencher at its 3-end. These two DNA strands are perfectly complementary, but with a 3-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the kinetics of ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

molecular biology↗

bioRxiv2020

Longitudinal analysis of the humoral response to SARS-CoV-2 spike RBD in convalescent plasma donors

Hema-Quebec, the blood supplier in the Province of Quebec, Canada, collects and tests convalescent plasma used in a clinical trial to determine the clinical efficacy of this product for the treatment of hospitalized COVID-19 patients. So far, we have collected 1159 plasma units from 282 COVID-19 convalescent donors. The presence of antibodies to the receptor binding domain (RBD) of SARS-CoV-2 spike protein in convalescent donors was established at the first donation. Seropositive donors were asked to donate additional plasma units every six days. Until now, 15 donors have donated at least four times and, in some cases, up to nine times. This allowed us to perform a longitudinal analysis of the persistence of SARS-CoV-2 RBD-specific antibodies in these repeat donors, with the first donation occurring 33-77 days after symptoms onset and donations up to 71-114 days after symptoms onset thereafter. In all donors, the level of antibodies remained relatively stable up to about 76 days after symptoms onset but then started to decrease more rapidly to reach, in some convalescent donors, a seronegative status within 100-110 days after symptoms onset. The decline in anti-RBD antibodies was not related to the number of donations but strongly correlated with the numbers of days after symptoms onset (r = 0.821). This suggests that de novo secretion of SARS-CoV-2 RBD antibodies by short-lived plasma cells stopped about 2-3 months after disease onset, an observation that has important implications for convalescent plasma collection and seroprevalence studies undertaken several months after the peak of infection.

immunology↗

bioRxiv2020

An experimentally evolved variant of RsmA confirms its central role in the control of Pseudomonas aeruginosa social motility

Bacteria can colonize a variety of different environments by modulating their gene regulation using two-component systems. The versatile opportunistic pathogen Pseudomonas aeruginosa has been studied for its capacity to adapt to a broad range of environmental conditions. The Gac/Rsm pathway is composed of the sensor kinase GacS, that detects environmental cues, and the response regulator GacA, that modulates the expression of a specific genes. This system, through the sRNA repressors RsmY and RsmZ, negatively controls the activity of the protein RsmA, which is centrally involved in the transition from chronic to acute infections by post-transcriptionally regulating several virulence functions. RsmA positively regulates swarming motility, a social surface behaviour. Through a poorly defined mechanism, RsmA is also indirectly regulated by HptB, and a {Delta}hptB mutant exhibits a severe swarming defect. Since a {Delta}hptB mutant retains all the known functions required for that type of motility, we used an experimental evolution approach to identify elements responsible for its swarming defect. After a few passages under swarming conditions, the defect of the {Delta}hptB mutant was rescued by the emergence of spontaneous single nucleotide substitutions in the gacA and rsmA genes. Since GacA indirectly represses RsmA activity, it was coherent that an inactivating mutation in gacA would compensate the {Delta}hptB swarming defect. However, the effect of the mutation in rsmA was unexpected since RsmA promotes swarming; indeed, using expression reporters, we found that the mutation that does not abolish its activity. Instead, using electrophoretic mobility shift assays and molecular simulations, we show that this variant of RsmA is actually less amenable to titration by its cognate repressor RsmY, supporting the other phenotypes observed for this mutant. These results confirm the central role of RsmA as a regulator of swarming motility in P. aeruginosa and identify residues crucial for RsmA function in social motility. Author summaryBacteria need to readily adapt to their environment. Two-component systems (TCS) allow such adaption by triggering bacterial regulation changes through the detection of environmental cues. The opportunistic pathogen Pseudomonas aeruginosa possesses more than 60 TCS in its genome. The Gac/Rsm is a TCS extensively studied for its implication in virulence regulation. This system regulates the transition between chronic and acute bacterial infection behaviours. To acquire a better understanding of this regulation, we performed a directed experimental evolution on a swarming-deficient mutant in a poorly understood regulatory component of the Gac/Rsm pathway. We observed single nucleotide substitutions that allowed restoration of a swarming phenotype similar to the wild-type behaviour. More specifically, mutations were found in the gacA and rsmA genes. Interestingly, the observed mutation in rsmA does not result in loss of function of the protein but rather alters its susceptibility to repression by its cognate interfering sRNA. Since modification in the RNA sequence of RsmA results in the rescue of swarming motility, we confirm the central role of this posttranscriptional repressor in this social lifestyle.

microbiology↗

bioRxiv2020

Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike

The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.

microbiology↗