Weak D type 42 accounts for an unusually high proportion of weak D phenotypes in Québec (Canada), which contrasts with other predominantly White populations. However, its prevalence in the general population is unknown. We estimated the prevalence of weak D type 42 and other common weak D phenotypes in Québec.

We previously estimated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies following the first pandemic wave at 2.23% in Québec, Canada. Following the much bigger second wave in fall 2020 and early 2021, we estimated the seroprevalence of anti-SARS-CoV-2 in Québec during the first months of 2021. Blood samples from regular, asymptomatic (for ≥ 14 days) donors were collected between January 25, 2021 and March 11, 2021. Anti-SARS-CoV-2 seropositivity was assessed using an enzyme-linked immunosorbent assay that captures antibodies directed against the receptor binding domain of the SARS-CoV-2 spike (and hence cannot discriminate between infection- and vaccine-induced seropositivity). Seroprevalence estimates were adjusted for regional distribution, age, and sex. Samples from 7924 eligible donors were analyzed, including 620 (7.8%) vaccinated donors and 7046 (88.9%) unvaccinated donors (vaccination status unknown for 258 (3.3%) donors). Overall, median age was 51 years; 46.4% of donors were female. The adjusted seroprevalence was 10.5% (95% CI = 9.7–11.3) in the unvaccinated population and 14.7% (95% CI = 13.8–15.6) in the overall population. Seroprevalence gradually decreased with age and was higher among donors who self-identified as having a racial/ethnic background other than white, both in the overall and in the unvaccinated populations. The seroprevalence of SARS-CoV-2 antibodies significantly increased in Québec since spring 2020, with younger persons and ethnic minorities being disproportionately affected. When compared with the cumulative incidence rate reported by public health authorities (i.e., 3.3% as of March 11, 2021), these results suggest that a substantial proportion of infections remain undetected despite improvements in access to COVID-19 testing.

Background The 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 Methods To 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 Conclusion We 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.

A substantial proportion of individuals infected with SARS-CoV-2 do not experience noticeable symptoms typical of COVID-19. Our objectives were to evaluate the impact of the first wave of the pandemic in Québec by measuring SARS-CoV-2 antibody seroprevalence in a convenience sample of healthy blood donors and to study the association between seropositivity and the occurrence of COVID-19 symptoms. The study design was a cross-sectional serological survey with a nested case-control study. Residual blood samples from donations collected between May 25 and July 9, 2020 (well before vaccination rollout) in the province of Québec were tested for anti-Spike RBD antibodies by ELISA. Seropositive donors and a control group of seronegative donors were questioned about prior COVID-19 symptoms. All qualified blood donors were eligible for participation. A total of 7691 blood donors were included in the study. After adjustments, the seroprevalence rate was 2.2% (95% CI 1.9–2.6). Seropositive donors reported one or more symptoms in a proportion of 52.2% (95% CI 44.2–60.1); this proportion was 19.1% (95% CI 13.4–26.1) among seronegative donors, suggesting that approximately 50–66% of all infections were asymptomatic. Univariate analysis of associations between symptoms and seropositivity revealed that except for rhinorrhea, all symptoms were significantly associated with seropositivity. Assuming that blood donors are fairly representative of the general adult population, this study shows that less than 3% of 18–69-year-olds have been infected during the first wave of the pandemic in the province of Québec. Our data also confirm that many infections escaped detection, including a substantial proportion that were asymptomatic.

ABSTRACTHéma-Québec, 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.

Perreault and colleagues examined antibody titers in sequential samples from serum donors recovering from COVID-19 and demonstrated that antibody titers fall over 3-4 months. These results have important implications for convalescent serum collection and seroprevalence studies.

In vitro-produced red blood cells (RBCs) could be used as cellular models for studying erythroid diseases or for providing reagent cells expressing specific blood group antigens. However, the non-fully mature state of cells achieved with current protocols is a significant barrier to their potential clinical applications. We aimed at modifying existing differentiation protocols in order to improve final erythroid maturation from human hematopoietic stem and progenitor cells (HSPCs). If successful, the method would facilitate the generation of mature erythrocytes, their preservation and allow phenotype evaluation. As a proof-of-concept using gene editing, we chose to model the “sickling” phenotype of sickle cell disease (SCD). After significant improvements of known methods, we devised an optimized culture medium for the efficient erythroid differentiation and maturation of adult HSPCs. The differentiation protocol yielded up to 95% of enucleation. More importantly, reticulocyte maturation yielded up to 80% of cells molecularly defined as mature erythrocytes devoid of any CD71 marker expression, total RNA, and mitochondria. Next, we have exploited a virus-free CRISPR-Cas9-based editing strategy to effectively introduce genetic variants in HSPCs combined with the erythroid differentiation protocol. We achieved 40% of precise gene modification within the beta-globin gene, with nearly 20% of the cells harboring the mutation on both alleles. Interestingly, when incubating cells in hypoxic conditions, we were able to visualize the “sickling” phenotype of the mature genetically-modified erythrocytes. In summary, we have successfully combined high-efficiency CRISPR-Cas9 editing of HSPCs with in vitro erythroid maturation to recapitulate SCD in vitro. This work can be applied to other genetic variants and cell types for the study of clinically relevant mutations. In vitro-produced red blood cells (RBCs) could be used as cellular models for studying erythroid diseases or for providing reagent cells expressing specific blood group antigens. However, the non-fully mature state of cells achieved with current protocols is a significant barrier to their potential clinical applications. We aimed at modifying existing differentiation protocols in order to improve final erythroid maturation from human hematopoietic stem and progenitor cells (HSPCs). If successful, the method would facilitate the generation of mature erythrocytes, their preservation and allow phenotype evaluation. As a proof-of-concept using gene editing, we chose to model the “sickling” phenotype of sickle cell disease (SCD). After significant improvements of known methods, we devised an optimized culture medium for the efficient erythroid differentiation and maturation of adult HSPCs. The differentiation protocol yielded up to 95% of enucleation. More importantly, reticulocyte maturation yielded up to 80% of cells molecularly defined as mature erythrocytes devoid of any CD71 marker expression, total RNA, and mitochondria. Next, we have exploited a virus-free CRISPR-Cas9-based editing strategy to effectively introduce genetic variants in HSPCs combined with the erythroid differentiation protocol. We achieved 40% of precise gene modification within the beta-globin gene, with nearly 20% of the cells harboring the mutation on both alleles. Interestingly, when incubating cells in hypoxic conditions, we were able to visualize the “sickling” phenotype of the mature genetically-modified erythrocytes. In summary, we have successfully combined high-efficiency CRISPR-Cas9 editing of HSPCs with in vitro erythroid maturation to recapitulate SCD in vitro. This work can be applied to other genetic variants and cell types for the study of clinically relevant mutations.

IVIg is used as an immunomodulatory agent in inflammatory disorders such as sepsis. IVIg also affects monocyte differentiation and functions, two processes in which microRNAs play a crucial role. Monocytes detect microorganisms through pathogen recognition receptors (PRRs) such as TLR4. MiR-146a has been shown to supress NF-κB and IRF3 activity, two key components of TLR4 signaling. To evaluate whether miR-146a is involved in the anti-inflammatory effects of IVIg, monocytes were treated with LPS or IVIg alone or, alternately, first activated with LPS followed by washing and addition of IVIg. MiR-146a, IRF3, TNF-α, IL-1β, IL-6, IL-10, IFN-β, TGF-β1 and IL-1Ra expression was analyzed by qPCR, while IRAK1, TRAF6 and IκBα expression was measured by Western blotting. We found that addition of IVIg to LPS-activated monocytes significantly upregulated the expression of miR-146a, which was associated with a significant reduction in the expression of its targets IRF3 and its regulated gene IFN-β. Furthermore, expression of IRAK1, TRAF6, and consequently NF-κB activation, was also reduced in LPS-activated monocytes following addition of IVIg, whereas TGF-β1, IL-10 and IL-1Ra were increased. Our results thus suggest that miR-146a is a mediator of IVIg effects in inflammatory disorders, point to an important role for miR-146a in the control of inflammation during sepsis and highlight a new mechanism by which IVIg exerts its anti-inflammatory effects in sepsis.

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

Complement dysregulation appears important in the etiology of many autoimmune and inflammatory diseases. IVIG, the C3 inhibitor compstatin and the humanized anti‐C5 antibody Eculizumab modulate complement through direct inhibition of protein activation. In an effort to decipher the mechanisms by which protein‐protein interactions modulate the activation of the complement protein C3 through the classical and alternate cascade, we investigated whether small peptides could interfere with the specific interaction between C3 and properdin. First, we verified that a 34 amino acid peptide, derived from C3, inhibits its interaction with properdin but cannot displace either C3 or properdin from aggregated IgG. Interestingly, a 9‐mer peptide that span within the original 34 amino acids is able to replicate the effect of the 34‐mer toward C3‐properdin interaction. Of utmost interest, further experiments demonstrate that this 9‐mer is also able to inhibit the binding of C3 to IgG. On a molar basis, the active peptide is as powerful as IVIG and an optimized cyclic peptide that inhibits the activation of C3, compstatin. Further analyses demonstrate that the active 9‐mer peptide is able to inhibit complement activation in sensitized red blood cells (SRBC) hemolysis assays. Altogether, these results showed that inhibitory effect of IVIG on in vitro complement activation can be mimicked, at least in part, by a 9‐mer peptide. A quantitative structure‐activity relationship (QSAR) was performed using the minimal active 9‐mer peptide. Linearity and the C‐terminus amino acids were shown to be critical for its activity, and further length reduction of the peptide strongly reduces its activity.We have identified a potent complement inhibitor with potential therapeutic applications in inflammatory and autoimmune diseases.

Investigation of the molecular processes which control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte associated pathogenesis. Adenovirus-mediated gene transfer provided a powerful tool to investigate these processes. We have previously demonstrated that adenoviral vector Ad5/F35 transduces plasma cell lines at a higher efficiency than primary B cells, owing to differences in intracellular trafficking. Given that phosphatases are effectors of intracellular trafficking, here we have analyzed the effects of a panel of phosphatase inhibitors on Ad5/F35 transduction efficiency in B lymphocytes in the present study. FACS analysis was conducted to determine Ad5/F35-EYFP transduction efficiency in lymphoid cells, including human primary B cells, following serine/threonine phosphatase (PSP) inhibitor treatment. We further used confocal microscopy to analyze intracellular trafficking and fate of CY3 labeled Ad5/F35 vectors, in PSP treated lymphoid cell. Finally, we analyzed the MAPK pathway by Western blot in PSP treated cells. Adenoviral transduction efficiency was unresponsive to inhibition of PP1 whereas inhibition of PP2A by cantharidic acid, or PP1 and PP2A by okadaic acid, substantially increased transduction efficiency. Importantly, confocal microscopy analyses revealed that inhibition of PP2A shut down adenovirus recycling. Moreover, inhibition of PP2A resulted in increased phosphorylation of AKT, ERK1/2 and MEK1/2. Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells following PP2A inhibition. Our results are in agreement with reports indicating that PP2A is involved in the formation of recycling vesicles and might be of interest for gene therapy applications.

2-Methoxyestradiol (2ME), an end-metabolite of 17β-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138− B lymphocytes into CD138+ cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 µM 2ME. Moreover, most of the converted cells were also CD27+ and secreted high levels of IgG (151 µg/106 cells/24 h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

Gene transfer applications with adenovirus (Ad) type 5 are limited by its native tropism, hampering their use in several cell types. To address this limitation, several Ad vectors bearing chimeric fiber have been produced to take advantage of the different cellular receptors used by other subgroups of Ads. In this study, we have compared the transduction efficiency of Ad5 and the chimeric Ad5/F35 in primary human B lymphocytes and B-cell lines as a function of the developmental stage. We found that transduction efficiencies of the two Ads differ independently of their targeted cellular receptor but are related to the intracellular localization of the virus. In efficiently transduced cells, Ads were localized in early endosomes or cytosol, whereas in poorly transduced cells they were localized within late endosomes/lysosomes. Finally, we demonstrate that treatment of cells with phosphatase inhibitors known to redirect endocytosis towards caveolae, increased Ad5/F35 transduction efficiency.

PAX5 is an essential transcription factor for the commitment of lymphoid progenitors to the B-lymphocyte lineage. PAX5 suppression results in retrodifferentiation of B lymphocytes to an uncommitted progenitor cell stage, whereas PAX5 suppression in mature B lymphocytes leads to further development into plasma cells. Here, we have analyzed the fate of plasma cell lines following PAX5 reexpression. Human B cell lines were infected with Ad5/F35 adenoviruses encoding either EYFP or PAX5. Expression analysis of specific plasma cell transcription factors (IRF4, Blimp-1 and XBP-1) suggests that PAX5 reexpression does not induce retrodifferentiation of plasma cells into B lymphocytes. Interestingly, the viability of RPMI-8226 and U266 multiple myeloma cell lines markedly declined at 4-7 days post-transduction, whereas other plasma cell lines maintained their viability. Apoptosis analysis through Annexin V measurement also revealed a higher level of apoptosis in PAX5-expressing myeloma cell lines. Finally, Western blot analysis of pro- and anti-apoptotic proteins revealed that the anti-apoptotic protein MCL-1 was down-modulated in PAX5-transduced multiple myeloma cell lines. In conclusion, our results show that the expression of PAX5 in plasma cell lines induces apoptosis exclusively in multiple myelomas. This might represent a potential therapeutic avenue in the treatment of multiple myeloma.

c-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.

The 60-kDa c-Src is the normal human cellular protein counterpart of the highly transforming v-src gene. Recently, c-Src activity was reported to increase in CD40-activated human B cells in the presence of IL-4, suggesting its involvement in proliferation. We report here that c-Src expression is detectable concomitant with the detection of Stat5b and, therefore, Stat5b may serve as a substrate for tyrosine phosphorylation by c-Src, inducing the activation of Stat5b and initiating a transcriptional pathway important for B cell expansion. To elucidate the exact role of c-Src in the proliferation of normal B cells, we undertook c-Src over-expression experiments. Recombinant adenoviruses Ad5/F35 vectors, which we previously reported as highly efficient for B cell transduction, encoding wild-type c-Src(c-Src/WT), constitutively active c-Src(c-Src/CA), dominant negative c-Src(c-Src/DN) or EYFP were constructed. B lymphocytes purified from human peripheral blood were activated with soluble CD154 in the presence or absence of IL-2, IL-4 and IL-10, and infected with the viruses. Real-time PCR and Western blot analysis revealed that vector-transferred c-Src were strongly expressed in infected B cells as early as 48 hours post infection. Kinase assays confirmed that vector-transferred c-Src/WT and c-Src/CA display a strong kinase activity whereas negligible kinase activity was detected with Ad5/F35-c-Src/DN. No significant variation of B cell expansion could be observed between uninfected cells, Ad/F35-EYFP and Ad5/F35-c-Src/CA or Ad5/F35-c-Src/WT infected cells, suggesting that B cell proliferation induced by endogenous c-Src already attains a maximum rate of expansion, which cannot be further enhanced by supplemental exogenous c-Src. In contrast, overexpression of c-Src/DN results in a 40% inhibition of B cell expansion. These results suggest that transgenic dominant negative c-Src may compete with endogenous c-Src resulting in a partial inhibition of a transcriptional pathway involved in B cell proliferation. In conclusion, our results confirm an important role for c-Src in the expansion of normal human B cells in vitro.