SARS-CoV-2 immunoassays in a predominantly vaccinated population: Performances and qualitative agreements obtained with two analytical approaches and four immunoassays.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys are typically analysed by applying a fixed threshold for seropositivity ('conventional approach'). However, this approach underestimates the seroprevalence of anti-nucleocapsid (N) in vaccinated individuals-who often exhibit a difficult-to-detect anti-N response. This limitation is compounded by delays between the onset of infection and sample collection. To address this issue, we compared the performance of four immunoassays using a new analytical approach ('ratio-based approach'), which determines seropositivity based on an increase in anti-N levels. MATERIALS AND METHODS: Two groups of plasma donors and four immunoassays (Elecsys total anti-N, VITROS total anti-N, Architect anti-N Immunoglobulin G (IgG) and in-house total anti-N) were evaluated. First-group donors (N = 145) had one positive SARS-CoV-2 polymerase chain reaction (PCR) test result and had made two plasma donations, including one before and one after the PCR test (median = 27 days post-PCR). Second-group donors (N = 100) had made two plasma donations early in the Omicron wave. RESULTS: Among first-group donors (97.9% vaccinated), sensitivity estimates ranged from 60.0% to 89.0% with the conventional approach, compared with 94.5% to 98.6% with the ratio-based approach. Among second-group donors, Fleiss's κ ranged from 0.56 to 0.83 with the conventional approach, compared with 0.90 to 1.00 with the ratio-based approach. CONCLUSION: With the conventional approach, the sensitivity of four immunoassays-measured in a predominantly vaccinated population based on samples collected ~1 month after a positive test result-fell below regulatory agencies requirement of ≥95%. The ratio-based approach significantly improved the sensitivities and qualitative agreement among immunoassays, to the point where all would meet this requirement.

Assessing introduction risk using species' rank-abundance distributions.

Mixed-species assemblages are often unintentionally introduced into new ecosystems. Analysing how assemblage structure varies during transport may provide insights into how introduction risk changes before propagules are released. Characterization of introduction risk is typically based on assessments of colonization pressure (CP, the number of species transported) and total propagule pressure (total PP, the total abundance of propagules released) associated with an invasion vector. Generally, invasion potential following introduction increases with greater CP or total PP. Here, we extend these assessments using rank-abundance distributions to examine how CP : total PP relationships change temporally in ballast water of ocean-going ships. Rank-abundance distributions and CP : total PP patterns varied widely between trans-Atlantic and trans-Pacific voyages, with the latter appearing to pose a much lower risk than the former. Responses also differed by taxonomic group, with invertebrates experiencing losses mainly in total PP, while diatoms and dinoflagellates sustained losses mainly in CP. In certain cases, open-ocean ballast water exchange appeared to increase introduction risk by uptake of new species or supplementation of existing ones. Our study demonstrates that rank-abundance distributions provide new insights into the utility of CP and PP in characterizing introduction risk.

Relationship between propagule pressure and colonization pressure in invasion ecology: a test with ships' ballast.

Increasing empirical evidence indicates the number of released individuals (i.e. propagule pressure) and number of released species (i.e. colonization pressure) are key determinants of the number of species that successfully invade new habitats. In view of these relationships, and the possibility that ships transport whole communities of organisms, we collected 333 ballast water and sediment samples to investigate the relationship between propagule and colonization pressure for a variety of diverse taxonomic groups (diatoms, dinoflagellates and invertebrates). We also reviewed the scientific literature to compare the number of species transported by ships to those reported in nature. Here, we show that even though ships transport nearly entire local communities, a strong relationship between propagule and colonization pressure exists only for dinoflagellates. Our study provides evidence that colonization pressure of invertebrates and diatoms may fluctuate widely irrespective of propagule pressure. We suggest that the lack of correspondence is explained by reduced uptake of invertebrates into the transport vector and the sensitivity of invertebrates and diatoms to selective pressures during transportation. Selection during transportation is initially evident through decreases in propagule pressure, followed by decreased colonization pressure in the most sensitive taxa.

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut.

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.

Analysis of the role of IL-21 in development of murine B cell progenitors in the bone marrow.

IL-21 plays a key role in the late stage of B cell development, where it has been shown to induce growth and differentiation of mature B cells into Ig-secreting plasma cells. Because IL-21R has also been reported on bone marrow (BM) B cell progenitors, we investigated whether IL-21R influenced earlier stages of B cell development. IL-21R is functional as early as the pro-B cell stage, and the strength of receptor-mediated signaling increases as cells mature. The addition of IL-21 to B cell progenitors in cell culture resulted in the accelerated appearance of mature B cell markers and was associated with the induction of Aid, Blimp1, and germline transcripts. We also found that stimulation of both IL-21R and CD40 was sufficient to induce the maturation of early B cell progenitors into IgM- and IgG-secreting cells. Consistent with a role for IL-21 in promoting B cell differentiation, the number of B220(+)CD43(+)IgM(-) pro-B cells was increased, and the number of mature IgM(hi)IgD(hi) cells was decreased in BM of IL-21R-deficient mice. We also report in this paper that IL-21 is expressed by BM CD4(+) T cells. These results provide evidence that IL-21R is functional in B cell progenitors and indicate that IL-21 regulates B cell development.

Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of European and North American genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcripts.

BACKGROUND: Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae, genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts. RESULTS: Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequences of segments 1, 2, 3, 5, and 7, and the 3' NCR sequences of segments 3 and 4 cRNA were 100% identical in the two genotypes, and the 3' NCR sequences of segment 5 cRNA was the most divergent, with a sequence identity of 77.2%. CONCLUSIONS: We report for the first time, the presence of intra-segment ISAV quasispecies, based on sequence variation in the NCR sequences of transcripts. In addition, this is the first report of a comprehensive unambiguous analysis of the 3' and 5' NCR sequences of all 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CAT/ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.

IL-21: an executor of B cell fate.

IL-21 is a type I cytokine that shares the common receptor gamma-chain with IL-2, IL-4, IL-7, IL-9, and IL-15. B cells are one of the lymphoid cell types whose development and function are regulated by IL-21. Depending on the interplay with costimulatory signals and on the developmental stage of a B cell, IL-21 can induce proliferation, differentiation into Ig-producing plasma cells, or apoptosis in both mice and humans. Alone and in combination with Th cell-derived cytokines IL-21 can regulate class switch recombination to IgG, IgA, or IgE isotypes, indicating its important role in shaping the effector function of B cells. This review highlights the role of IL-21 in B cell development, function, and disease and provides some perspectives on the future studies in this area.

Relative expression of matrix metalloproteinase-2 and -9 in synovial fluid from healthy calves and calves with experimentally induced septic arthritis.

OBJECTIVE: To identify changes over time in relative expression of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in synovial fluid from healthy calves and calves with experimentally induced septic arthritis. ANIMALS: 12 Holstein calves. PROCEDURES: In 7 calves, Escherichia coli was injected in the right tarsal joint on day 1. Joint lavage was performed on day 2, and calves were treated with ceftiofur from days 2 through 21. Synovial fluid samples were collected on days 1 (before inoculation), 2 (before joint lavage), 3, 4, 8, 12, 16, 20, and 24. In the remaining 5 calves, joint lavage was performed on day 2 and synovial fluid samples were collected from the left tarsal joint. Relative expression of MMP-2 and MMP-9 was determined by means of gel zymography. RESULTS: On day 1, MMP-2 was detected in all synovial fluid samples but MMP-9 was not detected. In calves with septic arthritis, values for relative expression of MMP-9 monomer and dimer were significantly increased on days 2 through 20 and days 2 through 24, respectively, and relative expression of MMP-2 was significantly increased on days 3 through 20. There were significant linear associations between relative expression of the monomer and dimer forms of MMP-9 and between neutrophil count and relative expression of the MMP-9 monomer and dimer forms. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that relative expression of MMP-9 and MMP-2 increased in synovial fluid from calves with experimentally induced septic arthritis, with relative expression remaining high for several days after infection.

Interleukin-21 regulates expression of key Epstein-Barr virus oncoproteins, EBNA2 and LMP1, in infected human B cells.

Epstein-Barr virus (EBV) persists for the life of the host by accessing the long-lived memory B cell pool. It has been proposed that EBV uses different combinations of viral proteins, known as latency types, to drive infected B cells to make the transition from resting B cells to memory cells. This process is normally antigen-driven. A major unresolved question is what factors coordinate expression of EBV latency proteins. We have recently described novel type III latency EBV+ B cell lines (OCI-BCLs) that were induced to differentiate into late plasmablasts/early plasma cells in culture with interleukin-21 (IL-21), mimicking normal B cell development. The objective of this study was to determine whether IL-21-mediated signals also regulate the expression of key EBV latent proteins during this window of development. Here we show that IL-21-reduced gene and protein expression of growth-transforming EBV nuclear antigen 2 (EBNA2) in OCI-BCLs. By contrast, the expression of CD40-like, latent membrane protein 1 (LMP1) strongly increased in these cells suggesting an EBNA2-independent mode of regulation. Same results were also observed in Burkitt's lymphoma line Jijoye and B95-8 transformed lymphoblastoid cell lines. The effect of IL-21 on EBNA2 and LMP1 expression was attenuated by a pharmacological JAK inhibitor indicating involvement of JAK/STAT signalling in this process. Our study also shows that IL-21 induced transcription of ebna1 from the viral Q promoter (Qp).

The road to licensure of a DNA vaccine.

The licensure of three DNA vaccines for animal health applications has provided renewed interest in the broader potential of this technology. At the very least, this will spur efforts to understand the reasons behind these successes and whether this information can be used to enable DNA vaccines for humans. This review maps the pathway to the licensure of the DNA vaccine against infectious hematopoietic necrosis virus in fish, and discusses the implications of this on the development of human DNA vaccines.

A novel approach to measure the contribution of matrix metalloproteinase in the overall net proteolytic activity present in synovial fluids of patients with arthritis.

Despite decades of research, only a very limited number of matrix metalloproteinase (MMP) inhibitors have been successful in clinical trials of arthritis. One of the central problems associated with this failure may be our inability to monitor the local activity of proteases in the joints since the integrity of the extracellular matrix results from an equilibrium between noncovalent, 1:1 stoichiometric binding of protease inhibitors to the catalytic site of the activated forms of the enzymes. In the present work, we have measured by flow cytometry the net proteolytic activity in synovial fluids (SF) collected from 95 patients with osteoarthritis and various forms of inflammatory arthritis, including rheumatoid arthritis, spondyloarthropathies, and chronic juvenile arthritis. We found that SF of patients with inflammatory arthritis had significantly higher levels of proteolytic activity than those of osteoarthritis patients. Moreover, the overall activity in inflammatory arthritis patients correlated positively with the number of infiltrated leukocytes and the serum level of C-reactive protein. No such correlations were found in osteoarthritis patients. Members of the MMP family contributed significantly to the proteolytic activity found in SF. Small-molecular-weight MMP inhibitors were indeed effective for inhibiting proteolytic activity in SF, but their effectiveness varied greatly among patients. Interestingly, the contribution of MMPs decreased in patients with very high proteolytic activity, and this was due both to a molar excess of tissue inhibitor of MMP-1 and to an increased contribution of other proteolytic enzymes. These results emphasize the diversity of the MMPs involved in arthritis and, from a clinical perspective, suggest an interesting alternative for testing the potential of new protease inhibitors for the treatment of arthritis.