The mucosal surfaces of all vertebrates have been exposed to similar evolutionary pressures for millions of years. In terrestrial vertebrates such as birds and mammals, the nasopharynx-associated lymphoid tissue (NALT) represents a first line of immune defence. Here we propose that NALT is an ancient arm of the mucosal immune system not restricted to terrestrial vertebrates. We find that NALT is present in rainbow trout and that it resembles other teleost mucosa-associated lymphoid tissues. Trout NALT consists of diffuse lymphoid cells and lacks tonsils and adenoids. The predominant B-cell subset found in trout NALT are IgT + B cells, similar to skin and gut. The trout olfactory organ is colonized by abundant symbiotic bacteria, which are coated by trout secretory immunoglobulin. Trout NALT is capable of mounting strong anti-viral immune responses following nasal delivery of a live attenuated viral vaccine. Our results open up a new tool for the control of aquatic infectious diseases via nasal vaccination.

Transportation of live fish is a common practice among aquaculture facilities. Many studies have previously reported how transport elicits physiological stress responses and increases disease susceptibility in farmed fish. The aim of this work is to investigate the changes that the skin of rainbow trout (Oncorhynchus mykiss) experiences due to stress. Since NaCl is commonly added to transport water as a stress mitigator, the effects of salt addition on the skin mucosa and skin-associated bacteria were also examined. Three experimental groups (Control, post-transport no salt (PTNS) and post-transport with salt (PTS)) were analyzed in a 5-hour transport acute stress model. Results indicate that the skin mucosa and the skin-associated bacteria are affected by transport stress. Total numbers of culturable skin-associated bacteria increased by ~10-fold and ~50-fold in the PTS and PTNS groups, respectively. Compared to controls, MUC2 expression was increased by 5-fold and 2-fold in the PTNS and PTS groups, respectively. Claudin-7, 8d and 12 expression levels were higher in both PTNS and PTS groups whereas antimicrobial peptide gene expression was lower than controls. Expression of the anti-inflammatory cytokine TGF-β but not IL-1β, IL-6 and TNF-α was up-regulated 2-3 fold in both the PTS and PTNS groups. The addition of salt diminished some of the physiological responses measured including the numbers of skin-associated bacteria. The responses recorded here appeared to be efficient at controlling bacterial translocation since stress did not lead to significant presence of bacteria in the liver or spleen of rainbow trout. When examining the ability of skin mucus to inhibit or promote growth of the bacterial pathogen Vibrio anguillarum, the skin mucus of PTS trout was more efficient at inhibiting V. anguillarum growth (20% inhibition) compared to control or PTNS mucus (11-12% inhibition). Our data clearly indicate the skin and skin microbiota of rainbow trout undergo important physiological responses during stress. The reduction in the magnitude of the skin responses recorded when salt was added to the transport water explains a new mechanism by which salt is an effective stress mitigator in some fish species. Aquaculture specialists will benefit from the present study by taking into consideration the importance of skin health during live transport.

J chain is a small polypeptide responsible for immunoglobulin (Ig) polymerization and transport of Igs across mucosal surfaces in higher vertebrates. We identified a J chain in dipnoid fish, the African lungfish (Protopterus dolloi) by high throughput sequencing of the transcriptome. P. dolloi J chain is 161 aa long and contains six of the eight Cys residues present in mammalian J chain. Phylogenetic studies place the lungfish J chain closer to tetrapod J chain than to the coelacanth or nurse shark sequences. J chain expression occurs in all P. dolloi immune tissues examined and it increases in the gut and kidney in response to an experimental bacterial infection. Double fluorescent in-situ hybridization shows that 88.5% of IgM+ cells in the gut co-express J chain, a significantly higher percentage than in the pre-pyloric spleen. Importantly, J chain expression is not restricted to the B-cell compartment since gut epithelial cells also express J chain. These results improve our current view of J chain from a phylogenetic perspective.

Background Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. Results 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). Conclusions SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

SUMMARY One of the most remarkable innovations of the vertebrate adaptive immune system is the progressive organization of the lymphoid tissues that leads to increased efficiency of immune surveillance and cell interactions. The mucosal immune system of endotherms has evolved organized secondary mucosal lymphoid tissues (O-MALT) such as Peyer’s patches, tonsils, and adenoids. Primitive semi-organized lymphoid nodules or aggregates (LAs) were found in the mucosa of anuran amphibians [1], suggesting that O-MALT evolved from amphibian LAs_250 million years ago [1–4]. This study shows for the first time the presence of O-MALT in the mucosa of the African lungfish, an extant representative of the closest ancestral lineage to all tetrapods. Lungfish LAs are lymphocyte-rich structures associated with a modified covering epithelium and express all IGH genes except for IGHW2L. In response to infection, nasal LAs doubled their size and increased the expression of CD3 and IGH transcripts. Additionally, de novo organogenesis of inducible LAs resembling mammalian tertiary lymphoid structures was observed. Using deep-sequencing transcriptomes, we identified several members of the tumor necrosis factor (TNF) superfamily, and subsequent phylogenetic analyses revealed its extraordinary diversification within sarcopterygian fish. Attempts to find AICDA in lungfish transcriptomes or by RT-PCR failed, indicating the possible absence of somatic hypermutation in lungfish LAs. These findings collectively suggest that the origin of O-MALT predates the emergence of tetrapods and that TNF family members play a conserved role in the organization of vertebrate mucosal lymphoid organs.

BackgroundDespite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation.Design and methodsWe performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants.ResultsWe demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo.ConclusionThe combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0319-0) contains supplementary material, which is available to authorized users.

Context: Emerging evidence shows correlation between the presence of neutralization antibodies (nAbs) and protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently available commercial serology assays lack the ability to specifically identify nAbs. An ELISAbased nAb assay (GenScript cPass neutralization antibody assay) has recently received emergency use authorization from the Food and Drug Administration (FDA). Objective: To evaluate the performance characteristics of this assay and compare and correlate it with the commercial assays that detect SARS-CoV-2 specific IgG. Design: Specimens from SARS-COV-2 infected patients (n=124), healthy donors obtained pre-pandemic (n=100), and from patients with non-COVID (coronavirus disease 2019) respiratory infections (n=92) were analyzed using this assay. Samples with residual volume were also tested on three commercial serology platforms (Abbott, EUROIMMUN, Siemens). Twenty-eight randomly selected specimens from patients with COVID-19 and 10 healthy controls were subjected to a Plaque Reduction Neutralization Test (PRNT). Results: The cPass assay exhibited 96.1% (95% CI, 94.9%–97.3%) sensitivity (at &gt;14 days post- positive PCR), 100% (95% CI, 98.0%–100.0%) specificity and zero cross-reactivity for the presence of non- COVID respiratory infections. When compared to the plaque reduction assay, 97.4% (95% CI, 96.2%–98.5%) qualitative agreement and a positive correlation (R2 =0.76) was observed. Comparison of IgG signals from each of the commercial assays with the nAb results from PRNT/cPass assays displayed &gt;94.7% qualitative agreement and correlations with R2=0.43/0.68 (Abbott), R2=0.57/0.85 (EUROIMMUN) and R2=0.39/0.63 (Siemens), respectively. Conclusions: The combined data support the use of cPass assay for accurate detection of the nAb response. Positive IgG results from commercial assays associated reasonably with nAbs presence and can serve as a substitute.

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. Tracking clonal behavior by proviral “zip codes” indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP- cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in the sense orientation of genes were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP- population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.