Signs of immunosenescence correlate with poor outcome of mRNA COVID-19 vaccination in older adults

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is effective in preventing COVID-19 hospitalization and fatal outcome. However, several studies indicated that there is reduced vaccine effectiveness among older individuals, which is correlated with their general health status1,2. How and to what extent age-related immunological defects are responsible for the suboptimal vaccine responses observed in older individuals receiving SARS-CoV-2 messenger RNA vaccine, is unclear and not fully investigated1,3–5. In this observational study, we investigated adaptive immune responses in adults of various ages (22–99 years old) receiving 2 doses of the BNT162b2 mRNA vaccine. Vaccine-induced Spike-specific antibody, and T and memory B cell responses decreased with increasing age. These responses positively correlated with the percentages of peripheral naïve CD4+ and CD8+ T cells and negatively with CD8+ T cells expressing signs of immunosenescence. Older adults displayed a preferred T cell response to the S2 region of the Spike protein, which is relatively conserved and a target for cross-reactive T cells induced by human ‘common cold’ coronaviruses. Memory T cell responses to influenza virus were not affected by age-related changes, nor the SARS-CoV-2-specific response induced by infection. Collectively, we identified signs of immunosenescence correlating with the outcome of vaccination against a new viral antigen to which older adults are immunologically naïve. This knowledge is important for the management of COVID-19 infections in older adults

Extracellular vesicles from pristane-treated CD38-deficient mice express an antiinflammatory neutrophil protein signature, which reflects the mild lupus severity elicited in these mice

In CD38-deficient (Cd38-/-) mice intraperitoneal injection of pristane induces a lupus-like disease, which is milder than that induced in WT mice, showing significant differences in the inflammatory and autoimmune processes triggered by pristane. Extracellular vesicles (EV) are present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin and/or tissue pathological situation. The aim of this study was to analyze the protein composition, protein abundance, and functional clustering of EV released by peritoneal exudate cells (PECs) in the pristane experimental lupus model, to identify predictive or diagnostic biomarkers that might discriminate the autoimmune process in lupus from inflammatory reactions and/or normal physiological processes. In this study, thanks to an extensive proteomic analysis and powerful bioinformatics software, distinct EV subtypes were identified in the peritoneal exudates of pristane-treated mice: 1) small EV enriched in the tetraspanin CD63 and CD9, which are likely of exosomal origin; 2) small EV enriched in CD47 and CD9, which are also enriched in plasma-membrane, membrane-associated proteins, with an ectosomal origin; 3) small EV enriched in keratins, ECM proteins, complement/coagulation proteins, fibrin clot formation proteins, and endopetidase inhibitor proteins. This enrichment may have an inflammation-mediated mesothelial-tomesenchymal transition origin, representing a protein corona on the surface of peritoneal exudate EV; 4) HDL-enriched lipoprotein particles. Quantitative proteomic analysis allowed us to identify an anti-inflammatory, Annexin A1- enriched pro-resolving, neutrophil protein signature, which was more prominent in EV from pristane-treated Cd38-/- mice, and quantitative differences in the protein cargo of the ECM-enriched EV from Cd38-/- vs WT mice. These differences are likely to be related with the distinct inflammatory outcome shown by Cd38-/- vs WT mice in response to pristane treatment. Our results demonstrate the power of a hypothesis-free and data-driven approach to transform the heterogeneity of the peritoneal exudate EV from pristanetreated mice in valuable information about the relative proportion of different EV in a given sample and to identify potential protein markers specific for the different small EV subtypes, in particular those proteins defining EV involved in the resolution phase of chronic inflammation.Proyecto del plan estatal, Ministerio de Ciencia e Innovacion PT13/0001/011CSIC PT17/0019/0010 PID2020-119567RB-I0

Detection of infectious pancreatic necrosis in a carrier population of rainbow trout, Oncorhynchus mykiss(Richardson), by flow cytometry

A non-lethal study of the disease status of adult rainbow trout, Oncorhynchus mykiss (Walbaum), suspected of being carriers of infectious pancreatic necrosis virus (IPNV) was carried out using purified leucocytes from pooled blood samples. Leucocytes were stained by indirect immunofluorcscence to detect IPN viral antigen and analysed by flow cytometry. Leucocytes from an IPN free source were also used as controls. Three populations of leucocytes were analysed: (1) leucocytes examined immediately following purification from blood, which gave positive results with 30¿58% of fluorescent cells: (2) purified leucocytes cultured for 7 days in medium at 15 °C. which gave a higher number of fluorescent cells, suggesting multiplication of IPNV; and (3) leucocytes co-cultured on CHSE-214 cell monolayers for 7 days at 15 °C, which amplified the number of infected leucocytes to more than 90% but delayed the result 7 days. Isolation and serological identification of the pathogen was carried out on CHSE-214 cells, which confirmed the positive results obtained by flow cytometry analysis. Further experiments are in progress to complete the applications of flow cytometry to salmonid virus studies

Proteomic analyses of two distinct exosome fractions present in peritoneal exudates from mice with pristane-induced lupus.

Background: Intraperitoneal injection of pristane to C57BL6/J WT mice provokes a strong inflammatory reaction with a massive influx of pro-inflammatory Ly6Chi monocytes and neutrophils to the peritoneum that ends up in overt autoimmunity (a murine lupus model of systemic lupus erythematosus (SLE) human disease). The inflammation is milder in CD38-deficient (Cd38-/-) and in double-deficient (Cd38-/- Art2-/-) mice, while it is similar to WT in Art2-deficient (Art2-/-) mice, which suggests an important role of CD38 on the onset of the disease. The aim of this work has been to isolate exosomes from peritoneal exudates (PE-EXO) of mice treated with pristane and to identify/quantify the proteins by mass spectrometry. Functional analyses were performed in silico with the use of FunRich and ClueGO software. Methods: Two types of PE-EXOs (Fr5-10 and Fr11-12) were isolated from pristane-treated mice by qEV size exclusion column methodology. Protein extracts were analyzed by LC-MS/MS. Protein identification was performed with ProteinScape 4.0 (Bruker) and MASCOT data searching using Swiss-Prot database. For relative quantification the emPAI-based method was used. The functional enrichment analysis was based on the latest publicly available data from multiple annotation and ontology resources that can be automatically accessed through FunRich and ClueGO. Results: Gene Ontology (GO) enrichment analyses of Fr5-10 and Fr11-12 PE-EXOs with FunRich showed that 65-75% of the identified proteins clustered in the GO term extracellular vesicular exosomes, while the comparative analyses with CluGO showed that these PE-EXOs were functionally quite distinct. Thus, Fr5-10 PE-EXOs were enriched in terms such as ¿regulation of myeloid leukocyte mediated immunity¿, ¿positive regulation of adaptive immune response¿, while Fr11-12 exosomes were enriched in terms such as ¿complement activation¿, and ¿regulation of humoral immune response¿. Interestingly the predominance of given GO terms in Fr5-10 vs Fr11-12 PE-EXOs seemed to vary with the extent of the inflammatory reaction to pristane, suggesting that these exosomes are representative of the distinct inflammatory cells that are recruited to the peritoneum. Conclusions: Proteomics with the help of appropriate bioinformatics tools such as ClueGO visualizes GO terms and pathways as networks, and groups them based on their biological role. This may contribute to a better diagnosis/prognosis of autoimmune diseases such as lupus. Keywords: exosomes; inflammation; lupus; bioinformatics; gene ontology; omics data analysis; biological networks

Semi-quantitative analysis of protein abundance in exosomes from peritoneal exudates in an experimental lupus model.

Background: Intraperitoneal injection of pristane to WT mice provokes a strong inflammatory reaction with a massive influx of pro-inflammatory Ly6Chi monocytes and neutrophils to the peritoneum (experimental lupus model). The inflammation is milder in CD38 deficient (Cd38-/-) mice. The aim of this work has been to isolate exosomes from peritoneal exudates (PE-EXO) of mice treated with pristane and to identify/quantify the proteins by mass spectrometry. Methods: PE-EXOs were isolated from pristane-treated mice by qEV size exclusion column methodology. Protein extracts were analyzed by LC-MS/MS and/or MALDI-TOF-MS/MS. Protein identification was performed with ProteinScape 4.0 (Bruker) and MASCOT data searching using Swiss-Prot database. For relative quantification the emPAI-based method was used. In addition to emPAI and molar fraction percentages, the fold change in the abundance of proteins identified was also calculated by dividing the molar percentage value for an individual protein in pristane-treated Cd38-/- mice with the cognate value in WT mice. The log2 value of the Cd38-/-/WT ratio was calculated and finally the absolute fold change calculated as 2log2 value. Results: Two weeks after pristane treatment 76 proteins were modulated by 1.3-fold or more in PE-EXO from Cd38-/- mice versus WT mice, representing 73% of the proteins that were common in PE-EXO from both types of mice: 26 proteins were significantly up-regulated, whereas 39 proteins were significantly down-regulated. Some of the down-modulated proteins in the Cd38-/- PE-EXOs, such as S100A9, may contribute to the migration of Ly6Chi monocytes and neutrophils to the inflamed peritoneum. Conclusions: emPAI is a reliable method for the semi-quantitative analysis of changes in protein abundance in exosomes from mice subjected to inflammatory/autoimmune challenges. These changes in protein abundance reflect the extent of the inflammatory reaction and may contribute to a better diagnosis/prognosis of autoimmune diseases such as lupus. Keywords: exosomes, inflammation, emPAI, relative quantification, expression proteomics

Proteomic analyses of exosome fractions present in peritoneal exudates from mice with pristane-induced lupus.

The aim of this work has been to isolate exosomes from peritoneal exudates (PE-EXO) of mice with pristane-induced lupus (a murine lupus model of systemic lupus erythematosus (SLE) human autoimmune disease) to identify/quantify the proteins by mass spectrometry and to group them into functional modules. Methods: Two pools of PE-EXOs were isolated from pristane-treated mice by qEV size exclusion column methodology (Fr5-10 and Fr11-12). Protein extracts were analyzed by LC-MS/MS. MS analysis was performed using an Amazon Speed ETD de Bruker, or a Q-Exactive mass spectrometer (Thermo Scientific). Protein identification was performed with ProteinScape 4.0 (Bruker), or Proteome Discoverer (version1.4.1.14) (Thermo), and MASCOT data searching using Swiss-Prot database. For relative quantification the emPAI-based method was used. The functional enrichment analysis was based on the latest publicly available data from multiple annotation and ontology resources that can be automatically accessed through FunRich and ClueGO. Results: Gene Ontology (GO) enrichment analyses of Fr5-10 and Fr11-12 PE-EXOs with FunRich showed that 65-75% of the identified proteins clustered in the GO term ¿extracellular vesicular exosomes¿, while the ClueGO analyses showed that the proteins clustered into functionally distinct GO terms. Thus, Fr5-10 PE-EXOs were enriched in terms such as ¿regulation of myeloid leukocyte mediated immunity¿, ¿positive regulation of adaptive immune response¿, while Fr11-12 exosomes were enriched in terms such as ¿complement activation¿, and ¿regulation of humoral immune response¿. Moreover, the predominance of given GO terms in PE-EXOs seemed to vary with the extent of the inflammatory/autoimmune reaction to pristane. Conclusions: Proteomic analyses, with the help of appropriate bioinformatics tools such as ClueGO, visualize GO terms and pathways as networks, and group them based on their biological role. This approach may contribute to a better diagnosis/prognosis of autoimmune diseases such as lupus. Keywords: exosomes; inflammation; lupus; bioinformatics; gene ontology; omics data analyses; biological networks