CHARACTERIZATION of the IMMUNE RESPONSE in PATIENTS with INFLAMMATORY IMMUNE-MEDIATED DISEASES on IMMUNOSUPPRESSIVE TREATMENT after ONE MONTH of SARS-COV-2 VACCINATION

Background: The relevance of studying immune response after SARS-CoV-2 vaccination in patients with infammatory immune-mediated diseases (IMIDs) represents a deep concern regarding the risk estimation and management of patients with these diseases on immunomodulatory drugs. It is well known that certain treatments as anti CD20 therapies results in a diminished immunogenicity against common vaccines but it is a scarce data regarding the cellular protection obtained upon vaccination between patients with different IMID and between different treatments. Objectives: To compare a potential detriment on cellular and antibody-mediated protection upon SARS-CoV-2 vaccination in patients with IMIDs treated with immunosuppressive drugs. Methods: We recruited 73 patients with rheumatoid arthritis-RA-(n=49), spondy-larthritis-SpA-(n=19), infammatory bowel disease-IBD-(n=5), idiopathic juvenile arthritis-IJA-(n=2) and heterogenous group composed of sclerodermia, lupus, uveitis(n=6). They were treated mainly with rituximab (n=27), TNFi (n=37) or JAKi (n=3). We collected data of age,sex, csDMARDs, previous SARS-CoV-2 infection, last RTX infusion and prednisone use. After one month of vaccination, we assessed the humoral response performing the Thermo Scientific EliA SARS-CoV-2-Sp1 IgG Test (positivity cut-off >0.70 IU/ml) which was also compared with the data with of 35 healthy controls. In addition, in 40 patients who had serum antibody levels under 100UI/ml, we analysed the cellular response by the use of the QuantiFERON SARS-CoV-2 Starter Pack (Quiagen). A cut-off value of 0.15 IU/ml discriminate between positive or negative cell-mediated immune responses. We compared differences among the different IMIDs and between the different immu-nosuppressive treatments through non-parametric test (p<0.05) Results: Regarding demographic characteristics of patients, older patients (>56 years) and female sex were factors which were associated with low titles of serum antibodies. Anti-spike IgG antibodies were present in an 86% of the IMIDs patients and in 100% healthy controls with signifcant different IgG titre (median [IQR]): 51[11-184] vs 700[440-940];p<0.0001. The differences between (median [IQR]) serum antibody levels were statistically different between IMID type: 33[1-138] in RA vs 94[34-191] in SpA vs 204[187-204] in IBD vs 133[61-204] in IJA vs 13[1.5-31.8] in the rest;p=0.04. Remarkably, patients with IBD who had the highest antibodies titles were the youngest compared with the other patients. Target of the therapy played also an important role in serum antibody levels being these: 3.6 [0.7-51] in RTX patients vs 156 [45-204] in TNFi vs 40 [18-58] in JAKi patients;p<0.0001. In those patients who the last infusion of rituximab was, at least, one year before vaccination presented CD19+ B cells detected by fow cytometry and anti-spike IgG antibodies as well. Cell-mediated responses to SARS-CoV-2 were positive in 33% of IMIDs patients, indeterminated in 3% and negative in 65% of the patients. Strikingly, out of the 33% positive patients, 85% were treated with RTX. A 61% of the RTX patients had inducible cell-mediated responses vs 14% of the patients treated with TNFi;p<0.01. On the other hand, there were not differences in cell-mediated responses between positive and negative antibody patients. Conclusion: Titres of serum antibodies against spike protein of SARS-CoV-2 were lower in IMIDs patients than in controls. Patients with RTX had lower rates of positivity humoral response as well as lower serum titles than patients treated with other therapies regardless the patients 'age. Neverthless, in those patients in whom RTX infusion was delayed because of vaccination they conserved a humoral response. On the other hand, more patients treated with RTX had inducible cell-mediated responses compared with patients with TNFi.

IMPROVED IMMUNOLOGIC RESPONSE TO COVID-19 VACCINE WITH PROLONGED DOSING INTERVAL IN HEMODIALYSIS PATIENTS

BACKGROUND AND AIMS: Vaccination against coronavirus disease 2019 (COVID- 19) can reduce disease incidence and severity. Dialysis patients demonstrate a delayed immunologic response to vaccines. We determined factors affecting the immunologic response to COVID-19 vaccines in hemodialysis patients. METHOD: All patients within a Swedish hemodialysis network, vaccinated with two doses of COVID-19 vaccine 2-8 weeks before inclusion, were eligible for this cross-sectional study. Severe adult respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein antibody levels were determined by the EliA SARS-CoV-2-Sp1 IgG test (Thermo Fisher Scientific, Phadia AB) and related to clinical and demographic parameters. Eighty-nine patients were included. RESULTS: Patients were vaccinated with two doses of Comirnaty (BNT162b2, 73%) or Spikevax (mRNA-1273, 23.6%). Three patients received combinations of different vaccines. Response rate (antibody titres >7 U/mL) was 89.9%, while 39.3% developed high antibody titres (>204 U/mL), 47 (43-50) days after the second dose. A previous COVID-19 infection associated with higher antibody titres [median (25th-75th percentile) 1558.5 (814.5-3763.8) U/mL versus 87 (26-268) U/mL;P = 0.002], while the time between vaccine doses did not differ between groups (P = 0.7). Increasing SARS-CoV-2 antibody titres were independently associated with increasing time between vaccine doses, decreasing serum calcium levels and previous COVID-19 (Table 1). CONCLUSION: In conclusion, a longer interval between COVID-19 mRNA vaccine doses, lower calcium and a previous COVID-19 infection were independently associated with a stronger immunologic vaccination response in hemodialysis patients. While the response rate was good, only a minority developed high antibody titres 47 (43-50) days after the second vaccine dose. (Table Presented).

MicroRNAs based regulation of cytokine regulating immune expressed genes and their transcription factors in COVID-19.

BACKGROUND: Coronavirus disease 2019 is characterized by the elevation of a broad spectrum of inflammatory mediators associated with poor disease outcomes. We aimed at an in-silico analysis of regulatory microRNA and their transcription factors (TF) for these inflammatory genes that may help to devise potential therapeutic strategies in the future. METHODS: The cytokine regulating immune-expressed genes (CRIEG) were sorted from literature and the GEO microarray dataset. Their co-differentially expressed miRNA and transcription factors were predicted from publicly available databases. Enrichment analysis was done through mienturnet, MiEAA, Gene Ontology, and pathways predicted by KEGG and Reactome pathways. Finally, the functional and regulatory features were analyzed and visualized through Cytoscape. RESULTS: Sixteen CRIEG were observed to have a significant protein-protein interaction network. The ontological analysis revealed significantly enriched pathways for biological processes, molecular functions, and cellular components. The search performed in the miRNA database yielded ten miRNAs that are significantly involved in regulating these genes and their transcription factors. CONCLUSION: An in-silico representation of a network involving miRNAs, CRIEGs, and TF, which take part in the inflammatory response in COVID-19, has been elucidated. Thus, these regulatory factors may have potentially critical roles in the inflammatory response in COVID-19 and may be explored further to develop targeted therapeutic strategies and mechanistic validation.

PABPC4 Broadly Inhibits Coronavirus Replication by Degrading Nucleocapsid Protein through Selective Autophagy.

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.

Porcine Epidemic Diarrhea Virus Inhibits HDAC1 Expression To Facilitate Its Replication via Binding of Its Nucleocapsid Protein to Host Transcription Factor Sp1.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.