Immunotherapy: OFF-THE-SHELF PARTIAL HLA MATCHING SARS-COV-2 ANTIGEN SPECIFIC T CELL THERAPY FOR SEVERE COVID-19 PATIENTS

Background & Aim: Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. Methods, Results & Conclusion(s): To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Our results show that SARSCoV- 2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. In conclusion, our findings show that SARSCoV- 2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients.Copyright © 2023 International Society for Cell & Gene Therapy

Structural Insights of the SARS-CoV-2 Nucleocapsid Protein: Implications for the Inner-workings of Rapid Antigen Tests

The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling tools. Epitope mapping based on structural data supported host-immune interactions in the C-terminal portion of the protein, while other regions revealed protein-protein interaction sites. Complementary modeling results suggested that N protein structures from known variants of concern (VOC) are nearly 100% conserved at specific antibody-binding sites. Collectively, these results suggest that rapid tests that target the nucleocapsid C-terminal domain should have similar accuracy across all VOCs. In addition, our combined structural modeling workflow may guide the design of immune therapies to counter viral processes as we plan for future variants and pandemics.

Gene Editing/Gene Therapies: BIOREACTOR RECOMBINANT PRODUCTION OF SARS-COV-2 VIRUS ANTIGENS IN CHO CELL CLONES BY USING NEW UCOE LENTIVIRAL VECTOR SYSTEM

Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy

Plant-derived active compounds as a potential nucleocapsid protein inhibitor of SARS-CoV-2: an in-silico study.

The coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2. This virus has a high mismatch repair proofreading ability due to its unique exonuclease activity, making it knotty to treat. The nucleocapsid protein can serve as a potential antiviral drug target, as this protein is responsible for multiple captious functions during the viral life cycle. Herein, we have investigated the potential to repurpose active antiviral compounds of plant origins for treating the SARS-CoV-2 infection. In the present study, we followed the molecular docking methodology to screen druggable natural plants' active compounds against the nucleocapsid protein of SARS-CoV-2. The virtual screening of all 68 compounds revealed that the top seven active compounds, such as withanolide D, hypericin, silymarin, oxyacanthine, withaferin A, Acetyl aleuritolic acid, and rhein, exhibit good binding affinity with druggable ADME properties, toxicity, and Pass prediction. The stability of the docked complexes was studied by conducting molecular simulations of 100 ns. MM-GBSA calculated the binding free energy uncovered that withanolide D, hypericin, and silymarin result in highly stable binding conformations in three different sites of the nucleocapsid protein. However, further investigation is needed in order to validate the candidacy of these inhibitors for clinical trials. HighlightsNatural plants' active compounds may aid in the inhibition of SARS-CoV-2 replication and COVID-19 therapeutics.Hypericin, silymarin, withanolide D, oxyacanthine, withaferin A, Acetyl aleuritolic acid, and rhein are effective against SARS-CoV-2 N protein.Studied natural plants' active compounds could be useful against COVID-19 and its associated organs comorbidities.ADMET properties of selected compounds favor these compounds as druggable candidates.Communicated by Ramaswamy H. Sarma.

Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals.

As there are limited data on B cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine.Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n=30), WT infected (n=30), delta infected (n=30), omicron infected+vaccinated (n=20) and Sinopharm (BBIBP-CorV) vaccinees (n=30). We then investigated the sensitivity and specificity of these immunodominant regions and analysed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274 to 297 and aa 365 to 388, were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

Infectious bronchitis virus nucleocapsid protein suppressed type I interferon production by interfering with the binding of MDA5-dsRNA and interacting with LGP2.

The type I interferon (IFN-I) is a critical component of the innate immune responses, and Coronaviruses (CoVs) from both the Alphacoronavirus and Betacoronavirus genera interfere with the IFN-I signaling pathway in various ways. Of the gammacoronaviruses that mainly infect birds, little is known about how infectious bronchitis virus (IBV), evades or interferes with the innate immune responses in avian hosts since few IBV strains have been adapted to grow in avian passage cells. Previously, we reported that a highly pathogenic IBV strain GD17/04 has adaptability in an avian cell line, providing a material basis for further study on the interaction mechanism. In the present work, we describe the suppression of IBV to IFN-I and the potential role of IBV-encoded nucleocapsid (N) protein. We show that IBV significantly inhibits the poly I: C-induced IFN-I production, accordingly the nuclear translocation of STAT1, and the expression of IFN-stimulated genes (ISGs). A detailed analysis revealed that N protein, acting as an IFN-I antagonist, significantly impedes the activation of the IFN-ß promoter stimulated by MDA5 and LGP2 but does not counteract its activation by MAVS, TBK1, and IRF7. Further results showed that IBV N protein, verified to be an RNA-binding protein, interferes with MDA5 recognizing double-stranded RNA (dsRNA). Moreover, we found that the N protein targets LGP2, which is required in the chicken IFN-I signaling pathway. Taken together, this study provides a comprehensive analysis of the mechanism by which IBV evades avian innate immune responses.

SARS-CoV-2 Nucleocapsid Protein Is a Potential Therapeutic Target for Anticoronavirus Drug Discovery.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, is a highly contagious positive-sense RNA virus. Its explosive community spread and the emergence of new mutant strains have created palpable anxiety even in vaccinated people. The lack of effective anticoronavirus therapeutics continues to be a major global health concern, especially due to the high evolution rate of SARS-CoV-2. The nucleocapsid protein (N protein) of SARS-CoV-2 is highly conserved and involved in diverse processes of the virus replication cycle. Despite its critical role in coronavirus replication, N protein remains an unexplored target for anticoronavirus drug discovery. Here, we demonstrate that a novel compound, K31, binds to the N protein of SARS-CoV-2 and noncompetitively inhibits its binding to the 5' terminus of the viral genomic RNA. K31 is well tolerated by SARS-CoV-2-permissive Caco2 cells. Our results show that K31 inhibited SARS-CoV-2 replication in Caco2 cells with a selective index of ~58. These observations suggest that SARS-CoV-2 N protein is a druggable target for anticoronavirus drug discovery. K31 holds promise for further development as an anticoronavirus therapeutic. IMPORTANCE The lack of potent antiviral drugs for SARS-CoV-2 is a serious global health concern, especially with the explosive spread of the COVID-19 pandemic worldwide and the constant emergence of new mutant strains with improved human-to-human transmission. Although an effective coronavirus vaccine appears promising, the lengthy vaccine development processes in general and the emergence of new mutant viral strains with a potential to evade the vaccine always remain a serious concern. The antiviral drugs targeted to the highly conserved targets of viral or host origin remain the most viable and timely approach, easily accessible to the general population, in combating any new viral illness. The majority of anticoronavirus drug development efforts have focused on spike protein, envelope protein, 3CLpro, and Mpro. Our results show that virus-encoded N protein is a novel therapeutic target for anticoronavirus drug discovery. Due to its high conservation, the anti-N protein inhibitors will likely have broad-spectrum anticoronavirus activity.

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly.

SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the NIDR, the serine/arginine rich motif (SRIDR), and the CIDR. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid-liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (NFL) assembles into ring-like architecture whereas the truncated SRIDR-CTD-CIDR (N182-419) promotes filamentous assembly. Moreover, LLPS droplets of NFL and N182-419 are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N182-419 droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic DELTAserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) DELTAserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain >=10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses. GCE4All Biomedical Technology Development and Dissemination Center was supported by National Institute of General Medical Science, OSU NMR Facility funded in part by the National Institutes of Health, the Medical Research Foundation at OHSU and the Collins Medical Trust, National Science Foundation EAGER, and by the M. J. Murdock Charitable Trust.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

MECHANISM AND RESISTANCE STUDIES OF SARS-CoV-2 MPRO INHIBITOR POMOTRELVIR (PBI-0451)

Background: The unprecedented scale of the COVID-19 pandemic and rapid evolution of SARS-CoV-2 variants underscores the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential viral enzyme required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir (PBI-0451) is a novel Mpro inhibitor currently completing phase 2 clinical trial. Here we describe the mechanism of action, broad activity against SARS-CoV-2 clinical isolates, combination studies with other SARS-CoV-2 inhibitors and favorable resistance profile of pomotrelvir. Method(s): The kinetic parameters of pomotrelvir Mpro inhibition and its interaction with nirmaltrevir were determined in a kinetic protease assay. The IC50s of pomotrelvir on mutant Mpro proteins were measured in an endpoint Mpro assay. Combination studies of pomotrelvir with remdesivir and molnupiravir were carried out in A549-hACE2 cells infected with SARS-CoV-2 NLuc virus. Activity against SARS-CoV-2 clinical variants was assessed by infection of A549-ACE2-TMPRSS2 cells followed by immunostaining of the viral nucleocapsid protein. Result(s): Pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro (Ki =2.7 nM). Binding of pomotrelvir and the Mpro inhibitor nirmatrelvir to the active site is mutually exclusive. In the SARS-CoV-2 NLuc assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. When the effect of Mpro substitutions previously selected in a resistance study of pomotrelvir were analyzed in an enzyme assay, only Mpro-N133H showed a significant increase in IC50 (45-fold). The catalytic efficiency of Mpro-N133H is reduced by 10-fold and the recombinant virus SARSCoV-2 (WA1) -N133H is not viable, suggesting that N133H has lower replicative fitness. Lastly, pomotrelvir exhibits broad activity against all SARS-CoV-2 clinical isolates tested to date, including five omicron variants. Conclusion(s): PBI-0451 is a potent competitive inhibitor of SARS-CoV-2 Mpro and is broadly active against SARS-CoV-2 clinical isolates including omicron variants. Results from inhibitor interaction studies support the potential combination of pomotrelvir with remdesivir and molnupiravir but not nirmatrelvir. Enzymatic characterization of in vitro selected pomotrelvir resistant variants indicates they either confer no resistance or have reduced fitness.

Modifications in SARS-CoV-2 N Linker Region Regulate RNA Interactions and Phase Separation

The N protein of the SARS-CoV-2 virion is critical for viral genome packaging via RNA binding and regulation of viral transcription at the replication-transcription complex (RTC). The N protein can be divided into five main domains, and the central region is the linker, which is predicted to be primarily disordered and has not been heavily studied. The linker is Serine-Arginine Rich, which is phosphorylated at multiple sites by host kinases during infection, thereby promoting the N protein's role in viral transcription. Phosphorylation is a critical process for the regulation of many cellular processes and can provide recognition sites for binding complexes. In a study that examined the recognition of the SARS-CoV-2 N protein by the human 14-3-3 protein, the linker was found to contain critical phosphosites for 14-3-3 binding. The goals of this project are to determine the structure, dynamics, and RNA interactions of the Serine-Arginine Rich linker region. To accomplish this, we performed Nuclear Magnetic Resonance spectroscopy (NMR) experiments to analyze the structure of the linker region of the N protein and its ability to bind viral RNA. NMR confirms predictions that the linker is not entirely unstructured and it is able to bind RNA. The linker region of the N protein with phosphoserine incorporated at S188 was also examined via an NMR titration experiment with 1-1000 RNA. Compared to wild type, the incorporation of phosphorylation decreases binding. Other biophysical techniques such as Analytical Ultracentrifugation (AUC) and Multi-Angle Light Scattering (MALS) are used to identify the association state of the linker and the size of the resulting protein-RNA complex. We are currently working to biophysically characterize the structure, dynamics, and viral RNA binding ability of a mutation found in the Delta and Omicron variants: the R203M linker, which have been shown to enhance viral infectivity. This work was supported by the NSF EAGER grant NSF/ MCB 2034446 and URSA Engage. Support to facilities includes the Oregon State University NMR Facility funded in part by NIH, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Comparative ELISA Detection of SARS-COV-2 Monoclonal Antibodies in Patients' Serum, Saliva, and Nasal Fluid in Iraq

Precise management of COVID-19 infection and global pandemic requires a precise assay of SARS-COV-2 antibodies. Spike protein and RBD specific serum monoclonal antibodies has been known to be elicited with the currently approved mRNA vaccine for COVID-19. Since combating SARS-CoV-2 infection begins as early at the viral entry port, mediated by mucosal immunity;whether the currently approved vaccines have the capacity to elicit mucosal immunity is still to be studied. Method;We conducted our research to detect SARS-Cov-2 IgA-RBD and IgG-NCP, in a set of a randomized three patients' groups (total 55 subjects, randomized into 20 (Non previously infected control, 20 fully vaccinated people from AlSalam teaching hospital vaccination ward upon receiving the vaccine doses, and 15 patients newly recovered from SARS-CoV-2 infection from AlShifaa quarantine hospital after remission from COVID-19 infection and preparing to discharge) in three fluid samples serum, saliva and nasal fluid, for a total of three samples per participant, to make a total of 165 samples to be tested, using a commercial immunoassay kit (Anti-SARS-CoV-2 ELISA RBD IgA, and Anti-SARS- CoV-2 NCP ELISA IgG) the study was designed to assess the immunoglobulins levels in each sample from different health status and anatomical locations to give the statistical difference and the correct conclusion regarding this study. Statistical analysis was performed using SPSS version 24, as well as Microsoft excel version (16.0.15028), test equations were performed via Mann-Whitney U Test, as well as The Kruskal-Wallis H test, to evaluate the non-gaussian distribution among the study group, for each of the outcomes, p < 0.05 was undertaken as statistically significant. Results;Our work showed that two doses of mRNA BNT162b2 vaccine Comirnaty (Pfizer/BioNTech, New York, NY, USA), elicited a robust anti SARS-CoV-2 nasal and salivary IgA and IgG monoclonal antibody protection (as compared to control 0.625 COI, the vaccinated subjects resulted in a higher median salivary concentration of IgG NCP values as 1.69 COI, with a p-value of 0.0001. same goes for median salivary concentration of IgA RBD with value of 2.875 ± 1.276, as compared to control median values of 0.667 ± 0.208), (as compared to control 0.462 ± 0.2369, the vaccinated subjects resulted in a higher median nasal concentration of IgG NCP values as 1.944 ± 0.694, with a p-value of 0.0001. Same goes for median nasal concentration of IgA RBD with value of 1.941 ± 0.53, as compared to control median values of 0.681 ± 0.226). Two injections 25 days a part shown to trigger a stronger titer of protective immunity as compared to single early shot, still it's less robust compared to the titers measured for the recovered subjects from COVID-19 infection. Taking into consideration that there was a lack of trustable ELISA based assays with specially designed kit for this purpose, focusing on nasal and salivary secretions, the current study that our pre investigational and investigational data are consistent. The results of this study indicates that a protective antigen specific nasal and salivary monoclonal antibody, can be triggered following proper vaccination regimen with mRNA COVID-19 vaccine, so this paves the way for using mucosal protective antibodies detection kits, as a suitable alternative option as a less invasive method compared to serum investigations, for detecting the protection against SARS-Cov-2 infection, induced by vaccine. The vaccinated subjects are significantly better tested via IgA in Saliva as compared to IgG, because of the higher median values for the IgA vs IgG, 2.875 ± 1.276 and 2.083 ± 1.610 respectively with a p-value of 0.008. in a similar state the IgA is the best test for the convalescent subjects in term of nasal fluid samples investigations, as the median value are comparatively higher than median value of IgG, 2.024 ± 0.520 and 1.786 ± 1.115 respectively. © 2023 Israa University Journal of Applied Science. All rights reserved.

Surfactant Protein B Reduces Respiratory Viral Infectivity in Human Lungs

Purpose of Study: COVID pneumonia caused by SARS-CoV-2 can result in a depletion of surfactant & lung injury, which resembles neonatal respiratory distress syndrome. Exogenous surfactant has shown promise as a therapeutic option in intubated hospitalized patients. Our preliminary data in human lung organoids (LOs) with a deficiency of surfactant protein B (SP-B) showed an increased viral load compared to normal LOs. Single cell RNA sequencing (scRNAseq) revealed that SP-B-deficient cells showed increased viral entry genes (ACE2 receptor) & dysregulated inflammatory markers emanating from the lung cells themselves. Our objective was to determine: (1) cell-specific transcriptional differences between normal & SP-B deficient human lung cells after infection with SARS-CoV-2 and (2) a therapeutic role of SP-B protein & surfactant in COVID-19 pneumonia. Methods Used: We used normal and SP-B mutant (homozygous, frameshift, loss of function mutation p.Pro133GlnfsTer95, previously known as 121ins2) human induced pluripotent stem cells (hiPSC) and differentiated them into 3D proximal lung organoids. The organoids were infected with the delta variant of SARS-CoV-2 for 24 hours at an MOI of 1. Infected and uninfected organoids were fixed in trizol in triplicate and underwent processing for bulk RNA sequencing. We tested for differentially expressed genes using the program DEseq. We also plated normal iPSC derived lung organoids as a monolayer and pre-treated them with 1mg/ml of Poractant alfa or 5 uM of recombinant SP-B protein. The delta strain of SARS-CoV-2 was added to the 96 wells at an MOI of 0.1 for one hour with shaking, then an overlay with DMEM/CMC/FBS was added and left on for 23 hours. The plate was fixed and stained for nucleocapsid (NC) protein. Summary of Results: Bioinformatic analysis of the bulk RNA sequencing data showed an increase in the multiple cytokines and chemokines in the SP-B mutant LOs compared to control. We also saw differential gene expression patterns in the SP-B mutant LOs including a reduction in SFTPC, FOXA2, and NKX2-1 and an increase in IL1A, VEGFA, PPARG and SMAD3. In the exogenous surfactant experiments, there was a decrease in total expression of viral NC in the Poractant alfa & rSP-B-treated cells compared to SARS-CoV-2 infection alone (p<0.001). Conclusion(s): Surfactant modulates the viral load of SARS-CoV-2 infection in the human lung. Deficiency in SP-B results in the dysregulation of the lung epithelial inflammatory signaling pathways resulting in worsening infections.

METFORMIN REDUCED SARS-CoV-2 VIRAL LOAD IN A PHASE 3 RANDOMIZED CLINICAL TRIAL

Background: Metformin has in vitro activity against SARS-CoV-2. In a published phase 3, quadruple-blinded, placebo-controlled randomized trial of outpatient COVID-19 therapy, metformin resulted in a 42% reduction in ER visits/hospitalizations/deaths by day 14, 58% reduction in hospitalizations/ death by day 28, and 42% reduction in Long Covid through 10 months. This analysis presents the results of viral load sampling performed during that clinical trial. Method(s): Covid-Out trial (NCT04510194) enrolled adults aged 30 to 85 within 3 days of a documented SARS-CoV-2 infection and < 7 days after symptom onset. The trial randomized 1323 participants to metformin (1000mg/day days 2-5;1500mg/day days 6 to 14), ivermectin, fluvoxamine, and/or exact-matching placebo in a 2x3 factorial trial design. Nasal swabs for viral load were an optional component, self-collected from the anterior nares on day 1, 5, and 10. Viral loads were measured via RT-qPCR using N1 and N2 targets in the SARSCoV- 2 nucleocapsid protein, with relative Ct values converted to absolute copy number via calibration to droplet digital PCR. A linear Tobit regression model was used to assess change over time while accounting for left censoring due to the viral load limit of detection. Results were adjusted for other treatment allocations within the factorial design, vaccination status, and baseline viral load. Repeated measures were accounted for using clustered standard errors within participants. Result(s): Samples were available from n = 945, 871, and 775 participants on days 1, 5, and 10, respectively. The mean change from baseline to followup was -0.64 log10 copies/mL (95%CI, -1.16 to -0.13) for metformin versus placebo, which equates to a 4.4-fold greater decrease. The mean change in SARS-CoV-2 from baseline to day 5 was -0.48 log10 copies/mL, and was -0.81 log10 copies/mL from baseline to day 10. The anti-viral effect increased with increased metformin dosing days 6-14. The antiviral effect was larger in those unvaccinated (mean -0.95 log copies/mL) than vaccinated (mean -0.39 log copies/mL). There was no change in viral load vs. placebo for ivermectin or fluvoxamine. Conclusion(s): Metformin lowered SARS-CoV-2 viral load in this quadrupleblinded, randomized clinical trial. The temporal relationship to dose titration suggests a dose-dependent effect. The magnitude of antiviral effect was similar to nirmatrelvir at day 5, greater than nirmatrelvir at day 10. Metformin is safe, widely available, and has few contraindications.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

Association between IgG responses against the nucleocapsid proteins of alphacoronaviruses and COVID-19 severity.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Heterologous chimpanzee adenovirus vector immunizations for SARS-CoV-2 spike and nucleocapsid protect hamsters against COVID-19.

Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines.

Dynamics of T cellular immunity to nucleocapsid protein (N-protein) of SARS-CoV- 2 assessed through 9 months after infection

Background: The nucleocapsid protein (N-protein) of SARS-CoV- 2 regulates transcription, replication and packaging of the viral genome. Potentially, each of the structural proteins can act as an antigen for the production of specific antibodies and T-cell stimulation. This study evaluates T-cellular immunity to N-protein after infection caused by the SARS-CoV- 2 for up to 9 months. Method(s): Patients who had COVID-19 in 2020 were divided by severity into 3 groups: mild (n = 41), moderate (n = 46), and severe (n = 29). Blood samples were taken 3 times: at 3, 6 and 9 months after infection. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained with Tag-it Violet vital dye. PBMCs were stimulated with PepTivator SARS-CoV- 2 ProtN (pool of peptides, covering the complete sequence of N-protein);unstimulated cells served as negative control. PBMCs were cultured in AIM-V media for 5-7 days. Cell colonies were detected by microscopy. Relative content (%) of proliferating live T cells (CD3+7AAD-) were evaluated by flow cytometry. Samples were counted positive for antigen-specific T cells (ASCs) to N-protein when both proliferation was >1% compared with negative control and microcolony were observed. Result(s): There were 58.5% positive samples with ASCs to the N-protein in mild, 60.9% in moderate and 48.3% in severe group in 3 months after recovery from COVID-19. There was no difference between groups at this point (p > 0.05, chi2). After 6 months results were: 26.7% positive samples in mild, 21.9% in moderate and 35.0 % in severe group. No differences between groups were observed after 6 months, but percentages of positive samples were less in mild and moderate groups when compared 3d and 6th months (p3-6 < 0.05, chi2). Drastic decreases of ASCs-positives in the mild group was observed after 9 month -only 4.2% (p6-9 < 0.05, chi2). Further decrease in moderate (25.0%) and severe (41.7%) groups was not observed after 9 months. Conclusion(s): T cell immunity to the N-protein of SARS-CoV- 2 was formed after mild, moderate and severe forms of COVID-19 with different effectiveness. The duration of immunity depends on the form of the disease and was stronger in severe group. By the 9th month, the number of positive samples in the group with mild COVID-19 was significantly lower when compared with the moderate and severe groups.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2.

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.

Coagulopathies after vaccination against SARS-CoV-2: The sole solution might lead to another problem

The common reported adverse impacts of COVID-19 vaccination include the injection site's local reaction followed by various non-specific flu-like symptoms. Nevertheless, uncommon cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) following viral vector vaccines (ChAdOx1 nCoV-19 vaccine, Ad26.COV2 vaccine) have been reported. This literature review was performed using PubMed and Google Scholar databases using appropriate keywords and their combinations: SARS-CoV-2, adenovirus, spike protein, thrombosis, thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia (VITT), NF-kappaB, adenoviral vector, platelet factor 4 (PF4), COVID-19 Vaccine, AstraZeneca COVID vaccine, ChAdOx1 nCoV-19 COVID vaccine, AZD1222 COVID vaccine, coagulopathy. The s and titles of each article were assessed by authors for screening and inclusion English reports about post-vaccine CVST and VITT in humans were also collected. Some SARS-CoV-2 vaccines based on viral vector, mRNA, or inactivated SARS-CoV-2 virus have been accepted and are being pragmatic global. Nevertheless, the recent augmented statistics of normally very infrequent types of thrombosis associated with thrombocytopenia have been stated, predominantly in the context of the adenoviral vector vaccine ChAdOx1 nCoV-19 from Astra Zeneca. The numerical prevalence of these side effects seems to associate with this particular vaccine type, i.e., adenoviral vector-based vaccines, but the meticulous molecular mechanisms are still not clear. The present review summarizes the latest data and hypotheses for molecular and cellular mechanisms into one integrated hypothesis demonstrating that coagulopathies, including thromboses, thrombocytopenia, and other associated side effects, are correlated to an interaction of the two components in the COVID-19 vaccine.Copyright © 2022, Iranian Pediatric Hematology and Oncology Society. All rights reserved.