Critical role of defensins in respiratory viral infection induced breast cancer progression

Respiratory viral infections (RVI) such as influenza and COVID19 impact the host systemic immune system along with causing deleterious chronic inflammatory responses and respiratory distress. While the role of chronic inflammation in cancer is well-established, the role of RVI on tumorigenesis is poorly defined. To study the role of RVI on breast cancer, we first infected murine respiratory epithelial cells (mRES) with murine sendai virus (mSV), an analog for human parainfluenza virus. These infected mRES were co-cultured with 4T1 murine breast cancer cells in 1:1 dilution on a single 2D plate and also in trans-well format. Both in co-culture and transwell culture we saw a 40- 80% (p<0.05) increased proliferation of breast cancer cells. Similarly, when 4T1 cells were treated with the supernatant collected from infected mRES cells in 1:5 dilution, also demonstrated a 2.3 fold increased breast cancer cell proliferation. The cytokine analysis from the supernatant collected from infected mRES cells demonstrated a 17-23 fold enhanced secretion of alpha/beta-defensins. Direct treatment of alpha-defensin (cyptidin-4, 10 pg/mL) and beta-defensin-3 (mBD3, 20 pg/mL) on 4T1 cells demonstrated enhanced expression of chemokine metastatic receptor, CXCR4 (4.3 fold), angiogenic factor, VEGF (12.8 fold) and cell division favoring factor, CDK2 (8.1 fold). Further, analysis of infected mRES cells demonstrated upregulation of toll-like receptor 2 (TLR2) and NODlike receptor protein 3 (NLRP3) expression. Interesting, co-cultured of infected mRES with syngeneic murine CD4 T cells induced exhaustion phenotype (PD1+ and CTLA4+ ) differentiation of CD4 T cells. Taken together, these data suggest that respiratory viral infections through induction of cancer cell proliferation and inhibiting anti-tumor adaptive immune responses promote breast cancer proliferation.

SARS-CoV-2 VARIANTS-DEPENDENT INFLAMMASOME ACTIVATION

Background: Innate immunity is the first line of defense in response to pathogens, which acts locally and also leads the stimulation of adaptive immunity through at least with IL-1beta secretion. It has been shown that SARSCoV- 2 infection triggered the NLRP-3 inflammasome activation and the IL-1beta secretion. The aim of this study was to analyze and compare the level of IL-1beta secretion that is one of the most important innate immunity cytokines, in monocyte-like cells infected with 6 different variants of the SARS-CoV-2. Method(s): Six SARS-CoV-2 variants (historical (B.1, D614G), Alpha, Beta, Gamma, Delta and Omicron BA.1) were isolated from COVID-19 hospitalized patients. Viral stocks were obtained by inoculation in Vero and Vero-TRMPSS2 cells. THP-1 monocyte-like cells were cultured with RPMI-hepes 10% FBS-0.05 mM 2-mercaptoethanol. A total of 5 x 104 of THP-1 cells was plated per well in 96-wells plate and differentiated with 10nM of PMA for 24h. Differenciated- THP-1 were first primed with LPS 1mug/ml for 2h and infected with different SARS-CoV-2 variants with a MOI 0.1. IL-1beta was measured by luminescence in the supernatant after 24 h of infection. Result(s): We analyzed and compared IL-1beta secretion between SARS-CoV-2 virus 6 sublineages after infection of monocytes like THP-1. We observed that THP-1 cells infected with SARS-CoV-2 variants presented a significantly higher IL-1beta secretion than non-infected cells. Moreover, some SARS-CoV-2 variants led to a stronger IL-1beta secretion, and particularly we observed a significantly higher level of IL-1beta cells infected with Omicron BA.1 sublineage compared to other variants. Indeed, Omicron BA.1 infected cells presented the higher IL-1beta secretion (median 385.7 pg/ml IQR[302.6-426.3]) follows by the Delta variants and the historical variants (median 303.6 [266.3-391.9] and 281.9 [207.2-410], respectively). Alpha, Beta and Gamma variants presented the lowest IL-1beta secretion (median 228.1 [192.5-276.4], 219.1 [185.1-354.2] and 211 [149.8- 228.8]). Conclusion(s): We observed the inflammasome activation for the 6 SARS-CoV-2 sublineages with a variation in level of IL-1beta secretion. Indeed, our results suggested that Omicron BA.1 was more recognized by the innate immune cells than other SARS-CoV-2, which could in part, with its upper respiratory tract tropism, possibly explain its less clinical virulence. Taking together, these results suggest that the innate immunity response and precisely, IL-1beta secretion pathways were activated in a SARS-CoV-2 variants-dependent manner.

Hiv Detection in Wastewater as a New Epidemiological Tool

Background: Wastewater represents a broad, immediate, and unbiased accounting of the pathgens in the population. We aimed to develop methods to track HIV in wastewater utilizing a viral detection pipeline adapted from platforms developed to track SARS-COV-2. Method(s): We used samples from 6 wastewater treatment plants in the Houston area. We focused on regions of higher prevalence and lower prevalence. First, employing wastewater processing and nucleic acid extraction methods described by our group to detect SARS-COV-2, we tested a single high and low prevalence site in triplicate with all 3 primer sets. nucleic acid extracts from HIV and SIV cell culture supernatants were used as controls. Next, in subsequent samples, RT-PCR reactions with detections were subjected to gel electrophoresis to determine the amplified product sizes. To further confirm HIV detection, we sequenced the RT-PCR products and compared the proportion of reads which mapped to the expected amplified product. In a later set of studies, we fractionated samples into supernatant and pellet. We further tested HIV presence by performing whole virome sequencing on the extracts from some samples that produced detections and mapped reads to published genomes. A crAssphage genome was used as a negative control. Result(s): Samples from all sites resulted in signal detection at least once. Only reactions with gag and pol primers appeared to amplify the expected product. Products from the HIV positive control mapped almost exclusively to the HIV genome (97-100% of reads), with a fraction of reads from the SIV negative control doing the same (16-18% of reads). The ltr and pol products did not map the HIV genome while gag products did (34-44% of reads). Among the fractionated sample, in total, 6 supernatant fractions produced no detection compared to 7 of 8 pellet fractions. The whole virome sequencing produced reads that mapped to the HIV genome with at least 8X depth coverage. The sample with the lowest Ct detection (26) yielded HIV coverage several logs greater than those samples with higher Ct detection (37). Reads from all samples mapped to at least 20% of the HIV genome. Conclusion(s): This work provides the first evidence that HIV can be detected in municipal wastewater systems and has the potential to be developed into a new public health tool.

THE SPIKE OF SARS-CoV-2 VARIANTS ALLOWS FOR MORE EFFICIENT COUNTERACTION OF BST2

Background: BST2/Tetherin is an interferon-stimulated gene with antiviral activity against enveloped viruses. Particularly, BST2 tethers virions at their site of assembly, preventing their release and spread. In addition to this primary role, BST2 is as an important bridge between the innate and adaptive immune system, since (i) BST2 routes tethered particles to lysosomes, which generates viral breakdown products that engage pattern recognition receptors;and (ii) trapped virions facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). In turn, viruses have evolved mechanisms to bypass BST2, either by targeting BST2 for proteasomal/lysosomal degradation or by removing BST2 from sites of virion assembly. However, the role of BST2 in SARS-CoV-2 replication, spread, evolution, and pathogenesis remains largely unknown. Method(s): The antiviral potential of BST2 against SARS-CoV-2 was investigated by infecting different SARS-CoV-2 isolates (Hong Kong, Alpha, Beta, Delta, and Omicron) in BST2+ and BST2- cells. Culture supernatants were collected to assess virion production by ELISA and infectivity by TCID50. Infected cells were analyzed by western blot and flow cytometry to examine viral and cellular protein levels, including BST2. Transfection of individual SARS-CoV-2 ORFs and mutagenesis studies allowed us to identify the genes that the virus uses to downregulate BST2. Immunoprecipitation assays revealed protein-protein interactions and changes in ubiquitination patterns. Experiments with proteasomal and lysosomal inhibitors furthered our mechanistic understanding of how SARS-CoV-2 counteracts BST2. Finally, fluorescence microscopy studies uncovered changes in the subcellular distribution of BST2 in SARS-CoV-2 infected cells. Result(s): While BST2 reduces virion release, SARS-CoV-2 has evolved to counteract this effect. Specifically, SARS-CoV-2 uses the Spike to interact with BST2, sequester the protein at perinuclear locations, and ultimately route it for lysosomal degradation. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that each variant is more efficient than the previously circulating strain at downregulating BST2 and facilitating virion production, and that mutations in the Spike account for their enhanced BST2 antagonism. Conclusion(s): As part of its adaptation to humans, SARS-CoV-2 is improving its capacity to counteract BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity and transmission.

Covid-19 Vaccination Impacts Functional Immune Responses and Plasma Proteome in Plwh

Background: The impact of COVID-19 infection or COVID-19 vaccination on the immune system of people living with HIV (PLWH) is unclear. We therefore studied the effects of COVID-19 infection or vaccination on functional immune responses and systemic inflammation in PLWH. Method(s): Between 2019 and 2021, 1985 virally suppressed, asymptomatic PLWH were included in the Netherlands in the 2000HIV study (NCT039948350): 1514 participants enrolled after the start of the COVID-19 pandemic were separated into a discovery and validation cohort. PBMCs were incubated with different stimuli for 24 hours: cytokine levels were measured in supernatants. ~3000 targeted plasma proteins were measured with Olink Explore panel. Past COVID-19 infection was proven when a positive PCR was reported or when serology on samples from inclusion proved positive. Compared were unvaccinated PLWH with and without past COVID-19 infection, and PLWH with or without anti-COVID-19 vaccination excluding those with past COVID-19 infection. Result(s): 471 out of 1514 participants were vaccinated (median days since vaccination: 33, IQR 16-66) and 242 had a past COVID-19 infection (median days since +PCR: 137, IQR 56-206). Alcohol, smoking, drug use, BMI, age, latest CD4 count and proportion with viral blips were comparable between groups. Systemic inflammation as assessed by targeted proteomics showed 89 upregulated and 43 downregulated proteins in the vaccinated participants. In contrast, individuals with a past COVID-19 infection display lower levels of 138 plasma proteins compared to the uninfected group (see figure). 'Innate immune system' and 'cell death' were upregulated in pathway analysis in vaccinated PLWH, but downregulated in COVID-19 infected participants. The increased systemic inflammation of the COVID-19 vaccinated group was accompanied by lower TNF-alpha and IL-1beta production capacity upon restimulation with a range of microbial stimuli, while production of IL-1Ra was increased. In COVID-19 infected PLWH only a reduced production of TNF-alpha to S. pneumonia was significant. Vaccinated PLWH also showed upregulation of platelet aggregation pathways. Conclusion(s): COVID-19 vaccination in PLWH leads to an increased systemic inflammation, but less effective cytokine production capacity of its immune cells upon microbial stimulation. This pattern is different from that of COVID-19 infection that leads to a decreased inflammatory profile and only minimal effects on cytokine production capacity. (Figure Presented).

TARGETING THE HOST-VIRUS INTERFACE TO BLOCK SARS-CoV-2 ASSEMBLY IN AIRWAY CELLS

Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.

Association of urinary mitochondrial DNA with COVID-19 related acute kidney injury, mitochondrial stress, and inflammation in renal transplant recipients

Introduction: ACE-receptors are profusely expressed in the renal cell, making it highly susceptible for severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) infection. After entering the cells, the virus induces high levels of cytokines, chemokines, and inflammatory responses, resulting neutrophilic infiltration, activation, and profuse reactive oxygen species (ROS) formation, leading to cellular necrosis and acute tubular injury. Proximal convoluted tube cell are rich in mitochondria and susceptible for developing acute kidney injury (AKI) due to mitochondrial stress. Early detection of AKI may helpful in its management, limiting the severity, avoiding nephrotoxic medicines and modifying the drug dose depending on renal function. Therefore, in the current study, we have determined the utility of urinary mitochondrial DNA (umt-DNA) and neutrophil gelatinase-associated lipocalin (NGAL) in predicting COVID-19-associated acute kidney injury (AKI) and mitochondrial stress and demonstrated the inflammatory response of urinary mt-DNA. Method(s): Live-related RTRs(n=66), who acquired SARS-CoV-2 infection and were admitted to a COVID hospital were included and subclassified into AKI (N=19) with > 25% spike in serum creatinine level from the pre-COVID-19 serum creatinine level, and non-AKI (N=47) whose serum creatinine value remained stable similar to the baseline value, or a rise of < 25% of the baseline values of pre-COVID-19. A 50ml urine sample was collected and umt-DNA and N-GAL was determined by the RT-PCR and ELISA methods respectively. A 10ml blood sample from 10 healthy volunteers was also collected for PBMC isolation and inflammatory response demonstration. A 1x106 PBMC was stimulated for 24hrs. with 1microg/ml of urinary DNA or TLR9 agonist CpG oligodeoxynucleotide (5'-tcgtcgttttcggcgc:gcgccg-3') in duplicate. Unstimulated PBMCs served as control. The gene expression of IL-10, IL-6, MYD88 was analyzed by the RT-PCR and IL-6, IL-10 level in supernatants by the ELISA. Result(s): Both the urinary mitochondrial gene ND-1 and NGAL level was significantly higher in AKI group compared to non-AKI. The mean ND-1 gene Ct in AKI group was (19.44+/-2.58 a.u) compared to non-AKI (21.77+/-3.60;p=0.013). The normalized ND-1 gene Ct in AKI was (0.79+/-0.11 a.u) compared to non-AKI (0.89+0.14;P=0.007). The median urinary NGAL level in AKI group was (453.53;range, 320.22-725.02, 95% CI) ng/ml compared to non-AKI (212.78;range, 219.80-383.06, 95%CI;p=0.015). The median urine creatinine normalized uNGAL was 4.78 (0.58-70.39) ng/mg in AKI group compared to 11.26 ng/mg (0.41-329.71) in non-AKI group. The area under curve of ND-1 gene Ct was 0.725, normalized ND-1 Ct was 0.713 and uNGAL was 0.663 and normalized uNGAL was 0.667 for detecting the AKI and mitochondrial stress. The IL-10 gene expression was downregulated in umt-DNA treated PBMCs compared to control (-3.5+/-0.40vs1.02+/-0.02, p<0.001). IL-6 and Myd88 gene expression was upregulated. The culture supernatant IL-10 and IL-6 level in umt-DNA treatment PBMCs vs control was 10.65+/-2.02 vs 30.3+/-5.47, p=0.001;and 200.2+/-33.67 vs 47.6+/-12.83, p=0.001 pg/ml respectively. Conclusion(s): Urinary mt-DNA quantification can detect the Covid19 associated AKI and mitochondrial distress with higher sensitivity than uNGAL in RTRs. Urinary mt-DNA also induces a robust inflammatory response in PBMCs, which may exacerbate the Covid19 associated allograft injury. No conflict of interestCopyright © 2023

Protein Blend Suppress Inflammatory Response from Bronchial Epithelial Cells Induced by SARS-Cov-2

Even after virus elimination, coronavirus disease 2019 (COVID-2019) leaves numerous sequelae. Growing evidence demonstrates that massive release of proinflammatory cytokines, which drives COVID-19 progression, severity, and mortality, remains elevated after acute phase of COVID-2019, playing a central role in the disease' sequelae. In this way, bronchial epithelial cells are the first cells hyperactivated by coronavirus-2 (SARS-Cov-2) leading to massive cytokine release, triggering leukocytes and other cells hyperactivation, mediating COVID-19 sequelae. So, proinflammatory cytokines are initiated by the host. Thus, this in vitro study tested the hypothesis that ImmuneRecovTM, a protein blend, could inhibit the hyperactivation of human bronchial epithelial cells (BEAS-2B) induced by SARS-Cov-2. BEAS-2B (5x104/mL/well) cells were co-cultivated with 1ml of blood of a SARS-Cov-2 infected patient for 4 hours and protein blend (1ug/mL) was added in the first minute of the co-culture. After 4 hours, the cells were recovered and used for analysis of cytotoxicity by MTT and for mRNA expression of IL-1beta, IL-6, IL-10. The supernatant was used to measure cytokines. SARS-Cov-2 incubation resulted in increased levels of IL-1beta and IL-6 by BEAS-2B cells (p<0.001). Treatment with the protein blend resulted in reduced levels of pro-inflammatory IL1beta and IL-6 (p<0.001), and increased the levels of anti-inflammatory IL-10 (p<0.001). Protein blend reduced SARS Cov-2-increased the mRNA expression of IL-1beta and IL-6, and increased the expression of IL-10 and IFN-gamma. In conclusion, protein blend presents important anti-inflammatory effects in the context of SARS-Cov-2 infection.

Delayed immunological reaction of Human Microvascular Lung Endothelium in response to Human Coronavirus 229E infection

Introduction: In the natural conditions first and major target for respiratory viruses (RVs) are epithelial cells. Nonetheless, recently we have demonstrated that RVs are able to infect not only epithelium, but also Human Microvascular Lung Endothelial Cells (HMVEC-L) which increased network is observed during severe asthma due to increased angiogenesis. Furthermore, on the surface of HMVEC-L we observed intense expression of aminopeptidase N (AP-N)- an entry receptor for Human Coronavirus 229E (HCoV-229E). Due to the facts, that possibility of being infected by HCoV-229E should be considered and there is no research based on this model the aim of this study was to assess the vulnerability of HMVEC-L to HCoV-229E infection. Method(s): HMVEC-L was incubated with HCoV-229E (MOI 0,1;1,0;3,0) for 3 hours, 3x PBS washed and cultured for 120 hours. In relevant time points (5;24;48;72;96 and 120h) viral copy number and mRNA expression of inflammatory, anti-viral and receptor factors were evaluated in Real-Time PCR. Confocal microscopy (CM) and flow cytometry (FACS) were used to measure AP-N surface expression. Result(s): FACS and CM confirmed intense surface expression of AP-N on HMVEC-L. HCoV-229E efficiently infected HMVEC-L (604 945,5 +/-194 930,2 viral copies/mul) in 48h cultures (MOI 0,1) and induced relatively late (between 72- 96h) mRNA expression of RANTES (1181,12);IL-6 (89,6);IFN-beta (53,7);OAS-1 (64,3);PKR (11,4) and TLR-3 (42,4). Increased mRNA expression was also accompanied by protein release to the supernatants. Conclusion(s): HCoV-229E may efficiently infect HMVEC-L and induce delayed inflammatory and anti-viral response.

The use of quantitative enzyme-linked immunosorbent assay for the determination of S-antigen concentration in whole-virion inactivated adsorbed coronavirus vaccines

The severe consequences and high mortality of COVID-19 prompted the development of a wide range of preventive vaccines. The first vaccines to be tested were developed in China and formulated as inactivated SARS-CoV-2 adsorbed on aluminium hydroxide. One of the quality indicators for inactivated adsorbed vaccines is the degree of adsorption, which can be used to control the content not only of non-adsorbed antigen, but also of specific antigen in one dose of a vaccine. The aim of the study was to investigate the possibility of desorbing SARS-CoV-2 antigen from formulated adsorbed vaccines and the possibility of measuring its concentration using the BioScan-SARS-CoV-2 (S) ELISA kit for SARS-CoV-2 S-protein content determination. Materials and methods: the study used four batches of BBIBP-CorV by CNBG, Sinopharm (China) and three batches of CoronaVac by Sinovac Biotech (China). The authors desorbed SARS-CoV-2 S antigen in accordance with monograph FS.3.3.1.0029.15 of the State Pharmacopoeia of the Russian Federation edition XIV (Ph. Rus.), and quantified it using the BioScan-SARS-CoV-2 (S) ELISA kit by Bioservice Biotechnology Co. Ltd. (Russia). Results: mean S-antigen concentrations in the desorbed samples ranged from 61 to 129 ng/mL for BBIBP-CorV and from 461 to 533 ng/mL for CoronaVac. Conclusions: the study demonstrated the possibility of specific SARS-CoV-2 antigen desorption from the surface of aluminium hydroxide using the Ph. Rus. method, as well as the possibility of S-antigen quantification in desorbed medicinal products and supernatants using the BioScan-SARS-CoV-2 (S) ELISA kit. The authors observed 3.6- to 8.7-fold difference between the S-antigen concentrations of the desorbed preparations by the two manufacturers.

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope.

The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.

SPECIALIZED PRO-RESOLVING MEDIATORS (SPMS) and INFLAMMATORY NETWORKS in PATIENTS AFFECTED by ADULT ONSET STILL'S DISEASE (AOSD) and COVID-19

Background: Uncontrolled systemic infammation characterizes COVID-19 and autoinfammatory diseases such as adult-onset Still's disease (AOSD). Biosynthesis of pro-resolving mediators (SPMs), i.e. lipoxins (LX), resolvins (Rv), pro-tectins (PD), and maresins (MaR), ensures infammation shutdown and tissue repair, limiting neutrophils recruitment and stimulating macrophages to remove apoptotic cells. Among protectins, reduction of PD1 was found in the lungs of mice infected with the H5N1 infuenza virus and experimental treatment with PD1 resulted in increased animals' survival (Morita M et al 2013). Objectives: We investigated the effects of SPMs in pathogenesis and clinical evolution of AOSD and compared these data with mild and severe COVID-19. Finally, we analyzed the potential role of PD1 in modulating the infammatory response of macrophages obtained from AOSD patients, COVID-19 patients and healthy donors (HDs). Methods: 21 patients hospitalized for COVID-19 (10 ICU and 11 hospitalized in medical clinical unit) and 13 patients with AOSD were enrolled. Plasma PD1 levels from patients and controls were analyzed by ELISA, and mono-cytes-derived macrophages were polarized into M1 and M2 phenotype. We analyzed the effect of PD-1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. Peripheral blood mononuclear cells (PBMCS) from 3 AOSD patients, 2 COVID-19 patients and 3 HDs were obtained. Next-generation deep sequencing was then performed to identify the differences in PBMCs transcripts profiles. Results: AOSD patients with systemic scored (SS) ≥1 showed an increase of PD1 levels compared to AOSD patients with lower systemic score (p=0.04) (Figure 1A). Similarly, plasma levels of PD1 were increased in COVID-19 patients independently from their clinical subsets, compared to HDs (p=0.02). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a signifcant increase of M2 polarization vs control (p<0.05) (Figure 1B). Furthermore, a signifcant release of IL-10 and CCL4 from M2 macrophages was observed when compared to control (p<0.05) (Figure 1C). In the transcriptomes from 3 AOSD patients (2 mild and 1 severe), 2 COVID-19 patients (1 mild and 1 ICU) and 2 HDs, we observed that genes involved in infammation, lipid catabolism and monocytes activation were spe-cifcally dysregulated in AOSD and COVID-19 patients when compared to HDs. Among them pla2g15, pla2g12a, pla2g2d, involved in mobilization of SPMs precursors, were signifcant upregulated in patients compared to HDs (p<.01, ;log2FoldChange;>1.2) (Figure 1D). The largest part of the genes involved in infammation, lipid catabolism, and monocytes activation are less expressed in AOSD patients when compared to COVID19 patients, as reported in Table 1. Conclusion: The counterbalance by SPMs during infammation is still a largely unexplored pathway. Our study suggests that an imbalance of SPMs in autoin-fammatory diseases as well as COVID-19. The modulation of SPMs as observed in our experiments, might represent a new possible therapeutic strategy during AOSD and COVID-19.

ANTI-TNF THERAPY for IMMUNE MEDIATED INFLAMMATORY DISEASES MAY BE ASSOCIATED with LOWER ANTIBODY LEVELS and VIRUS NEUTRALIZATION EFFICACY FOLLOWING SARS-COV-2 MRNA VACCINATION

Background: The impact of immunosuppressants on COVID-19 vaccination response and durability in patients with immune-mediated infammatory diseases (IMID) is yet to be fully characterized. Humoral response may be attenuated in these patients especially those on B cell depleting therapy and higher doses of corticosteroids, but data regarding other immunosuppressants are scarce. Objectives: We aimed to investigate antibody and T cell responses and durability to SARS-CoV-2 mRNA vaccines (BNT162b and/or mRNA 1273) in IMID patients on immunomodulatory maintenance therapy other than B-cell depleting therapy and corticosteroids. Methods: This prospective observational cohort study examined the immuno-genicity of SARS-CoV-2 mRNA vaccines in adult patients with IMIDs (psoriatic arthritis, psoriasis, infammatory bowel disease and rheumatoid arthritis) with or without maintenance immunosuppressive therapies (anti-TNF, methotrexate/azathioprine [MTX/AZA], anti-TNF + MTX/AZA, anti IL12/23, anti-IL-17, anti-IL23) compared to healthy controls. Automated ELISA for IgGs to spike trimer, spike receptor binding domain (RBD) and the nucleocapsid (NP) and T-cell release of 9 cytokines (IFNg, IL2, IL4, IL17A, TNF) and cytotoxic molecules (sFasL, GzmA, GzmB, Perforinin) in cell culture supernatants following stimulation with spike or NP peptide arrays were conducted at 4 time points: T1=pre vaccination, T2=me-dian 26 days after dose 1, T3=median 16 days after dose 2 and T4=median 106 days after dose 2. Neutralization assays against four SARS-CoV-2 variants (wild type, delta, beta and gamma) were conducted at T3. Results: We followed 150 subjects: 26 healthy controls and 124 IMID patients: 9 untreated, 44 on anti-TNF, 16 on anti-TNF with MTX/AZA, 10 on anti-IL23, 28 on anti-IL12/23, 9 on anti-IL17, 8 on MTX/AZA (Table 1). Most patients mounted antibody and T cell responses with increases from dose 1 to dose 2 (100% sero-conversion at T3) and some decline by T4, with variability within groups. Antibody levels and neutralization efficacy was lower in anti-TNFgroups (anti-TNF, anti-TNF + MTX/AZA) compared to controls and waned by T4 (Figure 1). T cell responses were not consistently different between groups. Pooled data showed a higher antibody response to mRNA-1273 compared to BNT162b. Conclusion: Following 2 doses of mRNA vaccination there is 100% seroconver-sion in IMID patients on maintenance therapy. Antibody levels and neutralization efficacy in anti-TNF group are lower than controls, and wane substantially by 3 months after dose 2. These fndings highlight the need for third dose in patients undergoing treatment with anti-TNF therapy and continued monitoring of immunity in these patient groups, taking into consideration newer variants and additional vaccine doses.

Antibody Detection of Vaccine-induced Secretory Effects(ADVISE): A Prospective Study of Lactating Mothers Who Receive Sars-Cov-2 Vaccination

Background: The CDC recommends SARS-CoV-2 (COVID-19) vaccination for all adults, but vaccine safety for breastfeeding dyads and potential effects on lactation remain incompletely understood. Breastmilk immunological responses also have not been fully elucidated after COVID vaccination, specifically the formation of secretory IgA antibodies against viral Spike protein. Methods: ADVISE (NCT04895475) is a prospective trial evaluating breastmilk antibodies among lactating women who receive COVID vaccination. Maternal and infant demographics, breastfeeding characteristics including exclusivity, and vaccination type and side-effects were recorded. Milk samples before vaccination, and weekly after Dose 1 and Dose 2, were delivered using a convenient drop-off system. Maternal blood samples at monthly intervals, along with optional infant samples using a finger-stick Mitra® microsampler device (Neoteryx), were also collected. Breastmilk was frozen at -80C until processing, and fat-free supernatant was tested for quantitative ELISA titers against SARS-CoV-2 Spike protein and neutralizing activity using a pseudo-virus blocking assay. Results: A total of 66 women were consented, 3 of whom withdrew before data or sample collection. Of the remaining 63 (55 White, 5 Black, 3 Other), the median maternal age was 34.7 years (range 23.2-42.7 years);36 received the Moderna vaccine series and 27 received the Pfizer series. The median infant age at enrollment was 6 months;most were born full-term except 3 at <32 weeks and 5 at 33-36 weeks. Three mothers (5%) reported vaccine-related lactation sideeffects including 2 with a temporary decrease in milk supply and 1 who reported transient blue discoloration of the milk. Secretory IgA Spike antibodies (1:4 dilution, OD >0.5) were detected in the initial breastmilk samples from 14% of mothers, almost all of whom had either documented COVID infection or vaccination during pregnancy. For women who received vaccination while breastfeeding, 78% of the lactating mothers had secretory IgA antibodies in breastmilk within 2 weeks, which then dropped in titer and prevalence until the booster vaccine dose (Figure). Maximum breastmilk IgA titers were not associated with specific maternal or infant characteristics, including vaccine manufacturer. IgG breastmilk antibodies were also detected after the first vaccine, but titers were consistently high and sustained after the second dose (Figure). In preliminary analyses, most breastmilk samples had measurable neutralizing activity. Blood from mothers (1:400 dilution) had variable IgA responses but consistent IgG antibodies against Spike protein. In contrast, blood from infants only contained detectable Spike IgG and IgA antibodies if their mothers had COVID infection or vaccination during pregnancy, with no evidence that breastmilk antibodies transfer into the infant circulation. Conclusion: COVID vaccination during lactation is well tolerated with few side-effects and generates a strong immune response. Secretory IgA antibodies are routinely detected in breastmilk and have viral neutralizing activity, supporting wider immunization among breastfeeding mothers.

Interferon-mediated Responses to Human Metapneumovirus

Background: Acute respiratory infection (ARI) is the leading infectious cause of pediatric death worldwide, comprising 15% of all deaths in children under 5 years old. Human metapneumovirus (HMPV) is a primary cause of ARI, and accounts for a major portion of ARI-related hospitalizations in infants and young children. Although nearly every person is infected with HMPV during early childhood, re-infections occur often, highlighting the difficulty in building long-term immunity. There are no approved vaccines or antiviral therapies. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways and potential therapeutic targets. Type I (IFN-α/β) and III interferons (IFN-λ) display antiviral activity against numerous respiratory viruses and are currently being investigated for therapeutic use in several respiratory infections including SARS-CoV-2. However, their roles in HMPV infection remain largely unknown. Our laboratory has previously shown that type I IFN is critical for HMPV pathogenesis, as loss of IFN-α/β signaling reduces lung inflammation and lessens HMPV disease severity in mice. Here, we describe distinct antiviral roles for type I and III IFNs during HMPV infection using an established mouse model. Methods: In vivo studies were conducted using mice lacking either the IFN-α/ β receptor (IFNAR-/-) or IFN-λ receptor (IFNLR-/-). Early immune responses to HMPV strains TN/94-49 and C2-202 were assessed by clinical disease scoring, plaque assay, Luminex immunoassay, and spectral cytometry of mouse lung samples. In vitro studies were performed using CMT 64-61 mouse bronchial epithelial cells. Responses to TN/94-49 and C2-202 were measured by qPCR, plaque assay, and Luminex immunoassay of cell lysates and supernatants. Results: IFNAR-/- mice exhibited lower clinical disease scores, reduced lung levels of inflammatory cytokines IL6, MIP-1α, and MCP-1, and decreased numbers of lung interstitial macrophages during HMPV infection, highlighting their critical role in HMPV immune-mediated pathogenesis. IFNLR-/- mice with intact IFNAR showed moderate clinical disease, higher lung levels of inflammatory cytokines IL-6, MCP-1, and IFN-γ, and increased lung interstitial macrophage recruitment. A reduction in HMPV disease was also recapitulated by IFNAR-neutralizing antibody treatment of IFNLR-/- mice. Interestingly, IFNLR-/- showed higher HMPV viral titers, while IFNAR-/- mice showed no differences or slightly lower viral titers, compared to wild-type mice. Moreover, IFN-λ pre-treatment of infected CMT 64-61 cells reduced HMPV viral titers and decreased supernatant levels of inflammatory cytokines IL-6, IL-1β, TNFα, and MCP-1. Conclusion: These findings suggest that type I IFN is necessary for HMPV pathogenesis, while type III IFN is critical for limiting HMPV replication in the lungs but does not contribute to HMPV inflammatory disease. This work uncovers key functional differences between type I and III IFNs during HMPV infection, an important feature of innate immune responses to HMPV that may be utilized to inform treatment.

Vitamin D reduces the binding effects of S1 (spike) and N (nucleocapsid) proteins of the SARS-CoV-2 virus to MRC-5 lung fibroblast cells

In this study, the in vitro role of vitamin D on cell proliferation and the toxicity of MRC-5 cells induced by the presence of S1 (spike) and N (nucleocapsid) proteins of SARS-CoV2 virus were investigated. Vitamin D was used in various treatment protocols for COVID-19 before vaccination. The S1 and N protein, produced by molecular biology techniques at the Faculty of Chemistry, University of Belgrade, within the CAPSIDO project, were used to stimulate MRC-5 cells during 24 and 48 h cell culture. S1 protein was used in a concentration from 6.0 x10-2ng/μl to 7.5x10-4 ng/μl, and N protein from 12.00 x10-2 ng/μl to15.00 x10-4ng/μl. Cells were analyzed morphologically as well as on the basis of MTT test. The cytokine IL-6, LDH and AST enzymes as well as potassium were determined in the supernatant to analyze the degree of cell membrane damage. The results showed significantly different effects of S1 protein in relation to concentrations and time of incubation. In addition, S1 showed most prominent effects compared to N protein. The effects of S1 protein showed a statistically significant increase in the release of intracellular enzyme AST and intracellular potassium, depending on the concentration and incubation time (ANOVA, p < 0.05), while LDH and IL-6 were not detected under the tested conditions. Vitamin D (VigantolR) (20,000 IU/mL) significantly reduced the release of AST and potassium after S1 protein-induced treatment, while it was less pronounced on the effects caused by N protein. These preliminary results indicated that S1 protein, most likely due to its specific structure relative to N protein, after binding to appropriate ACE receptors and without the presence of the whole viral particle, can lead to changes in target cells and induce their necrosis depending on concentration. Vitamin D appears to at least partially reduce the direct binding of S1 protein to target cells and reverse its effects.

Drug contamination in Baikal endemic amphipods

This study was aimed to detect trace amounts of drugs in littoral crustaceans of ancient Lake Baikal. For the first step, these drugs were presented by ibuprofen and azithromycin. Ibuprofen is widely used to reduce fever and pain. Azithromycin is an antimalarial drug, that was recently reported to be active against acute respiratory coronavirus syndrome. Endemic crustaceans related to species Eulimnogammarus verrucosus were collected near village B. Goloustnoe. Animals were homogenized with acetonitrile and shaken intensively. Then precipitation of proteins was performed with a solution of TCA, and centrifuged. The supernatant was removed and filtered in chromatographic vials. The analysis was performed using an HPLC Agilent 1290 Infinity coupled with Agilent 6470 Triple Quadrupole. Mobile phase A consisted of 0.1% formic acid in the water, and mobile phase B was presented by 100% acetonitrile. The registration of ions was performed in different modes, such as: Full scan, MRM, Product Ion. For reliable identification of drug contaminants, we used the pharmaceutical substances of azithromycin and ibuprofen. Thus, here we first time reliably detected drugs in the amphipods of Lake Baikal and demonstrated the contamination of the Baikal ecosystem by azithromycin and ibuprofen. It indicates the ability to accumulate the drug substances from wastewater by amphipods, and increasing anthropogenic load on the ancient lake.

Impaired immunity in elderly, vaccinated residents of an long-term care facility affected by a COVID-19 outbreak

The ongoing COVID-19 pandemic has hit long-term care facilities (LTCF), with outbreaks affecting both residents and health care workers (HCWs). Elderly persons have been prioritized in the implementation of vaccination programs. Here we investigated a COVID-19 outbreak, caused by the Beta variant (B.1.351) in a LTCF where residents and HCWs had received 2 doses of Comirnaty vaccine (Pfizer/BioNTech) until one month before the outbreak. Samples from 14 residents (SARS-CoV-2 PCR-negative: n = 8, PCR-positive: n = 6) and 10 HCWs (PCR-negative: n = 10) were collected at a median of 54 days following the second vaccine dose. IgG antibodies to SARS-CoV-2 spike glycoprotein and neutralizing antibody (NAb) titers were measured. Additionally, functional responses of PBMCs to SARS-CoV-2 spike and nucleocapsid proteins were investigated. We observed that Comirnaty induced higher IgG concentrations and NAb titers in HCWs compared to residents. PBMCs of HCWs responded vigorously to stimulation with SARS-CoV-2 spike glycoprotein, with the secretion of interferon gamma, granzyme B and perforin-1 into supernatants. In comparison, only 3 of 9 samples from residents showed positive cellular responses to spike glycoprotein. Group-level cellular responses directed at SARS-CoV-2 nucleoprotein remained low both in HCWs and in residents. Only 2 of 2 PCR-positive residents showed a positive response consistent with exposure to SARS-CoV-2 breakthrough infection. Our results show that elderly persons are at increased risk for breakthrough infection after vaccination. Weak vaccine-directed responses in the elderly need to be addressed in vaccination protocols.

Which East Asian herbal medicines can decrease viral infections?

Introduction In the light of the ongoing corona and influenza virus public health crisis, traditional East Asian herbal medicines with anti-viral activities might be an option in therapy. Methods Literature research on anti-viral effects of traditional East Asian medicine was performed. Results In patients with uncomplicated upper respiratory tract infection, treatment with Andrographis paniculata (Jap. Senshinren) extract resulted in 53% of improvement compared with placebo. When 158 common cold patients took 1.2 g of dried extract of A. paniculata for 5 days, nasal secretion, sore throat and sleep disorder were improved [1]. Ding Y et al. [2] demonstrated that mice infected with influenza A and treated with extract of A. paniculata improved body weight, lung function and showed reduced inflammation. In Japan, Kampo prescriptions like Maoto as well as its variant Maoto-ka-senshinren ([Tab. 1]), were examined for anti-viral activity [3] [4]. Nabeshima et al. [5] investigated Maoto for the treatment of influenza in a randomized trial in comparison with oseltamivir and zanamivir. No significant differences for total symptom score and no severe adverse events were found. In A549 cells infected with influenza A virus that were treated with Maoto, the virus titre in the supernatant, intracellular viral proteins and viral RNA were significantly reduced. Maoto also inhibited the uncoating of the influenza virus [6]. Conclusion In Japan, Maoto is regarded as a suitable medication for influenza. Maoto-ka-senshinren ([Tab. 1]) may present a promising therapy option for influenza and potentially COVID-19.

Activation of immune receptor RIG-I protects human differentiated epithelial cells of the upper respiratory tract from SARS-CoV-2

SARS-CoV-2 virus has evolved non structural proteins (NSLs) to avoid recogniton by innate immune receptors such as RIG-I which induces an antiviral type I IFN response. A proper type I IFN response is essential for an effective defense against SARS-CoV-2. The aim of this project is the evaluation of the effect of a prophylactic activation of RIG-I by a specific synthetic RNA ligand (5-triphosphate RNA, 3pRNA) to an antiviral state in cells that protects against SARSCoV- 2. We analysed the effect of activation of RIG-I by 3pRNA on SARS-CoV-2 infection in primary upper airway cells. Primary nasal epithelial cells (PNEC) and primary bronchial cells (NHBEC) were cultivated in air-liquid interface and differentiated to cilia bearing epithelial cells. Viral replication was analyzed by plaque assay and qPCR. Furthermore, interferon-stimulated genes and ACE2/TMPRSS2 were determined on the transcriptional level and IP-10 protein was quantified in supernatants by ELISA. Only PNEC in a fully differentiated state could be infected with SARS-CoV-2, while undifferentiated cells were not susceptible to infection with this virus. Upon stimulation with 3pRNA at 6h prior to infection, PNEC produced more than 10.000 pg/ml IP-10. Infection with SARS-CoV-2 significantly decreased the average of 3pRNA-induced IP-10 production. 3pRNA pretreatment of PNEC significantly reduced SARS-CoV-2 replication at 48 h by up to 99,7 % as evaluated by plaque assay and up to 90 % as measured by qPCR. These results demonstrate for the first time that RIG-I activation protects primary fully differentiated epithelial cells against SARS-CoV-2 replication. Our results support the concept that RIG-I-mediated prophylaxis is a promising strategy to mitigate SARS-CoV-2 infection.