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Mucins are a family of large glycoproteins that represent the major structural components of the mucus and are encoded by 20 different mucin genes. Mucin expression can be modulated by different stimuli. In this study, we analyzed four mucins (MUC2, MUC3, MUC13, and MUC17) in coculture of Caco‐2/HT29‐MTX cells to demonstrate the variation in gene expression in the presence of antioxidant compounds like chlorogenic acid, epicatechin gallate, and quercetin (apple, tea, and coffee polyphenols, respectively). coculture of Caco‐2/HT29‐MTX cells was treated with polyphenols, and the expression of four mucins was determined by reverse‐transcriptase PCR. In addition, the secretion levels of MUC2 were established by enzyme‐linked immunoassay (ELISA) analysis. The results showed that each polyphenol compound induces different expression patterns of the mucin genes. Statistically significant up‐regulation of MUC17 was observed following incubation with epicatechin gallate and quercetin. ELISA results did not prove any significant differences in protein levels of MUC2 after treatment by the polyphenol compounds. The polyphenols considered in this study may influence mucin secretion and act on diverse salivary substrates to change the barrier properties of mucins for mucus secretion in different ways. The effects of dietary polyphenols on mucin secretion and expression have been studied to some extent, for that reason the present study investigated the use of two human adenocarcinoma cell lines in co‐culture of Caco‐2/HT29‐MTX, as a model to mimic gut epithelium, in order to examine the effect of three dietary plant polyphenols (chlorogenic acid, epicatechin gallate, and quercetin) on the gene expression of four mucins. Our research highlights the insights into the extent to which the polyphenols may influence mucin secretion and act on mucus barrier properties.

In recent years, new direct-acting antivirals for hepatitis C virus (HCV) have been approved, but hepatitis C continues to pose a threat to human health. It is important to develop neutralizing anti-HCV antibodies to prevent medical and accidental infection, such as might occur via liver transplantation of chronic HCV patients and needle-stick accidents in the clinic. In this study, we sought to obtain anti-HCV antibodies using phage display screening. Phages displaying human hepatocellular carcinoma patient-derived antibodies were screened by 4 rounds of biopanning with genotype-1b and -2a HCV envelope E2 protein adsorbed to magnetic beads. The three antibodies obtained from this screen had reactivity against E2 proteins derived from both genotype-1b and -2a strains. However, in epitope analysis, these antibodies did not recognize linear peptides from an overlapping E2 epitope peptide library, and did not bind to denatured E2 protein. In addition, these antibodies showed cross-genotypic neutralizing activity against genotype-1a, -1b, -2a, and -3a cell culture-generated infectious HCV particles (HCVcc). Moreover, emergence of viral escape mutants was not observed after repeated rounds of passaging of HCV-infected cells in the presence of one such antibody, e2d066. Furthermore, injection of the e2d066 antibody into human hepatocyte-transplanted immunodeficient mice inhibited infection by J6/JFH-1 HCVcc. In conclusion, we identified conformational epitope-recognizing, cross-genotypic neutralizing antibodies using phage display screening. Notably, e2d066 antibody did not select for escape mutant emergence in vitro and demonstrated neutralizing activity in vivo. Our results suggested that these antibodies may serve as prophylactic and therapeutic agents.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.

Background: Since the onset of the global COVID-19 pandemic in early 2020, numerous studies have been conducted worldwide to understand our immune response to the virus and to vaccination. This study investigates the humoral response elicited by SARS-CoV-2 infection and by vaccination in the poorly studied population of food and retail workers. These occupations were classified as essential by the Public Health Agency of Canada, potentially placing this population at greater risk of infection. Such a risk requires access to reliable and adaptable serological assays that can be rapidly deployed to guide public health strategies. Here we investigate the benefits and limitations of applying adaptable, decentralized tests for population-level immune surveillance in response to a pandemic, even before centralized testing is available. Methods and findings: The 1.5-year study period spans from early 2021, when vaccination became available in this region, to mid-2022, following the emergence of the first Omicron variants. The cohort of 304 food and retail workers was recruited in the Québec City area. Participants attended five evenly spaced visits, providing blood samples as well as information on SARS-CoV-2 symptoms or risk factors, prior antigen or PCR test results and vaccination status, as well as work-related risk factors and protective measures. Parallel COVID-19 serological assays were performed using both a standardized chemiluminescent ELISA assay at the centralized platform operated in partnership with the Public Health Agency of Canada, and a semi-automated in-house colorimetric ELISA assay developed at our decentralized site. The YES/NO determination of SARS-CoV-2 vaccine seroconversion and/or infection events using the SARS-CoV-2 ancestral spike protein and nucleocapsid protein validated coherence of the centralized and decentralized assays. The flexibility of the decentralized assays allowed broadening the study to determine cross-reactivity of IgG directed against the spike protein of the SARS-CoV-2 Delta and Omicron VOCs, and IgM directed against the ancestral spike and nucleocapsid proteins. The nature of the data obtained in the decentralized assays allowed treatment with a recently developed mathematical transformation to obtain normal distribution, enabling ANOVA-Welsh statistical analysis. Although no significant differences were observed in humoral response as related to BMI, age, level of education, or chronic illnesses in this cohort of workers, statistically higher levels of vaccine-induced antibodies were observed for restaurant workers and hardware store workers in the early stages of the study, compared to workers in bars and grocery stores and in non-smokers versus smokers. Conclusions: This work highlights the importance of developing adaptable, decentralized tests for population-level immune surveillance in response to a pandemic, even before centralized testing is available. To our knowledge, no other study has reported such an extensive longitudinal investigation during key periods of the COVID-19 pandemic in a cohort of food and retail workers to analyze two types of immunoglobulin, three epitopes and antigens to three VOC. This study will inform strategies and measures to be implemented in the event of a future pandemic.

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, tick-borne viral disease. Humans are the only species known to develop illness after CCHF virus (CCHFV) infection, characterized by a nonspecific febrile illness that can progress to severe, often fatal, hemorrhagic disease. A variety of animals may serve as asymptomatic reservoirs of CCHFV in an endemic cycle of transmission. Seroepidemiological studies have been instrumental in elucidating CCHFV reservoirs and in determining endemic foci of viral transmission. Herein, we review over 50 years of CCHFV seroepidemiological studies in domestic and wild animals. This review highlights the role of livestock in the maintenance and transmission of CCHFV, and provides a detailed summary of seroepidemiological studies of wild animal species, reflecting their relative roles in CCHFV ecology.

The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm. A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard. Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT. The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited.

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.