Development of an AlphaLISA assay for sensitive and accurate detection of influenza B virus.

Objective: Influenza B virus (IBV) is highly contagious, spreads rapidly, and causes seasonal epidemic respiratory disease in the human population, especially in immunocompromised people and young children. Clinical manifestations in this high-risk population are often more severe than in immunocompetent hosts and sometimes atypical. Therefore, rapid, and accurate detection of IBV is important. Methods: An amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) was developed for detection of IBV by optimizing the ratio of IBV antibody-labeled receptor beads, streptavidin-conjugated donor beads and biotinylated IBV antibody, as well as the optimal temperature and time conditions for incubation. Assay sensitivity, specificity and reproducibility were evaluated. A total of 228 throat swab samples and inactivated influenza B virus were tested by AlphaLISA and lateral flow colloidal gold-based immunoassay (LFIA). Results: AlphaLISA produced the best results for detection of inactivated influenza B virus when IBV antibody-labeled acceptor beads were 50 µg/ mL, streptavidin-conjugated donor beads were 40 µg/mL, and biotinylated IBV antibody was 0.5 µg/mL at 37°C for 15-10 min. Under these conditions, AlphaLISA had a limit of detection of 0.24 ng/mL for the detection of influenza B nucleoprotein, did not cross react with other common respiratory viruses, and showed good reproducibility with inter-assay coefficient of variation (CV) and intra-assay CV < 5%. The results of 228 clinical throat swab samples showed good agreement between AlphaLISA and LFIA (Kappa = 0.982), and AlphaLISA showed better sensitivity than LFIA for detecting inactivated influenza B virus. Conclusion: AlphaLISA showed higher sensitivity and throughput in the detection of IBV and can be used for IBV diagnosis and epidemic control.

Improvement influenza vaccine immune responses with traditional Chinese medicine and its active ingredients.

The current influenza vaccines are unable to provide effective protection in many cases, like influenza viruses strain antigenic drift or shift, and the influenza continues to cause significant annual morbidity and mortality. Improving the immune response to influenza vaccination is an unmet need. Traditional Chinese medicine (TCM) and its active ingredients are commonly known to have immunomodulatory properties. We therefore compared influenza vaccination alone or formulated with Astragali Radix (Huangqi in Chinese), and several representative ingredients of TCM, including lentinan (polysaccharide), panax notoginseng saponins (saponin), breviscapine (flavone), andrographolide (terpenoid), and a Chinese herbal compound (kangai) for their potential to enhance immune responses to influenza vaccine in mice. We found that all these TCM-adjuvants were able to increase hemagglutination inhibition (HAI) antibody titers, splenocyte proliferation, splenic T cell differentiation, bone marrow dendritic cell maturity, and both Th1 and Th2 cytokine secretion of influenza vaccine to varying degrees, and that had the characteristics of no excessive inflammatory responses and bidirectional regulation simultaneously. Taken together, our findings show that Astragali Radix exerts a more comprehensive effect on vaccine immunity, on both innate and adaptive immunity. The effects of lentinan and andrographolide on adaptive immunity were more significant, while the effects of breviscapine on innate immunity were stronger, and the other two TCM adjuvants were weaker. As the first report of a comprehensive evaluation of TCM adjuvants in influenza vaccines, the results suggest that TCM and their active ingredients are good candidates for enhancing the immune response of influenza vaccines, and that suitable TCMs can be selected based on the adjuvant requirements of different vaccines.

Bidirectional and persistent immunomodulation of Astragalus polysaccharide as an adjuvant of influenza and recombinant SARS-CoV-2 vaccine.

Respiratory viral infections, such as coronavirus disease of 2019 (COVID-19) and influenza, cause significant morbidity and mortality and have become a worldwide public health concern with tremendous economic and societal burdens. Vaccination is a major strategy for preventing infections. However, some new vaccines have an unmet need for impairing responses in certain individuals, especially COVID-19 vaccines, despite ongoing vaccine and adjuvant research. Here, we evaluated the effectiveness of Astragalus polysaccharide (APS), a bioactive polysaccharide extracted from the traditional Chinese herb Astragalus membranaceus as an immune adjuvant to regulate the efficacy of influenza split vaccine (ISV) and recombinant severe acute respiratory syndrome (SARS)-Cov-2 vaccine in mice. Our data indicated that APS as an adjuvant can facilitate the induction of high levels of hemagglutination inhibition (HAI) titer and specific antibody immunoglobulin G (IgG) and confer protection against the lethal challenge of influenza A viruses, including increased survival and amelioration of weight loss in mice immunized with the ISV. RNA sequencing (RNA-seq) analysis revealed that the NF-κB and Fc gamma R-mediated phagocytosis signaling pathways are essential for the immune response of mice immunized with the recombinant SARS-Cov-2 vaccine (RSV). Another important finding was that bidirectional immunomodulation of APS on cellular and humoral immunity was observed, and APS-adjuvant-induced antibodies persisted at a high level for at least 20 weeks. These findings suggest that APS is a potent adjuvant for influenza and COVID-19 vaccines, and has the advantages of bidirectional immunoregulation and persistent immunity.

NLRP6 Induces Lung Injury and Inflammation Early in Brucella and Influenza Coinfection.

(1) Background: With the resurgence of brucellosis epidemics in China in recent years, the chances of a brucella coinfection with other common respiratory pathogens, such as the influenza virus, have increased dramatically. However, little is known about the pathogenicity or the mechanisms of brucella and influenza coinfections. (2) Methods: To clarify the interventions in the early stages of lung damage due to brucella and influenza coinfections, we evaluated the effect of the coinfection on disease progression and mortality using a coinfection model in WT mice and NLRP6-/- mice, and we verified the function of NLRP6 in infection and proinflammation. (3) Results: The coinfection induced significant respiratory symptoms, weight loss, and a high mortality rate in WT mice. Influenza in the coinfection group significantly increased brucella proliferation in a synergistic manner. Meanwhile, a histological examination showed severe lung tissue destruction and excessive inflammatory responses in coinfected WT animals, and the expression of NLRP6 and IL-18 was dramatically increased in the lung tissues. Furthermore, NLRP6 deletion attenuated lung injuries and inflammation, a reduced bacterial load, and decreased IL-18 protein expression. (4) Conclusions: Our findings indicated that NLRP6 plays a critical role and might be a promising potential therapeutic target for brucella-influenza coinfections.

Exploring the active ingredients and pharmacological mechanisms of the oral intake formula Huoxiang Suling Shuanghua Decoction on influenza virus type A based on network pharmacology and experimental exploration.

Objective: To investigate the active ingredients, underlying anti-influenza virus effects, and mechanisms of Huoxiang Suling Shuanghua Decoction (HSSD). Materials and methods: The therapeutic effect of HSSD were confirmed through the survival rate experiment of H1N1-infected mice. Then, the HSSD solution and the ingredients absorbed into the blood after treatment with HSSD in rats were identified by UPLC/Q-TOF MS, while the main contents of ingredients were detected by high performance liquid chromatography (HPLC). Next, a systems pharmacology approach incorporating target prediction, gene ontology (GO) enrichment, kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, and molecular docking were performed to screen out the active compounds and critical pathways of HSSD in treating influenza. According to prediction results, real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry assay were used to detect the mRNA and protein expression levels of critical targets in H1N1-infected mice lungs. Results: Huoxiang Suling Shuanghua Decoction improved the survival rate of H1N1-infected mice and prolonged the mice's lifespan. Besides, HSSD exerts an antivirus effect by decreasing the levels of hemagglutinin (HA) and nucleoprotein (NP) to inhibit the replication and proliferation of H1N1, reducing the lung pathological state, inhibiting the cell apoptosis in the lung, and regulating the abnormal responses of peripheral blood, including GRA, LYM, white blood cell (WBC), PLT, and hemoglobin (HGB). Then, 87 compounds in the HSSD solution and 20 ingredients absorbed into the blood after treatment with HSSD were identified. Based on this, combined with the network analysis and previous research on antivirus, 16 compounds were screened out as the active components. Moreover, 16 potential targets were predicted by network pharmacology analysis. Next, molecular docking results showed stable binding modes between compounds and targets. Furthermore, experimental validation results indicated that HSSD regulates the contents of Immunoglobulin A (IgA), Immunoglobulin M (IgM), and Immunoglobulin G (IgG) in serum, modulating the levels of IFN-γ, IL-6, IL-10, MCP-1, MIP-1α, and IP-10 in the lung tissue, and significantly decreasing the mRNA and protein expressions of TLR4, CD14, MyD88, NF-κB p65, HIF1 α, VEGF, IL17A, and IL6 in the lung tissue. Conclusion: Huoxiang Suling Shuanghua Decoction exerts an anti-influenza effect by affecting the expressions of mRNA and protein including TLR4, CD14, MyD88, NF-kB p65, HIF-1α, VEGF, IL17A, IL6, and inhibiting the accumulation of inflammation. Our study provided experimental pieces of evidence about the practical application of HSSD in treating influenza.