Authors: Sphamandla E Mtambo, Samuel C Ugbaja, Aganze G Mushebenge...

The H7N9 virus attaches itself to the human cell receptor protein containing the polysaccharide that terminates with sialic acid. The mutation of neuraminidase at residue E119 has been explored experimentally. However, there is no adequate information on the substitution with E119V in peramivir at the intermolecular level. Therefore, a good knowledge of the interatomic interactions is a prerequisite in understanding its transmission mode and subsequent effective inhibitions of the sialic acid receptor cleavage by neuraminidase. Herein, we investigated the mechanism and dynamism on the susceptibility of the E119V mutation on the peramivir-neuraminidase complex relative to the wildtype complex at the intermolecular level. This study aims to investigate the impact of the 119V substitution on the neuraminidase-peramivir complex and unveil the residues responsible for the complex conformations. We employed molecular dynamic (MD) simulations and extensive post-MD analyses in the study. These extensive computational investigations were carried out on the wildtype and the E119V mutant complex of the protein for holistic insights in unveiling the effects of this mutation on the binding affinity and the conformational terrain of peramivir-neuraminidase E119V mutation. The calculated total binding energy (ΔG) for the peramivir wildtype is -49.09 ± 0.13 kcal/mol, while the E119V mutant is -58.55 ± 0.15 kcal/mol. The increase in binding energy (9.46 kcal/mol) is consistent with other post-MD analyses results, confirming that E119V substitution confers a higher degree of stability on the protein complex. This study promises to proffer contributory insight and additional knowledge that would enhance future drug designs and help in the fight targeted at controlling the avian influenza H7N9 virus. Therefore, we suggest that experimentalists collaborate with computational chemists for all investigations of this topic, as we have done in our previous studies.

Topics: Neuraminidase

PubMed: 35268741
DOI: 10.3390/molecules27051640

Authors: Muchtaridi Muchtaridi, Rina Fajri Nuwarda, Emmy Hainida Khairul Ikram...

Neuraminidase (NA) is an enzyme that prevents virions from aggregating within the host cell and promotes cell-to-cell spread by cleaving glycosidic linkages to sialic acid. The best-known neuraminidase is the viral neuraminidase, which present in the influenza virus. Thus, the development of anti-influenza drugs that inhibit NA has emerged as an important and intriguing approach in the treatment of influenza. L. (GA) dried fruits (GAF) are used commercially as seasoning and in beverages. The main objective of this study was to identify a new potential neuraminidase inhibitor from GA. A bioassay-guided fractionation method was applied to obtain the bioactive compounds leading to the identification of garcinia acid and naringenin. In an enzyme inhibition study, garcinia acid demonstrated the highest activity when compared to naringenin. Garcinia acid had the highest activity, with an IC of 17.34-17.53 µg/mL or 91.22-92.21 µM against -NA, and 56.71-57.85 µg/mL or 298.32-304.31 µM against H1N1-NA. Based on molecular docking results, garcinia acid interacted with the triad arginine residues (Arg118, Arg292, and Arg371) of the viral neuraminidase, implying that this compound has the potential to act as a NA enzyme inhibitor.

Topics: Antiviral Agents; Enzyme Inhibitors; Fruit; Garcinia; Humans; Neuraminidase; Plant Extracts; Plant Leaves; Viral Proteins

PubMed: 35164214
DOI: 10.3390/molecules27030949

A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model.

Authors: Shirin Strohmeier, Fatima Amanat, Xueyong Zhu...

Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses. Influenza virus infections remain a high risk to human health, causing up to 650,000 deaths worldwide every year, with an enormous burden on the health care system. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed. Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Topics: Amino Acid Sequence; Animals; Antibodies, Viral; Antigenic Drift and Shift; Cross Reactions; Humans; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Influenza, Human; Measles virus; Mice; Mice, Inbred BALB C; Neuraminidase; Phosphoproteins; Protein Domains; Sequence Alignment; Vaccination; Viral Proteins

PubMed: 34809451
DOI: 10.1128/mBio.02241-21

Review

Authors: Sarah Creytens, Mirte N Pascha, Marlies Ballegeer...

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

Topics: Antibodies, Viral; Antigenic Drift and Shift; Antigens, Viral; Catalytic Domain; Cross Protection; Evolution, Molecular; Humans; Immunogenicity, Vaccine; Influenza Vaccines; Influenza, Human; Influenzavirus A; Influenzavirus B; Mutation; Nanoparticles; Neuraminidase; Vaccines, Synthetic; Viral Proteins

PubMed: 34868073
DOI: 10.3389/fimmu.2021.786617

Evolution and Antigenic Advancement of N2 Neuraminidase of Swine Influenza A Viruses Circulating in the United States following Two Separate Introductions from Human Seasonal Viruses.

Authors: Bryan S Kaplan, Tavis K Anderson, Jennifer Chang...

Two separate introductions of human seasonal N2 neuraminidase genes were sustained in U.S. swine since 1998 (N2-98) and 2002 (N2-02). Herein, we characterized the antigenic evolution of the N2 of swine influenza A virus (IAV) across 2 decades following each introduction. The N2-98 and N2-02 expanded in genetic diversity, with two statistically supported monophyletic clades within each lineage. To assess antigenic drift in swine N2 following the human-to-swine spillover events, we generated a panel of swine N2 antisera against representative N2 and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assay and antigenic cartography. The antigenic distance between swine and human N2 was smallest between human N2 circulating at the time of each introduction and the archetypal swine N2. However, sustained circulation and evolution in swine of the two N2 lineages resulted in significant antigenic drift, and the N2-98 and N2-02 swine N2 lineages were antigenically distinct. Although intralineage antigenic diversity was observed, the magnitude of antigenic drift did not consistently correlate with the observed genetic differences. These data represent the first quantification of the antigenic diversity of neuraminidase of IAV in swine and demonstrated significant antigenic drift from contemporary human seasonal strains as well as antigenic variation among N2 detected in swine. These data suggest that antigenic mismatch may occur between circulating swine IAV and vaccine strains. Consequently, consideration of the diversity of N2 in swine IAV for vaccine selection may likely result in more effective control and aid public health initiatives for pandemic preparedness. Antibodies inhibiting the neuraminidase (NA) of IAV reduce clinical disease, virus shedding, and transmission, particularly in the absence of neutralizing immunity against hemagglutinin. To understand antibody recognition of the genetically diverse NA in U.S. swine IAV, we characterized the antigenic diversity of N2 from swine and humans. N2 detected in swine IAV were derived from two distinct human-to-swine spillovers that persisted, are antigenically distinct, and underwent antigenic drift. These findings highlight the need for continued surveillance and vaccine development in swine with increased focus on the NA. Additionally, human seasonal N2 isolated after 2005 were poorly inhibited by representative swine N2 antisera, suggesting a lack of cross-reactive NA antibody-mediated immunity between contemporary swine and human N2. Bidirectional transmission between humans and swine represents a One Health challenge, and determining the correlates of immunity to emerging IAV strains is critical to mitigating zoonotic and reverse-zoonotic transmission.

Topics: Animals; Antigenic Variation; Antigens, Viral; Cross Reactions; Epitopes; Evolution, Molecular; Genetic Variation; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Influenza A Virus, H3N2 Subtype; Influenza A virus; Influenza, Human; Neuraminidase; Orthomyxoviridae Infections; Seasons; Swine; Swine Diseases; United States; Virus Shedding

PubMed: 34379513
DOI: 10.1128/JVI.00632-21

Authors: Ashley C Campbell, John J Tanner, Kurt L Krause...

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

Topics: Antiviral Agents; Drug Discovery; Drug Resistance, Viral; Enzyme Inhibitors; HEK293 Cells; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Molecular Conformation; Neuraminidase; Protein Binding; Viral Proteins

PubMed: 34696326
DOI: 10.3390/v13101893

Authors: María Del Mar Fernández-Arjona, Ana León-Rodríguez, María Dolores López-Ávalos...

The administration of microbial neuraminidase into the brain ventricular cavities of rodents represents a model of acute aseptic neuroinflammation. Ependymal cell death and hydrocephalus are unique features of this model. Here we demonstrate that activated microglia participates in ependymal cell death. Co-cultures of pure microglia with ependymal cells (both obtained from rats) were performed, and neuraminidase or lipopolysaccharide were used to activate microglia. Ependymal cell viability was unaltered in the absence of microglia or inflammatory stimulus (neuraminidase or lipopolysaccharide). The constitutive expression by ependymal cells of receptors for cytokines released by activated microglia, such as IL-1β, was demonstrated by qPCR. Besides, neuraminidase induced the overexpression of both receptors in ventricular wall explants. Finally, ependymal viability was evaluated in the presence of functional blocking antibodies against IL-1β and TNFα. In the co-culture setting, an IL-1β blocking antibody prevented ependymal cell death, while TNFα antibody did not. These results suggest that activated microglia are involved in the ependymal damage that occurs after the administration of neuraminidase in the ventricular cavities, and points to IL-1β as possible mediator of such effect. The relevance of these results lies in the fact that brain infections caused by neuraminidase-bearing pathogens are frequently associated to ependymal death and hydrocephalus.

Topics: Animals; Cell Death; Cells, Cultured; Ependyma; Interleukin-1beta; Lipopolysaccharides; Male; Microglia; Neuraminidase; Rats; Rats, Wistar

PubMed: 33757539
DOI: 10.1186/s12987-021-00249-0

Authors: Ning Ma, Xingjie Li, Hongyu Jiang...

Influenza A viruses cause severe respiratory illnesses in humans and animals. Overreaction of the innate immune response to influenza virus infection results in hypercytokinemia, which is responsible for mortality and morbidity. However, the mechanism by which influenza induces hypercytokinemia is not fully understood. In this study, we established a mouse-adapted H9N2 virus, MA01, to evaluate the innate immune response to influenza in the lung. MA01 infection caused high levels of cytokine release, enhanced pulmonary injury in mice, and upregulated CD83 protein in dendritic cells and macrophages in the lung. Influenza virus neuraminidase (NA) unmasked CD83 protein and contributed to high cytokine levels. Furthermore, we provide evidence that CD83 is a sialylated glycoprotein. Neuraminidase treatment enhanced lipopolysaccharide (LPS)-stimulated NF-κB activation in RAW264.7 cells. Anti-CD83 treatment alleviated influenza virus-induced lung injury in mice. Our study indicates that influenza virus neuraminidase modulates CD83 status and contributes to the "cytokine storm," which may suggest a new approach to curb this immune injury. The massive release of circulating mediators of inflammation is responsible for lung injury during influenza A virus infection. This phenomenon is referred to as the "cytokine storm." However, the mechanism by which influenza induces the cytokine storm is not fully understood. In this study, we have shown that neuraminidase unmasked CD83 protein in the lung and contributed to high cytokine levels. Anti-CD83 treatment could diminish immune damage to lung tissue. The NA-CD83 axis may represent a target for an interruption of influenza-induced lung damage.

Topics: Animals; Antigens, CD; Dendritic Cells; Female; Host-Pathogen Interactions; Immunity, Innate; Immunoglobulins; Influenza A Virus, H9N2 Subtype; Lung Injury; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Neuraminidase; Orthomyxoviridae Infections; Signal Transduction; Viral Proteins; Virulence

PubMed: 33177200
DOI: 10.1128/JVI.01753-20

Authors: Won Ho Yang, Julia S Westman, Douglas M Heithoff...

Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Typhimurium (), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4-dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.

Topics: Animals; Colitis; Disease Models, Animal; Female; Humans; Intestines; Male; Mice; Mice, Inbred C57BL; Neuraminidase; Recurrence; Salmonella Food Poisoning; Salmonella typhimurium; Toll-Like Receptor 4

PubMed: 34266954
DOI: 10.1073/pnas.2100937118

Authors: Junfang Wu, Mingming Zhao, Haoran Wei...

AIMS

The aim of this study was to investigate the effects of Neuraminidase inhibitors (NI) on COVID-19 in a retrospective study.

METHODS AND RESULTS

The study included an overall COVID-19 patients (n = 3267) and a 1:1 propensity score-matched patients (n = 972). The levels of plasma N-acetylneuraminic acid and neuraminidase expression were further evaluated in a panel of hospitalized and 1-month post-infection recovered COVID-19 subjects. The mortality rate in the overall patients was 9.6% (313/3267) and 9.2% (89/972) in the propensity-score matched patients. The NI treatment lowered the mortality rate (5.7% vs. 10.3%) and the critically ill conversion rate (14.1% vs. 19.7%) compare to those in the non-NI group in the overall patients and evaluated in the propensity score-matched patients when applying the multivariate Cox model for adjusting imbalanced confounding factors. Furthermore, NI treatment was associated with attenuated cytokine storm levels and acute heart injury but not liver or kidney injuries. Further analysis in a small panel of patients found the levels of N-acetylneuraminic acid and neuraminidase (dominantly the NEU3 isoform) were elevated in the hospitalized COVID-19 subjects and recovered at the 1-month post-infection stage, suggesting increasing desialylation in COVID-19 patients.

CONCLUSION

These results suggest that NI treatment is associated with decreased mortality in COVID-19 subjects, especially for those subjects with acute heart injury.

Topics: Antiviral Agents; COVID-19; Cardiovascular Diseases; Humans; N-Acetylneuraminic Acid; Neuraminidase; Retrospective Studies

PubMed: 35294004
DOI: 10.1093/ehjcvp/pvac018