In-silico evidence for enhancement of avian influenza virus H9N2 virulence by modulation of its hemagglutinin (HA) antigen function and stability during co-infection with infectious bronchitis virus in chickens

In the last few decades, frequent incidences of avian influenza (AI) H9N2 outbreaks have caused high mortality in poultry farms resulting in colossal economic losses in several countries. In Egypt, the co-infection of H9N2 with the infectious bronchitis virus (IBV) has been observed extensively during these outbreaks. However, the pathogenicity of H9N2 in these outbreaks remained controversial.
The current study reports isolation and characterization of the H9N2 virus recovered from a concurrent IBV infected broiler chicken flock in Egypt during 2011. The genomic RNA was subjected to RT-PCR amplification followed by sequencing and analysis. The deduced amino acid sequences of the eight segments of the current study H9N2 isolate were compared with those of Egyptian H9N2 viruses isolated from healthy and diseased chicken flocks from 2011 to 2013.
In the phylogenetic analysis, the current study isolate was found to be closely related to the other Egyptian H9N2 viruses. Notably, no particular molecular characteristic difference was noticed among all the Egyptian H9N2 isolates from apparently healthy, diseased or co-infected with IBV chicken flocks.
Nevertheless, in-silico analysis, we noted modulation of stability and motifs structure of Hemagglutinin (HA) antigen among the co-infecting H9N2 AI and the IBV and isolates from the diseased flocks. The findings suggest that the putative factor for enhancement of the H9N2 pathogenicity could be co-infection with other respiratory pathogens such as IBV that might change the HA stability and function.

Recombinant bluetongue virus with hemagglutinin epitopes in VP2 has potential as a labeled vaccine

Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact.
Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals.
To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2.
In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag.
Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(-/-) mice and sheep, and serological detection results of an anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags.
Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.

Functional Analysis of Botulinum Hemagglutinin (HA).

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most potent toxin and produced as a complex with non-toxic components. Food-borne botulism is caused by the ingestion of these BoNT complexes. Hemagglutinin (HA), one of the non-toxic components, is known to have lectin (carbohydrate binding) activity and E-cadherin-binding activity. These activities promote the intestinal absorption of BoNT.
To elucidate the mechanism of the onset of food-borne botulism, we focused on the role of HA in the intestinal absorption of BoNT. We describe the functional analysis methods for HA, including the expression of recombinant proteins, binding to glycoproteins and epithelial cells, and localization in mouse intestinal tissue.

Influenza Hemagglutinin Nanoparticle Vaccine Elicits Broadly Neutralizing Antibodies against Structurally Distinct Domains of H3N2 HA

Influenza vaccine effectiveness varies annually due to the fast evolving seasonal influenza A(H3N2) strain and egg-derived mutations-both of which can cause a mismatch between the vaccine and circulating strains.
To address these limitations, we have developed a hemagglutinin (HA)-based protein-detergent nanoparticle influenza vaccine (NIV) with a saponin-based Matrix-M™ adjuvant. In a phase 1 clinical trial of older adults, the vaccine demonstrated broadly cross-reactive A(H3N2) HA antibody responses.
Two broadly neutralizing monoclonal antibodies derived from NIV-immunized mice were characterized by transmission electron microscopy (TEM), antibody competition assays, fluorescence-activated cell sorting (FACS) analysis, and protein-protein docking. These antibodies recognize two conserved regions of the head domain, namely the receptor binding site and the vestigial esterase subdomain, thus demonstrating the potential for an HA subunit vaccine to elicit antibodies targeting structurally and antigenically distinct but conserved sites.
Antibody competition studies with sera from the phase 1 trial in older adults confirmed that humans also make antibodies to these two head domains and against the highly conserved stem domain. This data supports the potential of an adjuvanted recombinant HA nanoparticle vaccine to induce broadly protective immunity and improved vaccine efficacy.

Measuring influenza hemagglutinin (HA) stem-specific antibody-dependent cellular cytotoxicity (ADCC) in human sera using novel stabilized stem nanoparticle probes.

Generating vaccine that confers a complete protection is a major goal in designing a universal influenza vaccine. Currently, there is a considerable interest in the broadly neutralizing antibodies (bnAb) targeting the conserved HA stem region.
These antibodies have been shown to activate cellular immune responses, such as ADCC, in addition to their neutralization activity. We had previously demonstrated that immunization with H1-based stabilized stem (SS) nanoparticles (np) protects against heterosubtypic lethal H5N1 challenge, despite the absence of detectable neutralizing activity.
Utilizing these novel SS probes to develop an ADCC assay would help in understanding the mechanism of action of stem-specific antibodies, as well as evaluating future influenza vaccines.To develop a new protocol to assess the ADCC activity mediated by stem-directed antibodies in human sera using novel SS np probes. Human sera samples were screened for binding and ADCC activities to different influenza group 1 SS probes (H1, H2, and H5) using trimeric SS or multivalent SS-np (n = 8 trimers) formats.Initial screening revealed 63% (57/90) seroprevalence of anti-HA (H1) stem-epitope antibodies, as determined by the differential binding to HA SS and its corresponding epitope-mutant (Ile45Arg/Thr49Arg) probe.

HA Hemagglutinin (YPYDVPDYA)

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HA Hemagglutinin (YPYDVPDYA)

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HA Hemagglutinin (YPYDVPDYA)

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Hemagglutinin (HA Tag) Antibody

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Hemagglutinin (HA Tag) Antibody

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Hemagglutinin (HA Tag) Antibody

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HA Hemagglutinin (YPYDVPDYA)-HRP

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HA Hemagglutinin (YPYDVPDYA)-HRP

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OVA Conjugated Hemagglutinin (HA)

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OVA Conjugated Hemagglutinin (HA)

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OVA Conjugated Hemagglutinin (HA)

RPU50043-100ug Biomatik Corporation 100ug 475.2 EUR

OVA Conjugated Hemagglutinin (HA)

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HA Hemagglutinin (YPYDVPDYA)-FITC

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Human hemagglutinin,HA ELISA Kit

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Human hemagglutinin(HA)ELISA Kit

GA-E1801HM-48T GenAsia Biotech 48T 346.8 EUR

Human hemagglutinin(HA)ELISA Kit

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Human hemagglutinin,HA ELISA Kit

CN-04295H1 ChemNorm 96T 520.8 EUR
Using equimolar amounts, the multivalent presentation of HA SS on np induced significantly higher ADCC activity compared to the monovalent (trimer) SS probes (2-6 fold increase).
Further, ADCC activity was similarly reported against different group 1 influenza subtypes: H1, H2, and H5. Importantly, ADCC was mediated mainly by antibodies targeting the bnAb-epitope on the HA stem.We report on an assay to measure stem-specific ADCC activity using SS np probes.
Our results indicate high prevalence of HA-stem antibodies with cross-reactive ADCC activity. Such assay could be utilized in the assessment of next generation influenza vaccines.