Helper-Independent Live Recombinant Adenovirus Vector Expressing the Hemagglutinin-Esterase Membrane Glycoprotein

Yoo, DongWan,Yoo, Ick-Dong,Yoon, Young-Ho,Frank L.Graham,Lorne A. Babiuk 한국미생물 · 생명공학회 1992 Journal of microbiology and biotechnology Vol.2 No.3

The hemagglutinin-esterase glycoprotein (HE) gene of bovine coronavirus, coupled with a simian virus 40 early promoter and polyadenylation signal, was inserted into a human adenovirus transfer vector. The transfer vector was used to co-transfect 293 cells along with adenovirus genomic DNA. The hemagglutinin-esterase transcription unit was rescued into the adenovirus genome by homologous in vivo DNA recombination between the vector piasmid DNA and the adenovirus genomic DNA, and a recombinant adenovirus was isolated by several rounds of plaque assays. Thus the recombinant adenovirus carries the hemagglutinin-esterase gene in the early transcription region 3 (E3) of the adenovirus genome in the parallel orientation to the E3 transcription. The recombinant adenovirus synthesized the HE polypeptide in HeLa cells as demonstrated by immunopredpitation with anti-coronavirus rabbit antisera. The recombinant HE polypeptide could be labelled by [^3H]glucosamine, demonstrating that the recombinant HE was glycosylated. Cells expressing the HE polypeptide exhibited hemadsorption activity when incubated with mouse erythrocytes. The HE was transported to the plasma membrane as shown by the cell surface immunofluorescence, indicating that the recombinant HE polypeptide retained its biological activities. Potential for the use of infectious recombinant adenovirus as a live virus-vectored vaccine candidate for bovine coronavirus disease is discussed.

Attachment of flagellin enhances the immunostimulatory activity of a hemagglutinin-ferritin nano-cage

Lee, Emerson B.,Jeon, Hyung-Min,Kim, Chang-Ung,Park, Sang M.,Cho, Geunyoung,Kim, Hyun-Jin,Kim, Youngjin,Kim, Doo-Jin,Kim, Young S.,Lee, Hayyoung,Lee, Jie-Oh Elsevier 2019 Nanomedicine Vol.17 No.-

<P><B>Abstract</B></P> <P>Hemagglutinin (HA) displayed on a ferritin nano-cage has been shown to be effective in generating a potent immune response against a broad range of influenza infections. Here, we showed that conjugation of flagellin together with HA to the exterior surface of the ferritin cage greatly enhanced not only the humoral immune response in mice but also antigen-specific T cell responses that include Th1 cytokine secretion. The effect of flagellin remained essentially unchanged when the molar ratio of flagellin to HA was reduced from 1:1 to 1:3. Injection of the ferritin-HA-flagellin cage provided protection against lethal virus challenge in mice. We used a small immunoglobulin fragment V<SUB>L</SUB>12.3 as a convenient method for attaching HA and flagellin to the ferritin cage. This attachment method can be used for rapid screening of a variety of protein cages and nano-assemblies to identify the most suitable carrier and adjuvant proteins for the target antigen.</P> <P><B>Graphical Abstract</B></P> <P>We have shown that a small immunoglobulin fragment, V<SUB>L</SUB>12.3, as a convenient linker can be used for conjugating protein cages, antigens and adjuvants in this study. We showed that influenza hemagglutinin (HA) could be attached to ferritin and an artificial cage protein, I3-01, by this method. We also showed that Protective Antigen (PA) of <I>Bacillus anthracis</I> could be attached to the ferritin cage with high efficiency. We propose that this approach can be used to quickly screen a variety of protein cages and nano-assemblies to identify the best carrier and adjuvant proteins for desired antigens. To demonstrate the usefulness of our V<SUB>L</SUB>12.3 method in the vaccine development, we generated a ferritin-HA-flagellin nano-cage. We discovered that attachment of flagellin together with HA greatly enhanced not only total IgG level but also Th1 cytokine secretion and class switching to IgG2a in mice.</P> <P>[DISPLAY OMISSION]</P>

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

Lee, Y.J.,Jang, Y.H.,Kim, P.,Lee, Y.H.,Lee, Y.J.,Byun, Y.H.,Lee, K.H.,Kim, K.,Seong, B.L. 3M Company 2016 Virology Vol.491 No.-

<P>In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. (C) 2016 Elsevier Inc. All rights reserved.</P>

Monitoring trafficking and expression of hemagglutinin-tagged transient receptor potential melastatin 4 channel in mammalian cells

Eun Mi Hwang,Bo Hyun Lee,Eun Hye Byun,Soomin Lee,Dawon Kang,Dong Kun Lee,Min Seok Song,Seong-Geun Hong The Korean Society of Pharmacology 2023 The Korean Journal of Physiology & Pharmacology Vol.27 No.4

The TRPM4 gene encodes a Ca²⁺-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

최강석,계수정,전우진,박미자,김새로미,설희정,권준헌 대한수의학회 2013 Journal of Veterinary Science Vol.14 No.3

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 213 per 25 μL,which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4oC. The rHN-based HI assay specifically detected NDV antibodies,but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%,respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Ji-Ye Kim,Hyun-Jeong Lee,Soo-Jeong Kye,Saeromi Kim,Hee-Jung Seul,Sang-Eun Kim,Hee-Soo Lee,Suk-Chan Jung,Kang-Seuk Choi 대한미생물학회 2015 Journal of Bacteriology and Virology Vol.45 No.4

Detection and characterization of bovine-like coronaviruses from four species of zoo ruminants

Chung, J.Y.,Kim, H.R.,Bae, Y.C.,Lee, O.S.,Oem, J.K. Elsevier Scientific Pub. Co 2011 Veterinary microbiology Vol.148 No.2

Five coronaviruses (CoVs) were detected in diarrheal feces from four zoo ruminant species: one wisent (Bison bonasus), two Himalayan tahr (Hemitragus jemlahicus), one sitatunga (Tragelaphus spekii), and one nyala (Tragelaphus angasii). We sequenced and analyzed the spike (S) and hemagglutinin/esterase (HE) genes of these viruses and compared the nucleotide (nt) and deduced amino acid (aa) sequences with those of other bovine CoV (BcoV) strains. Comparison of the entire deduced aa sequences of the S and HE glycoproteins revealed no specific differences that would account for discrimination between bovine-like CoV strains from zoo ruminants and BcoVs strains. In addition, the 99.9% aa identity among the five CoV strains revealed that the ruminants were infected by the same strain. Phylogenetically, bovine-like CoVs belong to group 2a CoVs, which are related most closely to the BcoV strains recently isolated in Korea. These data suggest that cattle are potential reservoirs for CoVs that are capable of infecting zoo ruminants.

Purification and Characterization of Hemagglutinin from Hemolymph of Oxya chinensis

Kim, Jung-Kon,Jeong, Gie-Joon,Kim, Mi-Kyung 한국곤충학회 1993 Entomological Research Vol.23 No.4

벼메뚜기 체액 내에 존재하는 혈구응집소에 대해 다양한 동물세포를 이용하여 혈구응집특이성을 규명하고, affinity chromatography를 통해 정제한 결과 다음과 같은 결론을 얻었다. 1. 벼메뚜기의 체액은 사람의 적혈구(A.B.O)에 대해 높은 응집역가를 나타내었고, mouse, rat 적혈구에 대해서도 응집효과가 있었다. 특히, neuraminidase로 처리한 사람적혈구에 대해서는 높은 역가를 볼 수 있었다. 2. 탄수화물에 대한 혈구응집 반응에서 벼메뚜기 체액은 D-glucose, sucrose, latose, methyl $\beta$-D-glucopyranoside에 대해 응집저해 반응을 보였으나 D-galactose, D-mannose, fructose, fucose, methyl $\beta$-D-galactoside, D(+)-trehalose에 대해서는 반응을 보이지 않았다. 3. 벼메뚜기 체액으로부터 Sepharose-glucose affinity chromatography를 통해 정제된 혈구응집소는 non-denaturing PAGE에서 하나의 band로 확인되었고, SDS-PAGE에서 94kD과 96kD의 subunit로 구성되었음이 확인되었다. The hemagglutinin present in the hemolymph of Oxya chinensis is characterized in vitro and its hemolymphatic hemagglutinin is purified for further studies. The results are as follows : 1) The hemolymph of Oxya chinensis showed high hemagglutination titer toward human erythrocytes(ABO), and also agglutinate mouse and rat erythrocytes. Particularly, neuraminidase-treated human erythrocytes showed higher hemagglutination titer than untreated human erythrocytes. 2) In carbohydrate inhibition tests of hemagglutinin, hemolymph of Oxya chinensis is inhibited by D-glucose, sucrose, lactose and methyl ${\beta}$-D-glucopyranoide, but unaffected by D-galactose, D-mannose, fucose, fructose, methyl ${\beta}$-D-glucoside and D(+)-trehalose. 3) Hemagglutinin is also purified from hemolymph of Oxya chinensis by Sepharose-glucose affinity chromatography. The purity of the eluted protein examined by PAGE under nondenaturing conditions was a thick single band on the gel. The subunit molecular weight of purified hemagglutinin was estimated to be 94KD and 96KD, respectively. Consequently, the hemagglutinin was found to be a large complex of the two distinct protein subunits.

Mycoplasma passive hemagglutinin body titer의 증가를 동반한 돌발성 난청에 대한 임상적 고찰

임대준,김보형 건국대학교 의과학연구소 2001 건국의과학학술지 Vol.11 No.-

Background and Objectives : Mycoplasma pneumonia (MP) is a common etiological factor in variety of respiratory diseases. MP infection developing especially as a primary atypical pneumonia causes diverse complications involving other organs and tissues such as skin, mucosa, musculoskeletal, cardiovascular, gastrointestinal, urogenital and central nervous systems. In the auditory system, otitis externa, otitis media, and myringitis bullosa have been reported. Sensorineural hearing loss possibly caused by MP is infrequently reported. The purpose of this study was to investigate the clinical importance of mycoplasma passive hemagglutinin body titer. Materials and Method : In patients of sudden sensorineural hearing loss who have increased mycoplasma passive hemagglutinin body titer, control group was treated with prednisolone alone, and experimental group was treated with prednisolone and roxithromycin. Both groups were compared with degree of hearing improvement by pure tone audiometry. Results : Experimental group have a better hearing improvement than control group, but these difference was statistically not significant. Conclusion : The reports of sudden sensorineural hearing loss caused by MP was extremely rare, but we suggest that the incidence was higher than previously reported, and if proper diagnosis could be done through the mycoplasma passive hemagglutinin body titer, the prognosis will be better.

Comparison of antigenic mutation during egg and cell passage cultivation of H3N2 influenza virus

Yong Wook Park,Yun Hee Kim,Hwan Ui Jung,Oh Seok Jeong,Eun Ji Hong,Hun Kim,Jae Il Lee 대한백신학회 2020 Clinical and Experimental Vaccine Research Vol.9 No.1

Purpose: When influenza viruses are cultured in eggs, amino acid mutations of the hemagglutinin may occur through egg adaptation. On the other hand, when influenza viruses are cultured in animal cells, no antigenic mutation occurs unlike in eggs. Therefore, we examined whether the antigenic mutations actually occurred after passage of H3N2 (A/Texas/50/2012) virus up to 15 times in eggs and MDCK-Sky3851 cells. Materials and Methods: Prototype A/Texas/50/2012 (H3N2) influenza virus which was isolated from clinical patient, not passaged in egg, was obtained and propagated using the specific pathogen free egg and the MDCK-Sky3851 cell line up to 15 passage, and the changes in the antigen sequence of the influenza viruses were confirmed by gene sequencing and protein structure analysis. Results: In term of the hemagglutination titer of influenza virus, the reactivity to chicken and guinea pig red blood cell showed different results between egg propagated and cell propagated viruses. In the sequence analysis results for hemagglutinin and neuraminidase, no antigenic mutation was observed throughout all passages when cultured in MDCK-Sky3851 cells. On the other hand, mutations occurred in three amino acid sequences (H156R, G186S, S219F) in hemagglutinin up to 15 passages when cultured in eggs. Conclusion: H3N2 influenza virus cultured in eggs could lead mutations in amino acid sequence of hemagglutinin, distinct from the corresponding virus cultured in cells for which no antigenic mutation was observed. These findings suggest that cell culture is a more stable and effective way of production with lower risk of antigenic mutations for the manufacture of influenza vaccines.