Proteins often interact with each other to form complexes and play functional roles in almost all cellular processes. The study of protein-protein interactions is therefore critical to understand protein function and biological pathways. Affinity Purification coupled with Mass Spectrometry (AP-MS) is an invaluable technique for identifying the interaction partners in protein complexes. In this approach, the protein of interest is fused to an affinity tag, followed by the expression and purification of the fusion protein. The affinity-purified sample is then analyzed by mass spectrometry to identify the interaction partners of the bait proteins. In this chapter, we detail the protocol for tandem affinity purification (TAP) based on the use of the FLAG (a fusion tag with peptide sequence DYKDDDDK) and hemagglutinin (HA) peptide epitopes. The immunoprecipitation using dual-affinity tags offers the advantage of increasing the specificity of the purification with lower nonspecific-background interactions.
A molecular docking tool of AutoDock3.05 was evaluated for its ability to reproduce experimentally determined affinities of various sialic acid analogues toward hemagglutinin of influenza A virus. With the exception of those with a C6-modified glycerol side chain, the experimental binding affinities of most sialic acid analogues (C2, C4 and C5-substituted) determined by viral hemadsorption inhibition assay, hemagglutination inhibition assay and nuclear magnetic resonance correlated well with the computationally estimated free energy of binding. Sialic acid analogues with modified glycerol side chains showed only poor correlation between the experimentally determined hemagglutinin inhibitor affinities and AutoDock3.05 scores, suggesting high mobility of the glutamic acid side chain at the glycerol binding pocket, which is difficult to simulate using a flexi-rigid molecular docking approach. In conclusion, except for some glycerol-substituted sialic acid analogues, the results showed the effectiveness of AutoDock3.05 searching and scoring functions in estimating affinities of sialic acid analogues toward influenza A hemagglutinin, making it a reliable tool for screening a database of virtually designed sialic acid analogues for hemagglutinin inhibitors.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) viscerotropic-velogenic strain AF2240 was determined by direct RNA sequencing and by sequencing RT-PCR products. It encodes a single open reading frame of 581 amino acids with a calculated Mr of 63.8 kDa. The predicted sequence contains five asparagine glycosylation sites. Comparison of the AF2240 HN protein sequence with 13 other previously published sequences showed 88% homology. This HN protein is unique because it lacked the Arg 403 residue which is present in all of the other strains and cannot be grouped under the proposed three size classes of HN proteins in NDV.
Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.
Reassortant influenza A viruses bearing the H1 subtype of hemagglutinin (HA) and the N2 subtype of neuraminidase (NA) were isolated from humans in the United States, Canada, Singapore, Malaysia, India, Oman, Egypt, and several countries in Europe during the 2001-2002 influenza season. The HAs of these H1N2 viruses were similar to that of the A/New Caledonia/20/99(H1N1) vaccine strain both antigenically and genetically, and the NAs were antigenically and genetically related to those of recent human H3N2 reference strains, such as A/Moscow/10/99(H3N2). All 6 internal genes of the H1N2 reassortants examined originated from an H3N2 virus. This article documents the first widespread circulation of H1N2 reassortants on 4 continents. The current influenza vaccine is expected to provide good protection against H1N2 viruses, because it contains the A/New Caledonia/20/99(H1N1) and A/Moscow/10/99(H3N2)-like viruses, which have H1 and N2 antigens that are similar to those of recent H1N2 viruses.
Plasmid DNA (pDNA)-based vaccines have emerged as effective subunit vaccines against viral and bacterial pathogens. In this study, a DNA vaccine, namely plasmid internal ribosome entry site-HN/F, was applied in ovo against Newcastle disease (ND). Vaccination was carried out using the DNA vaccine alone or as a mixture of the pDNA and dextran-spermine (D-SPM), a nanoparticle used for pDNA delivery. The results showed that in ovo vaccination with 40 μg pDNA/egg alone induced high levels of antibody titer (P<0.05) in specific pathogen-free (SPF) chickens at 3 and 4 weeks postvaccination compared to 2 weeks postvaccination. Hemagglutination inhibition (HI) titer was not significantly different between groups injected with 40 μg pDNA + 64 μg D-SPM and 40 μg pDNA at 4 weeks postvaccination (P>0.05). Higher antibody titer was observed in the group immunized with 40 μg pDNA/egg at 4 weeks postvaccination. The findings also showed that vaccination with 40 μg pDNA/egg alone was able to confer protection against Newcastle disease virus strain NDIBS002 in two out of seven SPF chickens. Although the chickens produced antibody titers 3 weeks after in ovo vaccination, it was not sufficient to provide complete protection to the chickens from lethal viral challenge. In addition, vaccination with pDNA/D-SPM complex did not induce high antibody titer when compared with naked pDNA. Therefore, it was concluded that DNA vaccination with plasmid internal ribosome entry site-HN/F can be suitable for in ovo application against ND, whereas D-SPM is not recommended for in ovo gene delivery.
Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)-TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006-2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.
Avian influenza viruses (AIV) cause high morbidity and mortality among the poultry worldwide. Their highly mutative nature often results in the emergence of drug resistant strains, which have the potential of causing a pandemic. The virus has two immunologically important glycoproteins, hemagglutinin (HA), neuraminidase (NA), and one ion channel protein M2 which are the most important targets for drug discovery, on its surface. In order to identify a peptide-based virus inhibitor against any of these surface proteins, a disulfide constrained heptapeptide phage display library was biopanned against purified AIV sub-type H9N2 virus particles.
In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.
The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets.
Every year, influenza virus infections cause significant morbidity and mortality worldwide. They pose a substantial burden of disease, in terms of not only health but also the economy. Owing to the ability of influenza viruses to continuously evolve, annual seasonal influenza vaccines are necessary as a prophylaxis. However, current influenza vaccines against seasonal strains have limited effectiveness and require yearly reformulation due to the virus undergoing antigenic drift or shift. Vaccine mismatches are common, conferring suboptimal protection against seasonal outbreaks, and the threat of the next pandemic continues to loom. Therefore, there is a great need to develop a universal influenza vaccine (UIV) capable of providing broad and durable protection against all influenza virus strains. In the quest to develop a UIV that would obviate the need for annual vaccination and formulation, a multitude of strategies is currently underway. Promising approaches include targeting the highly conserved epitopes of haemagglutinin (HA), neuraminidase (NA), M2 extracellular domain (M2e) and internal proteins of the influenza virus. The identification and characterization of broadly neutralizing antibodies (bnAbs) targeting conserved regions of the viral HA protein, in particular, have provided important insight into novel vaccine designs and platforms. This review discusses universal vaccine approaches presently under development, with an emphasis on those targeting the highly conserved stalk of the HA protein, recent technological advancements used and the future prospects of a UIV in terms of its advantages, developmental obstacles and potential shortcomings.
In light of the limited protection conferred by current influenza vaccines, immunisation using universal influenza vaccines has been proposed for protection against all or most influenza sub-types. The fundamental principle of universal influenza vaccines is based on conserved antigens found in most influenza strains, such as matrix 2, nucleocapsid, matrix 1 and stem of hemagglutinin proteins. These antigens trigger cross-protective immunity against different influenza strains. Many researchers have attempted to produce the conserved epitopes of these antigens in the form of peptides in the hope of generating universal influenza vaccine candidates that can broadly induce cross-reactive protection against influenza viral infections. However, peptide vaccines are poorly immunogenic when applied individually owing to their small molecular sizes. Hence, strategies, such as combining peptides as multi-epitope vaccines or presenting peptides on vaccinia virus particles, are employed. This review discusses the clinical and laboratory findings of several multi-epitope peptide vaccine candidates and vaccinia-based peptide vaccines. The majority of these vaccine candidates have reached the clinical trial phase. The findings in this study will indeed shed light on the applicability of universal influenza vaccines to prevent seasonal and pandemic influenza outbreaks in the near future.
In the present study, we used nucleotide and protein sequences of avian influenza virus H5N1, which were obtained in Asia and Africa, analyzed HA proteins using ClustalX1.83 and MEGA4.0, and built a genetic evolutionary tree of HA nucleotides. The analysis revealed that the receptor specificity amino acid of A/HK/213/2003, A/Turkey/65596/2006 and etc mutated into QNG, which could bind with á-2, 3 galactose and á-2, 6 galactose. A mutation might thus take place and lead to an outbreak of human infections of avian influenza virus. The mutations of HA protein amino acids from 2004 to 2009 coincided with human infections provided by the World Health Organization, indicating a "low-high-highest-high-low" pattern. We also found out that virus strains in Asia are from different origins: strains from Southeast Asia and East Asia are of the same origin, whereas those from West Asia, South Asia and Africa descend from one ancestor. The composition of the phylogenetic tree and mutations of key site amino acids in HA proteins reflected the fact that the majority of strains are regional and long term, and virus diffusions exist between China, Laos, Malaysia, Indonesia, Azerbaijan, Turkey and Iraq. We would advise that pertinent vaccines be developed and due attention be paid to the spread of viruses between neighboring countries and the dangers of virus mutation and evolution.
Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.
In view of the risk of introduction of yellow fever into South-East Asia, comparative studies have been made of yellow fever vaccination in Malayan volunteers with a high prevalence of antibody to related viruses and in volunteers without related antibody. In a previous paper the neutralizing antibody responses of these volunteers were reported. The present paper describes the haemagglutinin-inhibiting (HI) antibody responses of the same groups of volunteers and discusses the relationship of these responses to the neutralizing antibody responses.The HI responses to yellow fever following vaccination closely paralleled the neutralizing antibody responses whether vaccination was subcutaneous or by multiple puncture. Volunteers with a high level of YF HI antibody due to infection with other group B viruses were found to be less likely to show a significant YF HI response than those without antibody. 90% of HI responses could be detected by the 21st day after vaccination.As with neutralizing antibody responses, volunteers given vaccine doses of 50-500 mouse intracerebral LD(50) subcutaneously gave greater responses than those given higher doses.
Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.
A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site (112)RRRKGF(117) and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.
The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.