Rapid Estimation of Binding Activity of Influenza Virus Hemagglutinin to Human and Avian Receptors

A critical step for avian influenza viruses to infect human hosts and cause epidemics or pandemics is acquisition of the ability of the viral hemagglutinin (HA) to bind to human receptors. However, current global influenza surveillance does not monitor HA binding specificity due to a lack of rapid and reliable assays. Here we report a computational method that uses an effective scoring function to quantify HA-receptor binding activities with high accuracy and speed. Application of this method reveals receptor specificity changes and its temporal relationship with antigenicity changes during the evolution of human H3N2 viruses. The method predicts that two amino acid differences at 222 and 225 between HAs of A/Fujian/411/02 and A/Panama/2007/99 viruses account for their differences in binding to both avian and human receptors; this prediction was verified experimentally. The new computational method could provide an urgently needed tool for rapid and large-scale analysis of HA receptor specificities for global influenza surveillance.National Key Project (2008ZX10004-013)National Institutes of Health (U.S.) (grant AI07443)Singapore-MIT Alliance for Research and TechnologyMassachusetts Institute of Technology. International Science and Technology Initiatives Global Seed FundNational Basic Research Program (973 Program) (2009CB918503)National Basic Research Program (973 Program) (2006CB911002

Characterization of mutations in the receptor binding site of influenza A viruses determining virus host, tissue, and cell tropisms using systems biology approaches

Influenza A viruses (IAVs) cause occasional pandemics and seasonal epidemics, thus presenting continuous challenges to public health. Vaccination is the primary strategy for the prevention and control of influenza outbreaks. The antigenicity matched high-yield seed strain is critical for the success of influenza vaccine. Currently, there are several limitations for the influenza vaccine manufacture: 1) the conventional methods for generating such strains are time consuming; 2) egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage; 3) vaccine seed viruses often do not grow efficiently in mammalian cell lines. Previous studies suggested that mutations in the receptor binding site (RBS) that locates at the globular head of the HA1 can change IAVs’ binding specificity, antigenicity, and yield and thus RBS would be an potential target for engineering vaccine seed strain. However, systematic analysis of the mutations on RBS affecting those viral phenotypes is lacking. Specifically, this dissertation has following aims: Firstly, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. The error-prone PCR- based reverse genetic system could also be applied to gain-ofunction studies for influenza virus and other pathogens; Secondly, in this dissertation, we identified an Y161F mutation in the hemagglutinin (HA) that enhanced the infectivity and thermostability of virus without changing its original antigenic properties which would prompted the development of cell-based vaccines; Thirdly, the molecular mechanisms underlying host adaption of equine-origin influenza A(H3N8) virus from horses to dogs are unknown. This dissertation identified that a substitution of W222L in the HA of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to sialyl Lewis X motifs, which were found abundantly in the submucosal glands of dog trachea but not in equine trachea. To summary, this dissertation investigated the role of RBS in IAVs biology and expanded the current knowledge toward IAV vaccine strain engineering, IAV host adaption and evolution

Two Glycosylation Sites in H5N1 Influenza Virus Hemagglutinin That Affect Binding Preference by Computer-Based Analysis

Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs

Glycan receptor binding determinants of Influenza A virus hemagglutinin

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references.An understanding of the factors involved in the human adaptation of influenza A viruses is critical for various aspects of influenza preparedness, including the development of appropriate surveillance measures, preventive strategies and effective treatments. A key step in influenza human adaptation is the acquisition of mutations in the viral coat glycoprotein, hemagglutinin (HA), which changes its binding specificity towards glycan receptors in the human upper respiratory epithelia (referred to as human receptors). In this thesis, determinants that mediate changes in HA-glycan receptor binding specificity are investigated, with focus on the molecular environments within and surrounding the glycan receptor binding site (RBS) of HA. The glycan receptor binding properties of HA from different influenza subtypes (H1N1, H2N2, H3N2 and H5N1) are studied using a combination of approaches including dose-dependent glycan binding, human tissue staining and structural modeling. Using these complementary analyses, it is shown in this thesis that the molecular interactions between amino acids in and proximal to the RBS (referred to as amino acid interaction networks), including those between the RBS and glycosylation at sites proximal to the RBS, and interactions between the RBS and the glycan receptor together govern the high affinity binding of HA to human receptors. The thesis is divided into three sections. First, the evolution of glycan receptor binding specificity of recent human-adapted H3 strains such as A/Fujian/411/02 and A/Panama/2007/99 is investigated, with implications on vaccine production in chicken eggs. Second, the determinants of glycan receptor binding affinity of potentially pandemic avian viruses is studied in the context of the recently circulating H2 A/Chicken/Pennsylvania/2004 and the highly pathogenic H5 A/Vietnam/1203/2004. Here it is shown that mutations which cause human adaptation of H2 do not increase human receptor binding affinity in H5, and the importance of amino acid interaction networks is implicated. Third, determinants that govern the high affinity human receptor binding of pandemic influenza HAs is investigated using the prototypic 1918 H1N1 HA as a model system. The roles of amino acid interaction networks and the molecular interactions between the RBS and glycosylation at sites proximal to the RBS in contributing to the high affinity human receptor binding of 1918 H1N HA are investigated. The approaches presented in this thesis to systematically investigate molecular interactions between HA and glycan receptors that impinge on quantitative HA-glycan receptor binding affinity offer a new angle towards studying determinants of human receptor binding specificity and affinity of influenza A virus HAs.by Xiaoying Koh.Ph.D

Truncated Sequences of Influenza Subtype H5 Haemagglutinin for Vaccination and Diagnostic Purposes: Avian influenza, Yeast expression, Peptide vaccination, recombinant Elisa

The highly pathogenic Avian Influenza subtype H5N1 can lead to 100 % mortality in chickens. The main issue in prevention of H5N1 is the development of efficient poultry vaccines. Influenza haemagglutinin (HA) derived recombinant polypeptides would not elicit an immune response against internal viral proteins. Thus HA polypeptide use facilitates differentiation between infected and vaccinated animals (DIVA). Serological tests using recombinant immune-dominant proteins devoid of non-specific moieties present in whole cell preparations might have higher sensitivity and specificity. In the present study, four non-overlapping sequences of different functional domains of influenza A virus subtype H5 virus (A / Thailand / 1 (Kan-1) / 2004) designated P1, P2, P5 and rHA1 were cloned and expressed in Pichia pastoris for vaccination and diagnosis purposes. The four polypeptides were expressed successfully in P. pastoris using peptone methanol (1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (v/v) methanol). P1, P2 and rHA1 polypeptides were purified using nickel affinity chromatography, whereas, P5 was purified using lectin affinity chromatography. Correct expression was analysed by SDS-PAGE and western blot, glycosylation analysis and MALDI-TOF.The immune responses of P1, P2 and rHA1 polypeptides were assessed in BALB/C mice. To enhance antibody response, recombinant polypeptides were mixed with the Gerbu adjuvant and injected subcutaneously. Vaccination of mice induced high subtype specific antibody titres in mice as analysed by Elisa (using recombinant antigens or whole H5N1 antigen) and Immunofluorescence assay (IFA) performed on Vero cells infected with H5 (A / Thailand / 1 (Kan-1) / 2004). The immunogenicity of P1, P2, P5 and rHA1 polypeptides was determined in commercial layer chickens. Results showed that P1, P2 and rHA1 polypeptides induced high subtype specific antibody titres in chickens as analysed by Elisa (using recombinant antigens or whole H5N1 antigen), IFA (performed on Vero cells infected with H5N1 A / Thailand / 1 (Kan-1) / 2004) and microneutralization test (µNT). However, P5 polypeptide was not immunogenic in chickens. Neutralizing antibodies could be detected in chicken sera immunized with P1, P2 and rHA1 polypeptides as analyzed with microneutralization test. IgY was analysed in egg yolk of chickens immunized with recombinant polypeptides. The IgY of chicken immunized with P1 and rHA1, transferred to the egg yolk was proportional to maternal serum IgY. However, IgY could not be detected in egg yolk of chickens immunized with P2 and P5 recombinant polypeptides. The more immunogenic polypeptides P1 and rHA1 were used in an recombinant Elisa (rElisa) for detection of influenza A subtype H5 in chickens and duck sera.The optimal antigen for the concentrations of rHA1, P1 was 50 ng / well, 50 ng / well. Analysis of 25 positive sera and 25 negative sera to H5 antibodies revealed that, the sensitivity of Western blot, whole H5N1 Elisa, agar gel immunodiffusion test (AGID), P1-Elisa and rHA1-Elisa was 100 %, 100 %, 52 %, 80 % and 100 %, respectively, while the specificity was 100 %, 100 %, 100 %, 72 %, and 100 %, respectively. Moreover, duck sera, with haemagglutination inhibiting titer ranged from 4 - 8 log2, were tested positive by rHA1 Elisa compared with negative duck sera. Further analysis of 179 serum samples with rHA1-Elisa in comparison with haemagglutination inhibition (HI) and commercial Elisa proved to be highly sensitive and specific. The agreement ratio between rElisa and HI was 84.9 % and between commercial Elisa (Flock check) and HI was 76.5 %. In conclusion, P. pastoris may allow development of an effective recombinant influenza vaccine based on truncated sequences of HA that might provide broader protection against H5 influenza viruses. The possibilities to use rHA1, P1 and P5 recombinant polypeptides as a vaccine against H5 influenza should be further studied. Also our study demonstrates the potential utility of recombinant Elisa as a tool for improvement of serological diagnosis of influenza A subtype H5 in chickens and ducks.Die hochpathogene aviäre Influenza des Subtyps H5N1 erreicht beim Ausbruch von Infektionen in Nutzgeflügelbeständen Mortalitätsraten von bis zu 100 %. Effektive und kostengünstige Impfstoffe werden benötigt, die möglichst auch eine Differenzierung zwischen geimpften Tieren und mit Wild-Virus infizierten Tieren zulassen. In diesem Zusammenhang könnten Peptid-Vakzine eine mögliche Alternative zu den herkömmlichen Impfstoffen darstellen, bei denen unter Verwendung des Vollvirus Antikörper gegen mehrere Virusproteine induziert werden. Außerdem, könnten rekombinante Antigene in serologischen Tests zur Diagnose von H5 Virus in Nutzgeflügel eingesetzt werden. Von dem Einsatz spezifischer rekombinanter Antigene ist eine Verbesserung der Serodiagnostik zu erwarten. In dieser Arbeit, wurden vier verkürzte Sequenzen des Hämagglutinins (P1, P2, P5 und rHA1) von Subtyp H5 (A / Thailand / 1 (Kan-1) / 2004) rekombinant in Pichia Pastoris exprimiert. Dazu erfolgten zunächst eine Klonierung in der Expressionsvektor pAOX und die Transformation von Pichia Pastoris. Die Expression wurde durch Methanol induziert. Der Nachweis der rekombinanten Fusionspeptiden mit C-terminalen Histidin-Tag erfolgte durch SDS-PAGE, Western Blot, Glycolysierungsanalyse, und MALDI-TOF. Der Histidin-Tag ermöglichte die Reinigung von P1, P2 und rHA1 mit Metall-Affinitätschromatographie. Polypeptid P5 hingegen wurde mittels Lectin-Affinitäts- chromatographie gereinigt. Balb/c Mäuse wurden mit Polypeptid P1, P2 bzw. rHA1, versetzt mit Gerbu Adjuvans, immunisiert. Zur Untersuchung der Immunantwort wurden die murinen Seren mittels Elisa (unter Verwendung rekombinanter Antigene oder Voll-H5N1 Antigen) sowie IFA (durchgeführt in Vero- Zellen infiziert mit A / Thailand / 1 (Kan-1) / 2004) analysiert. Dabei wurde die präferentielle Induktion von H5-spezifischen Antikörpern detektiert. Die Immunogenität der P1, P2, P5 und rHA1-Polypeptide wurde in kommerziellen Legehennen bestimmt. Seren wurden mit ELISA, IFA, und Mikroneutralizationstest (μNT) analysiert. Die ELISA-Ergebnisse zeigten, dass die Polypeptide P1, P2 und rHA1 hohe Subtyp-spezifische Antikörpertiter in Hühnern induzierten. Im µNT konnte nur ein niedriger neutralisierender Antikörpertiter nachgewiesen werden. Das P5- Polypeptid ist bei Hühnern nicht immunogen. Im Eigelb von Hühnern, die mit den rekombinanten Polypeptiden P1 und rHA1 immunisiert wurden, konnten H5-spezifische IgY Antikörper detektiert werden. Hühner, die mit P2 und P5 immunisiert wurden, zeigten keine IgY im Eigelb. Die rekombinanten Antigene P1 und rHA1 wurden im ELISA auf ihre potenzielle Eignung für die Serodiagnostik untersucht. Die optimale Antigenkonzentration war 50 ng / well. Die serologische Analyse von 25 positiven und 25 negativen Seren auf Antikörper gegen H5 zeigte, dass Sensitivität und Spezifität von Western Blot, Voll-H5N1 ELISA und rHA1-ELISA bei jeweils 100 % lagen. Bei Agargel- Immunodiffusiontest (AGID) lagen Sensitivität und Spezifität bei 52 % und 100 %, während im P1-Elisa lediglich eine Sensitivität von 80 % und eine Spezifität von 72 % erreicht wurden. Somit eignet sich rHA1 für die Anwendung in der Serodiagnostik. Bei der serologischen Untersuchung von 175 Hühnerseren wurde eine Überbestimmung zwischen rHA1-ELISA und Hämagglutinationshemmungstest (HAI) 84.9 % festgestellt, während diese zwischen dem kommerziellen ELISA (Flock Check) und HAI 76.5 % betrug. Die Ergebnisse zeigten, dass das Expressionssystem P. pastoris als Produktionssystem rekombinanter Antigene für die Serodiagnostik von H5 Influenza geeignet ist. Challenge-Versuche sind nötig, um die Eignung von rekombinanten Antigenen als möglichen Impfstoff gegen H5 Influenza zu untersuchen

Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactions

Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins

Functional studies on the influenza A virus M2 protein

The M2 protein of influenza A viruses is a homotetramer composed of four 97 amino acid subunits which form an ion permeable channel. It plays an important role both in the process of virus uncoating and in modulating the pH of the transport pathway which is necessary for haemagglutinin (HA) maturation. M2 was stably expressed in mouse erythroleukaemia (MEL) cells under the control of the inducible β-globin locus control region (LCR). The production of M2 peaked at 4-6 days post induction and was at a level comparable to that in virus-infected MDCK cells. The expressed M2 was structurally similar to that produced in virus-infected MDCK cells, in particular with regard to the formation and stability of the tetramer and was also phosphorylated and palmitoylated. The function of the M2 protein expressed in MEL cells was studied using three assays, 1) co-expression of HA and M2, 2) determination of intracellular pH and 3) measurements of ion conductance. Analysis of MEL cells showed that they provided a suitable environment for the co-expression of M2 and HA. Expression of M2 resulted in a decrease in intracellular pH, indicating that the M2 protein is responsible for the decrease in cytoplasmic pH during virus infection. Voltage clamp measurements showed that the expressed M2 formed a proton-selective channel which was specifically inhibited by rimantadine. Two features of the results indicated that the current was due to a proton conductance. At zero membrane potential, both the direction and magnitude of the current were dependent on the proton gradient and secondly the reversal potential was equal to the proton equilibrium potential. Neither the reversal potential nor the amplitude of the current were influenced by the presence of other small ions, including Na+, or K+ or CI-. These results are consistent with the biochemical role that M2 is perceived to play during virus infection. Structure/activity studies were also undertaken to identify residues in the transmembrane domain of the M2 protein which affected both the activation and thermal stability of the tetramer. In particular amino acid differences between the Weybridge and Rostock M2 proteins were studied

Development of novel virus vectors for influenza vaccination

The influenza virus, a member of the Orthomyxoviridae family, causes regular, large-scale morbidity and mortality in birds and humans and significant human suffering and economic loss. The primary aim of this study was to develop a novel influenza vaccine. Vaccines are an essential tool for the control of influenza because they increase resistance to infection, prevent illness and death and help to limit virus transmission to other birds and mammals, including humans. By reducing the environmental contamination of influenza virus in global poultry stocks, the risk of a new pandemic virus being generated by the human-avian link is diminished. Marek’s Disease is a common lymphoproliferative disease of poultry that is readily controlled worldwide using the live attenuated vaccine, CVI988. The Marek’s Disease Virus (MDV) CVI988 viral genome, available as a Bacterial Artificial Chromosome (BAC), forms viable infectious viral particles when transfected into Chicken Embryo Fibroblast (CEF) cells. Using BAC mutagenesis, two non-essential genes in the MDV CVI988 BAC (UL41 and US10), were identified and replaced by the low pathogenic influenza haemagglutinin 10 (H10) gene. These live recombinant MDV-H10 vectors will allow simultaneous vaccination against both pathogens. In addition, the non-essential genes were also replaced with GFP creating MDV-GFP constructs. Both genes were expressed initially using a CMV promoter, although this disrupted the MDV CVI988 BAC; a second promoter, PGK-1, proved more successful. A third MDV gene (UL50) was deleted, but severe attenuation prevented the incorporation of H10 into this open reading frame. Future work to test the MDV-HA constructs in vivo will be carried out in collaboration with the Istituto Zooprofilattico Sperimentale delle Venezie in Italy. In addition, development of MDV constructs containing multiple HA genes (H10 and H5) linked by the 2A polyprotein can be developed with the goal of establishing heterosubtypic immunity

Development of Inducible Anti-influenza Therapies

Influenza viruses continue to cause significant morbidity and mortality each year despite the development of vaccines and antiviral therapies targeting these viruses. The inherent ability of influenza viruses to accumulate mutations over time has led to the emergence of strains resistant to antiviral therapies. Furthermore, genetic reassortment creates antigenically diverse viruses, making it difficult to develop vaccines that yield broad protection. The objective of the following research studies is to develop two alternative approaches to current methods of antiviral therapeutics.;Six new siRNAs targeting influenza protein expression by RNA interference (RNAi) were characterized. Three siRNAs (M747, M776, M832) knocked down the expression of matrix protein 2 and attenuated influenza infection to a similar degree as MDCK cells treated with a previously published siRNA, M950. The three siRNAs (NS570, NS595, NS615) that target the nonstructural protein 1 and 2 genes promoted the expression of type I interferons, but were unable to attenuate the production of infectious virus. However NS595- and NS615-siRNAs promoted the production of defective interfering viruses. Another siRNA, M331, was able knock down the expression matrix 1 and matrix 2 and attenuate viral replication. Combination siRNA treatment was found to attenuate 20.9% more infectious virus than M950-siRNA treatment alone. Treatment with a single siRNA (M331, NS570, NS595, or NS615) that targets two protein coding sequences was able to knock down the expression of two proteins, thus enhancing the utilities of the siRNAs.;To further take advantage of RNAi as a mechanism to attenuate influenza infection, we developed an inducible anti-influenza therapy containing the influenza conserved promoter that expresses asRNAs only after influenza infection or in the presence of the influenza virus RNA-dependent RNA polymerase (RdRP). asRNA expression was restricted to pM950, pM776, pNS595, or pNA105 treated cells containing the RdRP. The asRNAs expressed from the inducible asRNA expression vectors (pM776 or pNS595) were 84- to 343-fold below the concentration needed to reduce influenza virus infection by RNAi, thus illustrating the need for improved expression kinetics. Limiting expression of asRNAs within influenza infected cells could potentially reduce the adverse effects and limitation of RNAi therapeutics.;In an attempt to reverse antigenic variation and attenuate influenza titer, we developed additional inducible anti-influenza therapies (pUC57 NF-NA and pUC57 F-NA), similar to the inducible asRNA expression vector, which express nonfunctional or functional neuraminidases (NF-NA or F-NA) upon influenza infection. The presence of vector expressed RdRP or influenza infection induced the expression of NF-NA and F-NA. Overexpression of NF-NA was originally hypothesized to attenuate influenza titer; however, NF-NA regained its sialidase activity after RdRP-mediated transcription. pUC57 NF-NA or F-NA transfected cells produced an RNA-intermediate regardless of the presence of the RdRP, whereas the polymerase was required for NF-NA mRNA and protein expression. Interestingly, reinfection of MDCK cells with the supernatant from pUC57 NF-NA or F-NA treated and influenza (N1 subtype) infected cells revealed that the naive MDCK cells generated N2 subtype viruses, indicating the induced N2 viral RNA could be packaged into progeny viruses forcing the N1 virus to become an N2 virus.;These studies demonstrate that RNAi can be an effective means to attenuate influenza infection. Furthermore, incorporation of the influenza conserved promoter into asRNA or neuraminidase expression vectors can be exploited to promote influenza infected cell-specific expression of anti-influenza molecules. This approach may impact the design and advancement of antiviral therapeutics by overcoming the limitations associated with RNAi and allow for current vaccines to protect against influenza infection by forcing influenza viruses to converge into a single subtype