ANALISIS MOLEKULER FILOGENETIK DAN STRUKTUR ANTIGENIC VIRUS AVIAN INFLUENZA SUBTIPE H5N1 ISOLAT LAMPUNG TAHUN 2008-2013

Penelitian ini bertujuan melakukan karakterisasi molekuler antigenic site terhadap isolat virus avian influenza (AI) Balai Penyidikan dan Pengujian Veteriner (BPPV) Regional III Lampung dari tahun 2008-2013. Amplifikasi RNA dilakukan dengan teknik reverse transcription polymerase chain reaction (RT-PCR) menggunakan 4 pasang primer referens dari Australian Animal Health Laboratory (AAHL) Geelong Australia (HA10, HA20, dan HA30) dan dilanjutkan dengan proses pengurutan. Analisis hasil pengurutan dengan menggunakan perangkat lunak MEGA versi 5.05 yang meliputi multiple alignment, deductive amino acids prediction, dan phylogenic tree analysis diperoleh hasil perbedaan genetik antar isolat Lampung dari tahun 2003-2013 ditemukan berkisar 1,1-9,1% dengan tingkat homologi mencapai 90,9-98,9%. Variasi genetik ditemukan adanya substitusi pada posisi 53 (R53K), 126 (D126E), 136 (P136), 138 (H138Q, dan H138L), 140 (R140K, R140S, dan R140N), 141 (S141P), dan 189 (K189R). Berdasarkan analisis filogenic tree isolat Lampung tahun 2008-2011 termasuk ke dalam clade 2.1.3. Analisis filogenik isolat AI tahun 2012-2013 yang menginfeksi unggas air mempunyai homologi sekitar 98,5-99,1% dibandingkan dengan isolat AI yang menginfeksi unggas air asal Jawa dan termasuk ke dalam clade 2.3.2.1

Immune escape mutants of highly pathogenic avian influenza H5N1 selected using polyclonal sera: Identification of key amino acids in the HA protein

Evolution of Avian Influenza (AI) viruses – especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype – is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number

Novel Cationic Pentablock Copolymers as a Nanovaccine Delivery Platform

The main objective of this project is the development of a safe and effective vaccine against respiratory pathogens including the highly pathogenic avian influenza (HPAI) H5N1. Amphiphilic pentablock copolymers based on Pluronic F127 and poly(2-diethylaminoethyl methacrylate (PDEAEM) have several characteristics that make them promising candidates as injectable vaccine carriers and adjuvants. Individual block copolymer molecules self-assemble into micelles in aqueous solutions. These micelles can be used to encapsulate protein for vaccine delivery. As the concentration of the block copolymer increases, the micelles form a temperature dependent gel and the length of the PDEAEM blocks control the dissolution rate of the gels. An injectable formulation can be designed to gel at physiological temperatures and form an antigenic depot. In addition, these biocompatible pentablock copolymers are based on pluronic, which is FDA approved as an injectable material. In order to design an efficacious next-generation vaccine against HPAI H5N1 several specific research goals were laid out including: (1) Design, synthesis, and optimization of the pentablock copolymer vaccine platform; (2) Evaluation of vaccine efficacy in vitro and in vivo; and (3) Immunization of mice with the pentablock copolymer vaccine to asses an immune response. Our block copolymer injectable delivery platform demonstrates the ability to sustain the release of antigen with minimal effects on protein stability or antigenicity and persist at the injection site. We have also successfully modified the polymers through an azide-alkyne click reaction to include mannose moieties that act as ligands for pattern recognition receptors on antigen presenting cells. These data coupled with the strong immune response demonstrate the potential for block copolymers for use simultaneously for injectable delivery and as a vaccine adjuvant platform

Isolation und Charakterisierung von rekombinanten Designer-Antikörpern mittels Phagen-Display zum Aufbau eines schnellen und subtyp-spezifischen Influenza-Detektionssystems

Influenza A/H5N1 viruses caused several panzootic outbreaks in the past. These viruses of avian origin have acquired a zoonotic potential by genetic changes and can cross species barriers including humans. There is an urgent need of reliable broad spectrum H5N1 diagnostics. The aim of this work is to identify H5 specific antibodies applicable in a subtype specific diagnostic detection and discrimination system for influenza viruses. Two single chain (sc)Fv antibody gene phage libraries were generated from chickens vaccinated and challenged with avian H5N1 influenza viruses from phylogenetically distant clades. For antibody selection two additional recombinant H5 antigens were applied. Twelve unique anti-H5 scFvs were isolated expressed in mammalian cells. All of them showed binding to H5 hemagglutinin proteins in ELISA and immunoblot assays without cross-reactivity with unrelated proteins. The majority of selected binders detected a large variety of H5 viruses in immunoperoxidase monolayer assays (IPMA) and showed no cross-reactivity with other influenza viruses of closely related HA-subtypes. For one antibody very broad binding specificity for H5 antigens from different strains and for two antibodies neutralizing activity was demonstrated. Epitope mapping revealed a six amino acid short epitope highly conserved among H5 influenza viruses. In H5 sandwich ELISA and membrane based immunodot blot assays the proof of principle of a rapid diagnostic assay,was demonstrated. Antibodies specifically stained influenza viruses of an H5 strain in electron microscopy. Thus, a successful immunization and selection strategy comprising four different H5 sources leading to several H5 specific antibodies and elucidating a highly conserved linear epitope specific for H5 influenza viruses has been demonstrated. Isolated antibodies and the highly conserved linear epitope in the H5 HA1 domain can be used in different applications e.g. influenza diagnostics, therapy and vaccination.Influenza A/H5N1-Viren haben in der Vergangenheit verschiedene Pandemien verursacht. Diese humanpathogenen Viren aviären Ursprungs können durch genetische Veränderungen Artbarrieren überwinden. Für H5N1 besteht ein dringender Bedarf an Breitspektrum-Diagnostika. Ziel dieser Arbeit war es, H5-spezifische Antikörper zu identifizieren, die in einem subtypspezfischen System zum Nachweis und zur Unterscheidung von Influenzaviren anderen Subtypes eingesetzt werden können. Nach Vakzinierung von Hühnern mit aviären H5N1-Viren aus phylogenetisch entfernten „Clades“ wurden in dieser Arbeit zwei (scFv) Phage-Display Antikörperbibliotheken hergestellt. Zur Selektion der Antikörper wurden zwei zusätzliche rekombinante H5-Antigene verwendet. Es wurden 12 unterschiedliche H5 spezifisch scFv-Fragmente isoliert und in Säugerzellen als bivalente IgG-ähnliche Antikörper (scFv-Fc-Format) exprimiert. Alle scFv-Fc-Antikörper binden beide H5-Antigene im Immunoblot und im ELISA. Kreuzreaktivität mit weiteren Proteinen zeigte sich nicht. Die Mehrheit der selektierten Binder erkannte eine große Bandbreite an H5-Viren und zeigte keine Kreuzreaktivität mit anderen eng verwandten Influenzaviren anderer HA-Subtypen. Ein Antikörper wies eine sehr breite Bindungsspezifität für H5. Zwei Antikörper zeigten neutralisierende Aktivität. Die Epitop-Analyse auf membrangebundenen Peptiden führte zur Aufklärung eines 6-Aminosäure kurzen, innerhalb der H5-Influenzaviren hoch konservierten, Epitops. Im H5-Sandwich-ELISA und einem membranbasierten Immuno-Dotblot-Assay konnte gezeigt werden, dass mit den isoierten Antikörpern der Aufbau eines schnellen diagnostischen Tests möglich ist. Im Elektronenmikroskop konnte die spezifische Bindung der Antikörper an die Virusoberfläche gezeigt werden. Somit führte eine erfolgreiche Immunisierungs- und Selektionsstrategie zur Isolierung mehrerer H5-spezifischer Antikörper und zur Aufdeckung eines hoch konservierten linearen, für H5-Influenzaviren spezifischen Epitopes

Expression of Influenza nucleoprotein in microalgal chloroplasts. Application for generation of edible vaccines

The World Health Organization (WHO), the National Institutes of Health, and UNESCO have stressed the need for a new generation of low-cost vaccines to promote vaccination programs in the poorest regions of the world. They especially emphasized the need for heat-stable vaccines to avoid the considerable expense in maintaining the cold-chain during production and distribution, and needle-free formulations to reduce the risk of opportunistic contamination as well as the need for qualified personnel. Influenza is one of the most significant diseases worldwide, being implicated in 4 major pandemics and the causative agent of ongoing yearly epidemics. Every year Influenza A viruses initiate outbreaks of respiratory tract infection resulting in unacceptable mortality especially in the immuno-compromised persons and the +65's, and morbidity that impacts significantly in workplace economies. Influenza H1N1 nucleoprotein (NP) sequence, codon optimized for C.reinhardtii chloroplast-specific regulatory sequences was designed as an epitope to induce immunity against Influenza. Wild type C. reinhardtii chloroplast genomes were transformed by NP transgene expressed under control of chloroplast-specific regulatory sequences, promotors and terminators. A large region of the tscA gene was used for integration into the chloroplast genome via homological recombination. Integration and expression of NP epitope containing transgene in C. reinhardtii chloroplast genomes was confirmed phenotypically by antibiotic spectinomycin selection and using a set of molecular biology techiques such as PCR, RT-PCR, DNA sequencing and ELISA. It is considered further research would be advantageous to confirm vaccine candidacy