2nd International Conference on Influenza September 12-13, 2016 Berlin, Germany

Jana Pokorna completed PhD in biochemistry from Charles University in Prague in 2013. She works as an Postdoctoral Fellow at the Institute of Organic Chemistry and Biochemistry ASCR, v.v.i.. Her research interests are activity, inhibition, drug and resistance development focusing on HIV protease and neuraminidase from the Influenza virus. She published 11 peer viewed papers and she is the author of 3 patents.

Influenza is an acute viral infection that can cause serious complications and death, especially among elderly individuals and patients at risk. Neuraminidase, which plays an essential role in virus replication, is the main influenza drug target. At present, two neuraminidase inhibitors (NAIs) are licensed worldwide for therapeutic and prophylactic uses (oseltamivir marketed as Tamiflu and zanamivir, Relenza) and two others have been authorized in various countries for the emergency treatment during pandemics. However, drug resistant viruses readily emerge because of the high mutation rate of their RNA dependent RNA polymerase. Indeed, resistance to oseltamivir, the most prescribed NAI, was detected not only during treatment and prophylaxis, but also in influenza virus variants in untreated individuals. Novel neuraminidase inhibitor resistance substitutions I223V and S247N alone or in combination with a major oseltamivir resistance mutation H275Y have been observed recently in the 2009 pandemic H1N1 viruses. We overexpressed the ectodomain of the wild type neuraminidase from the influenza virus A/California/07/2009 (H1N1) as well as recombinants containing H275Y, I223V, and S247N single mutation and the H275Y, I223V and H275Y, S247N double mutants in Drosophila Schneider S2 cells and purified them by one-step purification using a streptavidin derivative. In order to quantify the level of resistance we enzymologically characterized these enzymes with the set of in-house designed and synthesized derivatives of oseltamivir. Thermodynamic analyses of oseltamivir binding to neuraminidase monomutants were performed by protein microcalorimetry. Finally, we crystallized neuraminidase variants in complexes with oseltamivir to structurally explain the resistance mechanism.

Ya-Yzu Chang is a Public Health Officer of the Department of Division of Preparedness and Emerging Infectious Disease in Taiwan Centers for Disease Control. She is responsible for policy making of influenza prevention and control and has handled experiences on 2009 H1N1 pandemic influenza and H7N9 influenza in Taiwan.

The 2015-2016 influenza season, was a tough period for Taiwan, having 1932 confirmed severe and complicated influenza cases including 328 estimated deaths. Most severe cases (about 77%) were infected with influenza A (H1N1) pdm09 virus. The majority of severe complicated influenza cases and deaths were adult aged 50-64 years. The incidences among all age groups were highest compared to the same period in the last 3 years, especially in the 50-64 age group. The main attacked age group changed from 65 years above to 50-64 years, similar demographic pattern seen in 2009 H1N1 pandemic. The government-funded influenza vaccination program in 2015-2016, following the international consensus, mainly targeted the elders aged more than 65 years, children aged six months through elementary school students and people aged above 50 years with chronic medical conditions. As a result, most people aged 50-64 years have not received influenza vaccines. In addition, the coverage rate of people with chronic diseases was only about 9%. Due to these reasons, in 2016/17 season, we plan to increase the purchase of influenza vaccines and vaccination points, as well as the awareness of the public, to improve the vaccination coverage rates and subsequently lower influenza incidence among people with chronic diseases.

Milan Kožíšek is a senior scientist at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague in the group of Jan Konvalinka, Ph.D. He finished there his Ph.D. in 2010 after defense of the thesis “Overcoming drug resistence: The discovery, design and characterization of new nonpeptidic inhibitors of HIV-1 protease“. He is author of 29 papers in peer-reviewed international journals and 4 patents.

Influenza virus causes severe respiratory infections in birds and mammals that are responsible for up to half a million deaths of human beings worldwide each year. Two targets of therapeutic interventions in influenza life cycle, viral neuraminidase and M2 channel, are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target and design of its inhibitor. Recently, an interesting protein-protein interaction between two subunits of viral polymerase PA and PB1 has been identified as a new promising drug target. The fact that relatively few residues drive the binding and the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we have expressed and purified recombinant C-terminal part of the PA polymerase subunit with GST at its N-terminus from pandemic isolate A/California/07/2009 H1N1. The biotinylated peptide representing the N-terminal interacting part of PB1 subunit was synthesized by using a solid-phase synthesizer. The protein-protein interaction between PA and PB1 was then kinetically characterized using a surface plasmon resonance (SPR). Finally, we developed and optimized an assay for screening the compounds disrupting the interaction between polymerase subunits, based on the AlphaScreen technology and validated the assay that has the potential to be used in drug discovery.

Hyeon Jang have completed my degree of Master of Science in Medicine at the age of 27 years from Seoul National University. I am an associate II (research worker) for 2 years in GCC, a leading pharmaceutical company in ROK.

It is generally known that as people get older, immune responses diminish. A series of studies supports the decreased immune response to influenza vaccination in the elderly might cause severe respiratory complications associated with influenza infection. For this reason, Fluad (Seqirus) and Fluzone High Dose (Sanofi Pasteur), which were recently approved adjuvanted and high-dose (HD) influenza vaccines, respectively, are recommended for the elderly as they produce higher SCR and SPR than conventional influenza vaccines. Especially, manufacturing of HD influenza vaccines need an additional concentration step in which micro-porous membranes are usually applied. High concentration of HA (hemagglutinin) and low levels of surfactant residues should be achieved in HD vaccines. To establish the concentration process, contents of HA and surfactant residues were compared among 3 membranes of different manufacturers(Sartorius, Merck, and PALL). We found that HA concentration increased proportionally to the concentration factor, and the sequential dilution step decreased levels of surfactant residues. Meanwhile, cellulose acetate (CA) and polyether sulfone (PES) are commonly used as micro-porous membrane materials but the characteristics of each material and the interaction between concentrates and membranes may have different influences on the concentration capability of membranes. In our study, PES showed equivalent capability to CA in concentrating HA, but 2-fold higher capability than CA in decreasing surfactant residues

Jin-Wook Jang is currently pursuing Doctoral studies from Seoul National University, Republic of Korea.

Avian influenza (AI) vaccines for poultry are based on hemagglutinin (HA) proteins and protection is specific to the vaccine subtype. Since 2004, AI vaccine strains have been developed using reverse genetic systems. Recent pair-wise comparison with internal genes of A/chicken/Korea/01310/2011 (H9N2; 01310) and A/chicken/Korea/KBNP-0028/2000 (H9N2; 0028) revealed that recombinant PR8 viruses possessing the PB2 of 01310 or NS of 0028 decreased pathogenicity in mice, protected against PR8 challenge and increased replication efficiency in embryonated chicken eggs (ECEs). And the LPAI H5N1 recombinant virus containing PB2 of 01310 or NS of 0028 reduced pathogenicity in mice and had high replication efficiency in ECEs. In the present study, we generated PR8-derived H5N1 recombinant viruses which have HA and NA gene of H5N1 HPAI virus A/mandarin duck/Korea/K10-483/2010 (K10-483), PB2 of 01310 and NS of 0028. The reassorted H5N1 virus possessing PB2 of 01310 [rH5N1-PB2(01310)] showed significantly higher replication efficacy in ECEs than the control H5N1 recombinant virus that containing six internal genes of PR8 (rH5N1). In contrast, replication efficacy in MDCK cell of recombinant virus that is harboring PB2 of 01310 and NS of 0028 [rH5N1-PB2(01310)-NS(0028)] was significantly lower than that of rH5N1. All recombinant viruses did not cause body weight loss in mice, although only control rH5N1 virus replicated in the lungs of inoculated mice. Thus, the novel vaccine strains that containing PB2 and NS gene of LPAIV may be useful to develop safe and efficacious vaccines.

Aliou Barry holds a Doctorate degree in Medicine. He has obtained a Diploma of specialized studies at the Cheikh Anta Diop University of Dakar, Senegal in 2012. He has received a Master II Scholarship in Tropical Medicine, Public Health and Research account of University agency of the Francophonie which he has completed in 2014. He has worked as a public health doctor In-Charge of coordinating the influenza surveillance within the unity of infectious disease epidemiology at Institute Pastor of Dakar, Senegal. He is currently finalizing a university degree in epidemiology at the University of Bordeaux, France.

In Senegal, since 2012, a sentinel syndromic-based surveillance system was established with the main goal of rapidly identifying outbreaks and issuing alerts. We describe the steps involved in developing a sentinel surveillance system and the well-timed information it provides for improving public health decision-making. The Senegalese sentinel surveillance network is based on data for fever and diarrheal syndromes collected by sentinel general practitioners (SGP). The SGPs were expected to communicate at least once a day encrypted short messages (number of fever cases, rapid test confirmed Malaria, ILI and Dengue-like syndromes or Diarrheal disease) from mobile phone. Standard WHO case definitions are used to ensure comparability. Data are validated by the management team and analyzed daily at the IPD. This data transmission costs 750 FCFA, around US$1 per month per sentinel center. In 2015, the sentinel surveillance system included 17 health centers and identified four (4) outbreaks confirmed: Two with an increase in ILI indicators (Influenza AH1N1, H3N2), one with an increase in RDT-confirmed cases of malaria and one with an increase in diarrhea disease. Of the 181,955 visits to SGPs, 22% were related to fever syndromes. Of these 40,030 fever cases, 32% were related to influenza-like illness, 6% to dengue-like syndrome, 16% to malaria cases confirmed by a specific rapid diagnostic test and 4% to diarrhea. Senegal’s sentinel syndromic surveillance system represents the country’s first nationwide “real-time” surveillance system. It has proved the feasibility of improving disease surveillance capacity through innovative systems despite resource constraints.

Hind Bouguerra has completed her Medical studies from the Faculty of Medicine of Tunis, Tunisia. She has specialized in Preventive Medicine and Public Health. She has a Master of Biostatics, Epidemiology and Clinical Research degree from the Faculty of Medicine of Tunis. She has worked in the Laboratory of Epidemiology at Pasteur Institute of Tunis and in the National Observatory of New and Emerging Diseases of Tunisia, participating in many papers. She has worked mostly in epidemiological surveillance including influenza program in Tunisia which is supported by US/CDC, part of InPRIS project.

Introduction: Seasonal influenza continues to be a major public health problem worldwide. In fact, this acute viral infection is highly contagious and affects all ages. Although in most cases it is a minor illness, it may lead to severe complications and death especially in high risk populations. Purpose: To describe influenza severe cases and deaths in the season of 2015-2016 and compare it to previous seasons, to determine what are the influenza viruses currently circulating and which types have particular virulence in this season and to suggest recommendations to improve influenza control. Methods: This is a retrospective study based on data provided by the national influenza surveillance unit. It is a descriptive analysis of influenza surveillance data collected from the network of sentinel sites and national influenza center. Results & Discussion: Influenza surveillance for 2015-2016 lasted from week 40/2015 to week 18/2016. During this season, 96240 cases of ILI (Influenza-like illness) were collected representing 6.9% of total patients seen at ILI sites. Among these cases, 190 were severe and hospitalized. Their age ranged from 6 months to 73 years with an average of 46.5 years. The hospitalization rate was 0.19% and comparable to the previous season (0.2%). However, the lethality of these severe cases was significantly higher in 2015-2016 Season. In fact, 38 deaths were reported representing 20% (vs. 3% in 2014-2015 Season). The majority of them were men (57%). The average age was 46.9 years with extremes varying from 6 months to 73 years. The most affected age group was the 50- 65 year group. Most of the cases who died had risk factors (62.9%) especially diabetes, HTA and obesity. All the cases were not vaccinated. The virological analysis showed that 57% of severe cases and 77% of influenza deaths were infected with type A (H1N1) pmd09 virus. The rest of the deaths (23%) were due to A (H3N2) virus and only one death was due to virus B. During week 12, A (H1N1) was predominant and simultaneously the highest number of deaths was reported (10 deaths representing 26.3% of all influenza deaths). Conclusion: Comparing to last season 2014-2015, the influenza epidemics of 2015-2016 is considered similar in terms of number of ILI cases and hospitalization rate. However, the lethality of severe cases was significantly higher with 38 deaths reported this season. The type A (H1N1) pmd09 virus was responsible of most of severe cases and deaths, confirming its known virulence.

Sophie Buffin joined the Research department of Sanofi Pasteur in 2005 with a Master’s degree. She is currently a Ph.D. student at the University of Lyon.

To formulate inactivated influenza vaccines, the concentration of haemagglutinin (HA) must be accurately determined. The standard test currently used to measure HA in influenza vaccines is the single radial immunodiffusion (SRID) assay. The SRID assay is a cumbersome technique presenting a number of drawbacks such as low sensitivity and interference by some adjuvants. We developed a very simple, sensitive and rapid alternative HA assay using latex agglutination. The LAA uses the Spherotest® technology, which is based on the agglutination of HA-specific immunoglobulin-coated latex beads, which bind to the HA. The amount of HA in a sample can then be calculated from the level of bead agglutination by a simple absorbance measurement. A standard curve is generated using serially diluted HA reference protein. The results show that for monovalent A/H5N1 and A/H1N1 vaccines, the LAA demonstrated equivalent linearity, accuracy and precision as compared to the SRID assay. Moreover, unlike the SRID assay, LAA enables HA quantification in AlOOH-adjuvanted vaccines and in emulsion-based adjuvanted vaccines without interference. In addition, LAA was found to be more simple, rapid and sensitive than SRID. In conclusion, LAA may be useful to rapidly and accurately quantify the influenza HA protein in monovalent vaccines, especially in those formulated with low amounts of HA in the presence of an adjuvant.