Immune Responses in Acute and Convalescent Patients with Mild, Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway

RESEARCH ARTICLE Immune Responses in Acute and Convalescent Patients with Mild, Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway Kristin G.-I. Mohn1,2,5*, Rebecca Jane Cox1,3,5, Gro Tunheim4,5, Jan Erik Berdal6, Anna Germundsson Hauge7,8, Åsne Jul-Larsen1, Norwegian Pandemic Group¶, Bjoern Peters9, Fredrik Oftung4,5, Christine Monceyron Jonassen10,11,12☯, Siri Mjaaland4,5☯* OPEN ACCESS Citation: Mohn KG-I, Cox RJ, Tunheim G, Berdal JE, Hauge AG, Jul-Larsen Å, et al. (2015) Immune Responses in Acute and Convalescent Patients with Mild, Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway. PLoS ONE 10(11): e0143281. doi:10.1371/journal.pone.0143281 Editor: Florian Krammer, Icahn School of Medicine at Mount Sinai, UNITED STATES 1 The Influenza Centre, Department of Clinical Science, University of Bergen, Bergen, Norway, 2 Infectious Diseases Unit, Department of Internal Medicine, Haukeland University Hospital, Bergen, Norway, 3 Department of Research & Development, Haukeland University Hospital, Bergen, Norway, 4 Division of Infectious Disease Control, Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Oslo, Norway, 5 K.G. Jebsen Centre for Influenza Vaccine Research, Department of Clinical Science, University of Bergen, Bergen, and The Norwegian Institute of Public Health, Oslo, Norway, 6 Department of Infectious Diseases, Akershus University Hospital, Nordbyhagen, Norway, 7 Section for Virology, Department of Laboratory Services, Norwegian Veterinary Institute, Oslo, Norway, 8 Division of Infectious Disease Control, Norwegian Institute of Public Health, Oslo, Norway, 9 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, United States of America, 10 Genetic Unit, Department of Multidisciplinary Laboratory Medicine and Medical Biochemistry, Akershus University Hospital, Nordbyhagen, Norway, 11 Genetic Unit, Centre for Laboratory Medicine, Østfold Hospital Trust, Fredrikstad, Norway, 12 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway ☯ These authors contributed equally to this work. ¶ Membership of the Norwegian Pandemic Group is listed in the Acknowledgments. * (KGIM); (SM) Received: August 2, 2015 Accepted: November 3, 2015 Published: November 25, 2015 Copyright: © 2015 Mohn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The study was funded intramurally by the Influenza Centre at the University of Bergen, the Akershus University Hospital, and the Norwegian Institute of Public Health. Funding was received from the Bergen Clinical Vaccine Consortium and by the Ministry of Health and Care Services, Norway, the Norwegian Research Council Globvac program (220670), the Research Counsil Norway Bio bank (221122), the European Union (Univax 601738, Unisec 602012 and EU IMI115672 FLUCOP), Helse Abstract Increased understanding of immune responses influencing clinical severity during pandemic influenza infection is important for improved treatment and vaccine development. In this study we recruited 46 adult patients during the 2009 influenza pandemic and characterized humoral and cellular immune responses. Those included were either acute hospitalized or convalescent patients with different disease severities (mild, moderate or severe). In general, protective antibody responses increased with enhanced disease severity. In the acute patients, we found higher levels of TNF-α single-producing CD4+T-cells in the severely ill as compared to patients with moderate disease. Stimulation of peripheral blood mononuclear cells (PBMC) from a subset of acute patients with peptide T-cell epitopes showed significantly lower frequencies of influenza specific CD8+ compared with CD4+ IFNγ T-cells in acute patients. Both T-cell subsets were predominantly directed against the envelope antigens (HA and NA). However, in the convalescent patients we found high levels of both CD4+ and CD8+ T-cells directed against conserved core antigens (NP, PA, PB, and M). The results indicate that the antigen targets recognized by the T-cell subsets may vary according to the phase of infection. The apparent low levels of cross-reactive CD8+ T- PLOS ONE | DOI:10.1371/journal.pone.0143281 November 25, 2015 1 / 16 Low Levels of CD8+T-Cells in Hospitalized Patients with Pandemic H1N1 Vest and the K.G. Jebsen Centre for Research on Influenza Vaccines. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. cells recognizing internal antigens in acute hospitalized patients suggest an important role for this T-cell subset in protective immunity against influenza. Competing Interests: The authors have declared that no competing interests exist. Introduction During the 2009 influenza pandemic, young and otherwise healthy people experienced severe illness and mortality [1–4]. During the main wave of the pandemic in Norway, 1300 people were hospitalized, 200 patients received intensive care treatment, and 29 patients died [5]. Nevertheless, in hindsight, this pandemic was regarded as mild [6]. Post-pandemic studies have described the clinical picture, the risk factors associated with disease outcome, and effects of vaccines and antiviral medication [1,3,7–12]. Specific viral mutations and several host factors and underlying conditions, such as obesity and pregnancy, were identified and associated with increased disease severity [13–17]. People older than 65 years old experienced less severe infection, probably due to pre-existing cross-reactive immunity generated by previous H1N1 infections [18]. Seasonal vaccination or infection induces strain-specific neutralizing antibodies directed towards the viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HAspecific antibodies measured by the hemagglutination inhibition assay (HI) are defined as the primary correlate of protection against influenza in man (HI titers 40) [19]. However, strainspecific antibodies do not provide cross-protection against new epidemic or pandemic viruses [20]. Hence, due to the lack of protective antibodies, the novel A(H1N1)pdm09 virus spread rapidly worldwide. In contrast to antibodies, T-cells may mediate cross-protective immunity between strains due to recognition of epitopes from the conserved core antigens of the virus, which have a high degree of homology, e.g. (nucleoprotein (NP), the polymerases (PB1, PB2 and PA) and matrix (M) proteins. T-cells play important roles in coordinating and regulating the immune response against influenza [21]. CD4+ T-cells help B-ce (...truncated)