Truncation of NS1 Protein Enhances T Cell-Mediated Cross-Protection of a Live Attenuated Influenza Vaccine Virus Expressing Wild-Type Nucleoprotein

Current seasonal influenza vaccines have suboptimal effectiveness, especially in seasons dominated by viruses that do not match the vaccine. Therefore, finding new approaches to improve the immunogenicity and efficacy of traditional influenza vaccines is of high priority for public health. Licensed live attenuated influenza vaccine (LAIV) is a promising platform for designing broadly protective vaccines due to its ability to induce cross-reactive T-cell immunity. In this study, we tested the hypothesis that truncation of the nonstructural protein 1 (NS1) and the replacement of the nucleoprotein (NP) of the A/Leningrad/17 master donor virus with a recent NP, i.e., switching to 5:3 genome composition, could improve the cross-protective potential of the LAIV virus. We generated a panel of LAIV candidates differing from the classical vaccine by the source of NP gene and/or by the length of NS1 protein. We showed that NS1-modified LAIV viruses had reduced viral replication in the respiratory tract of mice, indicating a more attenuated phenotype compared to the LAIVs with full-length NS1. Most importantly, the LAIV candidate with both NP and NS genes modified induced a robust systemic and lung-localized memory CD8 T-cell response targeting more recent viruses, and better protected immunized mice against lethal challenge with a heterosubtypic influenza virus than the control LAIV variant. Overall, these data indicate that the 5:3 LAIVs with truncated NS1 may be beneficial for protection against heterologous influenza viruses and warrant further preclinical and clinical development.

[1]  D. van Baarle,et al.  A universal influenza mRNA vaccine candidate boosts T cell responses and reduces zoonotic influenza virus disease in ferrets , 2022, bioRxiv.

[2]  S. Ullah,et al.  Next generation live-attenuated influenza vaccine platforms , 2022, Expert review of vaccines.

[3]  G. Leroux-Roels,et al.  Randomized, Double-Blind, Reference-Controlled, Phase 2a Study Evaluating the Immunogenicity and Safety of OVX836, A Nucleoprotein-Based Influenza Vaccine , 2022, Frontiers in Immunology.

[4]  Diane J Post,et al.  A Research and Development (R&D) roadmap for influenza vaccines: Looking toward the future. , 2021, Vaccine.

[5]  L. Rudenko,et al.  Influenza vaccine: progress in a vaccine that elicits a broad immune response , 2021, Expert review of vaccines.

[6]  L. Rudenko,et al.  Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge , 2021, Viruses.

[7]  Yu-Jin Jung,et al.  Broad cross protection by recombinant live attenuated influenza H3N2 seasonal virus expressing conserved M2 extracellular domain in a chimeric hemagglutinin , 2021, Scientific Reports.

[8]  J. Nguyen-Van-Tam,et al.  Decline in Seasonal Influenza Vaccine Effectiveness With Vaccination Program Maturation: A Systematic Review and Meta-analysis , 2021, Open forum infectious diseases.

[9]  Manish M Patel,et al.  Effect of antigenic drift on influenza vaccine effectiveness in the United States - 2019-2020. , 2020, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[10]  Yu-Jin Jung,et al.  Generation and Characterization of Universal Live-Attenuated Influenza Vaccine Candidates Containing Multiple M2e Epitopes , 2020, Vaccines.

[11]  Irina Isakova-Sivak,et al.  Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses , 2020, Antiviral Research.

[12]  K. Subbarao,et al.  Live attenuated influenza vaccine. , 2020, Current topics in microbiology and immunology.

[13]  L. Rudenko,et al.  Insights into current clinical research on the immunogenicity of live attenuated influenza vaccines , 2020, Expert review of vaccines.

[14]  R. Albrecht,et al.  Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge , 2019, Front. Immunol..

[15]  J. Marvel,et al.  OVX836 a recombinant nucleoprotein vaccine inducing cellular responses and protective efficacy against multiple influenza A subtypes , 2019, npj Vaccines.

[16]  L. Rudenko,et al.  Sequential Immunization with Universal Live Attenuated Influenza Vaccine Candidates Protects Ferrets against a High-Dose Heterologous Virus Challenge , 2018, Vaccines.

[17]  A. Egorov,et al.  Enhancement of the immunogenicity of influenza a virus by the inhibition of immunosuppressive function of NS1 protein , 2018 .

[18]  L. Rudenko,et al.  Basics of CD8 T-cell immune responses after influenza infection and vaccination with inactivated or live attenuated influenza vaccine , 2018, Expert review of vaccines.

[19]  R. Cox,et al.  Improving influenza vaccines: challenges to effective implementation. , 2018, Current opinion in immunology.

[20]  Louise A. Carolan,et al.  Safety, immunogenicity and protection of A(H3N2) live attenuated influenza vaccines containing wild-type nucleoprotein in a ferret model. , 2018, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[21]  K. Kedzierska,et al.  Live Attenuated Influenza Vaccines engineered to express the nucleoprotein of a recent isolate stimulate human influenza CD8+ T cells more relevant to current infections , 2018, Human vaccines & immunotherapeutics.

[22]  Jan Kyncl,et al.  Estimates of global seasonal influenza-associated respiratory mortality: a modelling study , 2017, The Lancet.

[23]  S. Takamura Persistence in Temporary Lung Niches: A Survival Strategy of Lung-Resident Memory CD8+ T Cells , 2017, Viral immunology.

[24]  L. Rudenko,et al.  Immunogenicity and Cross Protection in Mice Afforded by Pandemic H1N1 Live Attenuated Influenza Vaccine Containing Wild-Type Nucleoprotein , 2017, BioMed research international.

[25]  L. Rudenko,et al.  Development and approval of live attenuated influenza vaccines based on Russian master donor viruses: Process challenges and success stories , 2016, Vaccine.

[26]  Nicholas S. Kelley,et al.  Variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies. , 2016, The Lancet. Infectious diseases.

[27]  R. Westermeier Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations , 2016 .

[28]  L. Rudenko,et al.  Reassortant viruses for influenza vaccines: is it time to reconsider their genome structures? , 2016, Expert review of vaccines.

[29]  R. Jacobi,et al.  Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets , 2015, PloS one.

[30]  Wenling Wang,et al.  Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice , 2015, Clinical and Vaccine Immunology.

[31]  R. Siliciano,et al.  Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations , 2015, Nature.

[32]  D. Marc Influenza virus non-structural protein NS1: interferon antagonism and beyond. , 2014, The Journal of general virology.

[33]  V. Egorov,et al.  Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins. , 2014, Virus research.

[34]  B. Ferko,et al.  Intranasal vaccination with a replication-deficient influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans. , 2014, Vaccine.

[35]  M. Zheng,et al.  Development of universal influenza vaccines based on influenza virus M and NP genes , 2014, Infection.

[36]  Zhiqiang Liu,et al.  Western Blot: Technique, Theory and Trouble Shooting , 2014, North American journal of medical sciences.

[37]  T. Braciale,et al.  Role of T cell immunity in recovery from influenza virus infection. , 2013, Current opinion in virology.

[38]  D. Kelvin,et al.  Extremely low vaccine effectiveness against influenza H3N2 in the elderly during the 2012/2013 flu season. , 2013, Journal of infection in developing countries.

[39]  K. Kedzierska,et al.  Nucleoprotein of influenza A virus is a major target of immunodominant CD8+ T‐cell responses , 2013, Immunology and cell biology.

[40]  X. Wu,et al.  The role of nasal IgA in children vaccinated with live attenuated influenza vaccine. , 2012, Vaccine.

[41]  Ryan A. Langlois,et al.  NS1-Truncated Live Attenuated Virus Vaccine Provides Robust Protection to Aged Mice from Viral Challenge , 2012, Journal of Virology.

[42]  O. Pleguezuelos,et al.  Synthetic Influenza vaccine (FLU-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled Phase I trial. , 2012, Vaccine.

[43]  M. Rose,et al.  Mucosal immunity and nasal influenza vaccination , 2012, Expert review of vaccines.

[44]  Tao Dong,et al.  Preliminary Assessment of the Efficacy of a T-Cell–Based Influenza Vaccine, MVA-NP+M1, in Humans , 2012, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[45]  N. Cox,et al.  Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2). , 2011, Virology.

[46]  A. Hill,et al.  Potent CD8+ T-Cell Immunogenicity in Humans of a Novel Heterosubtypic Influenza A Vaccine, MVA−NP+M1 , 2011, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[47]  R. Rappuoli,et al.  Influenza vaccine immunology , 2011, Immunological reviews.

[48]  E. Reinherz,et al.  Significant Impact of Sequence Variations in the Nucleoprotein on CD8 T Cell-Mediated Cross-Protection against Influenza A Virus Infections , 2010, PloS one.

[49]  T. Popow-Kraupp,et al.  A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1. , 2010, The Journal of infectious diseases.

[50]  Scott N. Mueller,et al.  Immunization with Live Attenuated Influenza Viruses That Express Altered NS1 Proteins Results in Potent and Protective Memory CD8+ T-Cell Responses , 2009, Journal of Virology.

[51]  J. Oxford,et al.  Preclinical Evaluation of a Replication-Deficient Intranasal ΔNS1 H5N1 Influenza Vaccine , 2009, PloS one.

[52]  F. Hayden,et al.  Report of the 5th meeting on the evaluation of pandemic influenza prototype vaccines in clinical trials: World Health Organization, Geneva, Switzerland, 12-13 February 2009. , 2009, Vaccine.

[53]  D. Voss,et al.  Development of a live-attenuated influenza B DeltaNS1 intranasal vaccine candidate. , 2009, Vaccine.

[54]  A. García-Sastre,et al.  The NS1 Protein of a Human Influenza Virus Inhibits Type I Interferon Production and the Induction of Antiviral Responses in Primary Human Dendritic and Respiratory Epithelial Cells , 2009, Journal of Virology.

[55]  T. Popow-Kraupp,et al.  Influenza B mutant viruses with truncated NS1 proteins grow efficiently in Vero cells and are immunogenic in mice. , 2009, The Journal of general virology.

[56]  John Steel,et al.  Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza , 2008, Journal of Virology.

[57]  L. Stitz,et al.  An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP , 2008, Virology Journal.

[58]  M. Katze,et al.  Functional Genomic and Serological Analysis of the Protective Immune Response Resulting from Vaccination of Macaques with an NS1-Truncated Influenza Virus , 2007, Journal of Virology.

[59]  Adolfo García-Sastre,et al.  Efficacy of intranasal administration of a truncated NS1 modified live influenza virus vaccine in swine. , 2007, Vaccine.

[60]  Larry R. Smith,et al.  Vaxfectin™-Formulated Influenza DNA Vaccines Encoding NP and M2 Viral Proteins Protect Mice against Lethal Viral Challenge , 2007, Human vaccines.

[61]  A. Osterhaus,et al.  Assessment of the extent of variation in influenza A virus cytotoxic T-lymphocyte epitopes by using virus-specific CD8+ T-cell clones. , 2007, The Journal of general virology.

[62]  D. Bruder,et al.  Cellular immunity and lung injury in respiratory virus infection. , 2006, Viral immunology.

[63]  Thomas M. Moran,et al.  Influenza Virus Evades Innate and Adaptive Immunity via the NS1 Protein , 2006, Journal of Virology.

[64]  R. Webby,et al.  Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs , 2005, Journal of Virology.

[65]  C. Walker,et al.  Mutational escape from CD8+ T cell immunity , 2005, The Journal of experimental medicine.

[66]  N. Bardiya,et al.  Influenza vaccines: recent advances in production technologies , 2005, Applied Microbiology and Biotechnology.

[67]  H. Katinger,et al.  Immunogenicity and Protection Efficacy of Replication-Deficient Influenza A Viruses with Altered NS1 Genes , 2004, Journal of Virology.

[68]  D. Levy,et al.  Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. , 1998, Virology.

[69]  A. Monto,et al.  Efficacy of live attenuated and inactivated influenza vaccines in schoolchildren and their unvaccinated contacts in Novgorod, Russia. , 1993, The Journal of infectious diseases.

[70]  B. Moss,et al.  Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[71]  Li-mei Chen,et al.  Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model. , 2017, Virology.

[72]  Caitlin E. Mullarkey,et al.  Coadministration of seasonal influenza vaccine and MVA-NP+M1 simultaneously achieves potent humoral and cell-mediated responses. , 2014, Molecular therapy : the journal of the American Society of Gene Therapy.

[73]  Nicholas S. Kelley,et al.  Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. , 2012, The Lancet. Infectious diseases.

[74]  A. García-Sastre,et al.  Attenuated influenza virus vaccines with modified NS1 proteins. , 2009, Current topics in microbiology and immunology.