Cell-Based Systems Biology Analysis of Human AS03-Adjuvanted H5N1 Avian Influenza Vaccine Responses: A Phase I Randomized Controlled Trial

Background Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood. Objective and Methods We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18–49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination. Results Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination. Conclusions Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed. Trial Registration ClinicalTrials.gov NCT01573312

[1]  E. De Gregorio,et al.  MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action , 2012, The Journal of Immunology.

[2]  R. Couch,et al.  Safety and immunogenicity of nonadjuvanted and MF59-adjuvanted influenza A/H9N2 vaccine preparations. , 2006, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[3]  W. Lim,et al.  Immunogenicity and safety of a two-dose schedule of whole-virion and AS03A-adjuvanted 2009 influenza A (H1N1) vaccines: a randomised, multicentre, age-stratified, head-to-head trial. , 2011, The Lancet. Infectious diseases.

[4]  R B Couch,et al.  Immunity to influenza in man. , 1983, Annual review of microbiology.

[5]  J. Haanen,et al.  Natural killer cell clones can efficiently process and present protein antigens. , 1991, Journal of immunology.

[6]  C. Samuel,et al.  Antiviral Actions of Interferons , 2001, Clinical Microbiology Reviews.

[7]  G. Ott,et al.  Recent advances in vaccine adjuvants: the development of MF59 emulsion and polymeric microparticles. , 1997, Molecular medicine today.

[8]  R. Tampé,et al.  A functionally defective allele of TAP1 results in loss of MHC class I antigen presentation in a human lung cancer , 1996, Nature Genetics.

[9]  M. Robinson,et al.  A scaling normalization method for differential expression analysis of RNA-seq data , 2010, Genome Biology.

[10]  M. Chignard,et al.  Cutting Edge: Innate Immune Response Triggered by Influenza A Virus Is Negatively Regulated by SOCS1 and SOCS3 through a RIG-I/IFNAR1-Dependent Pathway1 , 2008, The Journal of Immunology.

[11]  V. Kapur,et al.  Transcriptional response of human microglial cells to interferon-γ , 2005, Genes and Immunity.

[12]  D. Wiley,et al.  Identification of domain boundaries within the N‐termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin‐mediated increase in peptide loading of MHC class I , 2005, Immunology and cell biology.

[13]  D. Bernstein,et al.  Effects of adjuvants on the safety and immunogenicity of an avian influenza H5N1 vaccine in adults. , 2008, The Journal of infectious diseases.

[14]  J. Hanna,et al.  Novel APC-like properties of human NK cells directly regulate T cell activation , 2015, bioRxiv.

[15]  T. Honap,et al.  Alagarasu K*, Honap T*, Damle IM, Mulay AP, Shah PS, and Cecilia D (2013). Polymorphisms in the Oligoadenylate Synthetase Gene Cluster and its Association with Clinical Outcomes of Dengue Virus Infection. Infection, Genetics, and Evolution 14:390-395 , 2016 .

[16]  D. Taub,et al.  Alpha and beta chemokines induce NK cell migration and enhance NK-mediated cytolysis. , 1995, Journal of immunology.

[17]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[18]  David Hinkley,et al.  Bootstrap Methods: Another Look at the Jackknife , 2008 .

[19]  H. S. Kaye,et al.  Inactivated vaccines. 1. Volunteer studies with very high doses of influenza vaccine purified by zonal ultracentrifugation* , 1973, Postgraduate medical journal.

[20]  D. Carr,et al.  Dysregulation of CXCR3 Signaling due to CXCL10 Deficiency Impairs the Antiviral Response to Herpes Simplex Virus 1 Infection1 , 2008, The Journal of Immunology.

[21]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[22]  H. Matzkin,et al.  Accidental tenfold overdose of influenza vaccine: a clinical and serological study. , 1984, Israel journal of medical sciences.

[23]  Shuzhao Li,et al.  Systems vaccinology: learning to compute the behavior of vaccine induced immunity , 2012, Wiley interdisciplinary reviews. Systems biology and medicine.

[24]  O. Ramilo,et al.  Plasticity and Virus Specificity of the Airway Epithelial Cell Immune Response during Respiratory Virus Infection , 2012, Journal of Virology.

[25]  B. Efron.  Bootstrap Methods: Another Look at the Jackknife , 1979 .

[26]  J. Langley,et al.  Safety and cross-reactive immunogenicity of candidate AS03-adjuvanted prepandemic H5N1 influenza vaccines: a randomized controlled phase 1/2 trial in adults. , 2010, The Journal of infectious diseases.

[27]  Volker Brauer,et al.  Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels , 2009, Proceedings of the National Academy of Sciences.

[28]  Charlotte E. Egan,et al.  Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation. , 2011, International immunology.

[29]  S. Nishino,et al.  Hypocretin/orexin and narcolepsy: new basic and clinical insights , 2010, Acta physiologica.

[30]  J. Taubenberger,et al.  Influenza : the Mother of All Pandemics , 2022 .

[31]  A. Takashima,et al.  Neutrophil plasticity: acquisition of phenotype and functionality of antigen‐presenting cell , 2015, Journal of leukocyte biology.

[32]  Shinn-Jang Hwang,et al.  Immunogenicity and tolerability of an AS03(A)-adjuvanted prepandemic influenza vaccine: a phase III study in a large population of Asian adults. , 2009, Vaccine.

[33]  Mark Wolff,et al.  Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. , 2006, The New England journal of medicine.

[34]  Marc Lipsitch,et al.  Estimates of the Prevalence of Pandemic (H1N1) 2009, United States, April–July 2009 , 2009, Emerging infectious diseases.

[35]  M. Kanehisa,et al.  Computation with the KEGG pathway database. , 1998, Bio Systems.

[36]  M. Pizza,et al.  The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells , 2009, The Journal of Immunology.

[37]  D. Ostanin,et al.  Acquisition of Antigen-Presenting Functions by Neutrophils Isolated from Mice with Chronic Colitis , 2012, The Journal of Immunology.

[38]  P. Chomez,et al.  Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity. , 2011, Vaccine.

[39]  Helga Thorvaldsdóttir,et al.  Molecular signatures database (MSigDB) 3.0 , 2011, Bioinform..

[40]  Andrea A. Berry,et al.  Effect of Varying Doses of a Monovalent H7N9 Influenza Vaccine With and Without AS03 and MF59 Adjuvants on Immune Response: A Randomized Clinical Trial. , 2015, JAMA.

[41]  K. Takeda,et al.  Natural killer (NK)–dendritic cell interactions generate MHC class II-dressed NK cells that regulate CD4+ T cells , 2011, Proceedings of the National Academy of Sciences.

[42]  Janet M. Young,et al.  Rapid Evolution of PARP Genes Suggests a Broad Role for ADP-Ribosylation in Host-Virus Conflicts , 2014, PLoS genetics.

[43]  Mario Cortese,et al.  Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. , 2011, Vaccine.

[44]  E. Birney,et al.  Reactome: a knowledgebase of biological pathways , 2004, Nucleic Acids Research.

[45]  P M Guyre,et al.  Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-gamma, and IL-3. , 1993, Journal of immunology.

[46]  G. Superti-Furga,et al.  IFITs: Emerging Roles as Key Anti-Viral Proteins , 2014, Front. Immunol..

[47]  Shuzhao Li,et al.  Systems vaccinology: Probing humanity’s diverse immune systems with vaccines , 2014, Proceedings of the National Academy of Sciences.

[48]  Hidetoshi Shimodaira,et al.  Pvclust: an R package for assessing the uncertainty in hierarchical clustering , 2006, Bioinform..

[49]  J. Oxford,et al.  Influenza A pandemics of the 20th century with special reference to 1918: virology, pathology and epidemiology , 2000, Reviews in medical virology.

[50]  M. Zambon,et al.  Safety and immunogenicity of an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine: phase I randomised trial , 2006, The Lancet.

[51]  Taku Kambayashi,et al.  Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell? , 2014, Nature Reviews Immunology.

[52]  G. Leroux-Roels,et al.  Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial , 2007, The Lancet.

[53]  N. Garçon.  Preclinical development of AS04. , 2010, Methods in molecular biology.

[54]  Koichi S. Kobayashi,et al.  NLRC5: a key regulator of MHC class I-dependent immune responses , 2012, Nature Reviews Immunology.

[55]  D. Noah,et al.  Qualification of the Hemagglutination Inhibition Assay in Support of Pandemic Influenza Vaccine Licensure , 2009, Clinical and Vaccine Immunology.

[56]  Takuma Hayashi,et al.  The mutation in the ATP-binding region of JAK1, identified in human uterine leiomyosarcomas, results in defective interferon-γ inducibility of TAP1 and LMP2 , 2006, Oncogene.

[57]  H. Hauser,et al.  Distinct modes of action applied by transcription factors STAT1 and IRF1 to initiate transcription of the IFN-γ-inducible gbp2 gene , 2007, Proceedings of the National Academy of Sciences.

[58]  Yan Guo,et al.  A Cell-Based Systems Biology Assessment of Human Blood to Monitor Immune Responses after Influenza Vaccination , 2015, PloS one.

[59]  Julie G. Burel,et al.  Systems Approaches towards Molecular Profiling of Human Immunity. , 2016, Trends in immunology.

[60]  N. Cox,et al.  Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. , 1998, Science.

[61]  Tzeng-Ji Chen,et al.  Immunogenicity and safety of an AS03(A)-adjuvanted H5N1 influenza vaccine in a Taiwanese population. , 2011, Journal of the Formosan Medical Association = Taiwan yi zhi.

[62]  K. Hoschler,et al.  Trial of 2009 influenza A (H1N1) monovalent MF59-adjuvanted vaccine. , 2009, The New England journal of medicine.

[63]  P. Fisher,et al.  Fas-Associated Death Domain-Containing Protein-Mediated Antiviral Innate Immune Signaling Involves the Regulation of Irf71 , 2007, The Journal of Immunology.

[64]  J. Neefjes,et al.  Towards a systems understanding of MHC class I and MHC class II antigen presentation , 2011, Nature Reviews Immunology.

[65]  J. Haensler,et al.  AF03, an alternative squalene emulsion-based vaccine adjuvant prepared by a phase inversion temperature method. , 2012, Journal of pharmaceutical sciences.

[66]  J. L. La Montagne,et al.  Overview of clinical trials of influenza vaccines, 1976. , 1977, The Journal of infectious diseases.

[67]  Eva K. Lee,et al.  Systems Biology of Seasonal Influenza Vaccination in Humans , 2011, Nature Immunology.

[68]  W. Beyer,et al.  Antibody response after influenza immunization with various vaccine doses: a double-blind, placebo-controlled, multi-centre, dose-response study in elderly nursing-home residents and young volunteers. , 1993, Vaccine.

[69]  W. Keitel,et al.  Adjuvants for pandemic influenza vaccines. , 2009, Current topics in microbiology and immunology.

[70]  J. Ryan,et al.  Activating and Inhibitory Ly49 Receptors Modulate NK Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 10 and CXCL12 1 , 2003, The Journal of Immunology.

[71]  R. Childs,et al.  CXCL10-induced migration of adoptively transferred human natural killer cells toward solid tumors causes regression of tumor growth in vivo , 2015, Cancer Immunology, Immunotherapy.

[72]  Ann L Oberg,et al.  Systems biology approaches to new vaccine development. , 2011, Current opinion in immunology.

[73]  J. Nevins,et al.  Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[74]  L. Magder,et al.  The Guillain-Barré syndrome and the 1992-1993 and 1993-1994 influenza vaccines. , 1998, The New England journal of medicine.

[75]  E Lazzeri,et al.  Interferon-inducible protein 10, monokine induced by interferon gamma, and interferon-inducible T-cell alpha chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) alphabeta+ CD8+ single-positive T cells, TCRgammadelta+ T cells, and natural killer-type cells in hum , 2001, Blood.

[76]  Reiner Schulz,et al.  Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events , 2016, Nature Immunology.

[77]  M. Mulligan,et al.  Safety and immunogenicity of influenza A H5 subunit vaccines: effect of vaccine schedule and antigenic variant. , 2011, The Journal of infectious diseases.

[78]  Arnaud M. Didierlaurent,et al.  AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity1 , 2009, The Journal of Immunology.

[79]  Jeremy Seto,et al.  Antiviral Response Dictated by Choreographed Cascade of Transcription Factors , 2010, The Journal of Immunology.

[80]  R. Cherry,et al.  Interaction of HLA‐DR and CD74 at the cell surface of antigen‐presenting cells by single particle image analysis , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[81]  B. Pulendran,et al.  Immunogenomics and systems biology of vaccines , 2011, Immunological reviews.

[82]  Pak Chung Sham,et al.  Two-stage genome-wide association study identifies variants in CAMSAP1L1 as susceptibility loci for epilepsy in Chinese. , 2012, Human molecular genetics.

[83]  E. Remarque,et al.  Improvement of the immunoglobulin subclass response to influenza vaccine in elderly nursing-home residents by the use of high-dose vaccines. , 1993, Vaccine.

[84]  C. Denning,et al.  Immunization of Elderly People with High Doses of Influenza Vaccine , 1988, Journal of the American Geriatrics Society.

[85]  J. Banchereau,et al.  Gene expression patterns in blood leukocytes discriminate patients with acute infections. , 2007, Blood.

[86]  P. Brown,et al.  Gene expression patterns in human placenta. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[87]  R. Coffman,et al.  Vaccine adjuvants: putting innate immunity to work. , 2010, Immunity.

[88]  Mark J. Miller,et al.  Neutrophils Influence the Level of Antigen Presentation during the Immune Response to Protein Antigens in Adjuvants , 2010, The Journal of Immunology.

[89]  M. Eichelberger,et al.  FDA/NIH/WHO public workshop on immune correlates of protection against influenza A viruses in support of pandemic vaccine development, Bethesda, Maryland, US, December 10-11, 2007. , 2008, Vaccine.

[90]  D. Carr,et al.  Dysregulation of CXCR 3 Signaling due to CXCL 10 Deficiency Impairs the Antiviral Response to Herpes Simplex Virus 1 Infection 1 , 2008 .

[91]  Eva K. Lee,et al.  Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans , 2009, Nature Immunology.

[92]  N. Groth,et al.  MF59-adjuvanted versus non-adjuvanted influenza vaccines: integrated analysis from a large safety database. , 2009, Vaccine.

[93]  F. Ruben,et al.  Humoral and secretory antibody responses to immunization with low and high dosage split influenza virus vaccine , 2005, Archives of Virology.

[94]  M. Peiris,et al.  Human infection with influenza H9N2 , 1999, The Lancet.

[95]  R. Rappuoli,et al.  Vaccines with MF59 Adjuvant Expand the Antibody Repertoire to Target Protective Sites of Pandemic Avian H5N1 Influenza Virus , 2010, Science Translational Medicine.

[96]  I. McInnes,et al.  Murine neutrophils present Class II restricted antigen , 2008, Immunology letters.

[97]  M. Zambon,et al.  Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza , 2001, The Lancet.

[98]  S. Kakinuma,et al.  Antiviral effects of the interferon‐induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein , 2009, Hepatology.

[99]  W. Lim,et al.  Immunogenicity and safety of a two-dose schedule of whole-virion and AS03(A)-adjuvanted 2009 influenza A (H1N1) vaccines: a randomised, multicentre, age-stratified, head-to-head trial , 2011 .

[100]  Robert T. Chen,et al.  Guillain-Barré syndrome following influenza vaccination , 2004, Reactions Weekly.

[101]  J. Banchereau,et al.  Ribosomal protein mRNAs are translationally-regulated during human dendritic cells activation by LPS , 2009, Immunome research.

[102]  R. Couch,et al.  Increasing doses of purified influenza virus hemagglutinin and subvirion vaccines enhance antibody responses in the elderly , 1996, Clinical and diagnostic laboratory immunology.

[103]  G. Leroux-Roels,et al.  Priming with AS03 A-adjuvanted H5N1 influenza vaccine improves the kinetics, magnitude and durability of the immune response after a heterologous booster vaccination: an open non-randomised extension of a double-blind randomised primary study. , 2010, Vaccine.

[104]  W. Keitel,et al.  Safety and immunogenicity of an inactivated influenza A/H5N1 vaccine given with or without aluminum hydroxide to healthy adults: results of a phase I-II randomized clinical trial. , 2008, The Journal of infectious diseases.

[105]  D. Snydman.  Safety and Immunogenicity of an Inactivated Subvirion Influenza A (H5N1) Vaccine , 2007 .

[106]  T. Honap,et al.  Polymorphisms in the oligoadenylate synthetase gene cluster and its association with clinical outcomes of dengue virus infection. , 2013, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[107]  F. Kuper,et al.  Non‐clinical safety and biodistribution of AS03‐adjuvanted inactivated pandemic influenza vaccines , 2015, Journal of applied toxicology : JAT.

[108]  L. Trautmann,et al.  Solving vaccine mysteries: a systems biology perspective , 2011, Nature Immunology.

[109]  R. Rappuoli,et al.  Molecular and cellular signatures of human vaccine adjuvants , 2008, Proceedings of the National Academy of Sciences.

[110]  J. Haensler,et al.  Emulsion-based adjuvants for influenza vaccines , 2009, Expert review of vaccines.

[111]  S. Reed,et al.  Key roles of adjuvants in modern vaccines , 2013, Nature Medicine.

[112]  A. Ghaemmaghami,et al.  Mammalian Innate Resistance to Highly Pathogenic Avian Influenza H5N1 Virus Infection Is Mediated through Reduced Proinflammation and Infectious Virus Release , 2012, Journal of Virology.

[113]  David L Smith,et al.  The Potential for Respiratory Droplet–Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host , 2012, Science.

[114]  P. Kwan,et al.  The potential role of CAMSAP1L1 in symptomatic epilepsy , 2013, Neuroscience Letters.

[115]  Beibei Li,et al.  Nonstructural Protein 1 of Influenza A Virus Interacts with Human Guanylate-Binding Protein 1 to Antagonize Antiviral Activity , 2013, PloS one.

[116]  R. Couch,et al.  High doses of purified influenza A virus hemagglutinin significantly augment serum and nasal secretion antibody responses in healthy young adults , 1994, Journal of clinical microbiology.

[117]  Juliane C. Dohm,et al.  Substantial biases in ultra-short read data sets from high-throughput DNA sequencing , 2008, Nucleic acids research.

[118]  Michael G. Katze,et al.  Distinct RIG-I and MDA5 Signaling by RNA Viruses in Innate Immunity , 2007, Journal of Virology.

[119]  S. Hanabuchi,et al.  Phospholipid Scramblase 1 regulates Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells , 2012, Cell Research.

[120]  Shinn-Jang Hwang,et al.  Long-term booster schedules with AS03A-adjuvanted heterologous H5N1 vaccines induces rapid and broad immune responses in Asian adults , 2014, BMC Infectious Diseases.

[121]  Andrew J Link,et al.  Analyzing Proteomes and Protein Function Using Graphical Comparative Analysis of Tandem Mass Spectrometry Results* , 2006, Molecular & Cellular Proteomics.

[122]  P. Chomez,et al.  GlaxoSmithKline Adjuvant Systems in vaccines: concepts, achievements and perspectives , 2007, Expert review of vaccines.

[123]  J. L. Montagne,et al.  Summary of clinical trials of inactivated influenza vaccine - 1978. , 1983, Reviews of infectious diseases.

[124]  D. Vaughn,et al.  Development and evaluation of AS03, an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion , 2012, Expert review of vaccines.

[125]  G. Jackson,et al.  A new subunit influenza vaccine: acceptability compared with standard vaccines and effect of dose on antigenicity. , 1972, The Journal of infectious diseases.

[126]  Matthew D. Young,et al.  Gene ontology analysis for RNA-seq: accounting for selection bias , 2010, Genome Biology.