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Ferrara, Francesca (2015)
Languages: English
Types: Doctoral thesis
Subjects: RS

Classified by OpenAIRE into

mesheuropmc: virus diseases
Influenza viruses represent an important public health burden since they cause annual epidemics associated with severe illness and mortality in high-risk populations. Additionally, zoonotic influenza virus infections have potential to produce intermittent pandemics, which have led to millions of deaths globally. However, strategies to prevent influenza severity and spread can be implemented. It is known that antibodies against the haemagglutinin play a key role in protection from influenza virus infection, thus both seasonal and pandemic influenza vaccines aim to elicit such antibodies. Generally, they are directed against haemagglutinin globular head epitopes and are able to neutralize closely related influenza strains, but recently antibodies able to neutralize multiple influenza strains and subtypes have also been described. The discovery of these antibodies, primarily directed against the haemagglutinin stalk, has generated interest in understanding how they are generated and how widespread they are in the human population. Furthermore, eliciting such antibodies has become the aim of many ‘universal’ vaccine approaches. However, the study of these cross-reactive antibodies using classical serological assays is problematic since the current assays have been shown to be relatively insensitive in detecting them.\ud The main objective of this thesis was to study the presence and breadth of cross-reactive neutralizing responses in human populations. To overcome the limitations of current serological tests in detecting these responses, lentiviral pseudotype particles bearing the haemagglutinins of different influenza A subtypes and influenza B strains were used as surrogate antigens in neutralization assays. After the generation of these novel tools and the establishment of appropriate controls, pseudotype particle neutralization assays were employed to investigate cross-reactive antibody responses in pre- and post-vaccination sera. Next, the use of chimeric haemagglutinins, in which the globular head was substituted with the head of a different subtype, was incorporated into the pseudotype system. This allowed the differentiation between haemagglutinin head-directed and stalk-directed antibody responses. The ability to efficiently detect broadly neutralizing antibody responses, including those directed against the haemagglutinin stalk, shows that pseudotype particles are tools that should be further characterised and implemented to be used in sero-epidemiological studies and for ‘universal’ vaccine immunogenicity studies.
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    • CHAPTER 3 Production of low pathogenic avian and human influenza A
    • CHAPTER 4 Evaluation of reference antisera cross-reactivity for establishment
    • assays .................................................................................................................................119   4.1   Introduction ..................................................................................................................... 119   4.2   Material and methods ..................................................................................................... 120   4.2.1   Haemagglutinin-expressing plasmids and pseudotype production........................ 120   4.2.2   Reference sera........................................................................................................ 121   4.2.3   Pseudotype particle neutralization assays.............................................................. 122   4.2.4   Bioinformatic analysis ........................................................................................... 122   4.2.5   Statistical analysis.................................................................................................. 123   4.3   Results .............................................................................................................................. 124   4.4   Discussion and Conclusion ............................................................................................. 128  
    • CHAPTER 5 Use of influenza A pseudotypes to study heterosubtypic antibodies
    • 6.4   Discussion and Conclusion ............................................................................................. 212  
    • CHAPTER 7 Discussion ..................................................................................................221   7.1   Future work ..................................................................................................................... 223   7.1.1   Generation of other influenza pseudotypes, including influenza C and D ............ 223   7.1.2   Optimisation of the pseudotype production system............................................... 225   7.1.3   Study and prediction of haemagglutinin protease-mediated activation and cleavage ................................................................................................................. 226   7.1.4   Characterisation of pseudotype particles using microscopy and proteomics techniques .............................................................................................................. 226   7.1.5   Optimisation and standardisation of the pseudotype particle neutralization assay, including the establishment of reference standards and controls................ 227   7.1.6   Use of pseudotype particles in other serological/immunological assays............... 229   7.1.7   Application of influenza haemagglutinin pseudotype particles/lentiviral vectors in vaccine and gene therapies ................................................................................ 230  
    • CHAPTER 8 Conclusion .................................................................................................232  
    • APPENDIX .......................................................................................................................274   A.1   Additional Figures and Tables for Chapter 3............................................................... 274   A.2   Chapter 4 R codes ........................................................................................................... 297   A.2.1   Cross-reactivity map R code.................................................................................. 297   A.2.2   Percentage identity map R code ............................................................................ 297   A.3   Additional Figures and Tables for Chapter 5............................................................... 298   A.4   Additional Figures and Tables for Chapter 6............................................................... 312   A.5   Preliminary Experiments................................................................................................ 317   A.6   List of publications .......................................................................................................... 319  
    • Table 13: Primer names, restriction enzyme, and primer sequences used for Influenza A HA cloning............................................................................................................................ 86  
    • Table 16: Primer names and sequences used for A/black-headed gull/Sweden/2/1999 H16 mutagenesis ................................................................................................................... 92  
    • Table 18: OIE avian influenza reference antisera for HI assay, Agar Gel Immunodiffusion test, and Agar Gel Precipitation test......................................................................... 121  
    • Table 24: SCRs using cut-off values of 4-fold IC50 (SCR(4-fold)) or 2-fold IC50 increase (SCR(2-fold)) .................................................................................................... 163  
    • Table 25: SCRs using cut-off values of 4-fold IC50 (SCR(4-fold)) or 2-fold IC50 increase (SCR(2-fold)) .................................................................................................... 174  
    • Table 26: Primer names, restriction enzyme, and primer sequences used for Influenza B HA cloning into p.I.18 vector............................................................................................ 186  
    • Table 27: Primer names and sequences used for B/Brisbane/60/2008 HA mutagenesis ........ 188  
    • Table 28: Primer names and sequences used for B/Bangladesh/3333/2007 Kozak sequence mutagenesis ................................................................................................................. 189  
    • Appendix Table 1: Decision table for the production of influenza A pp ................................. 292  
    • Appendix Table 3: Quartiles and medians of the IC50 distributions of the 2007-2008 study after age- stratification............................................................................................... 304  
    • Appendix Table 4: Quartiles and medians of the IC50 distributions of the NCT00942071 clinical trial study ....................................................................................................... 306  
    • Appendix Table 5: Quartiles and medians of the IC50 distributions of the NCT00942071 clinical trial study after vaccine-regimen stratification .......................................... 308  
    • Appendix Table 6: Decision table for the production of influenza B pp ................................. 316  
    • Figure 22: Flow chart representing the subcloning of A/Korea/426/1968 H2 and A/Shanghai/2/2013 H7 into pI.18 vector..................................................................... 84  
    • Figure 23: Flow chart representing the cloning of A/Hong Kong/1073/1999 H9 into pI.18 vector ............................................................................................................................. 86  
    • Figure 25: Production of low pathogenic avian and human influenza A HA pp by four-plasmid co-transfection ....................................................................................... 90  
    • Figure 26: Site-direct mutagenesis of A/California/7/2004 H3 ................................................... 95  
    • Figure 27: Site-direct mutagenesis of A/Puerto Rico/8/1934 H1 ................................................ 96  
    • Figure 28: Role of HAT, TMPRSS2, and TMPRSS4 proteases in H1pp production............... 97  
    • Figure 29: Role of HAT, TMPRSS2, and TMPRSS4 proteases in H2pp production............... 98  
    • Figure 30: Role of HAT, TMPRSS2, and TMPRSS4 proteases in H3pp, H4pp, and H7pp production ..................................................................................................................... 99  
    • Figure 31: Role of HAT, TMPRSS2, and TMPRSS4 proteases in H8pp, H9pp, H10pp, and H11pp production....................................................................................................... 100  
    • Figure 32: Role of HAT, TMPRSS2, and TMPRSS4 proteases in H12pp, H13pp, and H14pp production ................................................................................................................... 101  
    • Figure 33: Western blot of A/duck/Italy/1447/2005 H1pp obtained using different proteases ...................................................................................................................................... 102  
    • Figure 34: Western blot of A/Udorn/307/1972 H3pp obtained using different proteases...... 103  
    • Figure 35: Site-direct mutagenesis of A/black-headed gull/Sweden/2/1999 H16 .................... 104  
    • Figure 36: Role of HAT, TMPRSS2, and TMPRSS4 proteases in the production of wild-type and mutated A/black-headed gull/Sweden/2/1999 H16pp ...................................... 104  
    • Figure 37: Production of A/Texas/05/2009 H1pp....................................................................... 105  
    • Figure 38: Site-direct mutagenesis K136N of A/Texas/05/2009 H1pp ..................................... 106  
    • Figure 39: Site-direct mutagenesis Q240R of A/Texas/05/2009 H1pp ..................................... 107  
    • Figure 40: Immunofluorescence of the wild-type A/Texas/6/2009 H1, and the K136N and Q240R mutants ........................................................................................................... 107  
    • Figure 41: Production of A/Wisconsin/67/2005 H3pp ............................................................... 108  
    • Figure 42: Immunofluorescence of the wild-type A/gull/Maryland/704/1977 H13................. 109  
    • Figure 43: Production of A/gull/Maryland/704/1977 H13pp .................................................... 110  
    • Figure 44: Cross-reactivity map of pp and reference sera based on IC50................................ 125  
    • Figure 45: Cross-reactivity map of pp and reference sera based on percentage of amino acid identity ......................................................................................................................... 127  
    • Figure 46: Schematic representation of chimeric HA cloning using Gibson Assembly ......... 137  
    • Figure 47: Amplification of the H1 stalk in the phCMV1 backbone (~5.3 kb) and of the H11 head (~640 bp)............................................................................................................. 144  
    • Figure 48: Colony PCR screening after Gibson Assembly of the chimeric HA ...................... 145  
    • Figure 49: Digestion screening of chimeric HA-encoding plasmids......................................... 146  
    • Figure 50: Production optimisation of the chimeric HA pp...................................................... 147  
    • Figure 51: Correlation of SRH and pp-NT................................................................................. 148  
    • Figure 52: IC50 of sera tested with A/Solomon Island/3/2006 H1 and A/Wisconsin/67/2005 H3 (A/Udorn/307/1972 N2) pp-NT assays ................................................................ 150  
    • Figure 53: IC50 of sera tested with A/New Caledonia/20/1999 H1 and A/Korea/426/1968 H2 pp-NT assays ............................................................................................................... 151  
    • Figure 54: IC50 of sera tested with A/Udorn/307/1972 H3 and A/duck/Czechoslovakia/1956 H4 pp-NT assays ......................................................................................................... 153  
    • Figure 55: IC50 of sera tested with A/Shanghai/2/2013 H7 and A/chicken/Italy/1082/1999 H7 pp-NT assays ............................................................................................................... 155  
    • Figure 56: IC50 of sera tested with A/Hong Kong/1073/1999 H9 and A/chicken/Germany/N49 H10 pp-NT assays ....................................................................................................... 156  
    • Figure 57: IC50 of sera tested with A/duck/Alberta/60/1976 H12, A/mallard/Astrakhan/263/1982 H14, and A/shearwater/West Australia/2576/1979 H15 pp-NT assays ....................................................................................................... 157  
    • Figure 58: Age-stratified IC50 of sera tested A/duck/Alberta/60/1976 H12, A/mallard/Astrakhan/263/1982 H14, and A/shearwater/West Australia/2576/1979 H15 pp-NT assays ....................................................................................................... 158  
    • Figure 59: IC50 of sera tested with A/South Carolina/1/1918 H1, A/duck/Memphis/546/1974 H11, and the chimeric HA pp-NT assays ................................................................. 160  
    • Figure 60: Age-stratified IC50 of sera tested with A/South Carolina/1/1918 H1, A/duck/Memphis/546/1974 H11, and the chimeric HA pp-NT assays .................. 161  
    • Figure 65: Comparison of TIV + placebo and TIV + MVA-NP+M1 vaccine combinations using pp-NT IC50 (1) ................................................................................................... 170  
    • Figure 66: Comparison of TIV + placebo and TIV + MVA-NP+M1 vaccine combinations using pp-NT IC50 (2) ................................................................................................... 171  
    • Figure 67: Comparison of TIV + placebo and TIV + MVA-NP+M1 vaccine combinations using pp-NT IC50 (3) ................................................................................................... 172  
    • Figure 68: Comparison of TIV + placebo and TIV + MVA-NP+M1 vaccine combinations using pp-NT IC50 (4) ................................................................................................... 173  
    • Figure 69: Gel electrophoresis of B/Bangladesh/3333/2007 and B/Brisbane/60/2008 HA amplicons..................................................................................................................... 194  
    • Figure 70: Gel electrophoresis of pI.18-B/Bangladesh/3333/2007 and pI.18-B/Brisbane/60/2008 HA colony PCR screenings............................................ 196  
    • Figure 71: Gel electrophoresis of positive clone digestions using EcoRI ................................. 197  
    • Figure 72: Site-direct mutagenesis of B/Brisbane/60/2008 HA................................................. 197  
    • Figure 73: Role of HAT, TMPRSS2, and TMPRSS4 proteases in B/Bangladesh/3333/2007 pp production ................................................................................................................... 198  
    • Figure 74: Role of HAT, TMPRSS2, and TMPRSS4 proteases in B/Hong Kong/8/1973 pp and B/Victoria/2/1987 pp production............................................................................... 199  
    • Figure 75: Role of HAT, TMPRSS2, and TMPRSS4 proteases in B/Yamagata/16/1988 pp and B/Florida/4/2006 pp production ................................................................................ 199  
    • Figure 76: Role of HAT, TMPRSS2, and TMPRSS4 proteases in B/Brisbane/60/2008 pp production ................................................................................................................... 200  
    • Figure 77: Western blot of B/Brisbane/60/2008 pp obtained using different proteases......... 201  
    • Figure 78: Electropherograms showing B/Bangladesh/3333/2007 HA Kozak sequence before and after mutagenesis................................................................................................. 201  
    • Figure 79: The role of influenza B Kozak sequence in pp production..................................... 202  
    • Figure 80: Transduction of HEK293T/17, MDCK, and A549 cells with Influenza B pp expressing firefly luciferase ....................................................................................... 203  
    • Figure 81: Transduction of HEK293T/17, MDCK, and A549 cells with influenza B/Florida/4/2006 pp expressing emGFP................................................................... 203  
    • Figure 82: Phylogenetic tree of the HAs used for pp production ............................................. 204  
    • Figure 83: Neutralization activity of NIBSC 11/136 anti-B/Brisbane/60/2008 HA serum and of the negative control .................................................................................................... 205  
    • Figure 84: Correlation of HI assay and pp-NT assay performed with B/Brisbane/60/2008 influenza strain ........................................................................................................... 206  
    • Figure 86: IC50 of sera tested with pp-NT assay reported in a Box-and-Whisker plot after stratification using vaccination regimens................................................................. 208  
    • Figure 87: Fold-increase in the IC50 titres of the TIV + placebo and the TIV + MVA-NP+M1 groups .......................................................................................................................... 209  
    • Figure 88: Amino acid alignment of B/Brisbane/60/2008, B/Hong Kong/8/1973, and B/Florida/4/2006 HAs................................................................................................. 210  
    • Figure 89: Differences between B/Brisbane/60/2008, B/Hong Kong/8/1973, and B/Florida/4/2006 HAs................................................................................................. 211  
    • Appendix Figure 1: Flow chart legend........................................................................................ 274  
    • Appendix Figure 2: Flow chart outlining the cloning of A/Solomon Islands/3/2006 H1 and A/Texas/05/2009 H1 HAs into pI.18 expression vector ........................................... 276  
    • Appendix Figure 3: Flow chart outlining the cloning of A/Brisbane/59/2007 H1 HA into pI.18 expression vector ........................................................................................................ 279  
    • Appendix Figure 4: Flow chart outlining the cloning of A/duck/Italy/1447/2005 H1 HA into pI.18 expression vector............................................................................................... 282  
    • Appendix Figure 5: Flow chart outlining the cloning of A/duck/Germany/1215/1973 H2 HA into phCMV1 expression vector................................................................................ 285  
    • Appendix Figure 6: Flow chart outlining the cloning of A/California/7/2004 H3 HA into pI.18 expression vector ........................................................................................................ 287  
    • Appendix Figure 7: Flow chart outlining the cloning of A/Wisconsin/67/2005 H3 HA into pI.18 expression vector............................................................................................... 289  
    • Appendix Figure 8: Flow chart outlining the cloning of A/turkey/Ontario/6118/1968 H8 HA into phCMV1 expression vector................................................................................ 291  
    • Appendix Figure 9: Comparison between 48 h and 72 h collections of A/duck/Czechoslovakia/1956 H4pp .......................................................................... 293  
    • Appendix Figure 11: TMPRSS2 expression profile................................................................... 295  
    • Appendix Figure 12: TMPRSS4 expression profile................................................................... 296  
    • Appendix Figure 13: Alignment of A/South Carolina/1/1918 H1, A/duck/Memphis/546/1974 H1, and of the combined chimeric HA ..................................................................... 298  
    • Appendix Figure 14: Alignment of the cloned chimeric HA Fw and Rev amino acid sequences with the assembled chimeric HA amino acid sequence........................................... 299  
    • Appendix Figure 15: IC50 of sera tested with H5 and H7 pp-NT assay ................................... 300  
    • Appendix Figure 16: Age-stratified IC50 of sera tested with H5 and H7 pp-NT assay........... 301  
    • Appendix Figure 17: Fold-increase in the IC50 titres of the TIV + placebo and the TIV + MVA-NP+M1 groups ................................................................................................. 311  
    • Appendix Figure 18: Flow chart outlining the cloning of B/Brisbane/60/2008 and B/Bangladesh/3333/2007 HAs into pI.18 expression vector.................................... 313  
    • Appendix Figure 19: Alignment of the B/Bangladesh/3333/2007 and B/Brisbane/60/200 HA sequences ..................................................................................................................... 313  
    • Appendix Figure 20: Alignment of the first 100 nucleotides of the circulating influenza B segment 4 (HA) ........................................................................................................... 314  
    • Appendix Figure 21: Alignment of the cloned B/Bangladesh/3333/2007 HA Fw and Rev sequences with the database amino acid sequence .................................................. 315  
    • Appendix Figure 22: HApp do not coat ELISA plates .............................................................. 317  
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