HHS Public Access Author manuscript Author Manuscript
Cell Host Microbe. Author manuscript; available in PMC 2017 June 08. Published in final edited form as: Cell Host Microbe. 2016 June 8; 19(6): 800–813. doi:10.1016/j.chom.2016.05.014.
Both Neutralizing and Non-neutralizing Human H7N9 Influenza Vaccine-induced Monoclonal Antibodies Confer Protection Carole J. Henry Dunand1,†, Paul E. Leon2,3,†, Min Huang1, Angela Choi2,3, Veronika Chromikova2, Irvin Y. Ho1, Gene S. Tan2, John Cruz4, Ariana Hirsh2, Nai-Ying Zheng1, Caitlin Mullarkey2, Francis A. Ennis5, Masanori Terajima5, John J. Treanor6, David J. Topham7, Kanta Subbarao8, Peter Palese2,9, Florian Krammer2,*, and Patrick C. Wilson1,*
Author Manuscript
1The
Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago, Chicago, IL 60637, USA 2Department
of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029,
USA 3Graduate
School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 4Department
of Pathology, University of Massachusetts Medical School, Worcester, MA 01655,
USA 5Division
of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA
Author Manuscript
6Division
of Infectious Disease, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA 7Center
for Vaccine Biology & Immunology, Department of Microbiology & Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
8Laboratory
of Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20852, USA 9Department
of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
SUMMARY Author Manuscript
*
Co-corresponding authors: (P.C.W.) [email protected] (F.K.) [email protected]. †Carole J. Henry Dunand and Paul E. Leon contributed equally to the manuscript. ACCESSION NUMBERS Antibody sequences were deposited in GenBank with accession numbers KU987551-KU987574. AUTHOR CONTRIBUTIONS C.J.H.D and P.E.L. designed and performed experiments, analyzed data, and wrote the manuscript, M.H., A.C., V.C., I.Y.H., G.S.T., J.C., A.H., N.Y.Z and C.M. performed experiments and/or made reagents, F.A.E. and M.T. designed and analyzed the CDL experiment, J.J.T., D.J.T and K.S. designed and performed the vaccine study and P.P., F.K. and P.C.W. designed and directed the study.
SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and one table, Supplemental Experimental Procedures and Supplemental References. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Henry Dunand et al.
Page 2
Author Manuscript
Pathogenic H7N9 avian influenza viruses continue to represent a public health concern and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination, and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly crossreactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines.
Author Manuscript
INTRODUCTION
Author Manuscript
Influenza epidemics result in 250,000–500,000 deaths annually (World Health Organization, 2014). Vaccination offers the most effective protection against infection but vaccines have to be reformulated every year due to antigenic drift. (Krammer and Palese, 2015). In addition to seasonal epidemics, influenza virus strains that are antigenically divergent can arise, leading sporadically to pandemics. The surface glycoprotein hemagglutinin (HA) is the main target of neutralizing antibodies (Kaur et al., 2011). Seasonal vaccination generally induces a narrow, strain-specific response against the highly variable head domain of HA, whereas broadly neutralizing antibodies specific to the more conserved stalk domain are typically rare (Henry Dunand et al., 2015; Krammer and Palese, 2013; Wilson and Andrews, 2012). Although in vitro neutralization traditionally correlates with protection against infection in humans (Couch and Kasel, 1983), recent work has highlighted the importance of nonneutralizing antibodies (Jegaskanda et al., 2013a; Krammer et al., 2014b; Terajima et al., 2015). A more complete understanding of all types of protective antibodies is critical for the improvement of existing influenza vaccines and the development of new ones.
Author Manuscript
A novel reassortant avian H7N9 virus crossed the species barrier and caused a zoonotic epidemic in China in 2013 (Gao et al., 2013; Watanabe et al., 2013). This virus reemerged in 2014 and 2015 in a seasonal pattern, causing morbidity and mortality in humans (World Health Organization, 2015). Although virus transmission occurs primarily through poultry exposure, its continuous circulation in poultry and the large number of sporadic human infections increase the chance of a new reassortment or the acquisition of mutations that could change the properties of the virus (Hu et al., 2014). To prevent H7N9 influenza infections, a live-attenuated A/Anhui/1/2013 H7N9 virus vaccine candidate was developed (Chen et al., 2014b) and evaluated in healthy individuals (Sobhanie et al., 2015). Induction of potent humoral immune responses with H7 vaccines has proven to be problematic due to the poor immunogenicity of novel avian HAs (Cox et al., 2009). Several studies using inactivated or live attenuated H7 vaccines (Couch et al., 2012; Cox et al., 2009; Karron et al., 2009; Rudenko et al., 2014; Talaat et al., 2009; Treanor et al., 2006) showed similarly modest results. However, an H7N7 live-attenuated virus vaccine led to long-term
Cell Host Microbe. Author manuscript; available in PMC 2017 June 08.
Henry Dunand et al.
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Author Manuscript
cross-reactive immune memory (Babu et al., 2014) and a strong recall response with highaffinity H7 head and stalk domain-specific serum antibodies (Halliley et al., 2015). Given that candidate H7N9 vaccines are currently being developed, it is vital to assess if protective antibodies are induced following vaccination, and additionally, to characterize the diversity of epitopes targeted on the HA protein.
Author Manuscript
In this study, we mapped the H7 HA antigenic sites using human monoclonal antibodies (mAbs) isolated from individuals who had received an H7N9 vaccine. Twelve mAbs with particularly high potency and/or breadth of reactivity to multiple influenza strains were chosen for in-depth characterization. These mAbs bound to various epitopes on the head and the stalk domains and were found to be both neutralizing and non-neutralizing in vitro. Importantly, passive transfer of both categories of mAbs protected mice in vivo against a stringent lethal challenge. This work identifies potential therapeutic mAbs and provides a better understanding of the antibody response to H7 viruses.
RESULTS Generation and characterization of human monoclonal antibodies after H7N9 immunization
Author Manuscript Author Manuscript
Four healthy individuals were primed with either one or two doses of a live attenuated coldadapted influenza A/Anhui/1/2013 (H7N9) vaccine (Chen et al., 2014b). The subjects were then boosted 12 weeks later with an inactivated virus vaccine based on the closely related A/ Shanghai/2/2013 (H7N9) strain (Sanofi Pasteur) (Figure 1A). Plasmablasts were isolated 7 days after administration of the inactivated vaccine and mAbs were cloned as previously described (Smith et al., 2009; Wrammert et al., 2008). Twenty mAbs bound A/Anhui/1/2013 (H7N9) HA by ELISA and were then screened using three criteria: HAI activity, neutralization activity, and cross-reactivity to diverse influenza A HAs (group 1 and group 2). Nine out of the 20 mAbs displayed neutralization activity, with 6 displaying HAI activity (Figure 1B). Half of the mAbs (10/20) were cross-reactive within group 2 HAs (H3 and H7) and/or between group 1 and 2 HAs (Figure 1B). Twelve influenza virus-positive mAbs with particularly high potency (HAI and microneutralization) and/or breadth were then chosen for full characterization: 22-3E05, 07-5B05, 07-5D03, 07-4D05, 07-5F01, 07-5G01, 07-4B03, 07-4E02, 07-5E01, 41-5D06, 41-5E04 and 24-4C01 (Figure 1C). Four of the mAbs (22-3E05, 07-5B05, 07-5D03, 07-5F01) were restricted in binding to H7 strains from the Eurasian lineage (H7N9 and H7N7). Four antibodies (07-5G01, 07-4B03, 07-4E02 and 07-4D05) bound to strains from both the Eurasian and the North American lineages (H7N3 and H7N1). Three of the 12 mAbs were broadly cross-reactive (07-5E01, 41-5D06 and 41-5E04) and bound to various HAs from group 1 and group 2 HAs (Figure 1C). None of them bound to influenza B HA (data not shown). The binding affinity of each mAb to A/ Shanghai/1/2013 (H7N9) HA was determined using biolayer interferometry (Table S1). The majority of the mAbs bound with subnanomolar affinities (KD values ranging from 5.27 × 10−9 M to 9.72 × 10−11 M) except 07-5G01 (KD = 2.32 × 10−8 M), 41-5D06 (KD = 8.36 × 10−8 M) and 24-4C01 (KD = 3.94 × 10−8 M).
In vitro HAI and neutralization activities were then determined. 07-5D03, 07-5F01, 07-5G01, 07-4B03, 07-4E02 and 07-4D05 displayed HAI and neutralization activities with A/Shanghai/1/2013 (H7N9) viruses, with 07-4B03, 07-4E02 and 07-4D05 being the more Cell Host Microbe. Author manuscript; available in PMC 2017 June 08.
Henry Dunand et al.
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Author Manuscript Author Manuscript
potent (minimum positive concentration 5) and they had similar scores compared to the neutralizing mAbs tested: 22-3E05, 07-5B05, 41-5E04, CR9114 and CR8020. We also observed that the HAI+ Neut+ mAbs were able to induce phagocytosis as efficiently as the HAI− ones (phagocytic scores between 4 and 7). Furthermore, in order to address the mechanism of protection for the non-neutralizing mAbs in vivo, we conducted a passive transfer experiment and tested mouse serum for reactivity to purified H7 HA at day 7 and 10 post-vaccination. We observed that the serum reactivity on day 7 was higher for the mice that received each of the non-neutralizing mAbs, compared to the mice treated with an irrelevant mAb (Figure S6D). At day 10, this difference was lost (data not shown). These results indicate that the non-neutralizing mAbs drive a faster endogenous response, likely through efficient immune complex formation and improved activation of helper T cells upon peptide presentation after phagocytosis. As these mAbs are not able to induce ADCC or CDL in vitro, uptake of immune complexes by phagocytes is most likely contributing to protection.
Cell Host Microbe. Author manuscript; available in PMC 2017 June 08.
Henry Dunand et al.
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Author Manuscript
DISCUSSION
Author Manuscript
In the present study, we characterize the protective response to an H7N9 vaccine through a high-resolution monoclonal antibody-based approach. Our results suggest that nonneutralizing antibodies, a class of antibodies typically not examined in assessments of vaccine efficacy, may contribute to protection in vivo. This is a noteworthy finding as clinical trials with vaccines based on pre-pandemic avian strains have been shown to be poorly immunogenic when efficacy is measured using the traditional HAI assay (Couch et al., 2012; Cox et al., 2009; Mulligan et al., 2014). Inducing high titers of HAI active antibodies with classical inactivated avian influenza vaccines has been especially challenging even when administered with strong adjuvants (Mulligan et al., 2014). We suggest that a proportion of protective immunity against H7 might be achieved by antibodies that are missed in a classical HAI assay. However, assessing which non-neutralizing antibodies contribute to protection and how to measure their significant contribution in vaccinees remains a difficult challenge. The characterization of the neutralizing mAbs allowed us to generate a detailed map of the antigenic sites on the H7 HA and reveal four previously uncharacterized epitopes. The Arg65 residue plays a role in the binding of two mAbs with diverse functionality (HAI+ versus HAI-). It has been previously shown that different angles of approach can in part explain why antibodies have distinct sensitivities to epitope mutations affecting their neutralization activity (Friesen et al., 2014; Tan et al., 2014). Importantly, escape mutations sometimes arise at positions that are not in the center of the antibody footprint but might change the footprint by network interactions. This has been shown for conformational epitopes of stalk-reactive mAbs (Henry Dunand et al., 2015; Tan et al., 2012).
Author Manuscript Author Manuscript
Our results strongly suggest that cross-reactive non-neutralizing mAbs target previously unrecognized epitopes on the HA protein. Analysis of somatic mutations in the heavy chain variable region reveal that the average number of mutations of the non-neutralizing mAbs (16.3 mutations ± 2) was significantly higher than the average of the head-reactive neutralizing ones (9.9 mutations ± 0.8) (p
Cell Host Microbe. Author manuscript; available in PMC 2017 June 08. Published in final edited form as: Cell Host Microbe. 2016 June 8; 19(6): 800–813. doi:10.1016/j.chom.2016.05.014.
Both Neutralizing and Non-neutralizing Human H7N9 Influenza Vaccine-induced Monoclonal Antibodies Confer Protection Carole J. Henry Dunand1,†, Paul E. Leon2,3,†, Min Huang1, Angela Choi2,3, Veronika Chromikova2, Irvin Y. Ho1, Gene S. Tan2, John Cruz4, Ariana Hirsh2, Nai-Ying Zheng1, Caitlin Mullarkey2, Francis A. Ennis5, Masanori Terajima5, John J. Treanor6, David J. Topham7, Kanta Subbarao8, Peter Palese2,9, Florian Krammer2,*, and Patrick C. Wilson1,*
Author Manuscript
1The
Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago, Chicago, IL 60637, USA 2Department
of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029,
USA 3Graduate
School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 4Department
of Pathology, University of Massachusetts Medical School, Worcester, MA 01655,
USA 5Division
of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA
Author Manuscript
6Division
of Infectious Disease, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA 7Center
for Vaccine Biology & Immunology, Department of Microbiology & Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
8Laboratory
of Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20852, USA 9Department
of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
SUMMARY Author Manuscript
*
Co-corresponding authors: (P.C.W.) [email protected] (F.K.) [email protected]. †Carole J. Henry Dunand and Paul E. Leon contributed equally to the manuscript. ACCESSION NUMBERS Antibody sequences were deposited in GenBank with accession numbers KU987551-KU987574. AUTHOR CONTRIBUTIONS C.J.H.D and P.E.L. designed and performed experiments, analyzed data, and wrote the manuscript, M.H., A.C., V.C., I.Y.H., G.S.T., J.C., A.H., N.Y.Z and C.M. performed experiments and/or made reagents, F.A.E. and M.T. designed and analyzed the CDL experiment, J.J.T., D.J.T and K.S. designed and performed the vaccine study and P.P., F.K. and P.C.W. designed and directed the study.
SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and one table, Supplemental Experimental Procedures and Supplemental References. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Henry Dunand et al.
Page 2
Author Manuscript
Pathogenic H7N9 avian influenza viruses continue to represent a public health concern and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination, and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly crossreactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines.
Author Manuscript
INTRODUCTION
Author Manuscript
Influenza epidemics result in 250,000–500,000 deaths annually (World Health Organization, 2014). Vaccination offers the most effective protection against infection but vaccines have to be reformulated every year due to antigenic drift. (Krammer and Palese, 2015). In addition to seasonal epidemics, influenza virus strains that are antigenically divergent can arise, leading sporadically to pandemics. The surface glycoprotein hemagglutinin (HA) is the main target of neutralizing antibodies (Kaur et al., 2011). Seasonal vaccination generally induces a narrow, strain-specific response against the highly variable head domain of HA, whereas broadly neutralizing antibodies specific to the more conserved stalk domain are typically rare (Henry Dunand et al., 2015; Krammer and Palese, 2013; Wilson and Andrews, 2012). Although in vitro neutralization traditionally correlates with protection against infection in humans (Couch and Kasel, 1983), recent work has highlighted the importance of nonneutralizing antibodies (Jegaskanda et al., 2013a; Krammer et al., 2014b; Terajima et al., 2015). A more complete understanding of all types of protective antibodies is critical for the improvement of existing influenza vaccines and the development of new ones.
Author Manuscript
A novel reassortant avian H7N9 virus crossed the species barrier and caused a zoonotic epidemic in China in 2013 (Gao et al., 2013; Watanabe et al., 2013). This virus reemerged in 2014 and 2015 in a seasonal pattern, causing morbidity and mortality in humans (World Health Organization, 2015). Although virus transmission occurs primarily through poultry exposure, its continuous circulation in poultry and the large number of sporadic human infections increase the chance of a new reassortment or the acquisition of mutations that could change the properties of the virus (Hu et al., 2014). To prevent H7N9 influenza infections, a live-attenuated A/Anhui/1/2013 H7N9 virus vaccine candidate was developed (Chen et al., 2014b) and evaluated in healthy individuals (Sobhanie et al., 2015). Induction of potent humoral immune responses with H7 vaccines has proven to be problematic due to the poor immunogenicity of novel avian HAs (Cox et al., 2009). Several studies using inactivated or live attenuated H7 vaccines (Couch et al., 2012; Cox et al., 2009; Karron et al., 2009; Rudenko et al., 2014; Talaat et al., 2009; Treanor et al., 2006) showed similarly modest results. However, an H7N7 live-attenuated virus vaccine led to long-term
Cell Host Microbe. Author manuscript; available in PMC 2017 June 08.
Henry Dunand et al.
Page 3
Author Manuscript
cross-reactive immune memory (Babu et al., 2014) and a strong recall response with highaffinity H7 head and stalk domain-specific serum antibodies (Halliley et al., 2015). Given that candidate H7N9 vaccines are currently being developed, it is vital to assess if protective antibodies are induced following vaccination, and additionally, to characterize the diversity of epitopes targeted on the HA protein.
Author Manuscript
In this study, we mapped the H7 HA antigenic sites using human monoclonal antibodies (mAbs) isolated from individuals who had received an H7N9 vaccine. Twelve mAbs with particularly high potency and/or breadth of reactivity to multiple influenza strains were chosen for in-depth characterization. These mAbs bound to various epitopes on the head and the stalk domains and were found to be both neutralizing and non-neutralizing in vitro. Importantly, passive transfer of both categories of mAbs protected mice in vivo against a stringent lethal challenge. This work identifies potential therapeutic mAbs and provides a better understanding of the antibody response to H7 viruses.
RESULTS Generation and characterization of human monoclonal antibodies after H7N9 immunization
Author Manuscript Author Manuscript
Four healthy individuals were primed with either one or two doses of a live attenuated coldadapted influenza A/Anhui/1/2013 (H7N9) vaccine (Chen et al., 2014b). The subjects were then boosted 12 weeks later with an inactivated virus vaccine based on the closely related A/ Shanghai/2/2013 (H7N9) strain (Sanofi Pasteur) (Figure 1A). Plasmablasts were isolated 7 days after administration of the inactivated vaccine and mAbs were cloned as previously described (Smith et al., 2009; Wrammert et al., 2008). Twenty mAbs bound A/Anhui/1/2013 (H7N9) HA by ELISA and were then screened using three criteria: HAI activity, neutralization activity, and cross-reactivity to diverse influenza A HAs (group 1 and group 2). Nine out of the 20 mAbs displayed neutralization activity, with 6 displaying HAI activity (Figure 1B). Half of the mAbs (10/20) were cross-reactive within group 2 HAs (H3 and H7) and/or between group 1 and 2 HAs (Figure 1B). Twelve influenza virus-positive mAbs with particularly high potency (HAI and microneutralization) and/or breadth were then chosen for full characterization: 22-3E05, 07-5B05, 07-5D03, 07-4D05, 07-5F01, 07-5G01, 07-4B03, 07-4E02, 07-5E01, 41-5D06, 41-5E04 and 24-4C01 (Figure 1C). Four of the mAbs (22-3E05, 07-5B05, 07-5D03, 07-5F01) were restricted in binding to H7 strains from the Eurasian lineage (H7N9 and H7N7). Four antibodies (07-5G01, 07-4B03, 07-4E02 and 07-4D05) bound to strains from both the Eurasian and the North American lineages (H7N3 and H7N1). Three of the 12 mAbs were broadly cross-reactive (07-5E01, 41-5D06 and 41-5E04) and bound to various HAs from group 1 and group 2 HAs (Figure 1C). None of them bound to influenza B HA (data not shown). The binding affinity of each mAb to A/ Shanghai/1/2013 (H7N9) HA was determined using biolayer interferometry (Table S1). The majority of the mAbs bound with subnanomolar affinities (KD values ranging from 5.27 × 10−9 M to 9.72 × 10−11 M) except 07-5G01 (KD = 2.32 × 10−8 M), 41-5D06 (KD = 8.36 × 10−8 M) and 24-4C01 (KD = 3.94 × 10−8 M).
In vitro HAI and neutralization activities were then determined. 07-5D03, 07-5F01, 07-5G01, 07-4B03, 07-4E02 and 07-4D05 displayed HAI and neutralization activities with A/Shanghai/1/2013 (H7N9) viruses, with 07-4B03, 07-4E02 and 07-4D05 being the more Cell Host Microbe. Author manuscript; available in PMC 2017 June 08.
Henry Dunand et al.
Page 4
Author Manuscript Author Manuscript
potent (minimum positive concentration 5) and they had similar scores compared to the neutralizing mAbs tested: 22-3E05, 07-5B05, 41-5E04, CR9114 and CR8020. We also observed that the HAI+ Neut+ mAbs were able to induce phagocytosis as efficiently as the HAI− ones (phagocytic scores between 4 and 7). Furthermore, in order to address the mechanism of protection for the non-neutralizing mAbs in vivo, we conducted a passive transfer experiment and tested mouse serum for reactivity to purified H7 HA at day 7 and 10 post-vaccination. We observed that the serum reactivity on day 7 was higher for the mice that received each of the non-neutralizing mAbs, compared to the mice treated with an irrelevant mAb (Figure S6D). At day 10, this difference was lost (data not shown). These results indicate that the non-neutralizing mAbs drive a faster endogenous response, likely through efficient immune complex formation and improved activation of helper T cells upon peptide presentation after phagocytosis. As these mAbs are not able to induce ADCC or CDL in vitro, uptake of immune complexes by phagocytes is most likely contributing to protection.
Cell Host Microbe. Author manuscript; available in PMC 2017 June 08.
Henry Dunand et al.
Page 9
Author Manuscript
DISCUSSION
Author Manuscript
In the present study, we characterize the protective response to an H7N9 vaccine through a high-resolution monoclonal antibody-based approach. Our results suggest that nonneutralizing antibodies, a class of antibodies typically not examined in assessments of vaccine efficacy, may contribute to protection in vivo. This is a noteworthy finding as clinical trials with vaccines based on pre-pandemic avian strains have been shown to be poorly immunogenic when efficacy is measured using the traditional HAI assay (Couch et al., 2012; Cox et al., 2009; Mulligan et al., 2014). Inducing high titers of HAI active antibodies with classical inactivated avian influenza vaccines has been especially challenging even when administered with strong adjuvants (Mulligan et al., 2014). We suggest that a proportion of protective immunity against H7 might be achieved by antibodies that are missed in a classical HAI assay. However, assessing which non-neutralizing antibodies contribute to protection and how to measure their significant contribution in vaccinees remains a difficult challenge. The characterization of the neutralizing mAbs allowed us to generate a detailed map of the antigenic sites on the H7 HA and reveal four previously uncharacterized epitopes. The Arg65 residue plays a role in the binding of two mAbs with diverse functionality (HAI+ versus HAI-). It has been previously shown that different angles of approach can in part explain why antibodies have distinct sensitivities to epitope mutations affecting their neutralization activity (Friesen et al., 2014; Tan et al., 2014). Importantly, escape mutations sometimes arise at positions that are not in the center of the antibody footprint but might change the footprint by network interactions. This has been shown for conformational epitopes of stalk-reactive mAbs (Henry Dunand et al., 2015; Tan et al., 2012).
Author Manuscript Author Manuscript
Our results strongly suggest that cross-reactive non-neutralizing mAbs target previously unrecognized epitopes on the HA protein. Analysis of somatic mutations in the heavy chain variable region reveal that the average number of mutations of the non-neutralizing mAbs (16.3 mutations ± 2) was significantly higher than the average of the head-reactive neutralizing ones (9.9 mutations ± 0.8) (p
