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Adeno‐associatedvirus‐vectoredinfluenza vaccineelicits neutralizingand Fcγreceptor‐activatingantibodies Daniel E DemmingerLisa WalzKristina Dietert2020 год

Adeno‐associated virus‐vectored influenza vaccine elicits neutralizing and Fcγ receptor‐activating antibodies
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Аннотация: Article12 March 2020Open Access Source DataTransparent process Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fcγ receptor-activating antibodies Daniel E Demminger Daniel E Demminger Unit 17—Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, Germany Search for more papers by this author Lisa Walz Lisa Walz Veterinary Medicine Division, Paul-Ehrlich-Institute, Langen, Germany Search for more papers by this author Kristina Dietert Kristina Dietert Department of Veterinary Medicine, Institute of Veterinary Pathology, Berlin, Germany Search for more papers by this author Helen Hoffmann Helen Hoffmann Department of Immunology, Interfaculty Institute for Cell Biology, Eberhard Karls University, Tübingen, Germany Search for more papers by this author Oliver Planz Oliver Planz Department of Immunology, Interfaculty Institute for Cell Biology, Eberhard Karls University, Tübingen, Germany Search for more papers by this author Achim D Gruber Achim D Gruber Department of Veterinary Medicine, Institute of Veterinary Pathology, Berlin, Germany Search for more papers by this author Veronika von Messling Veronika von Messling Veterinary Medicine Division, Paul-Ehrlich-Institute, Langen, Germany Search for more papers by this author Thorsten Wolff Corresponding Author Thorsten Wolff [email protected] orcid.org/0000-0001-7688-236X Unit 17—Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, Germany Search for more papers by this author Daniel E Demminger Daniel E Demminger Unit 17—Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, Germany Search for more papers by this author Lisa Walz Lisa Walz Veterinary Medicine Division, Paul-Ehrlich-Institute, Langen, Germany Search for more papers by this author Kristina Dietert Kristina Dietert Department of Veterinary Medicine, Institute of Veterinary Pathology, Berlin, Germany Search for more papers by this author Helen Hoffmann Helen Hoffmann Department of Immunology, Interfaculty Institute for Cell Biology, Eberhard Karls University, Tübingen, Germany Search for more papers by this author Oliver Planz Oliver Planz Department of Immunology, Interfaculty Institute for Cell Biology, Eberhard Karls University, Tübingen, Germany Search for more papers by this author Achim D Gruber Achim D Gruber Department of Veterinary Medicine, Institute of Veterinary Pathology, Berlin, Germany Search for more papers by this author Veronika von Messling Veronika von Messling Veterinary Medicine Division, Paul-Ehrlich-Institute, Langen, Germany Search for more papers by this author Thorsten Wolff Corresponding Author Thorsten Wolff [email protected] orcid.org/0000-0001-7688-236X Unit 17—Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, Germany Search for more papers by this author Author Information Daniel E Demminger1, Lisa Walz2, Kristina Dietert3, Helen Hoffmann4, Oliver Planz4, Achim D Gruber3, Veronika Messling2 and Thorsten Wolff *,1 1Unit 17—Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, Germany 2Veterinary Medicine Division, Paul-Ehrlich-Institute, Langen, Germany 3Department of Veterinary Medicine, Institute of Veterinary Pathology, Berlin, Germany 4Department of Immunology, Interfaculty Institute for Cell Biology, Eberhard Karls University, Tübingen, Germany *Corresponding author. Tel: +49 30 187542278; E-mail: [email protected] EMBO Mol Med (2020)12:e10938https://doi.org/10.15252/emmm.201910938 See also: JA Bengoechea (May 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The current seasonal inactivated influenza vaccine protects only against a narrow range of virus strains as it triggers a dominant antibody response toward the hypervariable hemagglutinin (HA) head region. The discovery of rare broadly protective antibodies against conserved regions in influenza virus proteins has propelled research on distinct antigens and delivery methods to efficiently induce broad immunity toward drifted or shifted virus strains. Here, we report that adeno-associated virus (AAV) vectors expressing influenza virus HA or chimeric HA protected mice against homologous and heterologous virus challenges. Unexpectedly, immunization even with wild-type HA induced antibodies recognizing the HA-stalk and activating FcγR-dependent responses indicating that AAV-vectored expression balances HA head- and HA stalk-specific humoral responses. Immunization with AAV-HA partially protected also ferrets against a harsh virus challenge. Results from this study provide a rationale for further clinical development of AAV vectors as influenza vaccine platform, which could benefit from their approved use in human gene therapy. Synopsis The study shows that immunization with adeno-associated virus vectors protects against divergent lethal influenza A virus infections. Protection correlated with induction of antibodies targeting the head and stalk regions of the viral protein HA, and activating FcγR-dependent responses. Adeno-associated virus (AAV) vectors expressing viral antigens were studied for the induction of immune responses protecting against challenge infection with influenza A viruses. Immunization of mice with AAV-vectored HA constructs elicited broadly reactive antibodies recognizing the HA-head and -stalk regions, respectively, and inducing Fcγ receptor-dependent responses. Immunized mice were protected against lethal homologous or heterologous viral challenges. Intranasal immunization with AAV-HA elicited protective responses towards a viral challenge also in ferrets. The paper explained Problem Current seasonal influenza vaccines show low effectiveness and protection is limited to the virus strains contained within the vaccine. High morbidity and mortality caused by seasonal influenza and the risk of emergence of pandemic and/or zoonotic virus strains emphasize the urgent need for a broadly reactive vaccine. Results AAV vectors were used to deliver influenza antigens to the lung of mice and ferrets to induce protective immunity. AAV vectors expressing HA, NP, or chimeric HA were shown to protect mice from challenge with divergent H1N1 virus strains. This was associated with the induction of non-neutralizing but FcγR-activating antibodies. AAV-HA was also shown to induce protective immunity in ferrets against a homologous H1N1 challenge. Impact The results of this work demonstrate that the AAV vectors are promising carriers for a broadly reactive influenza vaccine. The vectored expression of the antigen was shown to balance the immune response toward more conserved broadly reactive epitopes within the influenza virus antigens and to induce high levels of FcγR-activating antibodies. Furthermore, the licensure of AAV vector for human gene therapy could ease further clinical development of a vaccine. Introduction Influenza remains a severe public health threat. The infection is associated with high morbidity and mortality, especially in very young or very old individuals, and has thus considerable socio-economic impact (WHO, 2018b). Currently, influenza A viruses of the subtypes H1N1 and H3N2 as well as the influenza B virus lineages Victoria and Yamagata circulate in humans. Influenza viruses are genetically and antigenically highly variable, resulting in recurrent epidemics in humans ("flu season"). The main mechanism driving this variability is antigen drift caused by the accumulation of point mutations in the antigenic surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Moreover, reassortment between influenza A virus subtypes can generate even larger antigenic alterations, which may allow for a pandemic circulation in a naïve population (Palese, 2004). Influenza pandemics occur in unpredictable intervals. In 1918, the most devastating pandemic caused by an H1N1 virus, the "Spanish flu," claimed millions of deaths worldwide (Johnson & Mueller, 2002). The most recent influenza pandemic was triggered by a related H1N1 virus in 2009, which arose from the porcine reservoir and imposed a high burden of disease on public health (Fineberg, 2014). Currently, the most effective prophylaxis against influenza is immunization with trivalent or quadrivalent influenza vaccines, most of which contain inactivated antigenic components of current seasonal influenza A and B viruses. However, protection is mainly virus strain-specific, and the efficacy against non-matched strains is generally poor. Although a more broadly reactive live-attenuated influenza vaccine (LAIV) is available for children, its usefulness is limited in adults due to pre-existing immunity against previously encountered influenza viruses (Belshe et al, 2000). The majority of vaccine doses is produced in embryonated chicken eggs, a process which is time-consuming and necessitates that the vaccine composition is predictively defined well in advance of the seasonal epidemic (Gerdil, 2003). This elevates the risk for vaccine mismatch and loss of effectiveness if the actually circulating influenza viruses represent drift variants of the vaccine strains (Rondy et al, 2017). Seasonal influenza vaccination also does not provide protection against shifted or emerging zoonotic influenza A virus strains, e.g., H5N1 or H7N9, which can be associated with increased disease severity (WHO, 2018a). Hence, there is a generally accepted impetus for development of a novel broadly protective vaccine (Erbelding et al, 2018; Ortiz et al, 2018). The seasonal vaccine predominantly induces antibodies targeting epitopes at or surrounding the receptor binding site (RBS) within the globular HA-head region (Caton et al, 1982). Since these epitopes are highly variable, antibodies show limited cross-protection (Yu et al, 2008). Significantly, broadly protective antibodies recognizing conserved epitopes in the membrane proximal HA-stalk or in the HA-head region have been discovered in mice and humans recently (Wu & Wilson, 2017). The majority of broadly reactive HA antibodies does not interfere with receptor attachment (Brandenburg et al, 2013). They rather execute their protective effect via interference with later steps in the viral replicative cycle or via Fc-receptor (FcR)-mediated mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) (DiLillo et al, 2014). Vaccination with inactivated influenza antigen alone is not able to efficiently induce ADCC-activating antibodies in a non-human primate model (Jegaskanda et al, 2013). However, pre-existing ADCC-activating antibody titers induced following natural infection could be boosted in humans by inactivated vaccine, indicating that efficient priming is imperative to elicit such antibodies (Jegaskanda et al, 2016). Overall, the level of broadly reactive antibodies in infected or vaccinated individuals is low due to immunodominance of variable epitopes in the HA-head region (Ellebedy et al, 2014). However, substantial antigenic changes of the HA-head region, for example as consequence of antigenic shift, can lead to the expansion of HA-stalk antibodies, in which the stalk-specific recall response outcompetes the de novo response against the shifted head (Li et al, 2012). Immunization strategies employing chimeric HA (cHA) or headless HA thus make use of this phenomenon (Steel et al, 2010; Hai et al, 2012; Li et al, 2012; Impagliazzo et al, 2015; Yassine et al, 2015). However, since the production of these antigens either includes vaccine bulk production in eggs or requires technically challenging protein purification processes as well as adjuvants, alternative vaccine platforms offer an attractive development perspective (Ramezanpour et al, 2016; Grimm & Buning, 2017). Vaccinia- or adenovirus-based vectors currently represent the most widely used platforms in clinical vaccine trials (Ramezanpour et al, 2016). However, adeno-associated virus (AAV) vectors might be particularly suited as influenza vaccine carrier, since AAV is naturally replication-incompetent and apathogenic in humans, which was a prerequisite for licensure as the first gene therapy vector for use in humans (Grimm & Buning, 2017). The "gutless" AAV vectors can be produced readily in cell culture according to good manufacturing practice criteria, avoiding some of the aforementioned limitations regarding vaccine production (Tripp & Tompkins, 2014). Also, AAV vectors are re-administrable into the respiratory tract in the context of pre-existing immunity without need to change the vector capsid (Limberis & Wilson, 2006). Intriguingly, AAV vectors have been used for passive immunization of mice and ferrets via expression of broadly reactive HA-stalk antibodies in the respiratory tract (Balazs et al, 2013; Limberis et al, 2013a,b; Adam et al, 2014; Laursen et al, 2018). Furthermore, active vaccination with AAV vectors expressing internal (nucleoprotein (NP), matrix protein 1) and surface (HA) influenza virus antigens protected mice from challenge infection (Xin et al, 2001; Lin et al, 2009; Sipo et al, 2011). In the absence of neutralizing antibodies against heterologous HA, the protection against a non-matched influenza virus observed in that study was attributed to the presence of cross-reactive T cells. However, the quality and influence on protection of non-neutralizing antibodies were neither evaluated nor compared to an inactivated vaccine (Sipo et al, 2011). Furthermore, immunization with AAV-vectored cHA or headless HA antigens has not been tested, and there are no data as to the transferability of active AAV vector vaccination to the ferret model, thought to most accurately represent human influenza disease (Enkirch & von Messling, 2015). A detailed knowledge of the effects of the AAV vector on the immune response is needed to advance the AAV vector vaccine approach toward clinical development (de Vries & Rimmelzwaan, 2016). Here, we show that vaccination of mice with AAV-HA or AAV-cHA induced broadly protective antibodies. Notably, protection was associated with strong induction of FcγR-activating antibodies. Finally, we were able to show that three doses of an AAV-HA vaccine conferred protection in ferrets against an (H1N1)pdm strain, demonstrating the potential of the platform for further development. Results AAV9-vectors induce strong antigen expression in vitro We evaluated the potency of AAV vectors expressing influenza virus wild-type HA and NP, or cHA and headless HA antigens to confer broad protection from influenza virus challenge in comparison with an inactivated vaccine (Fig 1A). The AAV9 serotype used herein was isolated from human tissue and efficiently transduces respiratory tissue of mice and ferrets (Gao et al, 2004; Limberis & Wilson, 2006; Limberis et al, 2013a). All vaccine constructs were based on proteins encoded by the prototypic pandemic influenza virus A/California/7/2009 (H1N1)pdm (Cal/7/9) (Fig 1B). The cHA contained the stalk region of Cal/7/9 and head regions derived from influenza A virus subtypes H2 (cHA1), H10 (cHA2), or H13 (cHA3) that currently not circulate in humans. Furthermore, three different headless HA constructs were tested, either containing merely the deletion of the head region (headless HA, HL), or further modifications that increase their antigenicity (modified headless 1 and 2, mHL1 and mHL2; Fig 1B; Impagliazzo et al, 2015; Steel et al, 2010; Yassine et al, 2015). Initially, we assessed correct folding of the HA-stalk within the constructs with the prototypic conformational stalk antibody C179, as several broadly reactive antibodies recognize conformational epitopes (Fig 1C; Okuno et al, 1993). Wild-type HA and cHA3 displayed the C179 epitope, while cHA1 and mHL1 showed reduced binding to C179 (Fig 1C). cHA2, headless HA and mHL2 were only faintly detectable with C179 (Fig 1C). Of note, all cHA were detected by immune sera raised against the respective parental influenza virus HA subtype, i.e., H2, H10, or H13, indicating correct recapitulation of the structure of the respective HA head (Appendix Fig S1A and B). The order of the cHA for vaccination of animals was set according to C179-staining intensity, i.e., cHA3–cHA1–cHA2 (Appendix Table S1). Figure 1. (H1N1)pdm-based AAV-vectored antigens are strongly expressed in vitro 3D structure of a HA trimer (PDB 3UBE generated with PyMol). Each monomer consists of a HA1 (dark gray) and HA2 (light gray) subunit. The trimer can be divided into a membrane distal head which contains the RBS (yellow) and a proximal stalk region. HA and NP represent the Cal/7/9 wild-type proteins. Chimeric HA (cHA) 1 contains the head regions of H2 HA, cHA2 of H10 HA, and cHA3 of H13 HA, while they all contain the Cal/7/9 HA-stalk region. Headless HA (HL) contains a deletion in the HA-head region (dashed line). Modified headless HA (mHL1 and mHL2) contain stabilizing mutations (black boxes) and lack additional internal parts. All constructs were codon-optimized and carry a V5-tag at their C-terminus. Frequency of C179+ or V5-tag+ 293T cells 24 h after transfection of AAV vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3). Immunoblot of 293T cells 72 h after transduction with AAV vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3). Source data are available online for this figure. Source Data for Figure 1 [emmm201910938-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint AAV2-vectors transencapsidated into AAV9 capsids were produced, and integrity and identity of the AAV9 capsids were verified using the conformational capsid antibody ADK9 (Appendix Fig S1C and D; Sonntag et al, 2011). All vector stocks contained high amounts of encapsidated viral genomes (vg) and relatively low amounts of empty capsids (Appendix Fig S1E and F). Correspondingly, robust in vitro AAV vector transduction rates were achieved (Fig 1D and Appendix Fig S1G). AAV-HA, AAV-cHA, AAV-NP, and inactivated vaccine induced broadly reactive antibodies in mice To assess immunogenicity of the AAV vector vaccines, 50 μl PBS containing 1011 vg per mouse was applied equally to both nostrils three times in 3-week intervals before being challenged with influenza viruses. Control groups received either three times AAV-GFP or two times Cal/7/9 whole-inactivated virus (WIV) via the same route in order to be consistent with the application of the AAV-vector vaccines (Fig 2A, Appendix Table S1). Earlier analysis had shown that intranasally applied WIV vaccine elicits protective anti-influenza immune responses in mice (Bhide et al, 2019). As expected, all animals vaccinated with AAV vectors developed high anti-AAV9 IgG titers which continued to increase over time (Fig EV1A). Furthermore, AAV9-vector-neutralizing antibodies were induced, which correlated with total anti-AAV9 serum IgG titers (Fig EV1B and C). Figure 2. AAV-vectored vaccines and WIV induce broadly reactive antibodies A. Mice were intranasally given 1011 vg of AAV vector in 50 μl volume three times in 3-week intervals. 20 μg of Cal/7/9 WIV per 50 μl was given i.n. two times. Blood samples were taken at indicated time points (red drops). After influenza challenge, mice were monitored for survival and weight loss for 2 weeks before necropsy. B. Total IgG ELISA titers expressed as area under the curve (AUC) against homologous Cal/7/9 virus of pre-immune and pre-challenge sera of individual animals of the indicated vaccine groups (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11). Background reactivity of pooled AAV-GFP pre-challenge serum is shown as dashed line. ELISAs were performed in technical duplicates. C. IgA ELISA titers in post-challenge lung homogenates against Cal/7/9 of individual mice of the indicated vaccine groups (AAV-HA, -cHA, -GFP, -NP, WIV n = 11). of the Cal/7/9 (blue symbols) and PR8 low-dose (red symbols) challenge groups. ELISAs were performed in technical duplicates. D–G. Total IgG ELISA titers expressed as log10 of the mean AUC against indicated viruses in pre-challenge sera in AAV-HA (D, n = 18)-, AAV-cHA (E, n = 18)-, AAV-NP (F, n = 11)-, or WIV (G, n = 18)-immunized animals (gray area). Reactivity of sera of AAV-GFP-immunized animals is indicated as white area in the center of each web diagram. ELISAs were performed in technical duplicates. Data information: Statistical significance between pre-immune and pre-challenge serum was determined using Wilcoxon matched pairs test (###P < 0.001). Statistical significance between vaccine groups was determined using Kruskal–Wallis test with Dunn's multiple comparison testing (*P < 0.05, **P < 0.01, ***P < 0.001). Lines indicate mean. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Serum antibody responses against the AAV vector capsid and PR8-specific IgA Total IgG ELISA titer induction over vaccination period against AAV9-vector capsids in pooled sera (AAV-HA, -cHA, -GFP, WIV n = 18; AAV-NP n = 11; AAV-HL, -mHL1, -mHL2 n = 7 mice per group) at indicated time points. AAV9-vector-neutralizing antibody titers in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18; AAV-NP n = 11; AAV-HL, -mHL1, -mHL2 n = 7 mice per group, technical duplicates). Dotted line indicates the limit of detection (LOD) at a dilution of 1:100. Mean ± SD. Regression analysis of total AAV9-vector IgG ELISA titers and MN50 titers. Coefficient of correlation (r2) and P-value are shown. Total IgG ELISA titers expressed as AUC against homologous Cal/7/9 virus in pre-challenge serum pools (AAV-HA, -cHA, -GFP, WIV n = 18; AAV-NP n = 11; AAV-HL, -mHL1, -mHL2 n = 7 mice per group, technical duplicates) of the indicated vaccine groups. Mean ± SD. Immunofluorescence microscopy staining of MDCKII cells 48 h after transfection with pAAV plasmids expressing the indicated constructs. Cells were fixed, permeabilized, and stained with the indicated mouse pre-challenge serum pools or with anti-V5-tag antibody groups (n = 2, technical duplicates) (bar, 50 μm). Immunoblot analysis with lysates obtained from 293T 48 h after transfection with pAAV plasmids expressing the indicated constructs. Immunoblot was performed with the indicated pre-challenge mouse serum pools (top, AAV-HA n = 18, AAV-HL, -mHL1, -mHL2 n = 7 mice per group). Hereafter, membranes were stripped and stained with anti-V5-tag antibody (bottom). Position of detected protein bands is indicated to the right (bottom). Immunofluorescence microscopy staining of MCDKII cells 24 h after transfection with wild-type Cal/7/9 NP or HA and mHL1 plasmids with pre-challenge serum from AAV-NP or AAV-mHL1 + NP immunization groups (n = 2, technical duplicates) (bar, 50 μm). IgA ELISA titers against Cal/7/9 or PR8 virus in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18; AAV-NP n = 11 mice per group, technical duplicates). Mean ± SD. IgA ELISA titers in post-challenge lung homogenates of individual mice of the Cal/7/9 and PR8 low-dose challenge groups of the indicated vaccine groups against PR8. Statistical significance between vaccine groups was determined using Kruskal–Wallis test with Dunn's multiple comparison testing (**P < 0.01, ***P < 0.001). Lines indicate mean. ELISAs were done in technical duplicates. Source data are available online for this figure. Download figure Download PowerPoint The reactivity breadth of serum antibodies was tested against a panel of ten influenza viruses from both antigenic group 1 (H1N1, H2N3, H5N1, H13N6) and 2 (H3N2, H7N9, H10N7; Fig 2). Immunization with AAV-HA, AAV-cHA, AAV-NP, and WIV induced a significant increase of homologous Cal/7/9-specific serum IgG antibody titers compared to pre-immune serum titers (Fig 2B). Furthermore, AAV-HA- and AAV-NP-immunized mice had significantly higher titers against Cal/7/9 compared to AAV-cHA or WIV (Fig 2B). Interestingly, none of the AAV-vectored headless HA vaccines induced detectable influenza-specific antibodies or antibodies against the respective AAV-vectored antigen (Fig EV1D–F). This might be due to the lack of immunodominant epitopes in these antigens. Based on a report by Hessel et al (2014), we evaluated whether the combination of AAV-mHL with the highly immunogenic AAV-NP would induce HA-stalk antibodies. This, however, was not the case, and only NP reactive antibodies were induced (Fig EV1G). Groups receiving AAV-vectored headless HA were therefore not included in subsequent analyses. AAV-HA, AAV-cHA, AAV-NP, and WIV induced broadened antibody responses (Fig 2D–G). AAV-HA triggered a strong response mainly against H1N1 viruses, including pandemic H1N1 virus from 1918, but also H5N1 (Fig 2D). Although reacting weaker with Cal/7/9 and the 1918 pandemic H1N1 viruses, AAV-cHA sera reacted also with H5N1 and two of the cHA parental group 1 viruses (subtypes H2 and H13) (Fig 2E). Both, AAV-HA and AAV-cHA, did, however, not induce antibodies against group 2 viruses (Fig 2D and E). In contrast, AAV-NP induced a strong antibody response covering viruses from both antigenic HA groups, including subtypes H3N2 and H7N9, most likely due to the high conservation of NP (Fig 2F). Unexpectedly, WIV vaccination also induced broadly reactive antibodies covering several subtypes of group 1 and 2, though at lower intensities (Fig 2G). IgA antibodies confer protection to respiratory pathogens due to their high local abundance in the airway mucosa (Asahi et al, 2002). We determined pre-challenge levels of serum IgA (Fig EV1H) as well as post-challenge levels of IgA in lung homogenates (Figs 2C and EV1I). AAV-HA and AAV-NP immunization induced IgA antibodies in the serum as well as in the lung against Cal/7/9. In contrast, IgA was not or barely detectable in serum and lung, respectively, after AAV-cHA immunization. Only AAV-NP-immunized mice mounted serum and lung IgA antibodies against heterologous A/Puerto Rico/8/1934 (H1N1) (PR8) virus (Fig EV1H and I). Interestingly, increased levels of IgA were detectable in lung homogenates but not in sera of WIV-immunized mice against both Cal/7/9 and PR8 (Figs 2C, and EV1H and I). In summary, antibodies were induced at least against some group 1 HA including the subtypes H1N1 and H5N1 after immunization with AAV-HA or AAV-cHA, while AAV-NP and WIV immunization led to antibody responses reacting with influenza A viruses from both antigenic groups. Broadly reactive HA-specific antibodies are non-neutralizing in vitro To analyze the characteristics of the serum antibody responses, we initially determined hemagglutination inhibition (HAI) and neutralizing antibody titers. The level of HAI+ antibodies that block the RBS and interfere with attachment is a key parameter for evaluation of currently licensed inactivated vaccines. Neutralizing antibodies, though not necessarily binding directly to the RBS, can also inhibit later steps in the viral replication cycle as well (Brandenburg et al, 2013). To capture such effects, we performed microneutralization (MN) assays (He et al, 2015). HAI+ and MN+ antibodies against the homologous Cal/7/9 virus were found only in animals immunized with AAV-HA (Table 1). However, these antibodies were specific for Cal/7/9 and did not react with another H1N1 or H3N2 virus. Sera from AAV-cHA-immunized animals were MN+ and HAI+ against H13N6, i.e., the parental subtype of cHA3 used for prime immunization, but were negative for all other tested viruses including the parental subtypes of the cHA used for immunization 2 and 3 (H2, H10) (Appendix Table S1, Table 1). Intranasal WIV, as well as AAV-NP and AAV-GFP did not induce HAI+ or MN+ antibodies (Table 1). The lack of MN+ and HAI+ antibodies following WIV application depended on the intranasal immunization route, as intramuscular (i.m.) injection with the same vaccine preparation led to the expected robust induction of MN+ and HAI+ antibodies (Appendix Fig S2). These results indicate that immunization by AAV in our scheme elicited MN+ and HAI+ antibodies toward the HA-head domain of the virus used for prime immunization. Table 1. HAI and MN50 antibody titers (mouse study) AAV-HA AAV-cHA WIV AAV-NP AAV-GFP Pre (H1N1)pdm (A/Cal/7/9) HAI 640 < 40 < 40 < 40 < 40 < 40 MN50 2,794 < 40 < 40 < 40 < 40 < 40 H1N1 (A/PR/8/34) HAI < 40 < 40 < 40 < 40 < 40 < 40 MN50 < 40 < 40 < 40 < 40 < 40 < 40 H3N2 (A/X31) HAI < 40 < 40 < 40 < 40 < 40 < 40 MN50 < 40 < 40 < 40 < 40 < 40 < 40 H13N6 HAI < 40 160 < 40 < 40 < 40 < 40 MN50 < 40 1,047 < 40 < 40 < 40 < 40 H2N3 HAI < 40 < 40 < 40 < 40 < 40 < 40 H10N7 HAI < 40 < 40 < 40 < 40 < 40 < 40 AAV-HA, AAV-cHA, and WIV induced distinct HA-specific antibody profiles Recent work on humoral responses against HA has raised great interest into non-neutralizing antibodies that bind outside the canonical antigenic sites, but are capable of activating ADCC or other protective responses (Henry Dunand et al, 2016; Leon et al, 2016; Tan et al, 2016; Wu & Wilson, 2017). Hence, we investigated in more detail the epitopes of HA-specific antibodies in the mouse sera. We first analyzed via immunoblot the differential binding to HA1 and HA2 subunits of four different H1N1 viruses spanning more than 90 years of influenza virus evolution (Fig EV2A). HA1 contains the head region, whereas most of the stalk is located on HA2. All serum pools were diluted equally allowing to compare the relative abundances of antibodies recogn
Год издания: 2020
Авторы: Daniel E Demminger, Lisa Walz, Kristina Dietert, Helen Hoffmann, Oliver Planz, Achim D. Gruber, Veronika von Messling, Thorsten Wolff
Издательство: Springer Nature
Источник: EMBO Molecular Medicine
Ключевые слова: Influenza Virus Research Studies, Immune Cell Function and Interaction, Respiratory viral infections research
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