A single-shot adenoviral vaccine provides hemagglutinin stalk-mediated protection against heterosubtypic influenza challenge in mice

Conventional influenza vaccines fail to confer broad protection against diverse influenza A viruses with pandemic potential. Efforts to develop a universal influenza virus vaccine include refocusing immunity towards the highly conserved stalk domain of the influenza virus surface glycoprotein, hemagglutinin (HA). We constructed a non-replicating adenoviral (Ad) vector, encoding a secreted form of H1 HA, to evaluate HA stalk-focused immunity. The Ad5_H1 vaccine was tested in mice for its ability to elicit broad, cross-reactive protection against homologous, heterologous, and heterosubtypic lethal challenge in a single-shot immunization regimen. Ad5_H1 elicited hemagglutination inhibition (HI+) active antibodies (Abs), which conferred 100% sterilizing protection from homologous H1N1 challenge. Furthermore, Ad5_H1 rapidly induced H1-stalk-specific Abs with Fc-mediated effector function activity, in addition to stimulating both CD4+ and CD8+ stalk-specific T cell responses. This phenotype of immunity provided 100% protection from lethal challenge with a head-mismatched, reassortant influenza virus bearing a chimeric HA, cH6/1, in a stalk-mediated manner. Most importantly, 100% protection from mortality following lethal challenge with a heterosubtypic avian influenza virus, H5N1, was observed following a single immunization with Ad5_H1. In conclusion, Ad-based influenza vaccines can elicit significant breadth of protection in naive animals and could be considered for pandemic preparedness and stockpiling. Conventional influenza vaccines fail to confer broad protection against diverse influenza A viruses with pandemic potential. Efforts to develop a universal influenza virus vaccine include refocusing immunity towards the highly conserved stalk domain of the influenza virus surface glycoprotein, hemagglutinin (HA). We constructed a non-replicating adenoviral (Ad) vector, encoding a secreted form of H1 HA, to evaluate HA stalk-focused immunity. The Ad5_H1 vaccine was tested in mice for its ability to elicit broad, cross-reactive protection against homologous, heterologous, and heterosubtypic lethal challenge in a single-shot immunization regimen. Ad5_H1 elicited hemagglutination inhibition (HI+) active antibodies (Abs), which conferred 100% sterilizing protection from homologous H1N1 challenge. Furthermore, Ad5_H1 rapidly induced H1-stalk-specific Abs with Fc-mediated effector function activity, in addition to stimulating both CD4+ and CD8+ stalk-specific T cell responses. This phenotype of immunity provided 100% protection from lethal challenge with a head-mismatched, reassortant influenza virus bearing a chimeric HA, cH6/1, in a stalk-mediated manner. Most importantly, 100% protection from mortality following lethal challenge with a heterosubtypic avian influenza virus, H5N1, was observed following a single immunization with Ad5_H1. In conclusion, Ad-based influenza vaccines can elicit significant breadth of protection in naive animals and could be considered for pandemic preparedness and stockpiling. IntroductionSeasonal influenza virus epidemics cause significant annual mortality, estimated at 500,000–650,000 deaths worldwide. It has been >100 years since the 1918 H1N1 pandemic, which is believed to have resulted in the deaths of more than 40 million individuals.1Palese P. Influenza: old and new threats.Nat. Med. 2004; 10: S82-S87https://doi.org/10.1038/nm1141Crossref PubMed Scopus (461) Google Scholar Although subsequent pandemics (1957, 1968, and 2009) were less severe, there is an urgent need to develop a universal influenza vaccine capable of providing broad protection against seasonal and pandemic viruses.2Kerstetter L. Buckley S. Bliss C. Coughlan L. Adenoviral vectors as vaccines for emerging avian influenza viruses.Front. Immunol. 2021; 11: 607333https://doi.org/10.3389/fimmu.2020.607333Crossref PubMed Scopus (7) Google ScholarNeutralizing antibodies (NAbs) directed toward the head of the major surface glycoprotein, hemagglutinin (HA), can provide sterilizing protection against infection with influenza viruses. As such, HA is the main target for current seasonal influenza virus vaccines, including the inactivated influenza vaccine (IIV). There are currently 18 different influenza A virus (IAV) HA subtypes (H1–H18), which are phylogenetically subdivided into two main groups: group 1 (G1) and group 2 (G2).3Coughlan L. Palese P. Overcoming barriers in the path to a universal influenza virus vaccine.Cell Host Microbe. 2018; 24: 18-24https://doi.org/10.1016/j.chom.2018.06.016Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar The subtypes that circulate in humans and have caused pandemics in the past include H1, H2, and H3. However, there is ongoing concern that sporadic introduction of viruses bearing zoonotic HA subtypes (e.g., avian H5) into an immunologically naive human population could result in a severe pandemic.2Kerstetter L. Buckley S. Bliss C. Coughlan L. Adenoviral vectors as vaccines for emerging avian influenza viruses.Front. Immunol. 2021; 11: 607333https://doi.org/10.3389/fimmu.2020.607333Crossref PubMed Scopus (7) Google Scholar,4Pyankova O.G. Susloparov I.M. Moiseeva A.A. Kolosova N.P. Onkhonova G.S. Danilenko A.V. Vakalova E.V. Shendo G.L. Nekeshina N.N. Noskova L.N. Demina J.V. Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020.Euro Surveill. 2021; 26: 2100439https://doi.org/10.2807/1560-7917.ES.2021.26.24.2100439Crossref Scopus (12) Google ScholarHA is a homotrimeric protein composed of two domains, the receptor-binding head and a highly conserved stalk (or stem) domain. 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To achieve this, efforts are focused on (1) increasing vaccine breadth by redirecting immunity toward highly conserved epitopes common to diverse influenza viruses and (2) investigating egg-independent vaccine platforms that could overcome some of the current vaccine limitations.The high degree of intra-group similarity within the stalk domain of G1 HAs prompted efforts to refocus humoral immunity toward the stalk to elicit within-group, multi-subtype protection (e.g., H1 and H5). To date, pre-clinical studies have clearly demonstrated that sequential immunization (i.e., prime:boost) with novel immunogens, such as stabilized headless HAs21Steel J. Lowen A.C. Wang T.T. Yondola M. Gao Q. Haye K. García-Sastre A. Palese P. Influenza virus vaccine based on the conserved hemagglutinin stalk domain.mBio. 2010; 1 (e00018-10)https://doi.org/10.1128/mBio.00018-10Crossref PubMed Scopus (430) Google Scholar, 22Yassine H.M. Boyington J.C. McTamney P.M. Wei C.J. Kanekiyo M. Kong W.P. 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Med. 2019; 25: 962-967https://doi.org/10.1038/s41591-019-0463-xCrossref PubMed Scopus (70) Google ScholarTo develop a vaccine platform that is completely independent of egg-based manufacturing, we engineered an adenoviral (Ad) vector encoding a secreted, trimeric H1 HA. We assessed its ability to elicit cross-reactive, stalk-mediated cellular and humoral immunity and protection in mice following a single shot, comparing it head to head with conventional IIV-based vaccines in homologous (H1), heterologous (cH6/1), and heterosubtypic (H5) lethal challenge models.ResultsValidation of vaccine antigen expression and structural integrity in vitroInaccessibility or occlusion of stalk epitopes to B cell receptors by the HA head through shielding or steric hindrance, or as a result of membrane anchoring of HA on virions (such as on IIV), are mechanisms that have been proposed to explain the immunosubdominance of the HA stalk.31Andrews S.F. Huang Y. Kaur K. Popova L.I. Ho I.Y. Pauli N.T. Dunand C.J.H. Taylor W.M. Lim S. Huang M. Qu X. Immune history profoundly affects broadly protective B cell responses to influenza.Sci. Transl. Med. 2015; 7: 316ra192https://doi.org/10.1126/scitranslmed.aad0522Crossref PubMed Scopus (238) Google Scholar Therefore, we reasoned that expression of a secreted, trimeric form of H1 by a non-replicating Ad vector might maximize accessibility of cross-reactive stalk epitopes. First, we constructed a vaccine immunogen based on H1 (A/California/07/2009) in which the transmembrane domain was removed and replaced with a heterologous trimerization domain, fibritin foldon,32Tao Y. Strelkov S.V. Mesyanzhinov V.V. Rossmann M.G. Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.Structure. 1997; 5: 789-798https://doi.org/10.1016/s0969-2126(97)00233-5Abstract Full Text Full Text PDF PubMed Google Scholar previously used to engineer soluble secreted HA33Krammer F. Margine I. Tan G.S. Pica N. Krause J.C. Palese P. A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates.PLoS One. 2012; 7: e43603https://doi.org/10.1371/journal.pone.0043603Crossref PubMed Scopus (120) Google Scholar (Figure 1A ). Prior to engineering an Ad5-based vaccine expressing H1, we confirmed that H1 was successfully secreted into the supernatant and that it was the correct size (Figure 1B). When purified, we verified that the H1 immunogen was structurally intact and recognized by monoclonal antibodies (mAbs) that bind conformation-sensitive epitopes on the HA stalk, including human mAbs CR911434Dreyfus C. Laursen N.S. Kwaks T. Zuijdgeest D. Khayat R. Ekiert D.C. Lee J.H. Metlagel Z. Bujny M.V. Jongeneelen M. Van Der Vlugt R. Highly conserved protective epitopes on influenza B viruses.Science. 2012; 337: 1343-1348https://doi.org/10.1126/science.1222908Crossref PubMed Scopus (510) Google Scholar and FI6,35Corti D. Voss J. Gamblin S.J. Codoni G. Macagno A. Jarrossay D. Vachieri S.G. Pinna D. Minola A. Vanzetta F. Silacci C. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.Science. 2011; 333: 850-856https://doi.org/10.1126/science.1205669Crossref PubMed Scopus (854) Google Scholar as well as murine mAbs KB227Hai R. Krammer F. Tan G.S. Pica N. Eggink D. Maamary J. Margine I. Albrecht R.A. Palese P. Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes.J. Virol. 2012; 86: 5774-5781https://doi.org/10.1128/JVI.00137-12Crossref PubMed Scopus (200) Google Scholar and GG336Heaton N.S. Leyva-Grado V.H. Tan G.S. Eggink D. Hai R. Palese P. In vivo bioluminescent imaging of influenza a virus infection and characterization of novel cross-protective monoclonal antibodies.J. Virol. 2013; 87: 8272-8281https://doi.org/10.1128/JVI.00969-13Crossref PubMed Scopus (108) Google Scholar (Figures 1C and 1D). The H1 antigen was subsequently engineered into an Ad5-based vector, under the control of a cytomegalovirus (CMV) promoter to facilitate in vivo expression of secreted H1 from the Ad5_H1 vaccine construct following immunization of mice.A single-shot of Ad5_H1 induces humoral immune responses that are superior to a traditional H1-matched inactivated influenza vaccineFemale BALB/cJ mice (n = 10/group) were vaccinated intramuscularly (i.m.) with Ad5_H1 at doses of 106, 107, or 108 infectious units (IFUs) at day 0 (D0) (Figure 1E). A positive control group consisted of matched H1 IIV administered at 1.5 μg of HA per dose. This dose represents 1/10th the human dose and is a dose of IIV regularly used to confer sterilizing protection from homologous challenge in mice.26Nachbagauer R. Kinzler D. Choi A. Hirsh A. Beaulieu E. Lecrenier N. Innis B.L. Palese P. Mallett C.P. Krammer F. A chimeric haemagglutinin-based influenza split virion vaccine adjuvanted with AS03 induces protective stalk-reactive antibodies in mice.NPJ Vaccines. 2016; 1: 16015https://doi.org/10.1038/npjvaccines.2016.15Crossref PubMed Scopus (54) Google Scholar Mice in negative control groups were vaccinated with Ad5 encoding an irrelevant antigen, enhanced green fluorescent protein (EGFP) (Ad5_EGFP) at 108 IFUs, or with sterile PBS. Following a single vaccination, serum immunoglobulin G (IgG) antibody (Ab) responses were measured by enzyme-linked immunosorbent assay (ELISA) on D14 and D28 against full-length H1 protein, homologous to the H1 encoded by Ad5_H1 vaccine, and matched to the H1N1 IIV positive control (Figures 2A and 2B ). Anti-H1 responses were detected at D14 and subsequently peaked at D28 in a dose-dependent manner (geometric mean endpoint titer 1.4 × 106 for 108, 6.4 × 105 for 107, and 2.5 × 105 for 106 IFUs Ad5_H1). More importantly, when compared with mice vaccinated with 1.5 μg of H1 IIV, D14 geometric mean endpoint titers were ∼333-fold greater for 108 Ad5_H1 (p = 0.0033) and ∼148-fold greater for 107 Ad5_H1 (p = 0.0287). At D28, Ab titers elicited by Ad5_H1 (108) were ∼123-fold greater than those induced by H1N1 IIV (p = 0.0019). These data demonstrate that an Ad-based vaccine elicits rapid and strong Ab responses following a single immunization in mice, which is superior to serum IgG titers induced by a conventional, matched H1 IIV.Figure 2A single shot of Ad5_H1 confers superior protection to an H1-matched inactivated influenza vaccine (IIV) following homologous lethal pH1N1 influenza virus challengeShow full captionBALB/cJ female mice were immunized i.m. with a single shot of Ad5_H1 (A/California/07/2009) at doses of 108–106 infectious units (IFUs), a matched monovalent split-virion H1N1 IIV (BEI no. NR-20347) at 1.5 μg, or Ad5_EGFP, an irrelevant antigen control at 108 IFUs. (A–C) Humoral immune responses were measured at D14 or D28 post-immunization by (A and B) serum IgG ELISA against full-length recombinant H1 (A/California/07/2009; BEI no. NR-44074) and (C) hemagglutination inhibition (HI) assay against pH1N1 virus. For ELISA titers, the line on data points indicates the geometric mean, and the dashed gray line in (A) and (B) indicates the input serum dilution and represents the lower limit of detection (LLD). The line on data points in the HI assay (C) denotes median. The dashed black line in (C) indicates an HI titer ≥1:40, which is a correlate of protection in humans. (D–F) At D30 post-immunization, mice were challenged i.n. with 5× mLD50 (300 PFUs) wild-type pH1N1 diluted in sterile PBS (50 μL). Vaccine efficacy was assessed by measuring morbidity (weight loss), mortality (survival), and lung viral titers on D3 post-challenge (C+3). (D) Weight loss data show mean and SD. The humane endpoint was reached when mice lost 25% body weight. (F) For lung viral titers, the line on data points indicates the geometric mean and dashed line indicates the LLD (minimum of one plaque counted × dilution factor). Statistical analyses were performed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons against the H1N1 IIV vaccine or against irrelevant vaccine, Ad5_EGFP. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Survival significance thresholds were calculated using log rank (Mantel-Cox test), corrected for multiple comparisons against Ad5_EGFP using Bonferroni method with K = 4, ∗p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)An established absolute correlate of protection for influenza virus vaccines is an Ab hemagglutination inhibition (HI) titer of 1:40.37de Jong J.C. Palache A.M. Beyer W.E. Rimmelzwaan G.F. Boon A.C. Osterhaus A.D. Haemagglutination-inhibiting antibody to influenza virus.Dev. Biol. (Basel). 2003; 115: 63-73PubMed Google Scholar, 38Hannoun C. Megas F. Piercy J. Immunogenicity and protective efficacy of influenza vaccination.Virus Res. 2004; 103: 133-138https://doi.org/10.1016/j.virusres.2004.02.025Crossref PubMed Scopus (208) Google Scholar, 39Hobson D. Curry R.L. Beare A.S. Ward-Gardner A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses.J. Hyg. (Lond). 1972; 70: 767-777https://doi.org/10.1017/s0022172400022610Crossref PubMed Google Scholar Strain-matched, head-specific Abs with HI activity can confer sterilizing immunity and protection from homologous challenge. Therefore, we assessed the ability of Ad5_H1 to induce HI active Abs when compared with a matched H1N1 IIV (Figure 2C). Following a single shot of Ad5_H1, HI+ Ab titers in mouse serum against pH1N1 virus increased in a dose-dependent manner, with 100% of mice that received Ad5_H1 108 IFUs exhibiting an HI titer of ≥1:40, with a median titer of 1:80. Vaccination with Ad5_H1 at 107 IFUs induced HI+ Abs with a median titer of 1:40 and resulted in HI activity in 100% of mice. At the lowest dose of Ad5_H1 (106), vaccination induced HI active Abs in the serum of 70% of mice, with a median titer of 1:20. A single dose of H1N1 IIV administered i.m. elicited an HI of ≥1:40, while no HI titer activity was induced by negative control vaccines Ad5_EGFP or PBS.A single shot of Ad5_H1 confers superior protection to an H1-matched IIV following homologous pH1N1 lethal influenza virus challengeTo determine the protective efficacy of Ad5_H1 vaccine, we designed a challenge experiment to evaluate homologous (matched H1) protection following a single immunization (Figure 1E). Efficacy following homologous influenza virus challenge was assessed using three measures: (1) weight loss; (2) survival; and (3) viral lung titers. Mice were challenged with five times the median mouse lethal dose (mLD50) of wild-type pH1N1 influenza virus (A/Netherlands/602/2009). Following pH1N1 challenge, <6% mean weight loss was measured in all Ad5_H1-vaccinated groups (106–108 IFUs), comparable to mice vaccinated with a matched H1 IIV, a positive control for protection in this challenge model (Figure 2D). Survival after pH1N1 challenge was significantly higher in Ad5_H1 (100%) and H1 IIV groups (100%) when compared with mice vaccinated with Ad5_EGFP (∼28%) or unvaccinated PBS control animals (∼14%; Figure 2E). Vaccinated mice with median HI titer of ≥1:40 against pH1N1 had complete sterilizing protection, with no virus detected in the lungs at D3 following challenge (C+3) with pH1N1 (Figure 2F). Consistent with the dose-dependent effect of Ad5_H1 on HI+ Abs (Figure 2C), 2/3 mice that received the lowest dose of Ad5_H1 (106 IFUs) lacked complete sterilizing immunity, with virus titers of ∼104 plaque-forming units (PFUs)/mL detected in the lungs on D3 post-challenge (Figure 2F).Immunization with Ad5_H1, but not IIV-based vaccines, elicits H1-stalk-specific T cell responsesIn contrast to strain-specific immune responses, which are largely directed toward the immunodominant HA head, stalk-specific immunity can be broadly cross-reactive. It would therefore be desirable for a universal influenza vaccine candidate to elicit stalk-reactive immunity. It has been observed that IIV platforms do not elicit robust stalk responses following a single shot and are limited in their ability to induce robust T cell responses.40Sridhar S. Brokstad K.A. Cox R.J. 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