A universal influenza virus vaccine candidate confers protection against pandemic H1N1 infection in ferrets

Influenza viruses can cause severe disease and mortality in humans. Due to constant change in their immuno-dominant antigenic sites they can evade adaptive immune responses. Current seasonal influenza virus vaccines therefore require annual re-formulation and re-administration to confer protection from circulating viruses. Additionally, these vaccines cannot protect against novel pandemic influenza virus strains. Novel vaccination approaches attempt to refocus antibody responses towards more conserved domains like the hemagglutinin stalk. Antibodies against the stalk domain are broadly-reactive and can neutralize multiple influenza virus subtypes. However, the stalk domain is immuno-subdominant and not preferentially targeted by the immune system. In this study, we tested if a vaccination strategy based on influenza viruses expressing chimeric hemagglutinins (cH) that contain exotic, divergent head domains, but a conserved H1 stalk domain could induce cross-protective antibody responses in ferrets. We compared a heterologous live-attenuated virus (cH8/1N1) prime followed by an inactivated split virus (cH5/1N1) boost combination approach to two doses of split-virus vaccines (cH8/1N1/cH5/1N1) and the impact of adjuvant on the immune response. Additionally, a ‘standard of care’ control group received 2 rounds of a human trivalent influenza virus vaccine. We found that all universal influenza virus vaccination approaches were successful at inducing stalk-reactive antibody responses in serum. Virus replication was limited to the nasopharynx in the live attenuated/split vaccine groups and nasal wash titers were lower than in the \u27standard of care\u27 control group. No virus replication was detected in the lungs of attenuated/split vaccinated ferrets, while the \u27standard of care\u27 group had similarly high titers as an unvaccinated control group. Our findings demonstrate that - using a chimeric hemagglutinin based heterologous attenuated/split combination strategy - our candidate universal influenza virus vaccine can successfully protect ferrets from pandemic H1N1 infection. The data support further development of this vaccination approach and advancement into clinical trials

Generation of an international standard serum to measure influenza virus hemagglutinin stalk-reactive antibodies

Current efforts towards the development of a universal influenza virus vaccine rely on induction of effective long-term antibody responses against conserved regions of the influenza virus glycoproteins. The stalk domain of the hemagglutinin has been targeted for this purpose due to its high degree of conservation among numerous influenza subtypes and strains. Several vaccine candidates targeting this domain are in late pre-clinical or early clinical stage of development. Currently assays to measure stalk-based immunity are not standardized and no international standard is available. As various vaccine developers are generating data from pre-clinical and clinical studies, harmonization of assay read-outs will help in the comparison of experiments conducted in different laboratories and increase confidence in results. Moreover, researchers will aim to define correlates of protection for new vaccines targeting the hemagglutinin stalk domain. Joint efforts between the Krammer Laboratory at the Icahn School of Medicine at Mount Sinai and the National Institute for Biological Standards and Control (NIBSC) led to a collaborative project to generate an international standard human serum to measure hemagglutinin stalk-reactive antibodies. The WHO Expert Committee on Biological Standardization (ECBS) is currently evaluating this project for its endorsement to generate the first international standard for antibodies to the influenza virus hemagglutinin stalk domain. Please click Download on the upper right corner to see the full abstract

Seasonal influenza vaccine performance and the potential benefits of mRNA vaccines

Influenza remains a public health threat, partly due to suboptimal effectiveness of vaccines. One factor impacting vaccine effectiveness is strain mismatch, occurring when vaccines no longer match circulating strains due to antigenic drift or the incorporation of inadvertent (eg, egg-adaptive) mutations during vaccine manufacturing. In this review, we summarize the evidence for antigenic drift of circulating viruses and/or egg-adaptive mutations occurring in vaccine strains during the 2011-2020 influenza seasons. Evidence suggests that antigenic drift led to vaccine mismatch during four seasons and that egg-adaptive mutations caused vaccine mismatch during six seasons. These findings highlight the need for alternative vaccine development platforms. Recently, vaccines based on mRNA technology have demonstrated efficacy against SARS-CoV-2 and respiratory syncytial virus and are under clinical evaluation for seasonal influenza. We discuss the potential for mRNA vaccines to address strain mismatch, as well as new multi-component strategies using the mRNA platform to improve vaccine effectiveness

Vaccination with recombinant neuraminidase protects against influenza virus infection in mice

While the efficacy of most influenza virus vaccines is measured by the ability to induce antibodies against the hemagglutinin (HA), antibodies against the viral neuraminidase (NA) are also correlated with less severe disease in humans and animal models. Yet, neither the amount nor the enzymatic activity of NA is standardized in current seasonal vaccines, and the breadth of NA-based protection is unknown. In the present study, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to homologous, heterologous, or heterosubtypic virus challenge. Additionally, pre- and post-vaccination human serum samples from vaccinees that received TIV were studied to compare induction of antibodies against the HA and NA. Finally, the amounts of NA in 4 different vaccine formulations from 2013-2014 were quantified using ELISA. Mice immunized with N2 were 100% protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Mice immunized with B/Yamagata/16/88 NA were 100% protected from morbidity and mortality when lethally challenged with a recent Victoria lineage strain. In our human cohorts, the increase in endpoint titers against N1 NA post-vaccination was less robust than that against HA and, as our quantification data suggests, the N1 NA amounts in seasonal vaccine formulations is quite variable. To confirm the broad protective effects of anti-influenza B NA antibodies on a monoclonal level, a panel of mouse monoclonal antibodies was generated against influenza B virus NA; several of these displayed broad reactivity in ELISA to whole virus and recombinant NA and protected against lethal influenza B virus challenge in mice when delivered at a dose of 5 mg/kg prophylactically, or therapeutically, 48 hours post-infection. Analysis of the protective epitopes is currently in progress. The demonstrated protective capacity of anti-NA antibodies suggests that targeting the NA through vaccination may offer increased protection against influenza virus infection

Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by “exosome display,” through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5

A chimeric haemagglutinin-based universal influenza virus vaccine boosts human cellular immune responses directed towards the conserved haemagglutinin stalk domain and the viral nucleoprotein

Background The development of a universal influenza virus vaccine, to protect against both seasonal and pandemic influenza A viruses, is a long-standing public health goal. The conserved stalk domain of haemagglutinin (HA) is a promising vaccine target. However, the stalk is immunosubdominant. As such, innovative approaches are required to elicit robust immunity against this domain. In a previously reported observer-blind, randomised placebo-controlled phase I trial (NCT03300050), immunisation regimens using chimeric HA (cHA)-based immunogens formulated as inactivated influenza vaccines (IIV) −/+ AS03 adjuvant, or live attenuated influenza vaccines (LAIV), elicited durable HA stalk-specific antibodies with broad reactivity. In this study, we sought to determine if these vaccines could also boost T cell responses against HA stalk, and nucleoprotein (NP). Methods We measured interferon-γ (IFN-γ) responses by Enzyme-Linked ImmunoSpot (ELISpot) assay at baseline, seven days post-prime, pre-boost and seven days post-boost following heterologous prime:boost regimens of LAIV and/or adjuvanted/unadjuvanted IIV-cHA vaccines. Findings Our findings demonstrate that immunisation with adjuvanted cHA-based IIVs boost HA stalk-specific and NP-specific T cell responses in humans. To date, it has been unclear if HA stalk-specific T cells can be boosted in humans by HA-stalk focused universal vaccines. Therefore, our study will provide valuable insights for the design of future studies to determine the precise role of HA stalk-specific T cells in broad protection. Interpretation Considering that cHA-based vaccines also elicit stalk-specific antibodies, these data support the further clinical advancement of cHA-based universal influenza vaccine candidates. Funding This study was funded in part by the Bill and Melinda Gates Foundation (BMGF)

Immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase 1 clinical trial.

BackgroundInfluenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses.MethodsWe did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime-boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050.FindingsBetween Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2-5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis.InterpretationThe tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed.FundingBill & Melinda Gates Foundation

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1

A Live-Attenuated Prime, Inactivated Boost Vaccination Strategy with Chimeric Hemagglutinin-Based Universal Influenza Virus Vaccines Provides Protection in Ferrets: A Confirmatory Study

Influenza viruses cause severe diseases and mortality in humans on an annual basis. The current influenza virus vaccines can confer protection when they are well-matched with the circulating strains. However, due to constant changes of the virus surface glycoproteins, the vaccine efficacy can drop substantially in some seasons. In addition, the current seasonal influenza virus vaccines do not protect from avian influenza viruses of human pandemic potential. Novel influenza virus vaccines that aim to elicit antibodies against conserved epitopes like the hemagglutinin stalk could not only reduce the burden of drifted seasonal viruses but potentially also protect humans from infection with zoonotic and emerging pandemic influenza viruses. In this paper, we generated influenza virus vaccine constructs that express chimeric hemagglutinins consisting of exotic, avian head domains and a consistent stalk domain of a seasonal virus. Using such viruses in a sequential immunization regimen can redirect the immune response towards conserved epitopes. In this study, male ferrets received a live-attenuated vaccine virus based on the A/Ann Arbor/6/60 strain expressing a chimeric H8/1 (cH8/1) hemagglutinin, which was followed by a heterologous booster vaccination with a cH5/1N1 formalin inactivated non-adjuvanted whole virus. This group was compared to a second group that received a cH8/1N1 inactivated vaccine followed by a cH5/1N1 inactivated vaccine. Both groups showed a reduction in viral titers in the upper respiratory tract after the A(H1N1)pdm09 virus challenge. Animals that received the live-attenuated vaccine had low or undetectable titers in the lower respiratory tract. The results support the further development of chimeric hemagglutinin-based vaccination strategies. The outcome of this study confirms and corroborates findings from female ferrets primed with a A/Leningrad/134/17/57-based live attenuated cH8/1N1 vaccine followed by vaccination with an AS03-adjuvanted cH5/1N1 split virus vaccine 10