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The World Health Organization advices the use of a quadrivalent vaccine as prophylaxis against influenza, to prevent severe influenza-associated disease and -mortality, and to keep up with influenza antigenic diversity. Different small molecule antivirals to treat influenza have become available. However, emergence of drug resistant influenza viruses has been observed upon use of these antivirals. An appealing alternative approach to prevent or treat influenza is the use of antibody-based antivirals, such as conventional monoclonal antibodies and single-domain antibodies (sdAbs). The surface of the influenza A and B virion is decorated with hemagglutinin molecules, which act as receptor-binding and membrane fusion proteins and represent the main target of neutralizing antibodies. SdAbs that target influenza A and B hemagglutinin have been described. In addition, sdAbs directed against the influenza A virus neuraminidase have been reported, whereas no sdAbs targeting influenza B neuraminidase have been described to date. SdAbs directed against influenza A matrix protein 2 or its ectodomain have been reported, while no sdAbs have been described targeting the influenza B matrix protein 2. Known for their high specificity, ease of production and formatting, sdAb-based antivirals could be a major leap forward in influenza control., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xavier Saelens is named as an inventor on the following patent applications, which describe single-domain antibodies directed against H5N1 influenza virus HA and NA. WO2009/147248, WO2010/081856, and WO2014/090865., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The fate of tissue-resident memory CD4 T cells (Trm) has been incompletely investigated. Here we show that intranasal, but not parenteral, immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 Trm cells, which conferred strong protection against influenza virus infection in the lung. These cells rapidly expanded upon infection and effectively restricted virus replication as determined by CD4 T cell depletion studies. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e-tetramer-sorted CD4 T cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1 or Tregs) with extensive functional diversity and expression of gene markers signifying mature resident Trm cells (Cd69, Nfkbid, Brd2, FosB). Unexpectedly, the same TCR clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), regulatory and cytotoxic cells, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up-regulation of regulatory genes (Lag3, Tigit, Ctla4, Pdcd1) in M2e-specific Trm cells on day 8, indicating a tissue damage preventing function. Thus, contrary to current thinking, lung M2e-specific Th17 Trm cells are sufficient for controlling infection and for protecting against tissue injury. These findings will have strong implications for vaccine development against respiratory virus infections and influenza virus infections, in particular., (© 2022. The Author(s).)

RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3 -/- mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3 -/- mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3 -/- vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3 -/- mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3 -/- mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3 -/- mice. Likewise following IAV infection of Ripk3 -/- mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3 -/- mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3 -/- mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors., (© 2022. The Author(s).)

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase., Competing Interests: XS declares to receive funding from Sanofi Pasteur for research related to influenza vaccine development. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Creytens, Pascha, Ballegeer, Saelens and de Haan.)

In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy.

Background: Current human influenza vaccines lack the adaptability to match the mutational rate of the virus and therefore require annual revisions. Because of extensive manufacturing times and the possibility that antigenic alterations occur during viral vaccine strain production, an inherent risk exists for antigenic mismatch between the new influenza vaccine and circulating viruses. Targeting more conserved antigens such as nucleoprotein (NP) could provide a more sustainable vaccination strategy by inducing long term and heterosubtypic protection against influenza. We previously demonstrated that intranodal mRNA injection can induce potent antigen-specific T-cell responses. In this study, we investigated whether intranodal administration of mRNA encoding NP can induce T-cell responses capable of protecting against a heterologous influenza virus challenge., Methods: BALB/c mice were immunized in the inguinal lymph nodes with different vaccination regimens of mRNA encoding NP. Immune responses were compared with NP DNA vaccination via IFN-γ ELISPOT and in vivo cytotoxicity. For survival experiments, mice were prime-boost vaccinated with 17 µg NP mRNA and infected with 1LD50 of H1N1 influenza virus 8 weeks after boost. Weight was monitored and viral titers, cytokines and immune cell populations in the bronchoalveolar lavage, and IFN-γ responses in the spleen were analyzed., Results: Our results demonstrate that NP mRNA induces superior systemic T-cell responses against NP compared to classical DNA vaccination. These responses were sustained for several weeks even at low vaccine doses. Upon challenge infection, vaccination with NP mRNA resulted in reduced lung viral titers and improved recovery from infection. Finally, we show that vaccination with NP mRNA affects the immune response in infected lungs by lowering immune cell infiltration while increasing the fraction of T cells, monocytes and MHC II + alveolar macrophages within immune infiltrates. This change was associated with altered levels of both pro- and anti-inflammatory cytokines., Conclusions: These findings suggest that intranodal vaccination with NP mRNA induces cross-strain immunity against influenza, but also highlight a paradox of influenza immunity, whereby robust immune responses can provide protection, but can also transiently exacerbate symptoms during infection.

The influenza A virus matrix protein 2 ectodomain (M2e) is a universal influenza A vaccine candidate. Numerous studies in laboratory mice, but very few in natural influenza A virus hosts, have demonstrated that M2e-based vaccines can provide protection against any influenza A virus challenge. M2e-based immunity is largely accomplished by IgG and early stage clinical studies have demonstrated that the vaccine is safe. Yet M2e is considered a difficult target to develop as a vaccine: it does not offer sterilizing immunity and its mode of action relies on Fcγ receptor-mediated effector mechanisms, most likely in concert with alveolar macrophages. In a human challenge study with an H3N2 virus, treatment with a monoclonal M2e-specific human IgG was associated with a faster recovery compared to placebo treatment. If the universal influenza vaccine field incorporates this antigen into next generation vaccines, M2e could prove its merit when the next influenza pandemic strikes., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Microneedle arrays (MNAs) are a promising mean to administer vaccines. Without the need of highly trained personnel, MNAs can be applied to deliver vaccines into the dermis, which is well equipped to initiate potent immune responses. While vaccination using dissolving microneedle arrays has been extensively investigated, the use of solid nanoporous MNAs (npMNAs) to deliver vaccines remained largely unexplored. In this report we investigated whether npMNAs with an average pore size of 80 nm, can be used for influenza vaccination based on recombinant hemagglutinin (HA) protein of the 2009 pandemic H1N1 (pH1N1) virus. Fluorescently labeled HA loaded in the npMNAs was effectively delivered into the skin of mouse ears, as a result of a diffusion-based process. Compared to intramuscular immunization, intradermal HA vaccination of mice using npMNAs elicited high levels of HA antigen specific antibodies, with pH1N1 hemagglutination inhibition and neutralization activity. Moreover, mice vaccinated with pH1N1 HA loaded npMNAs were completely protected against a potentially lethal challenge with mouse adapted pH1N1 virus. These results illustrate that intradermal subunit vaccine immunization using npMNAs is a promising approach to facilitate effective vaccination., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Due to the high risk of an outbreak of pandemic influenza, the development of a broadly protective universal influenza vaccine is highly warranted. The design of such a vaccine has attracted attention and much focus has been given to nanoparticle-based influenza vaccines which can be administered intranasally. This is particularly interesting since, contrary to injectable vaccines, mucosal vaccines elicit local IgA and lung resident T cell immunity, which have been found to correlate with stronger protection in experimental models of influenza virus infections. Also, studies in human volunteers have indicated that pre-existing CD4 + T cells correlate well to increased resistance against infection. We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. To improve the protective ability of the self-adjuvanting fusion protein, CTA1-3M2e-DD, we incorporated it into porous maltodextrin nanoparticles (NPLs). This proof-of-principle study demonstrates that the combined vaccine vector given intranasally enhanced immune protection against a live challenge infection and reduced the risk of virus transmission between immunized and unimmunized individuals. Most importantly, immune responses to NPLs that also contained recombinant hemagglutinin (HA) were strongly enhanced in a CTA1-enzyme dependent manner and we achieved broadly protective immunity against a lethal infection with heterosubtypic influenza virus. Immune protection was mediated by enhanced levels of lung resident CD4 + T cells as well as anti-HA and -M2e serum IgG and local IgA antibodies.

Vaccines that direct the immune response towards conserved B cell epitopes of influenza viruses can provide broad protection. In many instances, this requires the design of vaccine antigens that stimulate the immune system to levels that far exceed the natural responses towards such antigens. Here we focus on the matrix protein 2 ectodomain (M2e) as a 'universal' influenza A vaccine candidate. Thanks to its small size and high solubility, M2e can be expressed and delivered in almost any format. Protection against experimental influenza A virus challenge by M2e-based vaccines has been demonstrated in natural host of influenza and clinical studies demonstrated that such vaccines are safe and immunogenic. M2e-specific antibodies protect mainly by Fc receptor-mediated antibody-dependent cellular phagocytosis activity, which is reminiscent to how antibodies directed against the hemagglutinin stalk protect in vivo. Fighting influenza with a broadly protective influenza vaccine will likely require a blend of conserved antigens. M2e deserves its place in such a blend., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We describe a novel live oral vaccine type. Conceptually, this vaccine is based on a non-lytic, recombinant filamentous bacteriophage that displays an antigen of interest. To provide proof of concept we used the amino-terminal part of a conserved influenza A virus epitope, i.e. matrix protein 2 ectodomain (M2e) residues 2 to 16, as the antigen of interest. Rather than using the phages as purified virus-like particles as a vaccine, these phages were delivered to intestinal Peyer's patches as a live bacterium-phage combination that comprises Escherichia coli cells that conditionally express invasin derived from Yersinia pseudotuberculosis. Invasin-expressing E. coli cells were internalized by mammalian Hep-2 cells in vitro and adhered to mouse intestinal microfold (M) cells ex vivo. Invasin-expressing E. coli cells were permissive for recombinant filamentous bacteriophage f88 that displays M2e and became persistently infected. Oral administration of the live engineered E. coli-invasin-phage combination to mice induced M2e-specific serum IgG antibodies. Mice that had been immunized with invasin-expressing E. coli cells that carried M2e2-16 displaying fd phages seroconverted to M2e and showed partial protection against challenge with influenza A virus. Oral delivery of a live vaccine comprising a bacterial host that is targeted to Peyer's patches and is persistently infected with an antigen-displaying phage, can thus be exploited as an oral vaccine., (Copyright © 2017. Published by Elsevier B.V.)

Introduction: Current influenza vaccines can prevent disease caused by influenza viruses but require annual administration and almost yearly reformulation. An attractive alternative approach would be to use a vaccine that provides broad and, ideally, lifelong protection against all influenza A and B virus strains. The extracellular domain of matrix protein 2 (M2e) of influenza A viruses is conserved and thus fits well in such a broadly protective vaccine. Areas covered: Recent advances in M2e vaccine design, the mode of action of M2e-based immunity and clinical progress of M2-based influenza vaccines. Expert commentary: Many M2e vaccine have been successfully tested for efficacy against a panel of divergent influenza viruses in animal models. More recently, clinical studies have been conducted with M2e vaccine candidates, which demonstrated their safety and immunogenicity in humans. Efficacy studies in humans are still needed to provide evidence that an M2e-based vaccine can protect against human influenza.

Influenza A virus is a pathogen that is feared for its capacity to cause pandemics. In this review, we illustrate the clinical evidence which support the theory that bacterial co-infection is a considerable risk factor for exacerbated disease during pandemic and seasonal influenza, including infection with influenza B viruses. We provide an overview of the multiple and diverse mechanisms that help explain how influenza creates an opportunity for replication of secondary bacterial infections. Influenza vaccines and pneumococcal vaccines are widely used and often in overlapping target groups. We summarize the evidence for a protective effect of influenza immunization against bacterial infections, and vice versa of pneumococcal vaccines against influenza-associated pneumonia and lethality. It is important that future implementation of broadly protective influenza vaccines also takes into account protection against secondary bacterial infection.

Background: Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use., Methodology/principal Findings: We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA) and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect., Conclusions/significance: Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza.

The ectodomain of influenza A matrix protein 2 (M2e) is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs). We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4(+) T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2) or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

Currently licensed influenza vaccines rely predominantly on the induction of strain-matched hemagglutination inhibition antibody responses. These vaccines have a proven record of safety and efficacy in preventing influenza-induced illness and complications. However, they do not confer protection to all vaccinated individuals, and the protection they afford is short-lived, particularly in older adults. Hemagglutination inhibition titers induced by these vaccines are considered correlates of protection, but recent data demonstrate that this is not always the case. It is clear that better insight is needed into the immune responses that correlate with protection against human influenza. Influenza vaccines that can induce cross-reactive cellular immune responses (CD4(+) and/or CD8(+) T-cell responses) might correct some of the shortcomings of currently used influenza vaccines. In the future, the use of infection-permissive and disease-modifying vaccines that allow for the induction of cross-reactive T-cell responses may become a valuable complement to the administration of trivalent inactivated influenza vaccines.

Considering the emergence of highly pathogenic influenza viruses and threat of worldwide pandemics, there is an urgent need to develop broadly-protective influenza vaccines. In this study, we demonstrate the potential of T7 bacteriophage-based nanoparticles with genetically fused ectodomain of influenza A virus M2 protein (T7-M2e) as a candidate universal flu vaccine. Immunization of mice with non-adjuvanted T7-M2e elicited M2e-specific serum antibody responses that were similar in magnitude to those elicited by M2e peptide administered in Freund's adjuvant. Comparable IgG responses directed against T7 phage capsomers were induced following vaccination with wild type T7 or T7-M2e. T7-M2e immunization induced balanced amounts of IgG(1) and IgG(2a) antibodies and these antibodies specifically recognized native M2 on the surface of influenza A virus-infected mammalian cells. The frequency of IFN-γ-secreting T cells induced by T7-M2e nanoparticles was comparable to those elicited by M2e peptide emulsified in Freund's adjuvant. Emulsification of T7-M2e nanoparticles in Freund's adjuvant, however, induced a significantly stronger T cell response. Furthermore, T7-M2e-immunized mice were protected against lethal challenge with an H1N1 or an H3N2 virus, implying the induction of hetero-subtypic immunity in our mouse model. T7-M2e-immunized mice displayed considerable weight loss and had significantly reduced viral load in their lungs compared to controls. We conclude that display of M2e on the surface of T7 phage nanoparticles offers an efficient and economical opportunity to induce cross-protective M2e-based immunity against influenza A.

Here we demonstrate that by using non-toxic fractions of saponin combined with CTA1-DD we can achieve a safe and above all highly efficacious mucosal adjuvant vector. We optimized the construction, tested the requirements for function and evaluated proof-of-concept in an influenza A virus challenge model. We demonstrated that the CTA1-3M2e-DD/ISCOMS vector provided 100% protection against mortality and greatly reduced morbidity in the mouse model. The immunogenicity of the vector was superior to other vaccine formulations using the ISCOM or CTA1-DD adjuvants alone. The versatility of the vector was best exemplified by the many options to insert, incorporate or admix vaccine antigens with the vector. Furthermore, the CTA1-3M2e-DD/ISCOMS could be kept 1 year at 4°C or as a freeze-dried powder without affecting immunogenicity or adjuvanticity of the vector. Strong serum IgG and mucosal IgA responses were elicited and CD4 T cell responses were greatly enhanced after intranasal administration of the combined vector. Together these findings hold promise for the combined vector as a mucosal vaccine against influenza virus infections including pandemic influenza. The CTA1-DD/ISCOMS technology represents a breakthrough in mucosal vaccine vector design which successfully combines immunomodulation and targeting in a safe and stable particulate formation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

Human influenza causes substantial morbidity and mortality. Currently, licensed influenza vaccines offer satisfactory protection if they match the infecting strain, but they come with significant drawbacks. These vaccines are derived from prototype viruses, containing the hemagglutinin of influenza viruses that are likely to cause the next epidemic. Their usefulness against a future pandemic, however, remains problematic. A vaccine based on the ectodomain of influenza matrix protein 2 (M2e) could overcome these drawbacks. M2e is highly conserved in both human and avian influenza A viruses. The low immunogenicity against natural M2e can be overcome by fusing M2e to an appropriate carrier such as Hepatitis B virus-derived virus-like particles. Such chimeric particles can be produced in a simple and safe bacterial expression system, requiring minimal biocontainment, and can be obtained in a pure form. Experiments in animal models have demonstrated that M2e-based vaccines induce protection against a lethal challenge with various influenza A virus subtypes. Furthermore, the production and use of an effective M2e-vaccine could be implemented at any time regardless of seasonality, both in an epidemic as well as in a pandemic preparedness program. In animal models, M2e-vaccines administered parenterally or intranasally protect against disease and mortality following challenge with various influenza A strains. Adjuvants suitable for human use improve protection, which correlates with higher anti-M2e antibody responses of defined subtypes. Recently, Phase I clinical studies with M2e-vaccines have been completed, indicating their safety and immunogenicity. Further clinical development of this universal influenza A vaccine candidate is being pursued in order to validate its protective efficacy in humans.

Influenza vaccines used today are strain specific and need to be adapted every year to try and match the antigenicity of the virus strains that are predicted to cause the next epidemic. The strain specificity of the next pandemic is unpredictable. An attractive alternative approach would be to use a vaccine that matches multiple influenza virus strains, including multiple subtypes. In this review, we focus on the development and clinical potential of a vaccine that is based on the conserved ectodomain of matrix protein 2 (M2) of influenza A virus. Since 1999, a number of studies have demonstrated protection against influenza A virus challenge in animal models using chemical or genetic M2 external domain (M2e) fusion constructs. More recently, Phase I clinical studies have been conducted with M2e vaccine candidates, demonstrating their safety and immunogenicity in humans. Ultimately, and possibly in the near future, efficacy studies in humans should provide proof that this novel vaccine concept can mitigate epidemic and even pandemic influenza A virus infections.

The possible emergence of a new influenza pandemic is considered a major threat for human health worldwide. Pandemics start by the introduction or reintroduction and spread in the human population of an influenza virus subtype against which almost nobody has protective immunity. Currently used influenza vaccines provide good protection only against antigenically matching influenza strains. However, neither the timing nor the subtype of the next pandemic virus is known. Therefore, different approaches are being pursued in anticipation of this pandemic threat. In the present article, we review approaches that aim to induce heterosubtypic immunity, that is, protection against challenge with influenza A viruses belonging to two or more subtypes. Experimental and epidemiological studies indicate that natural infection can provide some heterosubtypic immunity, possibly involving cellular immune responses directed against matrix and/or nucleoprotein as well as humoral responses against neuraminidase. Other approaches have focused on the use of conserved epitopes of the viral proteins, including matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Proof-of-concept of protection by these novel vaccines has been obtained in animal models, and promising results from several clinical trials have recently been reported. Demonstrating the efficacy of these new vaccines against a potential pandemic influenza endowed with human transmissibility remains a major challenge., (Copyright 2009 Prous Science, S.A.U. or its licensors. All rights reserved.)

The extracellular domain of influenza A virus matrix protein 2 (M2e) is strongly conserved. Therefore, vaccines based on M2e can induce broad-spectrum immunity against influenza. We have mainly used recombinant virus-like particles derived from Hepatitis B virus core (HBc) as carrier for efficacious presentation of the M2e antigen. Here, we address whether pre-existing HBc-specific immunity interferes with the protective immune response obtained by M2e-HBc vaccination. Anti-HBc antibodies were induced by immunizing mice with unsubstituted HBc virus-like particles in the presence of two different adjuvants. We demonstrate that pre-existing HBc-specific antibodies affect neither the induction of M2e-specific antibody responses to vaccination with M2e-HBc particles, nor the protective efficacy of the resulting response. These results suggest that vaccination with M2e-HBc can induce protective anti-M2e antibodies even in anti-HBc positive individuals. The implications of these findings are discussed in the context of the clinical development of an M2e-based universal influenza vaccine, which recently successfully completed a Phase I trial.

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzyme-linked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.

At present few vaccine candidates exists against potentially pandemic influenza virus infections. We provide compelling evidence that a targeted fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the matrix protein 2 (M2e) ectodomain epitope, CTA1-3M2e-DD, confers strong protective immunity against a potentially lethal challenge infection with influenza virus in mice. The formulation was highly effective for mucosal immunizations and promoted high M2e-specific serum IgG and mucosal IgA antibody titers and an hitherto unknown anti-M2e CD4 T cell immunity. This novel CTA1-3M2e-DD fusion protein combines adjuvant and a conserved influenza A antigen in a promising candidate for a universal anti-influenza vaccine.

M2 is the third integral membrane protein of influenza A. M2e, the extracellular, 23 amino acid residues of M2, has been remarkably conserved in all human influenza A strains. This prompted us to evaluate the use of M2e as a potential broad-spectrum immunogen in a mouse model for influenza infection. Genetic fusion of the M2e and hepatitis B virus core (HBc) coding sequences allowed us to obtain highly immunogenic virus-like particles. This M2e-HBc vaccine induced complete protection in mice against a lethal influenza challenge. Protective immunity was obtained regardless of the position of M2e in the M2e-HBc chimera at the amino-terminus or inserted in the immuno-dominant loop of the HBc protein. Increasing the copy number of M2e inserted at the N-terminus from one to three per monomer (240-720 per particle) significantly enhanced the immune response and reduced the number of vaccinations required for complete protection against a lethal challenge with influenza A virus. A series of M2e-HBc constructs was subsequently combined with CTA1-DD, a recombinant cholera toxin A1 derived mucosal adjuvant, to test its efficacy as an intranasally delivered vaccine. All hybrid VLPs tested with CTA1-DD completely protected mice from a potentially lethal infection and, in addition, significantly reduced morbidity. Overall, increased resistance to influenza challenge in the mice correlated with an enhanced Th1-type M2e-specific antibody response induced by vaccination. These results show that M2e is a valid and versatile vaccine candidate to protect against any strain of human influenza A.

Mucosal vaccination requires effective and safe adjuvants. We have evaluated the non-toxic adjuvant CTA1-DD for mucosal vaccination against influenza. CTA1-DD contains the enzymatically active CTA1 subunit of cholera toxin (CT) genetically fused to a gene encoding a dimer of the D-fragment from Staphylococcus aureus protein A. CTA1-DD only binds to Ig-receptor carrying cells of the immune system. Nasal administration of the universal influenza vaccine M2e-HBc in combination with CTA1-DD completely protected mice from a potentially lethal infection and significantly reduced morbidity. Sera of mice immunized with M2e-HBc + CTA1-DD revealed IgG subclass profiles consistent with an enhanced Th1-type immunity. When the vaccine was administered intraperitoneally, the adjuvant improved the M2e antibody titer in circulation, but did not significantly reduce the morbidity.

Influenza B viruses (IBV) are responsible for a considerable part of the burden caused by influenza virus infections. Since their emergence in the 1980s, the Yamagata and Victoria antigenic lineages of influenza B circulate in alternate patterns across the globe. Furthermore, their evolutionary divergence and the appearance of new IBV subclades complicates the prediction of future influenza vaccines compositions. It has been proposed that the addition of the neuraminidase (NA) antigen could potentially induce a broader protection and compensate for hemagglutinin (HA) mismatches in the current vaccines. Here we show that anti-NA and -HA sera against both Victoria and Yamagata lineages have limited inter-lineage cross-reactivity. When transferred to mice prior to infection with a panel of IBVs, anti-NA sera were as potent as anti-HA sera in conferring protection against homologous challenge and, in some cases, conferred superior protection against challenge with heterologous IBV strains. [ABSTRACT FROM AUTHOR]

Background: Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use. Methodology/Principal Findings: We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA) and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect. Conclusions/Significance: Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza. [ABSTRACT FROM AUTHOR]

Matrix protein (M)2 is an Influenza A, type III membrane protein with an extracellular domain (ectodomain of M2 [M2e]) of 23 amino acid residues, which is strongly conserved across virus strains. M2 fulfills an important biological function in the life cycle of the Influenza A virus and has been a target of antiviral drugs. M2e has generated much interest as a potential vaccine target, and a clinical M2e vaccine trial was initiated in 2007. The advantage of M2e compared with hemagglutinin, the prime antigen target in conventional influenza vaccines, is that its sequence is conserved. This means that a stable, efficacious and easily produced M2e-based vaccine would provide protection not only against drifting seasonal influenza epidemic strains, but would also make it possible to vaccinate in anticipation of an emerging pandemic. Furthermore, most reported M2e-based vaccines are produced by economical and safe technologies. IgG subtype antibodies directed against M2e can prevent death from influenza and reduce morbidity in animal models for influenza disease. The immunological mechanism that mediates protection by anti-M2e antibodies is not completely understood, but it probably involves antibody-mediated cellular cytotoxicity. This review summarizes the findings on M2e vaccine candidates and addresses some of the key unanswered questions about this promising Influenza A vaccine target: what is its likely mechanism of action? Which measurable parameters correlate with protection? And what can be expected from clinical use of an M2e-based vaccine? [ABSTRACT FROM AUTHOR]

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections. [ABSTRACT FROM AUTHOR]

Reports on the production of the head domain of recombinant neuraminidase of A/Victoria/375 influenza virus in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating factor signal sequence. Hyperglycosylation of the secreted neuraminidase with N-glycans of the high-mannose type with 30-40 mannose residues.

Background: Influenza virus infection predisposes to secondary bacterial pneumonia. Currently licensed influenza vaccines aim at the induction of neutralizing antibodies and are less effective if the induction of neutralizing antibodies is low and/or the influenza virus changes its antigenic surface. We investigated the effect of suboptimal vaccination on the outcome of post-influenza bacterial superinfection. Methods: We established a mouse vaccination model that allows control of disease severity after influenza virus infection despite inefficient induction of virus-neutralizing antibody titers by vaccination. We investigated the effect of vaccination on virus-induced host immune responses and on the outcome of superinfection with Staphylococcus aureus. Results: Vaccination with trivalent inactivated virus vaccine (TIV) reduced morbidity after influenza A virus infection but did not prevent virus replication completely. Despite the poor induction of influenza-specific antibodies, TIV protected from mortality after bacterial superinfection. Vaccination limited loss of alveolar macrophages and reduced levels of infiltrating pulmonary monocytes after influenza virus infection. Interestingly, TIV vaccination resulted in enhanced levels of eosinophils after influenza virus infection and recruitment of neutrophils in both lungs and mediastinal lymph nodes after bacterial superinfection. Conclusion: These observations highlight the importance of disease modulation by influenza vaccination, even when suboptimal, and suggest that influenza vaccination is still beneficial to protect during bacterial superinfection in the absence of complete virus neutralization. [ABSTRACT FROM AUTHOR]

Abstract Influenza represents a global public health threat. Currently available influenza vaccines are effective against strain-matched influenza A and B viruses but do not protect against novel pandemic viruses. Vaccine candidates that target conserved B or T cell epitopes of influenza viruses could circumvent this shortcoming. The conserved extracellular domain of matrix protein 2 (M2e) of influenza A is an example of such a broadly protective vaccine candidate. Protection by M2e-based vaccine candidates largely depends on M2e-specific IgG antibodies. Here we show that the M2e-specific IgG2a monoclonal antibody 65 (MAb 65) can reduce influenza A/Udorn/72 (H3N2) and A/Hong Kong/68 (H3N2) virus plaque formation. This effect was not observed with other influenza A virus strains tested. We further show that passive transfer of MAb 65 to mice can reduce viral loads in the upper and lower airways, which results in reduced transmission of A/Udorn/72 and A/Hong Kong/68 viruses to cohoused, unimmunized contact mice. Virus restriction by passively transferred Mab 65 was significantly less pronounced in Fcgr1 −/− Fcgr3 −/− mutant mice compared with wild type controls, suggesting that in vivo protection provided by MAb 65 depends on Fcγ receptor-mediated antibody effector mechanisms. We conclude that M2e-based antibody immune therapy has the potential to diminish influenza A virus replication in the immunized host as well as in exposed naïve contacts. Highlights • Broadly protective influenza vaccines could be valuable in case of a new influenza pandemic outbreak. • Replication of influenza A virus strains A/Udorn/72 and A/HK/68 is suppressed in vitro by a M2e-specific IgG2a monoclonal antibody. • The M2e-specific antibody reduced influenza A virus transmission in a mouse model. • In vivo , anti-M2e antibody mediated protection against influenza A/Udorn/72 and A/HK/68 relied on Fcγ Receptor I and -III. • An M2e-based antibody therapy could be used to suppress influenza virus transmission, e.g. during a pandemic situation. [ABSTRACT FROM AUTHOR]

Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in the case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long-lasting immunity are preferred. Because influenza virus-specific CD8+ T cells are directed mainly against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8+ T cells. To optimize the induction of CD8+ T cell responses, we made several modifications to NP, aiming at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve the induction of CD8+ T cell responses. We showed that NP with increased degradation rates improved CD8+ T cell activation in vitro if the amount of antigen was limited or if CD8+ T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and wild-type NP (NPwt), since NPwt already induced optimal CD8+ T cell responses. [ABSTRACT FROM AUTHOR]

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ-/-, FcγRI-/-, FcγRIII-/-, and (FcγRI, FcγRIII)-/- mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII-/- mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)-/- mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG. [ABSTRACT FROM AUTHOR]