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A universal influenza A vaccine based on the extracellular domain of the M2 protein
Abstract
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.
... 40 Unlike HA and NA, M2e-specific antibodies protect via FcγR-dependent mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), rather than direct virus neutralization. 39,41−43 Various carriers have been used to increase the immunogenicity of M2e vaccines, including hepatitis B core protein (HBc), 41 tobacco mosaic virus (TMV) coat protein, 44 keyhole limpet hemocyanin (KLH), 40 rotavirus NSP4, 45 GCN4, 46 bacterial flagellin, 47 liposomes, 48 polymers, 49−51 and gold nanoparticles (NPs). 52,53 Early human trials confirmed the immunogenicity and tolerance of M2e vaccines but also revealed several weaknesses. ...
... 52,53 Early human trials confirmed the immunogenicity and tolerance of M2e vaccines but also revealed several weaknesses. For example, an adjuvanted M2e-HBc fusion vaccine 41 induced a short-lived anti-M2e antibody response, and an M2e-flagellin fusion vaccine 47 caused undesirable side effects at higher doses. A vaccine combining M2e with cytotoxic T lymphocyte (CTL) epitopes 54 induced strong cellular immunity, but this response was narrow and slow, making it unsuitable for effectively mitigating a future influenza pandemic. ...
... Interestingly, DLS analysis of the SEC fraction (13−14 mL) of an mAb148-purified hM2e-5GS-FR sample indicated the presence of three particle size populations, suggesting that cluster formation is an intrinsic feature of hM2e SApNPs ( Figure S2B). Differential screening calorimetry (DSC) 41 was used to quantify the thermostability of these hM2e constructs. Thermograms were obtained for hM2e-5GS-1TD0 and hM2e-5GS-FR, which showed a melting temperature (T m ) of 72.5−72.8°C ...
The development of a cross-protective pan-influenza A vaccine remains a significant challenge. In this study, we designed and evaluated single-component self-assembling protein nanoparticles (SApNPs) presenting the conserved extracellular domain of matrix protein 2 (M2e) as vaccine candidates against influenza A viruses. The SApNP-based vaccine strategy was first validated for human M2e (hM2e) and then applied to tandem repeats of M2e from human, avian, and swine hosts (M2ex3). Vaccination with M2ex3 displayed on SApNPs demonstrated higher survival rates and less weight loss compared to the soluble M2ex3 antigen against the lethal challenges of H1N1 and H3N2 in mice. M2ex3 I3-01v9a SApNPs formulated with a squalene-based adjuvant were retained in the lymph node follicles over 8 weeks and induced long-lived germinal center reactions. Notably, a single low dose of M2ex3 I3-01v9a SApNP formulated with a potent adjuvant, either a Toll-like receptor 9 (TLR9) agonist or a stimulator of interferon genes (STING) agonist, conferred 90% protection against a lethal H1N1 challenge in mice. With the ability to induce robust and durable M2e-specific functional antibody and T cell responses, the M2ex3-presenting I3-01v9a SApNP provides a promising pan-influenza A vaccine candidate.
... The M2 protein, compared with other proteins encoded by the influenza A genome, is highly conserved [4,5]. M2 is a tetrameric membrane protein. ...
... M2 is a tetrameric membrane protein. Its extracellular domain (M2e) is a short (23 a.a.) peptide, the sequence of which is highly conserved in nearly all human isolates of the influenza A virus that circulated between 1918 and 2008 [2,5]. The use of M2e peptide would allow the development of a "universal" vaccine that could be efficient against a wide range of influenza strains. ...
... M2e is a poor immunogen and M2e-specific antibody responses are hardly induced following an infection [6]. However, anti-M2e immune responses could be enhanced by attaching M2e to a highly immunogenic carrier or adjuvant [5]. ...
Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15–30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant “universal” vaccine against influenza A produced in plants.
... The immunogenicity of native M2e is low, but it can be improved by fusing M2e to a highly immunogenic carrier or adjuvant. The first study in which M2e was presented on the surface of virus-like particles formed by the core antigen of the hepatitis B virus dates back to 1999 [33], and a number of further studies demonstrated that M2e-based vaccines could be highly immunogenic and protect against infection (reviewed in [34,35]). Examples of recent clinical trials of M2e-containing candidate vaccines include Uniflu (NCT03789539) and Tandiflu1 [36,37]. ...
... Likewise, the RBD region of the S protein of SARS-CoV-2 could be a promising target for the development of recombinant vaccines against COVID-19 [15,16]. Previous studies have shown that the immunogenicity of native М2е is poor, but it can be increased by its fusion to highly immunogenic carriers or adjuvants [33,34,49]. In this study, we developed a plant-based expression system for the production of the RBD and M2e linked to bacterial flagellin, known to be a potent mucosal adjuvant. ...
... The M2e peptide of the influenza A virus is one of the most promising candidates for the development of a recombinant influenza vaccine since its sequence is highly conserved in all human isolates and differs in only a few amino acids in strains of animal origin [33,48]. Likewise, the RBD region of the S protein of SARS-CoV-2 could be a promising target for the development of recombinant vaccines against COVID-19 [15,16]. ...
The development of recombinant vaccines against SARS-CoV-2 and influenza A is an important task. The combination of the conserved influenza A antigen, the extracellular domain of the transmembrane protein M2 (M2e), and the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (RBD) provides the opportunity to develop a bivalent vaccine against these infections. The fusion of antigens with bacterial flagellin, the ligand for Toll-like receptor 5 and potent mucosal adjuvant, may increase the immunogenicity of the candidate vaccines and enable intranasal immunization. In this study, we report the transient expression of RBD alone, RBD coupled with four copies of M2e, and fusions of RBD and RBD-4M2e with flagellin in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff. The yields of purified recombinant proteins per gram of fresh leaf tissue were about 20 µg for RBD, 50–60 µg for RBD-4M2e and the fusion of RBD with flagellin, and about 90 µg for RBD-4M2e fused to flagellin. Targeting to the endoplasmic reticulum enabled the production of glycosylated recombinant proteins comprising RBD. Our results show that plant-produced RBD and RBD-4M2e could be further used for the development of subunit vaccines against COVID-19 and a bivalent vaccine against COVID-19 and influenza A, while flagellin fusions could be used for the development of intranasal vaccines.
... Influenza vaccines based on the highly conserved extracellular domain of matrix protein 2 (M2e) of influenza A, have been proposed as possible alternatives for currently licensed influenza vaccines [2,3]. Immunization of laboratory mice with M2e displayed on a virus-like particle (VLP) protect against a potentially lethal influenza A virus (IAV) challenge [4]. This protection can be transferred by serum, requires a functional Fcγ receptor compartment and is mediated by antibody-dependent cellular phagocytosis [4,5]. ...
... Immunization of laboratory mice with M2e displayed on a virus-like particle (VLP) protect against a potentially lethal influenza A virus (IAV) challenge [4]. This protection can be transferred by serum, requires a functional Fcγ receptor compartment and is mediated by antibody-dependent cellular phagocytosis [4,5]. Phase I studies with M2e-based vaccine candidates have been completed, which suggested that such vaccine candidates are safe and immunogenic in healthy volunteers (e.g., NCT00819013) [6]. ...
... This finding suggests that besides the antibody production post immunization, other mechanisms contribute to effective vaccination which would be lacking in RIPK3deficient mice. Active vaccination with M2e-VLP decreases viral titers in the lungs upon IAV challenge [4]. Since RIPK3 is important for viral protection we examined whether viral titers would be different in vaccinated Ripk3-deficient and-proficient animals. ...
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.
... On the other hand, haemagglutination inhibiting antibodies were not detected after immunization in both groups. Moreover, to induce protective immunity, in most instances it is essential to expose the immune system to full proteins except few exceptions of foot and mouth disease virus (Clarke et al. 1987) and M2-peptide derived from influenza virus (Neirynck et al. 1999). HAI specific antibodies could be detected upon challenge (121.6 ± 38.4 and 128 ± 35.05 at 14 dpc respectively) for groups A1 and B1 which commensurates with rise in ELISA antibody titers (11,520 ± 1280 and 30,720 ± 8681.38). ...
... In immunized groups, disease severity and bodyweight reduction were more in the rHA1 group than the rHA1-phage group. The rHA1-phage group experienced transient bodyweight reduction of 1.18 ± 1.28% (3 dpc) which recovered on 4 dpc indicating protection conferred by vaccination which aligns with the previous observations made by Hashemi et al. (2012) wherein mice were protected against H1N1 induce protective immunity, in most instances it is essential to expose the immune system to full proteins except few exceptions of foot and mouth disease virus (Clarke et al. 1987) and M2-peptide derived from influenza virus (Neirynck et al. 1999). Moreover, the low sensitivity of the HI assay may have contributed to this result (Treanor et al. 2001). ...
Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen’s eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.
... Highly conserved target antigens that may serve as universal vaccine targets include influenza A and B nucleoproteins (NPs), matrix proteins 1 and 2 (M2) of influenza A, and the HA stem. NP induces broadly protective T cell responses (1) as well as non-neutraliz ing antibodies (2), while M2 induces broadly protective, non-neutralizing antibodies (3). These responses do not prevent viral entry, so a mild, transient infection is possible. ...
... The A/NP+M2-rAd vaccination resulted in lower viral titers at day 3 than vaccination to A/NP-Ad alone. This is understandable since M2 in the mixture adds to the protection provided by A/NP via different immune mechanisms (3,41), which might synergize with the responses to A/NP. However, it is unclear why the GrzB response was greater in mice vaccinated with A/NP+M2-rAd than with A/NP-Ad alone. ...
New approaches to vaccination are needed for protection against rapidly evolving viruses because emerging variant strains often evade neutralizing antibodies. Alternative strategies include targeting highly conserved antigens that change more slowly over time, which could provide the basis for universal vaccines. Recombinant adenovirus (rAd)-based vaccines expressing conserved influenza virus antigens nucleoprotein (NP) and matrix 2 (M2) provide powerful, long-lasting protection against challenge with diverse influenza strains, especially if administered intranasally. After intranasal administration of these rAd-based vaccines, animals are protected against morbidity and mortality in part by strong local T cell responses. However, concerns have been raised that these local responses could result in immune-mediated lung damage. In this study, we vaccinated mice intranasally with rAd vectors expressing influenza A/NP and/or M2, or influenza B/NP as a control, and then challenged them with an influenza A virus 15 months later. Protection persisted, as shown by accelerated viral clearance. Vaccination reduced the incidence and/or severity of histopathologic findings associated with influenza infection. Vaccination with A/NP+M2-rAd or A/NP-rAd reduced cytokine responses in the lungs following challenge. Granzyme B levels peaked and resolved earlier in animals vaccinated with A/NP+M2-rAd or A/NP-rAd than in other groups, consistent with a rapid CD8 T cell response that resolved more quickly. An influx of neutrophils, shown by a rise in neutrophil elastase, occurred in all groups following the challenge but returned to baseline more quickly in the two A/NP-vaccinated groups. Thus, vaccines inducing potent local immunity in the respiratory tract provided protection, rather than damage, to the lungs.
IMPORTANCE
Vaccines targeting highly conserved proteins can protect broadly against diverse viral strains. When a vaccine is administered to the respiratory tract, protection against disease is especially powerful. However, it is important to establish that this approach is safe. When vaccinated animals later encounter viruses, does reactivation of powerful local immunity, including T cell responses, damage the lungs? This study investigates the safety of mucosal vaccination of the respiratory tract. Non-replicating adenoviral vaccine vectors expressing conserved influenza virus proteins were given intranasally. This vaccine-induced protection persists for at least 15 months. Vaccination did not exacerbate inflammatory responses or tissue damage upon influenza virus infection. Instead, vaccination with nucleoprotein reduced cytokine responses and histopathology, while neutrophil and T cell responses resolved earlier. The results are promising for safe vaccination at the site of infection and thus have implications for the control of influenza and other respiratory viruses.
... The M2 ion channel is highly conserved and is essential for influenza virus budding and disassembly of the viral core. Despite a number of challenges, influenza virus matrix protein 2 ectodomain (M2e) has been proposed as a universal vaccination antigen (Kolpe et al., 2017;Neirynck et al., 1999). The surface-exposed amino-terminal ectodomain is not very immunogenic (Turley et al., 2011). ...
... The surface-exposed amino-terminal ectodomain is not very immunogenic (Turley et al., 2011). However, this issue was resolved by conjugating M2e to various carriers such as hepatitis B core protein or the Neisseria meningitides outer membrane complex protein (OMPC), complex branched synthetic structures, virus-like particles (De Filette et al., 2005, 2006aFan et al., 2004;Huleatt et al., 2008;Neirynck et al., 1999;Jegerlehner et al., 2004;Mozdzanowska et al., 2003). In vivo studies using age-and sex-matched BALB/c, C57BL/6, and DBA/2 mice have shown that a self-adjuvating fusion protein of M2e, CTA1-3M2e-DD, delivered using porous maltodextrin nanoparticles induced IgG-mediated protection against different influenza virus strains (Bernasconi et al., 2018). ...
Influenza A virus (IAV) infection is a contagious respiratory infection responsible for high morbidity and mortality rates across the planet. The human immune system contains a wide range of soluble activators, membrane-bound receptors, and regulators to eliminate IAVs. Despite these various immune mechanisms that neutralize IAV or restrict their replication, IAVs still have developed distinct strategies to evade the host immunity and establish a successful infection. Given the higher and continuous rate of mutations in IAVs, decades of research have focused on understanding the host's immune mechanisms against IAVs, and the evasion strategies employed by the virus to overcome the e host immune system. Future IAV pandemics or epidemics remain inevitable, and a greater understanding of the host-pathogen interaction involved is required to develop universal vaccines and treatment against IAV. Here, we review how the host immune system responds to IAV infection as well as the strategies employed by the IAV to evade the host immune surveillance. Furthermore, this review also focuses on the treatments and vaccines that have been developed to counter IAV infection.
... M2 also participates in virion assembly and virus budding [20,21]. Moreover, the M2e sequence is highly conserved among all known human influenza A viruses, which is key to the development of universal vaccines [22][23][24]. mAbs against M2e (14C2) have prevented viral replication of some influenza A strains in vitro and reduced lung virus titers in mice [25,26]. Various M2e mAbs have been developed that demonstrate prophylactic and therapeutic activity against the influenza virus [27][28][29][30][31][32]. ...
... However, these specific mAbs showed no-or partial neutralization activity. The main mechanisms are dependent on antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis [22,24,28]. A synthetic camel-derived single-domain antibody (V H H) reportedly neutralized the influenza A virus by specifically binding to the M2 protein [48]. ...
Flu disease, with high mortality and morbidity, is caused by the influenza virus. Influenza infections are most effectively prevented through vaccination, but it requires annual reformulation due to the antigenic shift or drift of hemagglutinin and neuraminidase proteins. Increasing resistance to available anti-influenza drugs was also recently reported. The M2 surface protein of the influenza virus is an attractive target for universal vaccine development as it is highly conserved and multifunctional throughout the viral life cycle. This study aimed to discover a single-chain variable fragment (scFv) targeting the M2 protein of influenza A H1N1/PR8, showing neutralizing activity through plaque inhibition in virus replication. Several candidates were isolated using bio-panning, including scFv and single-domain V L target M2 protein, which was displayed on the yeast surface. The scFv/V L proteins were obtained with high yield and high purity through soluble expression in E . coli BL21 (DE3) pLysE strains. A single-domain V L -M2-specific antibody, NVLM10, exhibited the highest binding affinity to influenza virions and was engineered into a bivalent format (NVL2M10) to improve antigen binding. Both antibodies inhibited virus replication in a dose-dependent manner, determined using plaque reduction- and immunocytochemistry assays. Furthermore, bivalent anti-M2 single-domain V L antibodies significantly reduced the plaque number and viral HA protein intensity as well as viral genome ( HA and NP ) compared to the monovalent single-domain V L antibodies. This suggests that mono- or bivalent single-domain V L antibodies can exhibit neutralizing activity against influenza virus A, as determined through binding to virus particle activity.
... Targeting NA, the second major protein in influenza virus, can prevent virus entry into the host cell, decrease disease severity, and inhibit virus replication [39]. Similar to HA and NA, the M2 protein is an integral surface protein of influenza virus, and the extracellular domain of M2 is highly conserved in influenza A [41]. Matrix protein (M) and nucleoprotein (NP) are the major internal proteins of the virus. ...
... As HA proteins are very variable and poorly conserved, a vaccine based on conserved structures such as the extracellular domain of M2 (eM2) or the HA stem may be an interesting alternative to HA-and NA-based vaccines. Indeed, early experiments displaying eM2 on HBcAg by genetic fusion [41,48,49] or Qβ by chemical conjugation [50] induced antibodies that could protect against multiple clades of influenza A viruses. The Qβbased M2e vaccine was also tested for its ability to induce protective antibodies upon intranasal administration. ...
Virus-like particles (VLPs) have become key tools in biology, medicine and even engineering. After their initial use to resolve viral structures at the atomic level, VLPs were rapidly harnessed to develop antiviral vaccines followed by their use as display platforms to generate any kind of vaccine. Most recently, VLPs have been employed as nanomachines to deliver pharmaceutically active products to specific sites and into specific cells in the body. Here, we focus on the use of VLPs for the development of vaccines with broad fields of indications ranging from classical vaccines against viruses to therapeutic vaccines against chronic inflammation, pain, allergy and cancer. In this review, we take a walk through time, starting with the latest developments in experimental preclinical VLP-based vaccines and ending with marketed vaccines, which earn billions of dollars every year, paving the way for the next wave of prophylactic and therapeutic vaccines already visible on the horizon.
... Although the globular head of HA induces neutralizing antibodies (Krammer and Palese, 2013;Mullarkey et al., 2016), it contains a large number of mutations and thus cannot be used to develop a universal vaccine. However, the HA stalk and the highly conserved extracellular matrix protein (M2e) are promising for the development of a universal vaccine for influenza viral infection due to their durability and stability (Neirynck et al., 1999;Cummings et al., 2014;Uranowska et al., 2014;Tsybalova et al., 2018). These subunit proteins of influenza induce broader neutralization and participate in either antibodydependent cellular phagocytosis or antibody-dependent cellular cytotoxicity, which subsequently eliminate the influenza virus or destroy the cells already infected (Mezhenskaya et al., 2019;Von Holle and Moody, 2019). ...
... The HA stalk and M2e remain conserved among the strains of influenza virus and are targets for universal vaccine candidates (Neirynck et al., 1999;Krammer and Palese, 2013;Cummings et al., 2014;Uranowska et al., 2014;Mullarkey et al., 2016;Tsybalova et al., 2018;Krammer, 2019). To elicit a potent and broad immune response, we fused HAs and/or M2e with EboGP M (E M) that can efficiently target DCs and macrophages. ...
A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.
... Consequently, strain-matched vaccines would not be available for a long time after a novel strain emerges. In contrast, immune responses directed against conserved antigens can provide cross-protection against different influenza viruses regardless of their HA and NA strain or even across subtypes (9)(10)(11). Thus, these antigens offer new vaccine targets, and vaccines based on them might be used off-the-shelf at the time of an unexpected outbreak or at other times to supplement strain-matched vaccines. ...
... June 2022 Volume 96 Issue 1210.1128/jvi.00320-22 ...
Universal influenza virus vaccines targeting antigens conserved among influenza A virus strains can protect from severe disease but do not necessarily prevent infection. Despite allowing low-level infection, intranasal immunization with adenovirus vectors expressing the conserved antigens influenza nucleoprotein (A/NP) and M2 reduces influenza virus transmission from vaccinated to unvaccinated contact mice.
... Nanocarriers have been used to increase the immunogenicity of recombinant M2e protein to induce potent cross-protective immune responses in mice (Petukhova et al., 2013). An earlier study working on a universal influenza vaccine had used HBcAg protein as a carrier for the M2e peptide (M2e-HBcAg) (Neirynck et al., 1999). The authors demonstrated that vaccination of mice with M2e-HBcAg resulted in high levels of protection against the lethal dose of influenza A viruses; the induced anti-M2e antibodies were also shown to be effective in a passive immunisation study (Neirynck et al., 1999). ...
... An earlier study working on a universal influenza vaccine had used HBcAg protein as a carrier for the M2e peptide (M2e-HBcAg) (Neirynck et al., 1999). The authors demonstrated that vaccination of mice with M2e-HBcAg resulted in high levels of protection against the lethal dose of influenza A viruses; the induced anti-M2e antibodies were also shown to be effective in a passive immunisation study (Neirynck et al., 1999). Subsequently, various liposomal carriers, as well as rotavirus and tobacco mosaic virus surface proteins, have been tested as carriers for M2e antigens to induce anti-M2e antibodies (Adler-Moore et al., 2017;Andersson et al., 2012;Petukhova et al., 2013). ...
Influenza A virus poses a serious threat to human health, causing more than 650,000 deaths annually. While flu vaccines are effective in preventing infection, high rates of antigenic changes of the virus often give rise to vaccine escape mutants. Therefore, a universal vaccine that is effective against different influenza strains will produce broader protection from the infection. Although multiple strategies have been applied, there is still no licensed universal influenza vaccine available on the market. Thus, the design and development of an efficient universal vaccine is in high demand in the influenza vaccine industry. In this chapter, we discuss the recombinant strategies used in the development of influenza vaccine based on major viral structural proteins, including hemagglutinin, neuraminidase, matrix protein 2 extracellular domain, nucleoprotein, and multimeric epitope peptide vaccines. Additionally, we examine the immune responses induced by recombinant antigens and several highly potent vaccine platforms used to enhance immunogenicity. Later in this chapter, the production of virosome-based influenza vaccine will also be explored. Here, we demonstrate the development of a virosome-like influenza vaccine nanoparticle, using recombinant HA2-HBcAg hybrid antigen and synthetic lipids via thin film hydration method. Finally, the scientific term ‘Recombinant Virosome’ is proposed for this additional vaccine category.
... Furthermore, because of its small size, M2e is poorly immunogenic per se. To overcome this issue various, carriers and delivery systems have been used; however, typically, any M2e vaccination regimen so far reported has involved at least two injections (20,(25)(26)(27)(28)(29)(30)(31)(32). Furthermore, a rapid decline in M2e-specific antibody titers has been observed with these vaccines, as illustrated in a recent clinical trial with the ACAM-FLU-A vaccine candidate (an M2e-hepatitis B virus core fusion protein) (33). ...
... Finally, earlier studies have demonstrated the cross-protective potential of M2e-based vaccine candidates against various influenza A virus subtypes (25,30,31). Consistently, sera from mice immunized with the Clec9A-M2e construct displayed significant binding to H3N2-infected cells (SI Appendix, Fig. S3A). ...
Significance
Although the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24–amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.
... For example, the external region of the M2 ion channel protein (M2e, the first twentyfour amino acids of the protein) is one such region, with a high degree of similarity across several influenza strains including H1N1, H3N2, and H2N2. [6][7][8] Excitingly, this region, more specifically M22-16, has been shown to contain a B cell epitope. [9][10][11] Subunit vaccines, which leverage individual antigenic components (e.g., M22-16) instead of using the entire pathogen, are an attractive option to decrease vaccine production time. ...
A significant problem with current influenza vaccines is their reliance on predictions of what will be the most prevalent strains for the upcoming season. Mismatches between predictions and reality in any given year can greatly reduce the overall efficacy of an immunization campaign. A universal influenza vaccine, which leverages epitopes conserved across many, if not all, strains of influenza, can reduce the need for such accurate forecasting. The ectodomain of the M2 ion channel protein is highly conserved and includes a B cell epitope in the M22-16 region, making it a potentially viable candidate as a universal influenza vaccine. Unfortunately, the use of free peptide antigens as vaccines comes with several disadvantages including poor stability and weak immunogenicity in vivo. However, integrating peptide antigens into nanoparticles can avoid some of those drawbacks. Previous studies have shown that micellar nanoparticles can be generated from peptides by conjugating them with a lipid or lipids. Specifically, hydrophobically-driven, selfassembled peptide amphiphile micelles comprised of Palm2K-peptide-(KE)4 have been found to be immunostimulatory. Unlike other peptides previously used for this purpose, the M22-16 peptide interestingly formed micelles without any peptidyl or lipid modifications. Because this unmodified peptide self-assembled on its own, it enabled the decoupling of the effect of micellization on immunogenicity from the incorporation of non-vaccine components such as the addition of a lipid moiety (Palm2K) and a zwitterion-like peptide block ((KE)4). The enclosed work shows that M22-16 peptidyl micelles had some characteristic differences in shape, critical micelle concentration, and secondary structure when compared to M22-16 peptide amphiphile micelles, which produced a few differences in murine antibody responses. These results suggest that peptide amphiphile micelles could be leveraged as a one-dose vaccine, while either micelle formulation induced strong immunological responses with a prime-booster immunization regimen.
... In previous studies, fusing M2e to various adjuvants or carriers enhanced its antigenicity and provided protection against lethal challenges (24). Many investigations have also demonstrated that M2e-based vaccines can generate immunity against different influenza subtypes (11,17,19,(25)(26)(27). Additionally, M2e vaccines have been suggested as a supplement to increase the crossprotection of conventional vaccines (24,28). ...
... Accumulating reports indicate that HA2 might be a major target for broad protection against influenza A viruses because HA2 can induce broadly active anti-hemagglutinin antibodies against influenza A viruses [16,55,56]. Moreover, M2e has been widely used as a candidate of recombinant vaccines for cross-protection against influenza A viruses [29]. M2e is limited in inducing a strong immune response because it is too small to have high immunogenicity. ...
Vaccination is the most effective method for preventing the spread of the influenza virus. Cell-based influenza vaccines have been developed to overcome the disadvantages of egg-based vaccines and their production efficiency has been previously discussed. In this study, we investigated whether treatment with forskolin (FSK), an adenylyl cyclase activator, affected the output of a cell-based influenza vaccine. We found that FSK increased the propagation of three influenza virus subtypes (A/H1N1/California/4/09, A/H3N2/Mississippi/1/85, and B/Shandong/7/97) in Madin-Darby canine kidney (MDCK) cells. Interestingly, FSK suppressed the growth of MDCK cells. This effect could be a result of protein kinase A (PKA)-Src axis activation, which downregulates extracellular signal-regulated kinase (ERK)1/2 activity and delays cell cycle progression from G1 to S. This delay in cell growth might benefit the binding and entry of the influenza virus in the early stages of viral replication. In contrast, FSK dramatically upregulated ERK1/2 activity via the cAMP-PKA-Raf-1 axis at a late stage of viral replication. Thus, increased ERK1/2 activity might contribute to increased viral ribonucleoprotein export and influenza virus propagation. The increase in viral titer induced by FSK could be explained by the action of cAMP in assisting the entry and binding of the influenza virus. Therefore, FSK addition to cell culture systems could help increase the production efficiency of cell-based vaccines against the influenza virus.
... Some monoclonal antibodies (e.g., 14C2) raised against the M2 protein could not block the virus's attachment with the host cells while limiting the virus load [17]. At the same time, some showed protection in the mouse model against a lethal dose of the influenza virus [18,19]. Interestingly, some monoclonal antibodies bind exclusively to the dimeric M2e, while others can bind to both monomeric and dimeric forms of M2e [20]. ...
Background
The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation.
Methods
The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging.
Results
The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells.
Conclusion
This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.
... Given the disparate biochemical features and space flexibility, the NA-derived peptide design strategy for inducing protective antibodies is essentially distinct from those classical protective peptides, like influenza matrix protein 2 ectodomain (M2e), 20 small hydrophobic protein ectodomain of respiratory syncytial virus (RSV), 21 membrane-proximal external region of human immunodeficiency virus-1 gp41, 22 Article ll OPEN ACCESS structure constraints, vaccinations with the conserved M2e presented by many immunogenic carriers readily induced M2e-specific cross-protective antibody responses. 24,25 Influenza A viral NA glycoprotein is highly mutated. ...
Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.
... The M2e fragment is part of the M2 protein that is exposed on virion surface and is highly conserved between influenza A subtypes, and is hence an attractive model for developing a universal vaccine [57]. Several studies showed that the M2e protein provide protection as a vaccine in animal models, either alone [87,96,97] or in combination with another influenza conserved protein, NP [98]. The NA protein was also explored as a vaccine candidate due to the fact that drift in HA and NA are uncorrelated, and elicited cross protection in immunized animals [99][100][101][102][103]. ...
The ongoing pandemic has clearly demonstrated that respiratory viruses can cause immense loss of lives and livelihoods. India is home to some of the world’s largest vaccine manufacturers and has successfully vaccinated a large fraction of its population to protect against COVID-19 disease caused by the sarbecovirus, SARS-CoV-2. However, much of this was reliant on formulations developed elsewhere, and before vaccines could be widely deployed, a large fraction of the population was infected. There is a clear need to develop and nurture indigenous expertise to design, develop and rapidly produce efficacious vaccines against respiratory viruses, including SARS-CoV-2 and influenza virus.
... In agreement with this, in this study, it was observed that intranasal immunization induced a lower M2 IgG titer than intraperitoneal immunization. Similar results were observed in a study that compared intranasal and intraperitoneal immunization routes using M2 and HBV core fusion protein, however, higher protective activity against lethal infection was observed in the intranasal immunization [12]. Some mice of CCFkH5 with sesame oil intranasal immunized group showed large weight loss in especially HKH5 virus challenge. ...
Vaccine efficacy of conventional influenza vaccines depend on the antigenic similarity between the selected vaccine strain and annual epidemic strain. Since the influenza virus evolves yearly, a vaccine which is independent from viral antigenic mutation is desired. We have developed chimeric cytokine (CC) and hemagglutinin (HA) incorporated virus-like particle (CCHA-VLP) as a universal influenza vaccine candidate. Using mouse models, it was shown that the vaccine provided broad-based protective activity against several types of human and avian influenza A viruses. In this report, nasal immunization and mixture form (CC- and HA-VLP) were tested to improve usability of this vaccine. Immunogenicity was evaluated by induction of IgG, IgA, and IFN-γ secreting cells. Protective activity was measured as mouse survival rate against lethal challenge with H1N1 and H5N1 viruses and against H3N2 virus by lung viral titer. Nasal immunization showed low immunogenicity and low protective efficacy, but the addition of a sesame oil adjuvant improved vaccine efficacy. Mixture form of CC- and HA-VLP showed comparable or higher vaccine efficacy when compared to the incorporated form, CCHA-VLP. These results contribute to improved usability, such as needle-less administration and easy HA subtypes alteration.
... Given the disparate biochemical features and space flexibility, the NA-derived peptide design strategy for inducing protective antibodies is essentially distinct from those classical protective peptides, like influenza matrix protein 2 ectodomain (M2e), 20 small hydrophobic protein ectodomain of respiratory syncytial virus (RSV), 21 membrane-proximal external region of human immunodeficiency virus-1 gp41, 22 Article ll OPEN ACCESS structure constraints, vaccinations with the conserved M2e presented by many immunogenic carriers readily induced M2e-specific cross-protective antibody responses. 24,25 Influenza A viral NA glycoprotein is highly mutated. ...
... The influenza virus membrane contains two critical proteins: haemagglutinin (HA) and neuraminidase (NA). They are crucial for the entry and release of the virus from infected cells [55]. Apart from these two proteins, other structural components, such as the RNA-binding matrix protein M1, the nucleoprotein (NP) that coats the viral RNA, and the ion channel M2 protein, can be recognized by our immune systems. ...
Infectious diseases are reported worldwide, and the emergence of highly mutated antibiotic-resistant strains is a major concern globally. Developing efficient vaccines is the only way to prevent and treat diseases effectively. Though developing conventional vaccines is an intricate and time-consuming process due to several rate-limiting steps, these vaccines help treat an array of existing diseases. There is a dire need for new forms of vaccines as many incidents of resistance are reported, and the efficacy of newly developed vaccines must be enhanced to treat the infections well in time. The human immune system fights against several infections utilizing antibody and the non–antibody-based immune mechanism, providing significant protection against identified pathogens. Nowadays, much effort is being made to develop vaccines focussing on the role of cellular responses to clear several complicated infections. This chapter concentrates on strategies for designing therapeutic protein-based vaccines and their diverse clinical and nonclinical applications.
... Conserved epitopes are epitopes that are relatively the same among different strains of a pathogen. For example, due to the multiple strains of influenza over the years, the means of developing a universal vaccine has been the use of the conserved epitopes on the hemagglutinin (HA stalk) or the matrix ectodomain (M2e) [98][99][100][101][102][103][104]. The strategy of using conserved epitopes has also been used in the development of preventative vaccines for HIV, dengue virus, Lassa fever virus (LASV), hepatitis virus and Kaposi's sarcoma-associated herpesvirus (KSHV) [105][106][107][108][109][110][111][112][113]. ...
Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2, which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants’ proteins, especially in the spike protein (SP) used for viral entry. These mutations, especially in the SP, enable the virus to evade immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of a vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase its immunogenicity.
... Many studies have shown that this highly conserved region represents a vaccine strategy that may aid in pandemic preparedness. Because the M2e sequence is highly conserved across the influenza virus, it is also a promising antigenic target for developing a universal vaccine [12,13]. ...
Background
As a result of antigenic drift, current influenza vaccines provide limited protection against circulating influenza viruses, and vaccines with broad cross protection are urgently needed. Hemagglutinin stalk domain and ectodomain of matrix protein 2 are highly conserved among influenza viruses and have great potential for use as a universal vaccine.
Methods
In this study, we co-expressed the stalk domain and M2e on the surface of cell membranes and generated chimeric and standard virus-like particles of influenza to improve antigen immunogenicity. We subsequently immunized BALB/c mice through intranasal and intramuscular routes.
Results
Data obtained demonstrated that vaccination with VLPs elicited high levels of serum-specific IgG (approximately 30-fold higher than that obtained with soluble protein), induced increased ADCC activity to the influenza virus, and enhanced T cell as well as mucosal immune responses. Furthermore, mice immunized by VLP had elevated level of mucosal HA and 4M2e specific IgA titers and cytokine production as compared to mice immunized with soluble protein. Additionally, the VLP-immunized group exhibited long-lasting humoral antibody responses and effectively reduced lung viral titers after the challenge. Compared to the 4M2e-VLP and mHA-VLP groups, the chimeric VLP group experienced cross-protection against the lethal challenge with homologous and heterologous viruses. The stalk domain specific antibody conferred better protection than the 4M2e specific antibody.
Conclusion
Our findings demonstrated that the chimeric VLPs anchored with the stalk domain and M2e showed efficacy in reducing viral loads after the influenza virus challenge in the mice model. This antibody can be used in humans to broadly protect against a variety of influenza virus subtypes. The chimeric VLPs represent a novel approach to increase antigen immunogenicity and are promising candidates for a universal influenza vaccine.
... However, the protective efficacy of these VLP vaccines was not investigated in earlier studies. The first chimeric VLP vaccine study to evaluate the protective efficacy of these immunogenic nanoparticles in animal models was reported by Neirynck et al. [14]. Here, the authors fused the 23 amino acid M2 extracellular peptide (M2e) of the influenza A virus to the hepatitis B virus core protein (HBc) to generate the VLPs. ...
With technological advancements enabling globalization, the intercontinental transmission of pathogens has become much easier. Respiratory viruses are one such group of pathogens that require constant monitoring since their outbreak leads to massive public health crises, as exemplified by the influenza virus, respiratory syncytial virus (RSV), and the recent coronavirus disease 2019 (COVID-19) outbreak caused by the SARS-CoV-2. To prevent the transmission of these highly contagious viruses, developing prophylactic tools, such as vaccines, is of considerable interest to the scientific community. Virus-like particles (VLPs) are highly sought after as vaccine platforms for their safety and immunogenicity profiles. Although several VLP-based vaccines against hepatitis B and human papillomavirus have been approved for clinical use by the United States Food and Drug Administration, VLP vaccines against the three aforementioned respiratory viruses are lacking. Here, we summarize the most recent progress in pre-clinical and clinical VLP vaccine development. We also outline various strategies that contributed to improving the efficacy of vaccines against each virus and briefly discuss the stability aspect of VLPs that makes it a highly desired vaccine platform.
... M2 is a transmembrane protein that forms a proton channel on the viral envelope (24). Because of its relatively high conservation among different flu strains, M2 is considered a potential target for the development of a universal flu vaccine (25,26). Our previous studies have shown that protein cargos can be recruited into ARMMs via either direct fusion to the ARRDC1 protein or to the WW domains that interact with the PPXY motifs of ARRDC1 (27). ...
Membrane proteins expressed on the surface of enveloped viruses are conformational antigens readily recognized by B cells of the immune system. An effective vaccine would require the synthesis and delivery of these native conformational antigens in lipid membranes that preserve specific epitope structures. We have created an extracellular vesicle-based technology that allows viral membrane antigens to be selectively recruited onto the surface of WW domain-activated extracellular vesicles (WAEVs). Budding of WAEVs requires secretory carrier-associated membrane protein 3, which through its proline-proline-alanine-tyrosine motif interacts with WW domains to recruit fused viral membrane antigens onto WAEVs. Immunization with influenza and HIV viral membrane proteins displayed on WAEVs elicits production of virus-specific neutralizing antibodies and, in the case of influenza antigens, protects mice from the lethal viral infection. WAEVs thus represent a versatile platform for presenting and delivering membrane antigens as vaccines against influenza, HIV, and potentially many other viral pathogens.
... Conserved epitopes are epitopes that are relatively the same among different strains of a pathogen. For example, due to the multiple strains of influenza over the years, the means of developing a universal vaccine has been with the use of the conserved epitopes on the hemagglutinin (HA stalk) or the matrix ectodomain (M2e) [88][89][90][91][92][93][94]. The strategy of using conserved epitopes has also been in the development for preventative vaccines for HIV, dengue virus, Lassa fever virus (LASV), hepatitis virus, and Kaposi's sarcoma-associated herpesvirus (KSHV) [95][96][97][98][99][100][101][102][103]. ...
Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2 which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants’ proteins, especially in the spike protein (S) used for viral entry. These mutations, especially in the S, enable the virus to evade the immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase their immunogenicity.
... The extracellular domain of the transmembrane protein M2 (M2e), 23 a.a. in size, is a promising conserved antigen of the influenza A virus [16]. Its sequence is highly conserved among human strains and has only a few amino-acid replacements in influenza A strains of animal origin [17][18][19]. The disadvantage of this peptide is its low immunogenicity [20]; however, it becomes highly immunogenic and provides protection against infection when fused to an adjuvant protein or carrier VLPs [21]. ...
Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes' presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300-400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80-100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.
... To induce cross-protection and immune response against influenza VLP vaccines, we constructed a CC molecule, in order of N-to C-terminus, as follows: full-length M2 protein of A/Aichi/2/1968(H3N2) (ectodomain: 23 amino acids; transmembrane domain: 19 amino acids; and cytoplasmic domain: 54 amino acids), linker region (Gly-FLAG tag-8 amino acids-Gly-Gly), NA protein of A/Aichi/2/1968(H3N2) (cytoplasmic domain: 6 amino acids; transmembrane domain: 31 amino acids; and stalk region: 39 amino acids and 2 glycine linkers), and IL-12 (p40 subunit: 313 amino acids; linker: 19 amino acids; and p35 subunit: 193 amino acids), as depicted in Fig 11A. The stalk region of HA and M2e have previously been reported as candidates for universal vaccines; mice immunized with a peptide corresponding to the HA stalk (Wang et al, 2010) and a fusion protein of the M2 ectodomain with hepatitis B core protein (Neirynck et al, 1999) were challenged with divergent subtypes of the influenza A virus. Based on these reports, we constructed a CC fusion gene but included the stalk region of NA rather than HA as we assumed that both NA and M2 ectodomains, albeit expressed as a fusion product, could be exposed to the extracellular space because they are type II and III transmembrane proteins, respectively. ...
The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.
... Many studies have shown that this highly conserved region represents a vaccine strategy that may aid in pandemic preparedness. Because the M2e sequence is highly conserved across the in uenza virus, it is also a promising antigenic target for developing a universal vaccine [12,13]. ...
Background
Due to antigenic drift, current influenza vaccines provide limited protection against circulating influenza viruses, and vaccines with broad cross protection are urgently needed. Hemagglutinin (HA) stalk domain and ectodomain of matrix protein 2 are highly conserved among influenza viruses and have great potential for use in a universal vaccine.
Methods
In this study, we co-expressed the stalk domain and M2e on the surface of cell membranes and generated chimeric and standard virus-like particles of influenza to improve antigen immunogenicity. Then, we immunized BALB/c mice through intranasal and intramuscular routes.
Results
Results showed that vaccination with VLPs elicited high levels of serum-specific IgG (approximately 30-fold higher than that obtained with soluble protein), and T cell and mucosal immune responses were enhanced. Furthermore, HA and 4M2e-specific IgA titers in mucosal and cytokine production increased in the VLP-immunized mice more than in the mice immunized with soluble protein. The VLP-immunized group exhibited long-lasting humoral antibody responses and effectively reduced lung viral titers after the challenge. In particular, the chimeric VLP group experienced cross-protection against the lethal challenge with homologous and heterologous viruses compared to the 4M2e-VLP and mHA-VLP groups. The antibody with the stalk domain conferred better protection than the 4M2e specific antibody.
Conclusion
Our findings demonstrated that the cVLPs anchored with the stalk domain and M2e showed efficacy in reducing viral loads after the influenza virus challenge in the mice model and could be used in humans to broadly protect against a variety of influenza virus subtypes. The chimeric VLPs represent a novel approach to increase antigen immunogenicity and are promising candidates for a universal influenza vaccine.
... A wider range of protection could be achieved if both the M2e and HA2 influenza domains were administered together to formulate a single vaccine candidate [73][74][75][76]. Moreover, if the above strategy can be combined with a carrier VLP or an adjuvant, it could enhance their immunogenicity even further [75][76][77]. Tomato plant hairy roots were used to express the HPV16 E7 protein fused with SAPKQ, a nontoxic form of the Saponaria officinalis saporin protein [78]. ...
... In this Research Topic, Creytens et al., discussed the potential of NA as a vaccine antigen, and Hansen et al., reported that repeated vaccination with seasonal influenza vaccines can boost and maintain NA-specific humoral immunity health care workers in a five-year longitudinal study. The highly conserved ectodomain of the influenza matrix 2 protein (M2e) has been suggested as a good universal vaccine epitope, and antibodies targeting M2e correlated with protection in preclinical and clinical settings (10,11). However, following natural infection, M2e-specific antibodies are not highly induced, and M2e is poorly immunogenic if not presented to the immune system with the help of a scaffold (12). ...
... Another promising antigenic target is the highly conserved extracellular domain of matrix 2 (M2e) protein in influenza A viruses 12,13 . M2e-based vaccines could provide broad cross protection against different strains and subtypes in mice [12][13][14][15] . Recombinant M2e vaccines were safe in phase 1 trials 13,16,17 . ...
We developed a new chimeric M2e and H3 hemagglutinin (HA) stalk protein vaccine (M2e-H3 stalk) by genetic engineering of modified H3 stalk domain conjugated with conserved M2e epitopes to overcome the drawbacks of low efficacy by monomeric domain-based universal vaccines. M2e-H3 stalk protein expressed and purified from Escherichia coli was thermostable, displaying native-like antigenic epitopes recognized by antisera of different HA subtype proteins and influenza A virus infections. Adjuvanted M2e-H3 stalk vaccination induced M2e and stalk-specific IgG antibodies recognizing viral antigens on virus particles and on the infected cell surface, CD4+ and CD8+ T-cell responses, and antibody-dependent cytotoxic cell surrogate activity in mice. M2e-H3 stalk was found to confer protection against heterologous and heterosubtypic cross-group subtype viruses (H1N1, H5N1, H9N2, H3N2, H7N9) at similar levels in adult and aged mice. These results provide evidence that M2e-H3 stalk chimeric proteins can be developed as a universal influenza A virus vaccine candidate for young and aged populations.
... This ectodomain is highly conserved and therefore, is favourable as a universal antigen for the prevention of human influenza (Fiers et al., 2009). As such, this antigen has been studied extensively in various insertion sites of HBC subsequent to the work of (Neirynck et al., 1999). As mentioned in Section 1.5.1.1, ...
Vaccination is currently the predominant tool in the prevention of infectious disease. Each year, an estimated 2-3 million lives are saved worldwide and infant mortality has been significantly reduced. Despite substantial recent advances, vaccine manufacturing can still be laborious owing to difficulties in development, lengthy clinical trials, and stringent regulations. In light of the SARS-CoV-2 (Covid-19) pandemic, the need for a development platform which can rapidly screen potential candidates and/or a vaccine scaffold capable of adaptability to new disease targets has never been more apparent. To meet this need, the breadth of vaccine types under exploration has rapidly expanded. DNA and RNA vaccines offer the opportunity for rapid manufacture but can be poorly immunogenic, whilst subunit vaccines can require complex processing. Virus-like particles (VLPs) have the potential to address these two factors. Tandem Core VLPs, expressed in the methylotrophic yeast Pichia pastoris, are an exciting alternative to current manufacturing methods. They have excellent potential, both as standalone vaccines for the virus from which they are derived, or as scaffolds for the display of foreign antigens. The hepatitis B core antigen (HBC) can spontaneously self-assemble, forming icosahedral particles that are inherently immunogenic. Tandem Core HBC VLPs have been genetically modified in the major insertion region (MIR) enabling surface display of up two epitopes of interest when assembled. For HBC VLPs to be considered a viable vaccine candidate, their bioprocessing must be optimized. Currently, there are various issues to address including problems with formation, solubility and immunogenicity, which are often clone dependent. In this work, Tandem Core VLPs, consisting of genetically linked HBC monomers carrying different epitopes in the MIR will be used to develop a high-throughput platform and explore the impact of different inserts on VLP production and processing. Influenza will be used as a model pathogen owing to its persistence as a public health threat. The aim of this work is to develop a vaccine platform, defined by Adalja et al., (2019) as “a technology in which the underlying, nearly identical mechanism, device, delivery vector or cell line was employed for [design of] multiple target vaccines”. The equipment and methods developed in this work were considered to enable: (1) thorough investigation of three HBC VLP candidates in an attempt to identify a universal bioprocess, irrespective of surface displayed epitopes; (2) formation of a small-scale high throughput platform which could be implemented for rapid screening of new disease targets or to allow fine-tuning of processes for epitope-dependent optimisation. After initial studies using an ‘empty’ HBC VLP (HBC-K1,K1), the ambr®250 modular was used to investigate upstream bioprocessing of three influenza specific candidates (HBC -HA2,3M2E, -LAH3,K1 and -3M2E,K1), exploring different fermentation induction strategies and to identify epitope related differences. Following this, the most readily soluble candidate (HBC-LAH3,K1) was selected for further upstream optimisation combining ambr® 250 experimentation with statistical Design of Experiments (DoE). An improved process was identified enabling an increase in VLP titre, a 34% increase in biomass compared to the initial condition, and a 6% decrease in process time compared to methanol induction. This process was then applied to the production of the alternative VLP constructs. The improved feeding regime resulted in higher biomass and soluble HBC yield for all three VLPs. Subsequent downstream process studies on the primary recovery of VLP candidates was then necessary to account for the reduced volumes associated with miniaturised fermentation studies, and to bridge the gap between upstream processing and purification. Building on previous work, a high-throughput, small scale cell disruption method was investigated using Adaptive focused acoustics®. A 96-well plate workflow was demonstrated, enabling suitable VLP release and recovery with a ~99.7% reduction in sample volume, in comparison to high pressure homogenisation (HPH). Finally, chromatography screening was undertaken using high-throughput PreDictor® plates to rapidly identify separation conditions for the various vaccine candidates. Studies were conducted to investigate suitable resins and binding/elution conditions and to determine the influence of the physicochemical properties of the displayed epitopes on separation performance. Multiple resins were identified as being suitable for VLP purification, and results were useful to manipulate chromatographic separation (5mL column scale) conditions for the VLPs to achieve improved product yield and purity profiles. Overall, this research suggests that a high-throughput vaccine development platform can be realised through the integration of numerous small-scale single-use equipment, techniques and methodologies. Namely, the use of the ambr®250 bioreactors, AFA® cell disruption in 96-well plates and 96-well PreDictor™ resin plates. Combined with statistical DoE, this platform can be used to rapidly optimise production and purification conditions for novel vaccine technologies such as HBC Tandem Core VLPs. The improved bioprocessing of these constructs paves the way for future vaccine candidates which exploit HBC as a vaccine scaffold. These findings have implications for reducing the time taken to develop vaccine manufacturing processes and prepare for disease outbreaks based on ‘Pathogen X’.
... 11 Previous animal studies have proven that M2e immunity could provide broad protection against both group 1 and 2 viruses, regardless of HA subtypes. [12][13][14][15] Clinical trials reported safety of M2e-based recombinant vaccines. 13,16,17 However, the efficacy of M2e immunity alone was lower than that of inactivated virus vaccination inducing neutralizing antibodies, when vaccinated mice were challenged with HA-matching homologous virus, probably due to non-neutralizing immune-mediated protection and limited B cell and T cell epitopes. ...
Hemagglutinin (HA) stem-based vaccines have limitations in providing broad and effective protection against cross-group influenza viruses despite being a promising universal vaccine target. To overcome the limited cross protection and low efficacy by HA stem vaccination, we genetically engineered a chimeric conjugate of thermostable H1 HA stem and highly conserved M2e repeat (M2e-H1stem), which was expressed at high yields in Escherichia coli. M2e-H1stem protein presented native-like epitopes reactive to antisera of live virus infection. M2e-H1stem protein vaccination of mice induced strong M2e- and HA stem-specific immune responses, conferring broadly effective cross-protection against both antigenically distinct group 1 (H1N1, H5N1, H9N2 subtypes) and group 2 (H3N2, H7N9 subtypes) seasonal and pandemic potential influenza viruses. M2e-H1stem vaccination generated CD4⁺ and CD8⁺ T cell responses and antibody-dependent cytotoxic cellular and humoral immunity, which contributed to enhancing cross-protection. Furthermore, comparable broad cross-group protection was observed in older aged mice after M2e-H1stem vaccination. This study provides evidence warranting further development of chimeric M2e-stem proteins as a promising universal influenza vaccine candidate in adult and aged populations.
... We have focused on the development of a vaccine that incorporates the extracellular region of the matrix 2 protein (M2e), which is a 97 amino acid ion channel located on the membrane of the influenza virus that plays an important role during viral entry (Lee et al., 2014). More importantly the M2e region is highly conserved among all the strains of influenza (Bessa et al., 2008) (Mozdzanowska et al., 2003) (Neirynck et al., 1999) (Turley et al., 2011). This characteristic of the M2e protein serves as a basis for the development of a potential universal vaccine. ...
M2e VLP was previously described as a vaccine that incorporates the extracellular region of the matrix 2 protein (M2e), which is highly conserved amongst all the strains of influenza. In this study, we analyzed activation status of dendritic cells (DCs) after exposure to M2e VLP, stimulating DCs with M2e VLP and co-culturing the stimulated DCs with T cells to observe innate and adaptive immune responses. The M2e VLP microparticle was prepared by encapsulating into a polymer matrix using the one-step spray drying method. Adjuvants Alhydrogel®, MPL-A® or AddavaxTM were used to enhance the DC stimulatory effects by the M2e VLP microparticle. The M2e VLP microparticle yield was found to be 92% and the encapsulation yield was around 84% with a size of approximately 2.78μm. There was no short-term cytotoxicity found in DCs and macrophages with concentrations up to 1500μg/mL of M2e VLP microparticle, however long-term exposure resulted in 25% decrease in viability of cells with concentrations more than or equal to 500μg/mL. The M2e VLP microparticle vaccine with Alhydrogel® and MPL-A® induced high levels of TNFα in both DCs and macrophages. The high levels of MHC I, II, CD28, B7-1, ICAM-1, LFA-1 expression and IL-12 release in the M2e VLP microparticle group with Alhydrogel® suggests that the M2e VLP vaccine with this adjuvant activated T cells via the Th2 pathway. The increased expression of MHC I, II, CD40, CD154, ICAM-1 and LFA-1 on DCs and the release of IL-12 in the M2e VLP microparticle culture of DCs with MPL-A® demonstrated that the M2e VLP vaccine with this adjuvant activated T cells via the Th1 pathway. The decrease in fluorescence in the Alhydrogel® and MPL-A® group illustrates the proliferation of T cells took place following exposure of DCs to the M2e VLP microparticle with these adjuvants. The M2e VLP microparticle exhibited higher stimulatory responses of DCs than the M2e VLP in suspension. Furthermore, the presence of Alhydrogel® and MPL-A® enhanced the stimulatory effects of DCs by the M2e VLP microparticle (MP) vaccine.
Influenza virus is a prominent cause of respiratory illness in humans. Current influenza vaccines offer strain-specific immunity, while provide limited protection against drifted strains. Broad-spectrum influenza vaccines can induce broad and long-term immunity, and thus is regarded as a future direction for the development of next-generation influenza vaccines. In this study, we have conceptualized a novel mRNA-based multi-antigen influenza vaccine consisting of three conserved antigens of influenza A virus, including the ectodomain of the M2 ion channel (M2e), the long alpha helix of haemagglutinin stalk region (LAH), and nucleoprotein (NP). The vaccine design aims to enhance its potency and promote the development of a future broad-spectrum influenza vaccine. Our mRNA-based vaccine demonstrated potent humoral and cellular responses throughout the time points of the murine model, inducing viral neutralizing antibodies, antibody-dependent cell cytotoxicity effect mediating antibodies and cross-reactive CD8+ T cell immune responses. The vaccine conferred broad protection against H1N1, H3N2, and H9N2 viruses. Moreover, the single-cell transcriptional profiling of T cells in the spleens of vaccinated mice revealed that the mRNA-based vaccine significantly promoted CD8+ T cells and memory T cells by prime-boost immunization. Our results suggest that the mRNA-based influenza vaccine encoding conserved proteins is a promising approach for eliciting broadly protective humoral and cellular immunity against various influenza viruses.
Viral protein nanoparticles (ViP NPs) such as virus-like particles and virosomes are structures halfway between viruses and synthetic nanoparticles. The biological nature of ViP NPs endows them with the biocompatibility, biodegradability, and functional properties that many synthetic nanoparticles lack. At the same time, the absence of a viral genome avoids the safety concerns of viruses. Such characteristics of ViP NPs offer a myriad of opportunities for the application of ViP NPs at several points across disease development: from prophylaxis to diagnosis and treatment. ViP NPs present remarkable immunostimulant properties, and thus the vaccination field has benefited the most from these platforms capable of overcoming the limitations of both traditional and subunit vaccines. This was reflected in the marketing authorization of several VLP- and virosome-based vaccines. Besides, ViP NPs inherit the ability of viruses to deliver their cargo to target cells. These delivery properties make them promising candidates as vectors for drug and gene delivery, and for diagnostic applications. In this review, we analyze the pharmaceutical applications of ViP NPs, describing the products that are commercially available or under clinical evaluation, but also the advances that scientists are making toward the implementation of ViP NPs in other areas of major pharmaceutical interest.
The influenza A virus causes substantial morbidity and mortality worldwide every year and poses a constant threat of an emergent pandemic. Seasonal influenza vaccination strategies fail to provide complete protection against infection due to antigenic drift and shift. A universal vaccine targeting a conserved influenza epitope could substantially improve current vaccination strategies. The ectodomain of the matrix 2 protein (M2e) of influenza is a highly conserved epitope between virus strains but is also poorly immunogenic. Administration of M2e and the immunostimulatory stimulator of interferon genes (STING) agonist 3'3'-cyclic guanosine-adenosine monophosphate (cGAMP) encapsulated in microparticles made of acetalated dextran (Ace-DEX) has previously been shown to be effective for increasing the immunogenicity of M2e, primarily through T-cell-mediated responses. Here, the immunogenicity of Ace-DEX MPs delivering M2e was further improved by conjugating the M2e peptide to the particle surface in an effort to affect B-cell responses more directly. Conjugated or encapsulated M2e co-administered with Ace-DEX MPs containing cGAMP were used to vaccinate mice, and it was shown that two or three vaccinations could fully protect against a lethal influenza challenge, while only the surface-conjugated antigen constructs could provide some protection against lethal challenge with only one vaccination. Additionally, the use of a reducible linker augmented the T-cell response to the antigen. These results show the utility of conjugating M2e to the surface of a particle carrier to increase its immunogenicity for use as the antigen in a universal influenza vaccine.
Presenting exogenous antigens on virus-like particles (VLPs) through "plug-and-display" decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on "plug-and-display" decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.
As a prospective influenza vaccination platform, a microneedle patch offers advantages such as self-administration and reduction of needle-phobia-associated vaccination avoidance. In an effort to design a broadly protective influenza vaccine we have previously developed a vaccine formulation containing the highly conserved ectodomain sequence of the M2 influenza protein (M2e) attached to the surface of gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). Our previous studies have used the intranasal route for vaccination and demonstrated broad protection from this vaccine. Here we asked the question whether the same formulation can be effective when administered to mice using microneedles. We demonstrate that the microneedles can be coated with AuNP-M2e + sCpG formulation, and the AuNPs from the coating can be readily resuspended without aggregation. The AuNPs were delivered with high efficiency into murine skin, and the AuNPs cleared the skin within 12 h of microneedle treatment. After vaccination, strong M2e-specific humoral and cellular responses were stimulated, and the vaccinated mice were 100% protected following a lethal challenge with influenza A/PR/8/34 (H1N1).
Introduction
Virus-like particles (VLPs) are similar in size and shape to their respective viruses, but free of viral genetic material. This makes VLP-based vaccines incapable of causing infection, but still effective in mounting immune responses. Noro-VLPs consist of 180 copies of the VP1 capsid protein. The particle tolerates C-terminal fusion partners, and VP1 fused with a C-terminal SpyTag self-assembles into a VLP with SpyTag protruding from its surface, enabling conjugation of antigens via SpyCatcher.
Methods
To compare SpyCatcher-mediated coupling and direct peptide fusion in experimental vaccination, we genetically fused the ectodomain of influenza matrix-2 protein (M2e) directly on the C-terminus of norovirus VP1 capsid protein. VLPs decorated with SpyCatcher-M2e and VLPs with direct M2 efusion were used to immunize mice.
Results and discussion
We found that direct genetic fusion of M2e on noro-VLP raised few M2e antibodies in the mouse model, presumably because the short linker positions the peptide between the protruding domains of noro-VLP, limiting its accessibility. On the other hand, adding aluminum hydroxide adjuvant to the previously described SpyCatcher-M2e-decorated noro-VLP vaccine gave a strong response against M2e. Surprisingly, simple SpyCatcher-fused M2e without VLP display also functioned as a potent immunogen, which suggests that the commonly used protein linker SpyCatcher-SpyTag may serve a second role as an activator of the immune system in vaccine preparations. Based on the measured anti-M2e antibodies and cellular responses, both SpyCatcher-M2e as well as M2e presented on the noro-VLP via SpyTag/Catcher show potential for the development of universal influenza vaccines.
The development of a cross-protective pan-influenza A vaccine remains a significant challenge. Here, we designed and characterized single-component, self-assembling protein nanoparticles (1c SApNPs) presenting the conserved extracellular domain of matrix protein 2 (M2e) from influenza A viruses of human and other hosts. Vaccination with tandem repeats of M2e (M2ex3) displayed on 1c-SApNPs demonstrated higher survival and lower weight loss compared to the soluble M2ex3 antigen against lethal challenges of H1N1 and H3N2 in mice. The mechanism of vaccine-induced adaptive immunity was also investigated in mice. Compared with the soluble M2ex3 antigen, the M2ex3 I3-01v9a 1c-SApNP formulated with a squalene-based adjuvant showed 672 times longer follicular retention, 31 times greater exposure within follicular dendritic cell networks, and up to 2.5 times stronger germinal center reactions in lymph nodes. By inducing robust and durable M2e specific functional antibody and T cell responses, the M2ex3-presenting I3-01v9a 1c-SApNP provides a promising pan-influenza A vaccine candidate.
ONE-SENTENCE SUMMARY
Protein nanoparticles displaying tandem M2e elicit robust and durable immunity that may protect against influenza A viruses of diverse origins.
The highly conserved matrix protein 2 ectodomain (M2e) of influenza viruses presents a compelling vaccine antigen candidate for stemming the pandemic threat of the mutation‐prone pathogen, yet the low immunogenicity of the diminutive M2e peptide renders vaccine development challenging. A highly potent M2e nanoshell vaccine that confers broad and durable influenza protectivity under a single vaccination is shown. Prepared via asymmetric ionic stabilization for nanoscopic curvature formation, polymeric nanoshells co‐encapsulating high densities of M2e peptides and stimulator of interferon genes (STING) agonists are prepared. Robust and long‐lasting protectivity against heterotypic influenza viruses is achieved with a single administration of the M2e nanoshells in mice. Mechanistically, molecular adjuvancy by the STING agonist and nanoshell‐mediated prolongation of M2e antigen exposure in the lymph node follicles synergistically contribute to the heightened anti‐M2e humoral responses. STING agonist‐triggered T cell helper functions and extended residence of M2e peptides in the follicular dendritic cell network provide a favorable microenvironment that induces Th1‐biased antibody production against the diminutive antigen. These findings highlight a versatile nanoparticulate design that leverages innate immune pathways for enhancing the immunogenicity of weak immunogens. The single‐shot nanovaccine further provides a translationally viable platform for pandemic preparedness.
The ectodomain of influenza matrix protein 2 (M2e) is a promising target for the development of universal prophylactic and therapeutic agents against influenza viruses of different subtypes. We constructed three M2e-specific monoclonal antibody variants, M2A1-1 (IgG1), M2A1-2a (IgG2a), M2A1-2b (IgG2b), which have the same Fab region targeting the M2e epitope but different isotypes, and compared their protective efficacy in influenza PR8-infected mice. We found that anti-M2e antibodies provided protection against influenza virus in a subtype-dependent manner, with the IgG2a variant providing significantly better protection with lower virus titers and milder lung injury than IgG1 and IgG2b isotypes. Additionally, we observed that the protective efficacy was dependent on the administration routes, with intranasal administration of antibody providing better protection than intraperitoneal administration. The timing of administration was also critical in determining the protective efficacy; while all the antibody isotypes provided protection when administered before influenza challenge, only IgG2a provided minimal protection when the antibodies were administered after virus challenge. These results provide valuable information for optimizing the therapeutics usage of M2e-based antibodies and furthering the development of M2e-based universal influenza vaccines.
The expeditious growth of modern medicine has proven to be indispensable for protein therapeutics. In some instances, protein therapeutics are the only known cure. Continuous efforts have been put in to enhance their production and efficacy. The advancement of protein engineering methods has aided in developing effective protein therapeutics with increased affinity, pharmacokinetics, pharmacodynamics, immunogenicity, and productivity. In terms of novel protein therapeutic platform technologies, different synthetic protein scaffolds are in the early phases of clinical trials. Protein therapeutics have immense potential to improve human health. As protein engineering efforts have grown and diversified, understanding the biophysical and biochemical properties of proteins and applying this information to developing effective pharmacological medications has become increasingly relevant. The overall purpose of the various methodologies for designing protein therapeutics is to better understand their role in medicine for proper care and diagnosis, leading to further innovation and development.
Therapeutic proteins are gaining importance in disease therapy because of their specificity, efficiency, safety, and reduced side effects. With the dawn of recombinant DNA technology, the genes for the therapeutic proteins can be cloned into various expression systems, thus eliminating the earlier practice of obtaining such proteins from animal or human sources. Various expression systems like bacterial, yeast, mammalian, and plant hosts have been successfully used to recombinantly produce therapeutic proteins. Each expression system has its own benefits and limitations, thus making the expression of all proteins in a single system impossible. Prokaryotic systems like E. coli are well established and widely used for production; however, when it comes to glycosylated proteins, the lack of a secretory system in prokaryotes makes them ineffective. For producing such proteins, eukaryotic systems, particularly mammalian expression systems, are better suited. We discuss the methods for recombinant production of major therapeutic proteins using different expression systems.
Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.
In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.
The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.
Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)
An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped
duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer el al. 1984, Nucl. Acids Res. 12, 9441–9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of
the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively,
the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By
means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of
mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins
without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.
Amantadine (1-aminoadamantane hydrochloride) is effective in the prophylaxis and treatment of influenza A infections. In tissue culture this selective, strain-specific antiviral activity occurs at relatively low concentrations (5 microM or less), which inhibit either the initiation of infection or virus assembly. The data reported here demonstrate that the basis of these actions is similar and resides in the virus-coded M2 membrane protein, the product of a spliced transcript of RNA segment 7. Mutations which confer resistance to amantadine are restricted to four amino acids within a hydrophobic sequence, indicating that the drug is targetted against the putative membrane-associated portion of the molecule. The influence of the virus haemagglutinin on the amantadine sensitivity of virus strains implies that the drug may interfere with interactions between these two virus proteins.
We have obtained high level synthesis in Escherichia coli of mature human fibroblast interferon using a plasmid vector that was designed to allow easy coupling of a DNA coding region
to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage λ 's leftward promoter. The activity
of the promoter can be regulated by temperature. Induced cells accumulated the interferon up to 4% of the total cellular protein.
The biological activity of the product amounted to 4 × 109 international units per litre of culture. The synthesis of human fibroblast interferon was shown to drastically inhibit the
growth rate of the bacterial host.
The Semliki Forest virus (SFV) expression system can be used to package recombinant RNA into infectious suicide particles. Such RNA encodes only the SFV replicase and the heterologous protein but no structural proteins of SFV, and it is thus deficient in productive replication. We demonstrate here that infection of C57BL/6 (H-2b) and BALB/c (H-2d) mice with recombinant SFV expressing the nucleoprotein (NP) of influenza virus (SFV-NP) resulted in efficient priming of influenza virus-specific CD8+ cytotoxic T-cell (CTL) responses. The generated CTLs lysed both homologous (A/PR/8/34) and heterologous (A/HK/68) influenza virus-infected, or peptide-coated, target cells to a similar degree as CTLs induced by wild-type influenza virus in a major histocompatibility complex class I-restricted fashion. As few as 100 infectious units of virus induced a strong CTL response. Induction of CTL by SFV-NP could also be achieved in CD4 gene-targeted mice, demonstrating the independence of the primary CTL response of CD4+ helper T cells. One immunization generated a CTL response that peaked after 1 week, and an additional booster injection generated a CTL memory, which was still detectable after 40 days. SFV-NP immunizations also generated high-titered IgG humoral responses that remained significant after several months. These results demonstrate that the recombinant SFV suicide system is highly efficient in antigen presentation and suggest that it may have a potential as a recombinant vaccine.
DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.
Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major histocompatibility complex-restricted 9-mer epitope of the Plasmodium yoelii circumsporozoite protein or the nucleoprotein of influenza virus induces a large epitope-specific CD8(+) T-cell response. This immunization also elicits a high degree of protection against infection with malaria or influenza A virus.
In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.
We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.
We have investigated the potential of the conserved transmembrane M2 protein of influenza A/Ann Arbor/6/60 virus, expressed by a baculovirus recombinant, to induce protective immunity in mice. Vaccination of mice with shortened the duration of virus shedding and protected mice from a lethal infection with A/Ann Arbor/6/60 virus but not B/Ann Arbor/1/55 virus, suggesting that the protection was mediated by an M2-specific mechanism. Serum antibodies were detected which reacted with synthetic peptides defining three antigenic determinants located on both the external N- and internal C-termini of the M2 protein. Furthermore, vaccination with M2 protected mice from death following a lethal challenge with the heterologous A/Hong Kong/68 (H3N2) virus. These results demonstrate the potential to elicit heterosubtypic immunity to type A influenza viruses through vaccination with a conserved transmembrane protein.
To be effective as vaccines, most monomeric proteins and peptides either require chemical coupling to high molecular weight carriers or application together with adjuvants. More recently, recombinant DNA techniques have been used to insert foreign epitopes into proteins with inherent multimerization capacity, such as particle-forming viral capsid or envelope proteins. The core protein of hepatitis B virus (HBcAg), because of its unique structural and immunological properties, has gained widespread interest as a potential antigen carrier. Foreign sequences of up to approximately 40 amino acid residues at the N terminus, 50 or 100 amino acids in the central immunodominant c/e 1 epitope region of HBcAg, and up to 100 or even more residues at the C terminus, did not interfere with particle formation. The humoral immunogenicity of inserted epitopes is determined by the immunogenicity of the peptide itself and its surface exposure, and is influenced by the route of application. The probably flexible and surface-exposed c/e1 region emerged as the most promising insertion site. When applied together with adjuvants approved for human and veterinary use, or even without adjuvants, such chimeric particles induced B and T cell immune responses against the inserted epitopes. In some cases neutralizing antibodies, cytotoxic T cells and protection against challenge with the intact pathogen were demonstrated. Major factors for the potentiated immune response against the foreign epitopes are the multimeric structure of chimeric HBcAg that results in a high epitope density per particle, and the provision of T cell help by the carrier moiety. Beyond its use as subunit vaccine, chimeric HBcAg produced in attenuated Salmonella strains may be applicable as live vaccine.
The infection of eggs, cell cultures or mice with a mixture of amantadine-resistant and amantadine-sensitive strains of influenza virus resulted in the transfer of amantadine-resistance or sensitivity between strains. The response of a recombinant virus to amantadine was not related to either of its surface antigens. Resistance to amantadine was transferred as an all-or-none character. It is concluded that amantadine-resistance is a useful genetic marker for influenza viruses.
Influenza A virus recombinants derived from "resistant" and "sensitive" parental viruses were examined for susceptibility to inhibition by amantadine. Correlation of gene constellation and amantadine susceptibility revealed that the gene coding for M protein influences sensitivity or resistance to amantadine. All recombinants which derived an M protein from an amantadine-resistant parent were found to be resistant to amantadine. All amantadine-sensitive recombinants derived an M gene from the amantadine-sensitive parent. However, a few amantadine-resistant recombinants which derived an M gene from the sensitive parent were also isolated, suggesting that the expression of amantadine sensitivity in these recombinants may be influenced by other genes.
The genetic composition of several recombinant strains, produced by mixed infection with an amantadine-sensitive and an amantadine-resistant influenza virus, have been compared with their response to amantadine. It is concluded that transfer of resistance or sensitivity to amantadine is determined by a single gene, that coding for the matrix protein.
THE manufacture of predefined specific antibodies by means of permanent
tissue culture cell lines is of general interest. There are at present a
considerable number of permanent cultures of myeloma cells1,2
and screening procedures have been used to reveal antibody activity in
some of them. This, however, is not a satisfactory source of monoclonal
antibodies of predefined specificity. We describe here the derivation of
a number of tissue culture cell lines which secrete anti-sheep red blood
cell (SRBC) antibodies. The cell lines are made by fusion of a mouse
myeloma and mouse spleen cells from an immunised donor. To understand
the expression and interactions of the Ig chains from the parental
lines, fusion experiments between two known mouse myeloma lines were
carried out.
The evidence presented shows that the M2 protein of influenza A viruses exists in infected cells as a homotetramer composed of two disulfide-linked dimers held together by noncovalent interactions. The amphiphilic nature of the transmembrane alpha-helical domain is consistent with the protein forming a transmembrane channel with which amantadine, the specific anti-influenza A drug, interacts. Together these features provide a structural basis for the hypothesis that M2 has a proton translocation function capable of regulating the pH of vesicles of the trans-Golgi network, a role important in promoting the correct maturation of the hemagglutinin glycoprotein.
The oligomeric structure of the influenza A virus M2 integral membrane protein was determined. On SDS-polyacrylamide gels under nonreducing conditions, the influenza A/Udorn/72 virus M2 forms disulfide-linked dimers (30 kDa) and tetramers (60 kDa). Sucrose gradient analysis and chemical cross-linking analysis indicated that the oligomeric form of M2 is a tetramer consisting of either a pair of disulfide-linked dimers or disulfide-linked tetramers. In addition, a small amount of a cross-linked species of 150-180,000 kDa, which the available data suggest contains only M2 polypeptides, was observed. The role of M2 cysteine residues in disulfide bond formation and their role in forming oligomers were examined by converting each of the two extracellular and single cytoplasmic cysteine residues to serine residues and expressing the altered M2 proteins in eukaryotic cells. Removal of either one of the N-terminal cysteines at residues 17 or 19 indicated that tetramers formed that consisted of a pair of noncovalently associated disulfide-linked dimers, suggesting that each of the cysteine residues is equally competent for forming disulfide bonds. When both cysteine residues were removed from the M2 N-terminal domain, no disulfide-linked forms were observed. When solubilized in detergent this double-cysteine mutant lost reactivity with a M2-specific mAb and exhibited an altered sedimentation pattern on sucrose gradients. However, chemical cross-linking of this double-cysteine mutant in membranes indicated that it can form tetramers. Taken together, these data suggest that disulfide bond formation, although not essential for oligomeric assembly, stabilizes the M2 tetramer from disruption by detergent solubilization.
The M2 protein of influenza A virus is expressed on the surfaces of infected cells, and a monoclonal antibody to this protein inhibits plaque enlargement of sensitive influenza A viruses without reducing plaque titer (S.L. Zebedee and R.A. Lamb, J. Virol. 62:2762-2772, 1988). In the current study, passively transferred monoclonal antibody to M2 reduced the level of replication of influenza A virus but not of influenza B virus in the lungs of mice. These experiments demonstrated that antibody to a protein conserved among influenza A virus subtypes inhibits virus growth in vivo.
Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant. The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.
The influenza A virus M2 protein is an integral membrane protein of 97 amino acids that is expressed at the surface of infected cells with an extracellular N-terminal domain of 18 to 23 amino acid residues, an internal hydrophobic domain of approximately 19 residues, and a C-terminal cytoplasmic domain of 54 residues. To gain an understanding of the M2 protein function in the influenza virus replicative pathway, we produced and characterized a monoclonal antibody to M2. The antibody-binding site was located to the extracellular N terminus of M2 as shown by the loss of recognition after proteolysis at the infected-cell surface, which removes 18 N-terminal residues, and by the finding that the antibody recognizes M2 in cell surface fluorescence. The epitope was further defined to involve residues 11 and 14 by comparing the predicted amino acid sequences of M2 from several avian and human strains and the ability of the M2 protein to be recognized by the antibody. The M2-specific monoclonal antibody was used in a sensitive immunoblot assay to show that M2 protein could be detected in virion preparations. Quantitation of the amount of M2 associated with virions by two unrelated methods indicated that in the virion preparations used there are 14 to 68 molecules of M2 per virion. The monoclonal antibody, when included in a plaque assay overlay, considerably showed the growth of some influenza virus strains. This plaque size reduction is a specific effect for the M2 antibody as determined by an analysis of recombinants with defined genome composition and by the observation that competition by an N-terminal peptide prevents the antibody restriction of virus growth.
Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.
We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein. The synthetic gene contains 27 unique internal restriction sites. Thereby, it can easily be mutagenized by replacement of rather short restriction fragments. A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome. Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites. In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein. It forms particles closely resembling native HBV cores. After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products. The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle.
BALB/c mice immunized with graded doses of chromatographically purified hemagglutinin (HA) and neuraminidase (NA) antigens derived from A/Hong Kong/1/68 (H3N2) influenza virus demonstrated equivalent responses when HA-specific and NA-specific serum antibodies were measured by enzyme-linked immunosorbent assays (ELISAs). Antibody responses measured by hemagglutination inhibition or neuraminidase inhibition titrations showed similar kinetic patterns, except for more rapid decline in hemagglutination inhibition antibody. Injection of mice with either purified HA or NA resulted in immunity manifested by reduction in pulmonary virus following challenge with virus containing homologous antigens. However, the nature of the immunity induced by the two antigens differed markedly. While HA immunization with all but the lowest doses of antigen prevented manifest infection, immunization with NA was infection-permissive at all antigen doses, although reduction in pulmonary virus was proportional to the amount of antigen administered. The immunizing but infection-permissive effect of NA immunization over a wide range of doses is in accord with results of earlier studies with mice in which single doses of NA and antigenically hybrid viruses were used. The demonstrable immunogenicity of highly purified NA as a single glycoprotein without adjuvant offers a novel infection-permissive approach with potentially low toxicity for human immunization against influenza virus.
The emergence of influenza A viruses which had acquired resistance to rimantadine during a clinical trial (C. B. Hall, R. Dolin, C. L. Gala, D. M. Markovitz, Y. Q. Zhang, P. H. Madore, F. A. Disney, W. B. Talpey, J. L. Green, A. B. Francis, and M. E. Pichichero, Pediatrics 80:275-282, 1987) provided the opportunity to determine the genetic basis of this phenomenon. Analysis of reassortant viruses generated with a resistant clinical isolate (H3N2) and the susceptible influenza A/Singapore/57 (H2N2) virus indicated that RNA segment 7 coding for matrix and M2 proteins conferred the resistant phenotype. Resistant viruses isolated from seven patients each contained a single change in the nucleotide sequence coding for the M2 protein which resulted in substitutions in amino acid 30 (two viruses) or 31 (five viruses) in the transmembrane domain of the molecule. These changes occurred in locations identified in influenza viruses selected for resistance to amantadine in tissue culture and indicate a common mechanism of action of the two compounds in cell culture and during chemotherapeutic use.
The influenza A virus M2 protein is expressed abundantly at the cell surface, and in addition to the hemagglutinin (HA) and neuraminidase (NA), is a third virus-specific membrane protein. M2 has an internal hydrophobic membrane anchorage domain and associates with the same cellular membrane fractions as HA and NA. Trypsin treatment of infected cells and immunoprecipitation with site-specific antisera indicate that a minimum of 18 NH2-terminal amino acids of M2 are exposed at the cell surface. Ten NH2-terminal residues are conserved in all strains of influenza A virus for which sequences are available. Antibodies can recognize M2 on the cell surface and therefore it may be an infected-cell surface antigen. We discuss properties of M2 that match it to the elusive major target molecule on influenza A virus-infected cells for cross-reactive cytotoxic T cells.
The complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amiino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Hav1 strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.
We have explored the possibility that an animal viral reservoir contained a direct ancestor gene for the H3 hemagglutinin type present in influenza A viruses in humans since 1968. For this purpose, the duck/Ukraine/1/63 hemagglutinin gene was cloned and sequenced. From the comparison of its complete primary structure with that of several human H3 hemagglutinins as well as those of an H2 and an H7 hemagglutinin, we conclude that the duck/Ukraine/63 hemagglutinin sequence fully corroborates its previous identification by immunological and other methods as belonging to the H3 subtype. Moreover, the duck/Ukraine/63 amino acid sequence is more closely related structurally and presumably antigenically to the human Aichi/68 hemagglutinin, which formed the beginning of the H3N2 pandemic in humans, than to that of Victoria/75, which has undergone an additional 7 year drift period in humans. This observation could best be explained by a common ancestor hemagglutinin gene for duck/Ukraine/63 and human Aichi/68. On the basis of silent, accumulated base changes, we estimate that the strain carrying this postulated common progenitor hemagglutinin gene was circulating in the period 1949-1953 in the animal reservoir. This relatively recent divergence, as well as the closer kinship between the duck/Ukraine/63 and the human Aichi/68 hemagglutinin, as compared with the later Victoria/75, strongly suggests that the influenza A virus of the H3N2 subtype circulating in the human population since 1968 has derived its hemagglutinin gene from a strain in the animal reservoir. Undoubtedly, this occurred by reassortment between previously present human H2N2 virus and this animal strain. These results provide support at the molecular level for the general idea that the wide variety of influenza viruses known to be present in animals can serve as a gene reservoir for human influenza A viruses.
A family of plasmid cloning vectors has been constructed that make use of the leftward promoter () of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of is fully repressed at low temperature by a thermolabile repressor product of the gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under control.
RNA segment 7 of the influenza A virus genome codes for at least two proteins, M1 and M2, which are synthesized from separate mRNA species. Sequence analysis of the M2 mRNA has shown that it contains an interrupted sequence of 689 nucleotides. The approximately 51 virus-specific nucleotides comprising the 5'-end leader sequence of the M2 mRNA are the same as those found at the 5' end of the colinear M1 mRNA. Following the leader sequence of the M2 mRNA, where is a 271-nucleotide body region that is 3' coterminal with the M1 mRNA. Another small potential mRNA (mRNA3) related to RNA segment 7 has been found. mRNA3 has a leader sequence of approximately 11 virus-specific nucleotides that are the same as the 5' end of the M1 and M2 mRNAs, followed by an interrupted sequence of 729 nucleotides, and then a body region of approximately 271 nucleotides that is the same as that of the M2 mRNA. The nucleotide sequences found at the junctions of the interrupted sequences in M2 mRNA and mRNA3 are similar to those found at the splicing points of intervening sequences in eukaryotic mRNAs. In addition, both mRNAs contain 10-15 heterogeneous nonviral nucleotides at their 5' ends that appear to be derived from cellular RNAs used for priming the transcription of viral RNAs. Because the 5'-end sequences of the M1 mRNA and the M2 mRNA are the same and share the 5'-proximal initiation codon for protein synthesis, the first nine amino acids would be the same in the M1 and M2 protein and then the sequences would diverge. The approximately 271-nucleotide body region of the M2 mRNA can be translated in the +1 reading frame, and the sequence indicates that M1 and M2 overlap by 14 amino acids. The coding potential of the mRNA3 is for only nine amino acids, and these would be identical to the COOH-terminal region of the membrane protein (M1).
DNA fragments from hepatitis B virus (HBV) have been inserted into
plasmid vectors and cloned in Escherichia coli1, and some of
these bacterial clones have been reported to synthesize hepatitis B core
antigen (HBcAg)1,2. Material from a derivative of one
HBcAg-synthesizing clone is being evaluated in our laboratory and
elsewhere3,4 as a serological reagent for use in various
assays, including immune electron microscopy, to detect anti-HBcAg
antibody. It has been shown by gel exclusion chromatography that the
bacterial HBcAg is in an aggregated form3. We now report that
the E. coli HBcAg preparation contains Small round particles similar in
appearance to the viral cores previously seen in material obtained from
HBV-infected humans5-7 and chimpanzees8.
The genetic composition of 11 high-yielding recombinants of influenza virus was determined by polyacrylamide gel electrophoresis
of the 32P-Iabeled RNAs obtained from the recombinants and their parental viruses. The high-yield recombinants that were selected for
potential use as vaccine strains contained the surface hemagglutinin and neuraminidase antigens of the low-yielding parental
viruses. The increased growth capacity of the recombinants is associated with the presence of genes derived from the high-yielding
laboratory strain A/Puerto Rico/8/34. Although increased growth capacity in these recombinants could not be attributed to
specific genes or gene combinations, all of the high-yielding recombinants examined derived the Mgene from the A/Puerto Rico/8/34
parent.
A DNA copy of the gene coding for the influenza A/Aichi/2/68 haemagglutinin protein was cloned in the plasmid pBR322 and the complete nucleotide sequence determined. Comparison of this primary structure and the deduced amino acid sequence with the haemagglutinin gene and protein of strains belonging to the same (H3) subtype and to different subtypes, of both human (H2) and avian (Hav1) origin, documents further at the molecular level the two independent modes of antigenic variation of the virus--drift and shift.
A recombinant baculovirus expressing the M2 protein from influenza A/Ann Arbor/6/60 (H2N2) virus (AA60 virus) was constructed. The expressed M2 protein was recognized by a monoclonal antibody specific for the M2 protein and comigrated with the M2 protein from cells infected with AA60 virus on SDS-polyacrylamide gels. Immunofluorescence studies indicated that the expressed M2 protein was present on the surface of Spodoptera frugiperda (Sf9) cells infected with the recombinant baculovirus. Immunoassays using the expressed M2 protein were able to detect antibodies to the M2 protein in serum samples from humans and ferrets infected with influenza A viruses.
Cytotoxic T lymphocytes (CTLs) specific for conserved viral antigens can respond to different strains of virus, in contrast
to antibodies, which are generally strain-specific. The generation of such CTLs in vivo usually requires endogenous expression
of the antigen, as occurs in the case of virus infection. To generate a viral antigen for presentation to the immune system
without the limitations of direct peptide delivery or viral vectors, plasmid DNA encoding influenza A nucleoprotein was injected
into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific CTLs and protection from a subsequent
challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass
loss, and increased survival.
The immune system can remember, sometimes for a lifetime, the identity of a pathogen. Understanding how this is accomplished has fascinated immunologists and microbiologists for many years, but there is still considerable debate regarding the mechanisms by which long-term immunity is maintained. Some of the controversy stems from a failure to distinguish between effector and memory cells and to define their roles in conferring protection against disease. Here the current understanding of the cellular basis of immune memory is reviewed and the relative contributions made to protective immunity by memory and effector T and B cells are examined.
The N2 neuraminidase gene of A/Victoria/3/75 influenza virus was engineered to encode a secretable protein (NAs) by replacing the natural N-terminal membrane anchor sequence with the cleavable signal sequence of the corresponding influenza hemagglutinin gene. Soluble NAs was expressed by a baculovirus/insect cell system and accumulated in the medium at levels between 6 and 8 microgram ml-1. A combination of biochemical and standard chromatographic techniques allowed the purification of NAs to homogeneity. Cross-linking analysis indicated that NAs was partly recovered as an authentic tetrameric protein, while the remaining fraction was composed of dimeric molecules and small amounts of monomeric NAs. Purified NAs was supplemented with low-reactogenic adjuvants and used to immunize mice. After a challenge infection with a lethal dose of homologous mouse-adapted X47 influenza virus, vaccinated animals showed resistance against severe disease symptoms and were protected from lethality. Based on the results of a passive immunization experiment, it may be concluded that performed antibody plays a central role in the mechanism by which vaccination with NAs confers viral protection.
Since early this century, various substances have been added to vaccines and certain formulations have been devised in an attempt to render vaccines more effective. Despite a plethora of options, only aluminium salts have gained acceptance as human vaccine adjuvants and even veterinary vaccines are largely dependent upon the use of aluminium salts. Currently, many new vaccines are under development and there is a desire to simplify vaccination schedules both by increasing the number of components per vaccine and decreasing the number of doses required for a vaccine course. New, more effective adjuvants will be required to achieve this.
Influenza virus neuraminidase was chromatographically extracted from A/Johannesburg/33/94 (H3N2) and used to supplement conventional monovalent H3JHN2JH inactivated influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinin (HA) and neuraminidase (NA) equivalent for each antigen to titers in animals immunized with either antigen alone. Homotypic infection was suppressed and greater reduction in viral replication was observed following heterotypic infectious challenge than was observed following the non-supplemented vaccine. There was no evidence of suppression of the immune response to the HA despite the presence of high amounts of NA in the vaccine. Supplementation of conventional inactivated influenza vaccine with NA takes advantage of the equivalent immunogenicity of dissociated HA and NA, to produce a more balanced immune response to both surface antigens, without the antigenic competition tht occurs after immunization with conventional vaccine or infection. These studies in a mouse model system suggest that supplementation of current inactivated influenza vaccines offers the prospect of improved immunization of humans against influenza.
The ability of plasmid DNA encoding various influenza viral proteins from the A/PR/8/34 (H1N1) virus to protect against influenza was compared in BALB/c mice. The plasmid DNA encoded hemagglutinin (HA), neuraminidase (NA), matrix protein (M1), nucleoprotein (NP) or nonstructural protein (NS1) in a chicken beta-actin-based expression vector (pCAGGS). Each DNA was inoculated twice 3 weeks apart at a dose of 1 microgram per mouse by particle-mediated DNA transfer to the epidermis (gene gun). Seven days after a second immunization, mice were challenged with the homologous virus and the ability of each DNA to protect mice from influenza was evaluated by decreased lung virus titers and increased survival. Mice, given HA- or NA-expressing DNA, induced a high level of specific antibody response and protected well against the challenge virus. On the other hand, mice given M1-, NP-, or NS1-DNA failed to provide protection, although M1- and NP-DNAs did induce detectable antibody responses. These results indicate that both HA- and NA-expressing DNAs for the surface glycoproteins are most protective against influenza from among the various viral protein-expressing DNAs used here.
I-Adamantanamine (amantadine) causes a selective, reproducible, dose-related inhibition of influenza infections in tissue
culture, chick embryos, and mice. The compound is not virucidal and appears to act by interfering with the penetration of
the host cell by the virus. In influenza infections of mice, greatest efficacy occurs with treatment at the time of infection;
however, there is significant antiviral activity with treatment delayed up to 72 hours after infection. Virus inhibition is
not complete and survivors are immune to a challenge infection with the original infecting virus.
Textbook of Influenza
- K G Nicholson
- R G Webster
- A J Hay
Nicholson, K.G., Webster, R.G. & Hay, A.J. (eds.). Textbook of Influenza (Blackwell Science, Oxford, 1998).
Complete structure of the hemagglutinin gene from the human in-fluenza A/Victoria
- Min Jou
Min Jou, W. et al. Complete structure of the hemagglutinin gene from the human in-fluenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell 19, 683–696 (1980).
Antiviral activity of 1-adamantanamine
- W L Davies
- WL Davies
Site-directed mutagenesis of virtually any plasmid by eliminating a unique site
- W P Deng
- J A Nickolov
- WP Deng