Recombinant M1 Research Articles

Molecular and Cellular Characterization of the Glutathione Transferases Involved in the Olfactory Metabolism of the Mammary Pheromone.

Odorant metabolizing enzymes, considered as critical olfactory perireceptor actors, control the odor molecules reaching the olfactory epithelium by biotransforming them. As an odorant, the mammary pheromone, i.e., 2-methylbut-2-enal (2MB2), emitted in the milk of lactating female rabbits triggers typical nipple searching-grasping behavior through orocephalic movements in newborn rabbits but not in weaned rabbits. We previously showed that 2MB2 perception is significantly modified when its glutathione transferase-dependent olfactory metabolism is affected in newborns. Here, enzymatic assays of the recombinant enzymes GSTA1, M1, and P1 revealed the activity of these enzymes toward the mammary pheromone. Histological experiments revealed strong expression of the GSTA class restricted to the Bowman glands and of GSTP1 in the nuclei of sustentacular cells. Moreover, some modulations of GSTs have been demonstrated, including a significant increase in GSTP1 expression (2-fold in mRNA, p value < 0.001; protein, p value: 0.031) after 45 min of mammary pheromone exposure at 10-2 g/mL and an increase in GSTA expression in weaned rabbits compared with newborn rabbits (3-fold in mRNA, p value: 0.011; protein, p value: 0.001). Our results provide new insights into the activity, cellular expression, and modulation of the mammary pheromone GST-metabolizing enzymes and clues about their olfactory function.

Investigating the impact of M1 protein from Group A Streptococcus on fibrin clot formation, structure and fibrinolytic potential

Background: Fibrin formation is an essential part of innate immunity, sealing off infections to limit bacterial spreading.The surface-anchored M1 protein is a major virulence determinant of Group A streptococcus (GAS). During early infection M1 is cleaved from the cell surface by SpeB, a streptococcal protease regulated by CovR/S. M1 forms a supramolecular complex with fibrinogen however the impact on fibrin formation is not known. Methods: The effects of recombinant M1 (rM1) were assessed in fibrin clots made from plasma or purified fibrinogen incubated with thrombin, by confocal microscopy and scanning electron microscopy. Clotting and lysis profiles (with plasminogen activators and plasminogen) were investigated kinetically using thromboelastography (ROTEM). Results: rM1 (0.47-60 μg/ml) increased clotting rates to produce heterogeneous clots with irregular fibre bundles and compacted fibrin. Formation of the protective fibrin biofilm was also disrupted by rM1. Furthermore, mechanical strength of fibrin clots was reduced with increasing rM1 concentrations and was undetectable above 15.5 μg/ml. Purified and plasma clots formed with rM1 were more susceptible to lysis by plasmin, with a 1.4 – 2-fold reduction in lysis times. Conclusions: At sites of GAS infection, cleaved M1 may bind to fibrinogen generating fibrin clots which: lack the protective film at the clot surface; are mechanically weaker; and are less resistant to lysis by plasmin. GAS strains of M1-type are commonly associated with invasive infections; the impact of M1 on fibrin structure could contribute to the severity of GAS infection by compromising the fibrin barrier that limits bacterial proliferation and migration.

Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

Costimulation is required for optimal T cell activation, yet it is unclear whether poxviruses dedicatedly subvert costimulation during infection. Here, we report that the secreted M2 protein encoded by cowpox virus (CPXV) specifically interacts with human and murine B7.1 (CD80) and B7.2 (CD86). We also show that M2 competes with CD28 and CTLA4 for binding to cell surface B7 ligands, with stronger efficacy against CD28. Functionally, recombinant M2 and culture supernatants from wild-type (WT) but not M2-deficient (∆M2) CPXV-infected cells can potently suppress B7 ligand-mediated T cell proliferation and interleukin-2 (IL-2) production. Furthermore, we observed increased antiviral CD4 and CD8 T cell responses in C57BL/6 mice challenged by ∆M2 CPXV compared with WT virus. These differences in immune responses to ∆M2 and WT CPXV were not observed in CD28-deficient mice. Taken together, our findings define a mechanism of viral sabotage of T cell activation that highlights the role of CD28 costimulation in host defense against poxvirus infections.

Residue Mutations in Murine Herpesvirus 68 Immunomodulatory Protein M3 Reveal Specific Modulation of Chemokine Binding.

The M3 protein (M3) encoded by murine gammaherpesvirus 68 (MHV-68) is a unique viral immunomodulator with a high-affinity for a broad spectrum of chemokines, key mediators responsible for the migration of immune cells to sites of inflammation. M3 is currently being studied as a very attractive and desirable tool for blocking the chemokine signaling involved in some inflammatory diseases and cancers. In this study, we elucidated the role of M3 residues E70 and T272 in binding to chemokines by examining the effects of the E70A and T272G mutations on the ability of recombinant M3, prepared in Escherichia coli cells, to bind the human chemokines CCL5 and CXCL8. We found that the E70A mutation enhanced binding of M3 to CCL5 two-fold but had little effect on its binding to CXCL8. In contrast, the T272G mutation was found to be important for the thermal stability of M3 and significantly decreased M3's binding to both CCL5 (by about 4×) and CXCL8 (by about 5×). We also constructed in silico models of the wild-type M3–CCL5 and M3–CCL8 complexes and found substantial differences in their physical and chemical properties. M3 models with single mutation E70A and T272G suggested the role of E70 and T272 in binding M3 protein to chemokines. In sum, we have confirmed that site-directed mutagenesis could be an effective tool for modulating the blockade of particular chemokines by M3, as desired in therapeutic treatments for severe inflammatory illnesses arising from chemokine network dysregulation.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract.Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking.Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50–100μg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction.Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

A Deletion of Signal Peptide in Recombinant Murine Gamma-herpesvirus 68 M3 Protein Enhances its Binding Affinity to CCL5 Chemokine and Increases its Yield from Insect Cells

Objective: The M3 protein encoded by murine gamma-herpesvirus 68 (MHV-68) was the first secreted protein identified in herpesvirus. This protein is unique in its ability to bind a broad-spectrum of chemokines from all four subfamilies, thus it has been proposed to be a potential gene therapy candidate for controlling the overactive inflammatory responses of some human inflammatory diseases. Methods: We prepared MHV-68 M3del protein with a deletion of its signal peptide (M3del) and full-length MHV-68 M3 protein (M3) using a baculovirus-mediated insect cell expression system and confirmed their specificity by Western blot using newly prepared mouse monoclonal anti-M3 antibody 1/27. Binding affinity of both proteins to human CCL5 and CXCL8 chemokine was examined by ELISA. Results: M3del and M3 displayed affinity to both chemokines tested. M3 del concentration sufficient to bind 25% of CCL5 was two times smaller than that of M3 (IC25=3.5 nmol/l vs. 6.1 nmol/l). In contrary, the values of IC25 for CXCL8 for M3del and M3 were comparable (IC25=13.8 nmol/l vs. 13.3 nmol/l. We have found that the absence of signal peptide strongly affects the yield of recombinant M3 protein. The yields of M3del and M3 were in an unbalanced ratio of 67 to 1. Conclusions: The results suggest that the absence of signal peptide in recombinant MHV-68 M3 protein allows increased binding activity of the protein to CCL5 but not to CXCL8, and even a very large increase in its yield from insect cells.

A novel muscarinic receptor-independent mechanism of KCNQ2/3 potassium channel blockade by Oxotremorine-M

Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels.

Electrophysiological evidence showing muscarinic agonist–antagonist activities of N-desmethylclozapine using hippocampal excitatory and inhibitory neurons

The atypical antipsychotic clozapine is widely used for treatment-resistant schizophrenic patients. Clozapine and its major active metabolite, N-desmethylclozapine (NDMC), have complex pharmacological properties, and interact with various neurotransmitter receptors. There are several biochemical studies reporting that NDMC exhibits a partial agonist profile at the human recombinant M1 muscarinic receptors. However, direct electrophysiological evidence showing the ability of NDMC to activate native M1 receptors in intact neurons is poor. Using rat hippocampal neurons, we previously demonstrated that activation of muscarinic receptors by a muscarinic agonist, oxotremorine M (oxo-M), induces a decrease in outward K+current at −40mV. In the present study, using this muscarinic current response we assessed agonist and antagonist activities of clozapine and NDMC at native muscarinic receptors in intact hippocampal excitatory and inhibitory neurons. Suppression of the oxo-M-induced current response by the M1 antagonist pirenzepine was evident only in excitatory neurons, while the M3 antagonist darifenacin was effective in both types of neurons. Muscarinic agonist activity of NDMC was higher than that of clozapine, higher in excitatory neurons than in inhibitory neurons, sensitive to pirenzepine, and partially masked when co-applied with clozapine. Muscarinic antagonist activity of clozapine as well as NDMC was not different between excitatory and inhibitory neurons, but clozapine was more effective than NDMC. These results demonstrate that NDMC has the ability to activate native M1 receptors expressed in hippocampal excitatory neurons, but its agonist activity might be limited in clozapine-treated patients because of the presence of excessive clozapine with muscarinic antagonist activity.

Hydroxycinnamic acid amides from Scopolia tangutica inhibit the activity of M1 muscarinic acetylcholine receptor in vitro

Scopolia tangutica Maxim (S. tangutica) extracts have been traditionally used as antispasmodic, sedative, and analgesic agents in Tibet and in the Qinghai province of China. Their active compositions are however poorly understood. We have recently isolated five new hydroxycinnamic acid (HCA) amides along with two known HCA amides, one cinnamic acid amide from these extracts. In this study, we evaluate their abilities to inhibit carbacol-induced activity of M1 muscarinic acetylcholine receptor along with the crude extracts. Chinese hamster ovary cells stably expressing the recombinant human M1 receptor (CHO-M1 cells) were employed to evaluate the anticholinergic potentials. Intracellular Ca2+ changes were monitored using the FLIPR system. Five HCA amides as well as the crude S. tangutica extract displayed dose-dependent inhibitory effects against M1 receptor. These findings demonstrate that HCA amides are part of the M1 receptor-inhibiting principles of S. tangutica. Since blockade of parasympathetic nerve impulse transmission through the inhibition of the M1 receptor lessens smooth muscle spasms, our findings provided a molecular explanation for the traditional use of S. tangutica against spasm.

Cell penetrable human scFv specific to middle domain of matrix protein-1 protects mice from lethal influenza.

A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1) is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD) and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN). The PEN-scFv (transbody), when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

Soluble M3 proteins of murine gammaherpesviruses 68 and 72 expressed in Escherichia coli: analysis of chemokine-binding properties.

M3 protein of murine gammaherpesvirus 68 (MHV-68) was identified as aviral chemokine-binding protein 3 (vCKBP-3) capable to bind abroad spectrum of chemokines and their receptors. During both acute and latent infection MHV-68 M3 protein provides aselective advantage for the virus by inhibiting the antiviral and inflammatory response. Aunique mutation Asp307Gly was identified in the M3 protein of murine gammaherpesvirus 72 (MHV-72), localized near chemokine-binding domain. Study on chemokine-binding properties of MHV-72 M3 protein purified from medium of infected cells implied reduced binding to some chemokines when compared to MHV-68 M3 protein. It was suggested that the mutation in the M3 protein might be involved in the attenuation of immune response to infection with MHV-72. Recently, Escherichia coli cells were used to prepare native recombinant M3 proteins of murine gammaherpesviruses 68 and 72 (Pančík et al., 2013). In this study, we assessed the chemokine-binding properties of three M3 proteins prepared in E. coli Rosetta-gami 2 (DE3) cells, the full length M3 protein of both MHV-68 and MHV-72 and MHV-68 M3 protein truncated in the signal sequence (the first 24 aa). They all displayed binding activity to human chemokines CCL5 (RANTES), CXCL8 (IL-8), and CCL3 (MIP-1α). The truncated MHV-68 M3 protein had more than twenty times reduced binding activity to CCL5, but only about five and three times reduced binding to CXCL8 and CCL3 when compared to its full length counterpart. Binding of the full length MHV-72 M3 protein to all chemokines was reduced when compared to MHV-68 M3 protein. Its binding to CCL5 and CCL3 was reduced over ten and seven times. However, its binding to CXCL8 was only slightly reduced (64.8 vs 91.8%). These data implied the significance of the signal sequence and also of asingle mutation (at aa 307) for efficient M3 protein binding to some chemokines.

The in vitro and in vivo profile of aclidinium bromide in comparison with glycopyrronium bromide

This study characterised the in vitro and in vivo profiles of two novel long-acting muscarinic antagonists, aclidinium bromide and glycopyrronium bromide, using tiotropium bromide and ipratropium bromide as comparators. All four antagonists had high affinity for the five muscarinic receptor sub-types (M1–M5); aclidinium had comparable affinity to tiotropium but higher affinity than glycopyrronium and ipratropium for all receptors. Glycopyrronium dissociated faster from recombinant M3 receptors than aclidinium and tiotropium but more slowly than ipratropium; all four compounds dissociated more rapidly from M2 receptors than from M3 receptors. In vitro, aclidinium, glycopyrronium and tiotropium had a long duration of action at native M3 receptors (>8 h versus 42 min for ipratropium). In vivo, all compounds were equi-potent at reversing acetylcholine-induced bronchoconstriction. Aclidinium, glycopyrronium and ipratropium had a faster onset of bronchodilator action than tiotropium. Aclidinium had a longer duration of action than glycopyronnium (time to 50% recovery of effect [t½ offset] = 29 h and 13 h, respectively); these compare with a t½ offset of 64 h and 8 h for tiotropium and ipratropium, respectively. Aclidinium was less potent than glycopyrronium and tiotropium at inhibiting salivation in conscious rats (dose required to produce half-maximal effect [ED50] = 38, 0.74 and 0.88 μg/kg, respectively) and was more rapidly hydrolysed in rat, guinea pig and human plasma compared with glycopyrronium or tiotropium. These results indicate that while aclidinium and glycopyrronium are both potent antagonists at muscarinic receptors with similar kinetic selectivity for M3 receptors versus M2, aclidinium has a longer dissociation half-life at M3 receptors and a longer duration of bronchodilator action in vivo than glycopyrronium. The rapid plasma hydrolysis of aclidinium, coupled to its kinetic selectivity, may confer a reduced propensity for systemic anticholinergic side effects with aclidinium versus glycopyrronium and tiotropium.

Survey of Human Antibody Responses to Influenza Virus Matrix Protein 2 by Use of a Sensitive Flow Cytometric Method

Conventional influenza vaccines are designed to elicit antibodies to strainspecific antigens, leaving a public health gap when novel viruses break out unexpectedly. As examples, strain-matched vaccine became available too late in the pandemic of 2009 to protect against the fall wave, and drift viruses are sometimes divergent enough to cause vaccine failure (eg, A/Sydney in 1997). Available vaccines are also inadequate to protect against various zoonotic strains, including avian influenza A(H5N1), influenza A(H7N7), and most recently influenza A(H7N9). For this reason, there is much interest in developing vaccines based on conserved influenza virus features that can provide protection regardless of strain. These are usually designed for influenza A virus, but such vaccines can also be made for influenza B virus. Universal influenza vaccines can induce immune protection dependent upon antibody or T-cell responses or both, and the target antigens explored have included nucleoprotein, matrix proteins (M1 and M2), the hemagglutinin (HA) stem, polymerase PB1, and other antigens, as reviewed previously [1]. M2, the focus of Zhong et al in the current issue of the Journal, has long been known as a target of antibodies that reduce viral replication and spread [2, 3]. Vaccines based on many forms of M2 (fusion proteins, M2 multiple antigenic peptides, peptide conjugates, and M2 expressed from viral vectors) provide protective immunity in animals [4–10], and some have been tested in humans. A clinical trial of recombinant M2 shows that this antigen is immunogenic when administered with adjuvant [11]. Human anti-M2 antibodies induced by natural infection have been reported, but data are scanty. Black et al reported that 6 of 17 pairs of acute-phase and convalescent-phase serum specimens showed increased anti-M2 activity by enzyme-linked immunosorbent assay (ELISA), while 12 of 17 convalescent-phase serum specimens demonstrated some signal by Western blot [12]. In a study by Feng et al, an increase in anti-M2 activity was found for 11 of 24 such serum pairs [13]. In that study, of the antibodies detectable by assay on cell surface tetrameric M2, only a minority also recognized M2e peptide. Thus, the majority of antibodies appeared to be conformational [13]. These antibodies may be biologically very important, so assays on native M2 are needed. However, measuring antibodies to native M2 has presented technical problems, with high background encountered in cell surface ELISA. To overcome these problems, Zhong et al developed a flow cytometric assay (M2-FCA) using a panel of 293FT transfected cell lines (M2-293FT) stably expressing full-length tetrameric forms of M2 from various viral strains [14]. With this sensitive assay, some mouse antibodies recognize strain-specific epitopes and some see epitopes cross-reactive among viral strains [14]. The present article uses the assay to analyze collections of human sera. Transfection efficiency and expression levels are normalized through the use of a positive human serum pool as a control. Unlike most serological assays in which a titer reflects the dilution at which a defined end point is reached, the unit of M2 antibody is defined for a single (1:40) dilution of serum. This allows large numbers of sera to be analyzed within a single run, but does not measure titers or other antibody properties revealed by dilution series. Results for healthy donors of different ages showed that antibodies to M2 were found in a higher percentage of, and at higher levels, in adults aged ≥40 years, compared with younger donors. If anti-M2 antibodies were present, they usually recognized both seasonal and swine-origin M2. Received 5 December 2013; accepted 5 December 2013. Correspondence: Suzanne Epstein, PhD, OCTGT, CBER, FDA, 1401 Rockville Pk, HFM-730, Rockville, MD 20852-1448 (suzanne.epstein@fda.hhs.gov). The Journal of Infectious Diseases Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2013. This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: 10.1093/infdis/jit813

A Synthetic Peptide Comprising Heterodimeric Epitopes of M Protein Elicits Mice Protective Immunity against H5N1 Influenza a Virus

Background: The high degree of conservation of the M2 protein of influenza A virus and its cross-reactive immunity that decreased the severity of diseases caused by influenza virus in animal models make it a prime candidate for a universal influenza vaccine. Studies showed that recombinant HA, NA and M2 proteins were poorly immunogenic and required multiple doses or the inclusion of adjuvant for improved immunogenicity and efficacy. Methods: A synthetic peptide PEP-M2 comprising heterodimeric epitopes was employed to immunize mice in the presence or absence of a 'K' type CpG ODN adjuvant. Results: Mice vaccinated with PEP-M2 peptide elicited strong IgG ELISA antibody titers to this peptide and increased protection (with survival rates raised from 25% to 60%) for mice challenged by lethal influenza A virus (NIBRG-14, 1000TCID50). The CpG ODN, used as an adjuvant for PEP-M2, elicited significantly higher antibody titers in mice. Further, PEP-M2 plus CpG ODN promoted in immunized mice higher survival rate (83.3%) and slower weight loss than PEP-M2 alone in lethal challenge assay. Conclusions: Results from this study revealed that peptide comprising heterodimeric epitopes of M Protein could elicit in mice protective immunity against influenza A virus in the presence of CpG ODN adjuvant.

On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts

Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment.The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant β2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay.Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with β2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities.Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.

Vaccination induced antibodies to recombinant avian influenza A virus M2 protein or synthetic M2e peptide do not bind to the M2 protein on the virus or virus infected cells

BackgroundInfluenza viruses are characterized by their highly variable surface proteins HA and NA. The third surface protein M2 is a nearly invariant protein in all Influenza A strains. Despite extensive studies in other animal models, this study is the first to describe the use of recombinant M2 protein and a peptide coding for the extracellular part of the M2 protein (M2e) to vaccinate poultry.MethodsFour groups of layer chickens received a prime-boost vaccination with recombinant M2 protein, M2e, a tetrameric construct from M2e peptide bound to streptavidin and a control tetrameric construct formulated with Stimune adjuvant.ResultsWe determined the M2-specific antibody (Ab) responses in the serum before vaccination, three weeks after vaccination and two weeks after booster, at days 21, 42 and 56 of age. The group vaccinated with the M2 protein in combination with Stimune adjuvant showed a significant Ab response to the complete M2 protein as compared to the other groups. In addition an increased Ab response to M2e peptide was found in the group vaccinated with the M2e tetrameric construct. None of the vaccinated animals showed seroconversion to AI in a commercial ELISA. Finally no Ab’s were found that bound to M2 expressed on in vitro AI infected MDCK cells.ConclusionAlthough Ab’s are formed against the M2 protein and to Streptavidin bound M2e peptide in a tetrameric conformation these Ab’s do not recognize of M2 on the virus or on infected cells.

Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication

BackgroundNovel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies.MethodsFully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking.ResultsHuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages.ConclusionsHuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.

Sublingual immunization with M2-based vaccine induces broad protective immunity (P4301)

Abstract Background: M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.

Enhanced Influenza VLP vaccines comprising matrix-2 ectodomain and nucleoprotein epitopes protects mice from lethal challenge

The matrix protein 2ectodomain (M2e) of the influenza A virus is a rational target antigen candidate for the development of a universal influenza virus-like particle (VLP) vaccine. In this study, a recombinant M2 protein with three tandem copies of M2e (3M2e), nucleoprotein (NP) epitopes and hepatitis B virus core (HBc), were expressed in Escherichia coli and purified by column chromatography. Mice immunized with 3M2e-NP-HBc in combination with an oil-in-water SP01 adjuvant produced robust M2e specific antibodies and cellular immune responses. Most importantly, the 3M2e-NP-HBc VLP vaccine provided enhanced protection against a lethal challenge with pandemic 2009 H1N1 and HPAI H5N1 virus through increased survival rates, a significant decrease in viral replication, and obvious alleviation of histopathological lung changes in challenged mice. Our results imply that a cellular immune response to NP is a plausible mechanism mediating this enhanced protection. These findings suggest that 3M2e-NP-HBc VLP has great potential as the basis development of a broadly protective influenza vaccine.

Purification of recombinant M3 proteins of murine gammaherpesviruses 68 and 72 expressed in Escherichia coli

M3 proteins of 44 kDa of murine gammaherpesviruses 68 and 72 (MHV-68, MHV-72) were identified as herpesvirus vCKBP-3, soluble inhibitors of the host chemokine network providing a selective advantage for the virus by inhibiting the antiviral and inflammatory response during both acute and latent infection. The MHV-72 M3 protein was found to contain a single mutation (Asp307Gly) near its chemokine-binding domain and differ from that of MHV-68 in the ability to bind some human chemokines. In this study, we optimized the expression of his-tagged M3 proteins of MHV-68 and MHV-72 in Escherichia coli and their purification by Ni-NTA chromatography under both denaturing and native conditions. The integrity of the N-terminus of the MHV-72 M3 protein was verified by partial sequencing. The results showed that E. coli is useful for the preparation of native, recombinant M3 proteins of murine gammaherpesviruses in sufficient quantity and purity for further biological studies.