Patent Application
20120213820
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The present invention relates to a pharmaceutical composition for the treatment and prevention of a rhinovirus infection.
Rhinoviruses are nonenveloped viruses containing a single-strand RNA genome within an icosahedral capsid. Rhinoviruses belong to the family of Picornaviridae, which includes the genera Enterovirus (polioviruses, coxsackieviruses groups A and B, echoviruses, numbered enteroviruses, parechoviruses) and Hepatovirus (hepatitis A virus). More than 110 serotypes have been identified.
Rhinoviruses are usually transmitted by aerosol or direct contact. The primary site of inoculation is the nasal mucosa, although the conjunctiva may be involved to a lesser extent. Rhinovirus attaches to respiratory epithelium and locally spreads, wherein the major human rhinovirus receptor is inter-cellular adhesion molecule-1 (ICAM-1). The natural response of the human defense system to injury involves ICAM-1, which supports the binding between endothelial cells and leukocytes. Rhinovirus takes advantage of the ICAM-1 by using it as a receptor for attachment.
A local inflammatory response to the virus in the respiratory tract may lead to nasal discharge, nasal congestion, sneezing, and throat irritation. Damage to the nasal epithelium does not occur, and inflammation is mediated by the production of cytokines and other mediators.
Histamine concentrations in nasal secretions do not increase. By days 3-5 of the illness, nasal discharge may become mucopurulent from polymorphonuclear leukocytes that have migrated to the infection site in response to chemoattractants, such as interleukin-8. Nasal mucociliary transport is markedly reduced during the illness and may be impaired for weeks. Both secretory immunoglobulin A and serum antibodies are involved in resolving the illness and protecting from reinfection.
Common colds caused by rhinovirus infection are most frequent from September to April in temperate climates. Rhinovirus infections, which are present throughout the year, account for the initial increase in cold incidence during the fall and for a second incidence peak at the end of the spring season. Several studies demonstrate the incidence of the common cold to be highest in preschool- and elementary school-aged children. An average of 3-8 colds per year is observed in this age group, with an even higher incidence in children who attend daycare and preschool. Because of the numerous viral agents involved and the many serotypes of rhinoviruses, younger children having new colds each month during the winter season is not unusual. Adults and adolescents typically have 2-4 colds per year.
The most common manifestation of rhinovirus, the common cold, is mild and self-limited. However, severe respiratory disease, including bronchiolitis and pneumonia, may occur rarely.
Since early attempts to prevent rhinovirus infections by vaccination have not been successful (Mc Cray et al. Nature 329: 736-738 (1987); Brown et al. Vaccine 9: 595-601 (1991); Francis et al. PNAS USA 87: 2545-2549 (1990)), the current rhinovirus treatment is limited to a symptomatic treatment with analgesics, decongestants, antihistamines and antitussives. Due to the diversity of rhinovirus serotypes and the lack of cross-protection during reinfection with heterologous serotypes a successful prevention by vaccination is considered impossible (Bardin P G, Intern. Med. J. 34 (2004): 358-360). Therefore, the development of respective pharmaceutical compounds is mainly focused on the development of antiviral molecules, such as interferons and synthetic anti-rhinovirus compounds, which could be used therapeutically as well as prophylactically.
WO 2008/057158 relates to vaccines comprising rhinovirus neutralizing immunogen peptides derived from the C-terminal region of the capsid protein VP1 of human rhinovirus. However, some of the peptides disclosed therein are able to induce the formation of antibodies directed to a broad member of rhinovirus serotypes.
In the EP 0 358 485 T cell epitope containing peptides of the VP2 capsid protein of rhinovirus serotype 2 having less than 40 amino acid residues are disclosed.
It is an object of the present invention to provide for the first time a pharmaceutical formulation to be used as vaccine for the treatment or prevention of rhinovirus infections.
The present invention relates to a pharmaceutical composition comprising at least one peptide consisting of a minimum of 8 and a maximum of 50 amino acid residues comprising amino acid residues 1 to 8 of a rhinovirus capsid protein selected from the group consisting of VP1, VP2, VP3 and VP4.
It turned out that peptides derived from rhinovirus capsid proteins VP1, VP2, VP3 and VP4 which comprise the first 8 N-terminal amino acid residues of said capsid proteins are able to induce the in vivo formation of antibodies directed to rhinovirus particles. The at least one peptide may comprise in total 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues. Thus, the at least one peptide comprises amino acid residues 1 to 8, preferably 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, 1 to 49 or 1 to 50 of rhinovirus capsid proteins VP1, VP2, VP3 or VP4.
These peptides can be used in a respective composition in preventing and/or treating a rhinovirus infection.
Another aspect of the present invention relates to a pharmaceutical composition comprising at least one polypeptide (protein) comprising an amino acid sequence consisting of a stretch of at least 80 consecutive amino acid residues of at least one full-length capsid protein of a rhinovirus for preventing and/or treating a rhinovirus infection.
It surprisingly turned out that the administration of at least one polypeptide comprising an amino acid sequence consisting of a stretch of at least 80 consecutive amino acid residues of at least one full-length capsid protein of a rhinovirus induces in an individual the formation of antibodies directed to rhinoviruses, in particular to the capsid proteins of rhinoviruses.
The polypeptides and peptides of the composition of the present invention induce—as mentioned above—the formation of antibodies, in particular, the formation of IgA. IgA plays an important role in mucosal immunity. More IgA is produced in mucosal linings than all other types of antibody combined. In its secretory form, IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, intestinal juice and secretions from the respiratory epithelium. It is also found in small amounts in blood. It was surprisingly found that antibodies of the IgA class are predominantly formed (compared to other anti-body classes) when the peptides according to the present invention are administered to an individual. This shows that the peptides of the present invention allow a superior protection against rhinovirus infections since the primary infection route of rhinoviruses is the respiratory tract, in particular the mucous membranes thereof, and IgA is known to play a critical role in mucosal immunity. The stretch of consecutive amino acid residues may preferably consist of at least 90, 100, 110, 120, 150, 200, 250, 260, 270, 280, 290, or even of all amino acid residues of the at least one full-length capsid protein. In a particular preferred embodiment of the present invention the stretch of consecutive amino acid residues comprises at least 90, in particular 100, amino acid residues.
Rhinoviruses are composed of a capsid that contains four viral proteins VP1, VP2, VP3 and VP4. VP1, VP2, and VP3 form the major part of the protein capsid. Therefore, the preferred capsid protein is VP1, VP2 or VP3.
In a particular preferred embodiment the rhinovirus capsid protein is VP1, preferably VP1 of human rhinovirus 89. A particular preferred polypeptide to be used in the composition of the present invention consists of or comprises the following amino acid sequence: MNPVENYIDSVLNEVLVVPNIQ PSTSVSSHAAPALDAAETGHTSSVQPEDMIET RYVITDQTRDETSIESFLGRSGCIAMIEFNTS SDKTEHDKIGKGFK (SEQ ID NO:7)(amino acid residues 1 to 100 of VP1 of human rhinovirus 89).
Exemplary capsid proteins to be used according to the present invention include VP1 proteins of human rhinovirus strains 1, in particular 1A and 1B, 2, 3, 6, 14, 15, 16, 18, 23, 25, 29, 35, 37, 44, 54, 72, 83, 86, 89, 92 and C. The respective amino acid sequences are identified in the following table:
The most preferred capsid proteins are derived from human rhinovirus 89.
According to a preferred embodiment of the present invention the rhinovirus is a rhinovirus strain selected from the group consisting of rhinovirus strain 89 and rhinovirus strain 14.
Of course, in order to further enhance the crossreactivity of the vaccine of the present invention, one or more other capsid proteins of one or more other rhinovirus serotypes can be used in said vaccine (e.g. VP1 of human rhinovirus 89 in combination with VP1 of human rhinovirus 14).
The capsid proteins of rhinovirus strain 89 and rhinovirus strain 14 show cross reactivity with most of the about 100 known rhinovirus strains. The administration of a polypeptide of the present invention derived from one of said rhinovirus strains induces the formation of antibodies, in particular of IgA, directed against most of the human rhinovirus serotypes. Therefore, it is especially preferred to use polypeptides derived from the capsid proteins of said rhinovirus strains.
According to a particular preferred embodiment the capsid protein is of human rhinovirus 89 and comprises the following amino as well as nucleic acid sequence:
CAT ATG GGC GCC CAG GTG TCT CGT CAG AAC GTC GGC
According to a preferred embodiment of the present invention the amino acid residues 1 to 8 of the rhinovirus capsid protein VP1 have amino acid sequence NPVENYID (SEQ ID NO:50).
The sequence information given herein and known in the prior art allows to determine the peptides preferably used in the present invention. The respective amino acid ranges are mentioned above.
According to a particularly preferred embodiment of the present invention the at least one peptide is selected from the group consisting of NPVENYIDSVLNEVLVVPNIQPSTSVSSHAA (SEQ ID NO:48) and NPVENYIDSVLNEVLVVPNIQ (SEQ ID NO:49).
According to another preferred embodiment of the present invention the peptide according to the present invention is fused or coupled to a carrier.
Suitable carriers include but are not limited to Limulus polyphemus hemocyanin (LPH), Tachypleus tridentatus hemocyanin (TTH), and bovine serum albumin (BSA), tetanus toxoid and diphtheria toxin, DHBcAg, polyribotol ribosyl phosphate (PRP), PncPD11, Maltose Binding Proteins (MBP) and nanoparticle formulations. In one embodiment, a suitable immunogenic carrier protein is Keyhole Limpet Hemocyanin (KLH).
In order to stimulate the immune system and to increase the response to a vaccine, the composition of the present invention comprises at least one at least one pharmaceutical excipient and/or adjuvant.
According to a particularly preferred embodiment of the present invention the adjuvant is alum, preferably aluminum phosphate or aluminum hydroxide, or carbohydrate based particles (CBP).
In order to increase the efficacy of the formulation according to the present invention, all kinds of adjuvants may be used. Preferred adjuvants are, however, aluminum based compounds. Other usable adjuvants include lipid-containing compounds or inactivated mycobacteria. PBC are known, for instance, from the EP 1 356 826.
Alum is known as a Th2 driving adjuvant resulting in the formation of IgG molecules. However, it was surprisingly found that the use of alum in combination with the at least one polypeptide of the present invention results in an induction of IgA rather than IgG. The induction of IgA is particularly advantageous, because IgA is a secretory immunoglobulin found in mucosal secretions and is therefore a first line of defense against an incoming virus.
Generally, adjuvants may be of different forms, provided that they are suitable for administration to human beings. Further examples of such adjuvants are oil emulsions of mineral or vegetal origin, mineral compounds, such as aluminium phosphate or hydroxide, or calcium phosphate, bacterial products and derivatives, such as P40 (derived from the cell wall of Corynebacterium granulosum), monophosphoryl lipid A (MPL, derivative of LPS) and muramyl peptide derivatives and conjugates thereof (derivatives from mycobacterium components), alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, etc. (see, e.g., Gupta R. K. et al. (Vaccine 11:293-306 (1993)) and Johnson A. G. (Clin. Microbiol. Rev. 7:277-289)).
According to another preferred embodiment of the present invention said formulation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said polypeptide. The polypeptide of the present invention is administered to a mammal in these amounts. However, the amount of polypeptide applied is dependent on the constitution of the subject to be treated (e.g. weight). Furthermore, the amount to be applied is also dependent on the route of administration.
According to a further preferred embodiment of the present invention the composition is adapted for intradermal, intramuscular, subcutaneous, oral, rectal, vaginal or epicutaneous administration.
Preferred ways of administration of the formulation of the present invention include all standard administration regimes described and suggested for vaccination in general (oral, transdermal, intraveneous, intranasal, via mucosa, rectal, etc). However, it is particularly preferred to administer the molecules and proteins according to the present invention subcutaneously or intramuscularly.
Another aspect of the present invention relates to a peptide as defined above. In short, the peptide of the present invention consists of a minimum of 8 and a maximum of 50 amino acid residues comprising amino acid residues 1 to 8 of a rhinovirus capsid protein selected from the group consisting of VP1, VP2, VP3 and VP4.
A further aspect of the present invention relates to the use of a polypeptide or peptide as defined above for the manufacture of a medicament for the prevention and/or treatment of a rhinovirus infection.
The medicament is preferably administered intradermally, intramuscularly, subcutaneously, orally, rectally, vaginally or epicutaneously by applying for instance patches.
Human rhinoviruses (HRVs) are the primary cause of acute respiratory tract illness (ARTI) and upper respiratory tract (URT) infections, generally known as the common cold. However, this virus can also replicate in the lower respiratory tract contributing to more severe airway dysfunctions. A significant and increasing body of evidence demonstrates that HRV is responsible for ˜50% of asthma exacerbations and is one of the factors that can direct an infant immune system toward an asthmatic phenotype. Further evidence for HRV involvement in asthma is based on the seasonality of exacerbations. HRV infections occur throughout the year but usually with peaks in spring and autumn. Strong correlations have also been found between seasonal patterns of upper respiratory infections and hospital admissions for asthma.
There is no obvious pattern to the symptoms of HRV infections, so it devolves to the diagnostic laboratory only in order to confirm the presence of HRVs. Disappointingly, the routine screening for HRV strains occurs infrequently because testing is not always available or HRV infection is considered to be harmless. Currently the diagnosis of rhinovirus infections is mainly performed by direct detection of virus by PCR-based methods but positive results are seldom characterized beyond the genus level and are usually reported as ‘respiratory picornaviruses’. The commonly used serodiagnosis based on the strain-specific neutralization of the infection is also impractical for large population studies. Therefore, there exists a need for improving serological techniques for the diagnosis of HRV infections and to determine whether other respiratory diseases such as asthma have been triggered by human rhinoviruses.
Therefore, another aspect of the present invention relates to a method for diagnosing in vitro a rhinovirus infection in a mammal comprising the steps of:
Yet another aspect of the present invention relates to a method for diagnosing in vitro a rhinovirus infection in a mammal comprising the steps of:
Antibodies directed to the capsid proteins (in particular to VP1) of rhinovirus strains 89 and 14 are surprisingly able also to bind to capsid proteins of a broad variety of rhinovirus strains. This surprising fact is used to diagnose a rhinovirus infection caused by any rhinovirus strain in a mammal, preferably in a human. Therefore, the method of the present invention allows to diagnose a rhinovirus infection independent from a specific serotype. The at least one polypeptide has the characteristics as defined above.
The at least one polypeptide according to the present invention is preferably immobilized on a solid support. This allows to bind the antibodies binding to said at least one polypeptide to a solid support and to detect whether the sample analyzed comprises antibodies directed to rhinoviral capsid proteins. The presence of such antibodies allows the diagnosis of a rhinovirus infection.
In the method according to the present invention IgA, IgG, IgM and/or IgE are preferably measured.
According to a preferred embodiment of the present invention the sample is a blood sample, preferably serum or plasma, a sputum sample, neural lavage fluid sample or tear sample.
According to a particularly preferred embodiment of the present invention the capsid protein is VP1, VP2, VP3 or VP4.
Another aspect of the present invention relates to a method for diagnosing in vitro a respiratory disease associated with a rhinovirus infection in a mammal comprising the steps of:
It was found that the presence of antibodies of a specific class/isotype directed to rhinovirus VP1, VP2, VP3 and VP4 polypeptide indicates what kind of respiratory disease an individual may suffer from. Therefore the determination of the antibody class and the antibody specificity allows to diagnose a respiratory disease in an individual. Means and methods for determining the presence of antibodies binding to a specific target are known in the art. Also the determination of the isotype/class of an antibody is known to a person skilled in the art.
According to a preferred embodiment of the present invention the sample is a blood sample, preferably serum or plasma, a sputum sample, neural lavage fluid sample or tear sample.
The fragment of the VP1, VP2, VP3 and/or VP4 polypeptide consists preferably of a minimum of 8 and a maximum of 50 amino acid residues comprising amino acid residues 1 to 8 of a rhinovirus capsid protein selected from the group consisting of VP1, VP2, VP3 and VP4.
According to another preferred embodiment of the present invention the rhinovirus is rhinovirus strain 89 and the capsid protein is VP1.
The amino acid residues 1 to 8 of the rhinovirus capsid protein have preferably amino acid sequence NPVENYID (SEQ ID NO:50).
The fragment is preferably selected from the group consisting of NPVENYIDSVLNEVLVVPNIQPSTSVSSHAA (SEQ ID NO:48) and NPVENYIDSVLNEVLVVPNIQ (SEQ ID NO:49).
The present invention is further illustrated by the following figures and examples, yet, without being restricted thereto.
Blood samples were taken in winter 2006 (win06), spring 2007 (spr), summer 2007 (sum), autumn 2007 (aut) and winter 2007 (win07). Antibody titer was measured by ELISA experiments. ELISA plates (Nunc Maxisorb, Denmark) were coated with 5 μg/ml of VP1 (of rhinovirus strain 89) and incubated with mouse sera diluted 1:50. All experiments were performed in doublets and mean OD were calculated. Bound antibodies were detected with monoclonal mouse anti-mouse human IgA antibodies (BD Pharmingen, San Diego, Calif., USA) diluted 1:1000, and then with rat anti-mouse IgG POX-coupled antibodies (Amersham Bioscience) diluted 1:2000. OD was measured at 405 nm and 490 nm in an ELISA reader (Dynatech, Germany).
The antibody titer varies from season to season and from patient to patient. This leads to the conclusion that exposure to rhinoviruses can be determined by VP1 (
A healthy volunteer was vaccinated with a formulation containing the whole VP1 molecule, a rhinoviral protein, adsorbed to Al(OH)3 (20 μg/injection). This vaccine was injected subcutaneously in the upper arm of the subject three times (Day 0, 21, 42). Before the first vaccination and at days 65, 79, 91, 98 and 119 blood was taken to analyze the development of the antigen-specific immune response.
In
In order to determine the VP1-specific IgA response, a group of five mice was immunized subcutaneously with 5 μg of VP1 antigen adsorbed to aluminum hydroxide in three-week intervals. Serum samples were taken from the tail veins on the day before the first immunization (0) and after the second immunization (6w). VP1-specific IgA antibody levels were determined by ELISA. Plates were coated with 5 μg/ml of the VP1 protein and incubated overnight with mouse serum diluted 1:500. Bound IgA was detected with monoclonal rat anti-mouse IgA antibodies diluted 1:1000 and subsequently with goat anti-rat IgG PDX-coupled antibodies diluted 1:2000, respectively. OD was measured at 405 nm and 490 nm. All ELISA experiments were performed in duplicates, and the mean values were calculated.
Although immunization with recombinant VP1 protein induced VP1-specific IgA response in mice, the increase of antibody level after 6 weeks was not significant (
Materials and Methods
Construction of expression vectors containing the VP1 cDNA of HRV14 or HRV89
The plasmid containing the whole genome of HRV14 (33) was used as a template for the amplification of 14VP1 (VP1 of HRV14) by PCR. The following primers were used:
The restriction sites (NcoI, EcoRI) are underlined. The cDNA coding for the 14VP1 coding region (data base # AY355195) was inserted into the NcoI and EcoRI sites of plasmid pET23d (Novagen, Merck Bioscience, Germany).
Virus stocks of strain 89 were obtained from the collection of the Institute of Virology, Medical University of Vienna. Viral RNA was prepared from cell culture supernatants using the QIAamp viral RNA kit (Qiagen, Germany) and RNase inhibitor (Boehringer GmbH, Germany) was added to a final concentration of 0.01 U/μl. The 89VP1 cDNA (VP1 of HRV89) was amplified by RT-PCR using a SuperScript One Step RT PCR Kit from Invitrogen (USA) using the following primers:
The restriction sites (EcoRI, Asel) are underlined. The cDNA coding for the complete 89VP1 coding region (data base # AY355270) was subcloned into the NdeI and EcoRI site of a pET17b vector (Novagen, Merck Bioscience, Darmstadt, Germany).
Expression and Purification of Recombinant 89VP1 and 14VP1
Recombinant 89VP1 and 14VP1 were expressed in E. coli BL21(DE3) (Stratagene, USA). Protein synthesis was induced for 5 hours at 37° C. with 1 mM IPTG and the recombinant proteins were purified from the inclusion body fraction after solubilization in 6 M guanidinium hydrochloride, 100 mM NaH2PO4, 10 mM Tris, pH 8 using a Ni-NTA affinity matrix (Qiagen, Hilden, Germany). The proteins were washed with washing buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea pH 5.9) and eluted with the same buffer at pH 3.5. Protein preparations were dialyzed against buffers with decreasing urea concentration and finally against H2Odd. Protein purity and concentration were checked by SDS-PAGE and Coomassie blue staining.
Synthetic peptides and peptide conjugates HRV14-derived peptides (PVP1A, PVP1B and PVP3A) were synthesized on the Applied Biosystems (USA) peptide synthesizer Model 433A using a Fmoc (9 fluorenyl methoxy carbonyl) strategy with HBTU [2-(1H-Benzotriazol-1-yl)1,1,3,3 tetramethyluronium hexafluorophosphat] activation. The following peptides were purified to >90% purity by preparative HPLC and their identity was verified by mass spectrometry:
Purified peptides were coupled to KLH using an Imject Maleimide Activated Immunogen Conjugation Kit (Pierce, USA) according to the manufacturer's instruction.
Immunization of Mice and Rabbits
Rabbit antibodies against 14VP1, 89VP1, PVP1A, PVP1B or PVP3A were obtained by immunizing rabbits (Charles River, Kisslegg, Germany). Groups of five mice were also immunized subcutaneously with 5 μg of 89VP1 adsorbed to Alum in three-week intervals and bled from the tail veins.
ELISA Experiments
5 μg/ml 89VP1 or 14VP1 were coated to ELISA plates. The mouse sera were diluted 1:500 and the rabbit sera 1:103-1:106. Antigen-specific IgG1 mouse antibodies were detected with 1:1000 diluted alkaline phosphatase-coupled mouse monoclonal anti-mouse IgG1 antibodies (Pharmingen). Antigen-specific rabbit IgG anti-bodies were developed with a 1:2000 dilution of donkey anti-rabbit IgG peroxidase-coupled antibodies (Amersham Bioscience). The ODs corresponding to bound antibodies were measured at 405 nm and 490 nm for rabbit antibodies and at 405 nm and 450 nm for mice antibodies in an ELISA reader (Dynatech, Germany).
Reactivity of Anti-VP1 Antibodies with Blotted Rhinovirus Extract and Rhinovirus
Cell culture supernatants from HRV-infected HeLa cells were centrifuged in a bench fuge (15.000 rpm, 10 min, 20° C.) to remove insoluble particles. Then, 0.5 ml PEG (40% v/v polyethylene glycol 6000, 2.4% w/v NaCl, pH 7.2) was added to 2 ml of virus-containing supernatant. The solution was incubated at 4° C. over night and then centrifuged at 2,300× rpm for 45 minutes in a bench fuge at RT. The pellet was re-suspended in 100 μl PBS and lysed in 50 μl SDS sample buffer. 10 μl of this HRV14 protein extract and 0.5 μg purified 14VP1 were separated by 12% SDS PAGE and blotted onto nitrocellulose membranes. Identically prepared blots were incubated with 1:500 dilutions of rabbit anti-14VP1 antibodies or the corresponding pre-immune Ig. Bound antibodies were detected with 125I-labelled donkey anti-rabbit IgG and visualized by autoradiography.
For immunogold electron microscopy, 4.2 μl aliquots of the re-suspended viral precipitate were pipetted onto carbon-coated, plasma-cleaned copper grids and air-dried. After 5 minutes, remaining liquid was removed with a piece of filter paper. The grids were then incubated face down (moist chamber at room temperature) in the following buffers: First, PBS containing 1% (w/v) BSA at pH 7.4 and then Tris buffer containing 1% (w/v) BSA at pH 8.2.
Then the following incubation steps were done: (a) 5% (w/v) BSA, 5 min; (b) protein G-purified anti-VP11 g or pre-immune Ig adjusted to an OD280 nm of 0.6, 45 min; (c) 6× PBS buffer, 5 seconds each; (d) 6× Tris buffer, 5 seconds each; (e) goat anti-rabbit Ig coupled to colloidal gold particles with a diameter of 10 nm (Plano, Wetzlar, Germany), diluted 1:20 in Tris buffer, 30 min; (f) 6× Tris buffer, 5 seconds each; (g) 6× distilled water, 5 seconds each. After labelling, negative staining was performed by pipetting a saturated solution of uranyl acetate on the grids. After 1 minute, surplus negative stain was removed with a wet filter paper. The grids were then dried on air and viewed in a Philips EM 410 transmission electron microscope equipped with a high resolution CCD camera. Micrographs were taken at a magnification of 165,000× or 240,000×.
HRV Neutralization Test
Rhinovirus stocks and the HRV-sensitive “Ohio” strain of HeLa cells (Stott E J and Tyrrell D A, Arch. Gesamte Virusforsch. 1968; 23:236-244.) were used. HeLa cells were seeded in 24 well plates and grown to approximately 90% confluence. In a first set of experiments, 300 μl aliquots of HRV14 (100 TCID50) in medium were incubated for 2 h at 37° C. with 300 μl of rabbit anti-sera (anti-14VP1, anti-PVP1A, anti-PVP1B or PVP3A) or the corresponding pre-immune sera (undiluted or diluted 1:2-1:32) and added to the cells. MEM-Eagle medium (Invitrogen, USA) containing 1% FCS and 40 mM MgCl2 was used as a diluent in the experiments. Plates were incubated at 34° C. in a humidified 5% CO2 atmosphere and viable cells were stained with crystal violet after three days. Cross-neutralization tests were carried out in 96 well plates; HeLa cells were seeded in minimal essential medium (MEM) containing 2% fetal calf serum, 30 mM MgCl2, and 1 mM glutamine (infection medium) and grown over night at 37° C. to about 70% confluency. HRVs (100 TCID50 in 100 μl infection medium) were mixed with 100 μl of the respective undiluted antiserum and serial twofold dilutions thereof in the same medium. After incubation for 3 h at 37° C., the cells were overlaid with these solutions and incubation was continued at 34° C. for 3 days. The medium was removed and cells were stained with crystal violet (0.1% in water) for 10 min. After washing with water, the plate was dried, the stain was dissolved in 30 μl 1% SDS under shaking for 1 hour and cell protection was quantified as OD at 560 nm in a plate reader.
Results
Expression and Purification of Recombinant VP1 Proteins from HRV89 and HRV14
Recombinant VP1 of HRV89 (89VP1;
89VP1 and 14VP1 induce a VP1-specific immune response in animals. Immunization of rabbits with recombinant 89VP1 and 14VP1 induced VP1-specific IgG responses (
Reactivity of Antibodies Raised Against Recombinant VP1 Proteins Toward Virus-Derived VP1 and Entire Virions
The reactivity of antibodies induced by immunization with recombinant VP1 proteins with natural, virus-derived VP1 and whole virus was studied by immunoblotting and electron microscopy, respectively. As a representative example, binding of rabbit anti-14VP1 antibodies and of pre-immune Ig to nitrocellulose-blotted HRV14 proteins and 14VP1 is shown
Immunization of Rabbits with Recombinant 14VP1 Yields Higher 14VP1- and 89VP1-Specific Antibody Titers than Immunization with KLH-Coupled HRV14-Derived Peptides
Antisera were raised against KLH-coupled peptides which have been earlier described as possible vaccine candidates. The anti-peptide antisera contained high titers of peptide-specific anti-bodies (PVP1A:10−3; PVP1B:10−5; PVP3A:10−5). However, in comparison with antisera raised against recombinant 14VP1, they reacted only weakly with the 14VP1 protein and showed weak cross-reactivity with 89VP1 (
14VP1-Specific Antibodies Inhibit HRV Infection of HeLa Cells Better than Peptide-Specific Antibodies
Next, it was investigated whether rabbit IgG antibodies raised against recombinant 14VP1 protein can inhibit HRV infection of HeLa cells. Results from one set of cell protection experiments performed with HRV14 are shown in
Also the ability of antibodies raised against complete 14VP1 with antibodies raised against 14VP1-derived peptides for protection of the cells against viral infection was analyzed. Serial dilutions (undiluted or diluted 1:2-1:32) of anti-14VP1, -PVP1A, -PVP1B or -PVP3A antisera were incubated together with HRV14 and added to HeLa cells. The ability to inhibit cell infection of all three anti-peptide antisera was comparable amongst each other. A clear reduction in CPE was seen at a dilution of 1:8 with anti-PVP1A and anti-PVP1B and at a dilution of 1:4 with anti-PVP3A. A similar degree of inhibition of infection (i.e., partial CPE) was obtained with the anti-14VP1 antiserum up to dilution of 1:32. This suggests that the latter antiserum was approximately 8-fold more potent in inhibiting viral infections (Table 1).
In table 1 the neutralization of infection by antibodies raised against 14VP1 and HRV14 derived peptides is shown. A dilution of anti-14VP1, anti-PVP1A, anti-PVP1B or anti-PVP3A anti-bodies (undiluted or diluted 1:2-1:32) were preincubated with 100 TCID50 HRV14 and added to HeLa cells. Virus neutralizations and cytopathic effects (CPE) observed are indicated: +++: complete neutralization; ++: minimal CPE; +: partial CPE; +/−: almost complete CPE; − complete CPE.
Antibodies Raised Against Recombinant VP1 Proteins Show Cross-Protection Against Distantly Related HRV Strains.
A vaccine protecting against rhinovirus infections may be useful to reduce rhinovirus-induced asthma exacerbations. The HRV-derived VP1 capsid protein was investigated as a potential vaccine antigen for several reasons. The work of Rossmann et al., elucidating the crystal structure of HRV14, demonstrates that VP1 is critically involved in HRV binding to its receptor on human epithelial cells. It was found that five copies of VP1 form a depression, called canyon and that the ICAM-1 receptor binds into the central part of this canyon. Furthermore, studies of spontaneous mutations in the viral coat led to the identification of four neutralizing immunogenic (NIm) sites on the surface of HRV14. Additional investigations revealed that antibodies to two of the four antigenic sites which are located on the VP1 protein blocked cellular attachment.
The complete VP1 proteins from HRV89 and HRV14, which belong to the phylogenetically distant species HRV-A and HRV-B, respectively, were expressed in E. coli and purified afterwards. Using the ClustalW program for alignment (http://www.ebi.ac.uk/clustalw) only a 45% nucleotide and 41% amino acid identity could be found between 89VP1 and 14VP1. Recombinant 14VP1 and 89VP1 were purified via a C-terminal His-tag by Nickel affinity chromatography in a single step procedure. Immunization of mice and rabbits with recombinant 14VP1 as well as 89VP1 proteins led to the development of VP1-specific anti-body responses recognizing natural VP1 from the virus and even intact virus as demonstrated by immunogold electron microscopy.
The antibody responses obtained with the VP1 proteins were compared with those induced by HRV14 VP1- and VP3-derived peptides which had been earlier described as vaccine candidates and with those obtained with a peptide PVP1B located at the C-terminus of the VP1 protein, being part of the ICAM-1 attachment site in HRV14. It was found that the anti-HRV14 VP1 antisera reacted much stronger with VP1 than the anti-peptide antisera and exhibited a higher neutralization titer. The higher neutralization capacity of the antibodies raised against the complete proteins is most likely due to the fact that the antiserum raised against the complete protein recognizes several different epitopes on the VP1 protein and hence may exhibit a higher avidity than the peptide-specific antibodies.
There is a relatively low degree of sequence identity of 45% at the nucleotide and 41% at the amino acid level between 89VP1 and 14VP1. Yet it was found that antibodies raised against the recombinant VP1 proteins from each of these strains inhibited the infection of cultured HeLa cells by a variety of different rhinovirus strains belonging to the major and minor group. The latter finding may be important because it indicates that it may be possible to engineer a broadly cross-protective and effective vaccine against HRV by combining VP1 proteins from a few rhinovirus strains. The efficacy of such a vaccine may be also improved by the addition of other capsid proteins such as VP2, VP3, and/or VP4. The latter one has recently gained attention as it has also elicited cross-protection.
Major advantages of a vaccine based on recombinant rhinovirus capsid proteins are that the vaccine antigens can be easily produced under controlled conditions by large scale recombinant expression in foreign hosts, such as E. coli at reasonable costs. A broadly cross-protective HRV vaccine may be especially useful for the vaccination of patients suffering from rhinovirus-induced asthma attacks and may thus reduce asthma exacerbations.
The cDNAs coding for VP1, VP2, VP3 and VP4 of HRV89 were codon optimized for Escherichia coli and synthetically synthesized with the addition of six histidine residues at the 3′ end. The complete genes were inserted into the NdeI/XhoI fragment of multiple cloning site of pET-27b (ATG Biosynthetics, Germany). The resulting constructs are referred to as vectors p89VP1, p89VP2, p89VP3 and p89VP4 and gene products VP1, VP2, VP3 and VP4. The DNA sequences of VP1, VP2, VP3 and VP4 were confirmed by nucleotide sequencing and double digestion and are shown in
In order to achieve the expression of recombinant capsid proteins, Escherichia coli strains BL21 (DE3) were transformed with p89VP1, p89VP2, p89VP3 or p89VP4, respectively, and plated on LB plates containing 100 μg/ml kanamycin. A single colony was used to inoculate 250 ml LB medium containing 100 mg/L kanamycin. This culture was grown to an optical density (600 nm) of 0.6 and the protein expression was induced by adding IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation at 3500 rpm at 40 C for 10 min. Purification was performed with the Qiagen protocol using Ni-NTA. The cell pellet was resuspended under denaturing conditions in 10 ml 6M guanidine hydrochloride for 4 hours. After centrifugation (20 min, 18 000 rpm) the supernatant was incubated with 2 ml Ni-NTA for an additional 2 hours. The suspension was then loaded onto a column, washed twice with 10 ml wash buffer (8 M Urea, 100 mM NaH2PO4, 10 mM Tris-HCl pH 6.1) and then eluted with 12 ml elution buffer (8 M Urea, 100 mM NaH2PO4, 10 mM Tris-HCl pH 3.5. Renaturation was achieved after dialysis with decreasing molarity of urea.
Purity and size of the recombinant proteins were analyzed by SDS-PAGE as shown in
The cDNA coding for VP1 was used as a template for the amplification of VP1-derived epitopes by PCR (Table I). The EcoRI and BamHI restriction sites (underlined in Table I) of the pMalc4X vector were used for the insertion of PCR products down-stream of the malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of MBP fusion proteins (New England BioLabs). The insertion of cDNAs for VP1-derived epitopes was confirmed by nucleotide sequencing and the gene products are referred to as Ep—1, Ep—2 and Ep—3 (
M N P V E N Y I D S V L N E V L V V P N I Q P S T
Recombinant fusion proteins (Ep—1, Ep—2, Ep—3) were expressed in E. coli strain BL21 (DE3) as described in Example 6. The purification was performed using MBP's affinity for maltose. The inclusion body fraction was solubilized with 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-HCl and dialyzed against the Column Buffer (20 mM Tris, 200 mM NaCl, 1 mM EDTA pH=7.4). The clear lysate was loaded onto an equilibrated amylose resin affinity column, washed twice with 60 ml Column Buffer and eluted with 20 ml Column Buffer containing 10 mM Maltose.
Identity of the fusion proteins was confirmed by Western blot analysis using anti-VP1 rabbit antiserum.
To investigate the occurrence of VP1-, VP2- VP3- and VP4-specific antibodies in human blood, enzyme linked immunosorbent assay (ELISA) was performed. ELISA plates (Nunc) were coated with 5 μg/ml of recombinant rhinovirus-derived capsid proteins (VP1, VP2, VP3, VP4) and human serum albumin (HSA) was used as a control. The whole blood from 57 HRV-positive patients was diluted 1:50. Bound human IgA, IgM, IgG1 and IgG2 (BD Pharmingen) 1:1000 were detected with sheep anti-mouse peroxidase-coupled (Amersham Bioscience) 1:2000. The optical value (OD 405 nm) is displayed on the y-axis and corresponds to the level of VP1-, VP2—VP3- and VP4-specific antibodies in human blood (
In order to evaluate whether recombinant capsid proteins of HRV89 and VP1-derived epitopes cross-react with a variety of different rhinovirus strains, ELISA plates were coated with 5 μg/ml of each antigen. Guinea pig sera raised against twenty seven rhinovirus strains, belonging to different species and different receptor groups, were diluted 1:1000. Antigen-specific IgG were detected with 1:2000 diluted goat anti-guinea pig peroxidase-coupled antibodies (Jackson ImmunoResearch). The OD's corresponding to bound antibodies were measured at 405 nm and 490 nm in an ELISA reader. Anti-HRV89 serum and anti-quinea pig serum were used as controls (Sigma) (
A high anti-VP1 IgG titer could be detected in sera raised against almost a half of the strains tested and an enhanced anti-Ep—1 IgG titer was found in sera with a high anti-VP1 antibody level. These findings have important implications for the diagnosis of HRV infections, especially in the context of airway diseases, because they show that VP1 and its epitopes located mostly within the N-terminus of the entire protein are recognized not only by anti-guinea pig sera raised against the major group but also by sera raised against the minor group rhinoviruses.
In order to investigate whether it is possible to find a correlation between VP1-, VP2-, VP3- and VP4-specifc antibody responses and different clinical manifestations, a single variant analysis using the ‘Mann-Whitney’ test was used (p values <0.05 were considered positive). The following clinical data were considered:
A significant statistical connection was found among:
Next, a multi-variant analysis was performed. Basically, in this test clinical data were grouped with various ways and then compared with the antibody values like in the single variant tests. The only 2 groups that gave a correct hypothesis (p <0.05) were the following:
Group 1:
asthma/bronchiolitis/convulsion/croup
Group 2:
Asthma, bronchiolitis, viral positive PCR, convulsion, croup
Group 2 produced various statistical significant results. These were mostly affected by the presence of the viral double infection factor which seemed to be very important throughout the single and multi-variant analyses. For VP2-specific IgM there was a connection between viral double infection and convulsion, while for VP1-specific IgA a relationship between viral double infection and asthma was found.
Furthermore, it was found that antibody levels might be used as a biological marker for the HRV/Influenza double infection.
Based on these results, it is assumed that it will be possible to develop serological tests for the diagnosis of rhinovirus infections and their association with respiratory illnesses.
Recombinant VP1 of the HRV89 has been found to be the most immunologically important surface protein in human blood samples (
The amino acid sequences referred to in this example and in
It was investigated whether rabbit IgG antibodies raised against recombinant VP1, VP2, VP3 and VP4 capsid proteins can inhibit HRV infection of HeLa cells. Results from one set of experiments are shown in
This suggests that not only antibodies raised against VP1, but also VP2 and VP4, are able to protect HeLa cells from HRV infection (
In order to confirm the specificity of the rabbit anti-HRV14VP1 and anti-HRV89VP1 anti-sera an ELISA experiment using purified recombinant VP1 proteins from HRV14 and 89 as well as three recombinant fragments of HRV89-derived VP1 was performed. It has been found that anti-HRV14VP1 antibodies cross-reacted with VP1 and three VP1 fragments spanning aa 1-100 (see example 12), aa 101-200 and aa 201-293 but had a much lower titer than the anti-HRV89VP1 antibodies. In this context, it is noteworthy, that the anti-VP1 anti-sera obtained by immunization with VP1 from HRV14 and HRV89 differentially reacted with these recombinant fragments of 89VP1. It thus appears that this latter anti-serum contains IgGs reacting with many more epitopes than the anti-serum raised against 14VP1. Furthermore, it confirms the assumption that using VP1-derived fragments it is possible to detect anti-bodies directed against distantly related rhinovirus species.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AT10/00416 | 11/2/2010 | WO | 00 | 4/27/2012 |
