Source: https://patents.google.com/patent/US7399840?oq=3798359
Timestamp: 2018-06-24 13:02:51
Document Index: 456776197

Matched Legal Cases: ['art 1', 'art 1', 'art 1', 'art 3', 'art 3', 'art 3']

US7399840B2 - Proteosome influenza vaccine - Google Patents
US7399840B2
US7399840B2 US10771737 US77173704A US7399840B2 US 7399840 B2 US7399840 B2 US 7399840B2 US 10771737 US10771737 US 10771737 US 77173704 A US77173704 A US 77173704A US 7399840 B2 US7399840 B2 US 7399840B2
US10771737
US20040156867A1 (en )
Live attenuated cold adapted (CAV) influenza vaccines conventionally have been used via the nasal route in humans. These influenza strains are genetic reassortants combining the HA and NA genes of the current strains of flu virus with the 6 genes encoding the other internal and structural proteins from an influenza donor virus adapted to grow at lower temperatures (25° C.) thereby allowing only minimal replication in the nasopharyngal respiratory tract. These vaccines have the advantage of inducing protective immune responses similar to those elicited by natural infection with influenza, including induction of secretory IgA in the nasal washes, interferon gamma production in restimulated PMNC's and activation of CTL specific for internal viral proteins that may broaden the cross-reactivity against viruses within the same sub-type. CAV influenza vaccines are close to commercialization and have been demonstrated to be well-tolerated and immunogenic in children and healthy adults. In recent studies in healthy children, one or two doses of CAV flu vaccine have been shown to induce equivalent systemic antibody as injectable split flu vaccines. The ability of a single dose of CAV to induce >80% protection in seronegative children is an advantage over injectable split vaccines that require two immunizations to achieve similar protection in this age group. While pre-existing circulating antibodies in healthy adults and the elderly prevent efficient seroconversion in these age groups (see below), CAV's have been demonstrated to significantly reduce the number of febrile illnesses, days lost at work and visits to healthcare providers compared with placebo. In the elderly, CAV's in combination with an injectable split subunit vaccine significantly reduced laboratory documented influenza compared to placebo.
EXAMPLE 1 Production of Proteosomes
Outer membrane protein proteosome preparations were purified from Group B type 2 Neisseria meningitides by extraction of phenol-killed bacterial paste with a solution of 6% Empigen BB (EBB) (Albright and Wilson, Whithaven, UK) in 1 M calcium chloride followed by precipitation with ethanol, solubilization in 1% EBB-Tris/EDTA-saline and then precipitation with ammonium sulfate. The precipitates were re-solubilized in the 1% EBB buffer, dialyzed and stored in 0.1% EBB at −70° C. A flow chart of the process (Flowchart 1) is shown on the following pages. Proteosomes may also be prepared by omitting the ammonium sulfate precipitation step to shorten the process (Flowchart 1A). An alternative process that is also successful is shown in Flowchart 1B.
EXAMPLE 2 Preparation of Influenza Antigen (Influenza HA or Flu-HA) Containing Quantified Amounts of Influenza Hemagglutinin (HA)
EXAMPLE 3 Preparation of Proteosome-Influenza HA Vaccine
Portions of stock influenza split product antigens were complexed to and formulated with proteosomes using diafiltration/ultrafiltration methods described in Flowchart 3 or by using dialysis. For either method, the influenza split product was dissolved in saline buffered solution containing the desired detergent e.g. Empigen BB (EBB) at 1% or, at 0.1%-2% of EBB or other suitable detergent depending on the type of detergent used and was then mixed with proteosomes in the saline buffered 1% Empigen solution (or other appropriate detergent at appropriate concentrations as described above) at various proteosome:HA (wt/wt) ratios ranging from 4:1 to 8:1 including 1:4, 1:1, 2:1, 4:1 and 8:1. To remove Empigen, the mixture was then subjected to ultrafiltration/diafiltration technology as described in the Flowchart 3 or was exhaustively dialyzed across a dialysis membrane with a 10,000 Molecular Weight cut-off (MWCO) or functionally similar membranes with MWCO ranges of 1,000-30,000 against buffered saline for 1-2 weeks at 4° C. exchanging at least 500 parts buffer each day.
Multivalent vaccines may be prepared by making individual monovalent proteosome vaccines and then combining them at the required proportions prior to final formulation and fill. Multivalent preparations may also be formulated by pooling individual antigens in the desired proportions and formulating the mixture with proteosomes as outlined in Flowchart 3. Vaccines were passed through membrane filters of 0.8 μm pore size and stored at 4° C. prior to and during the immunizations.
One day prior to the first immunization randomly selected mice were pre-bled. BALB/c mice were immunized intranasally or intramuscularly on days 1 and 21 with antigens in volumes of 25 or 100 μl respectively containing between 0.3 and 10 μg HA A/Taiwan/1/86 or A/Beijing/262/95 as split influenza antigen or A/Texas/36/91 as baculovirus recombinants, alone or formulated with proteosomes (proteosome-flu vaccine or proteosome-rHA) at proteosome:HA ratio's at complex initiation of 1:4, 1:1, 2:1, 4:1 and 8:1 wt/wt. In some examples control mice were given a single intranasal immunization with either phosphate buffered saline or 0.04 LD50 mouse-adapted live influenza A/Taiwan/12/86 on day 1. Animals were bled on days 20 and 35 via the orbital sinus vein or by cardiac puncture. Nasal and lung lavage samples were taken on day 35. The lungs of each mouse were surgically exposed and a canula inserted in the trachea. Using a syringe containing phosphate buffered saline supplemented with 0.1% bovine serum albumin and protease inhibitors (0.2 mM AEBSF, 1 μg/ml Aprotinin, 3.25 μM Bestatin and 10 μM Leupeptin), 1 nasal lavage sample (approximately 1 ml) and 2 lung lavage samples (2×1 ml) were collected. The lung lavage fluids were combined and lavage fluids from individual animals vortexed and centrifuged to remove cell debris and supernatants stored at −70° C. until assayed by ELISA.
EXAMPLE 5 This Example Describes the Serum Hemagglutination Inhibition Assay (HAI)
Prior to determination of HAI activity, mouse or human sera were heated at 56° C. to inactivate complement. Elimination of non-specific agglutination was achieved by treating mouse sera with receptor destroying enzyme (RDE). To 0.1 ml of serum was added 0.4 ml of RDE (100 units/ml) for 12 to 18 hr at 37° C. Three hundred ml of sodium citrate (2.5%) was added for 30 min at 56° C. to inactivate the RDE. The sample volume was made up to 1 ml with PBS (to give final sample dilution of 1:10). Two-fold serial dilutions of each sample were tested for their ability to inhibit the agglutination of 0.5% chick red blood cells by A/Taiwan/1/86 virus in a standard HAI assay.
EXAMPLE 6 This Example Describes the Serum ELISA Assay to Measure Specific Anti Flu Antibodies in Sera, in Lung and Nasal Cavity Fluids
Sera were collected after each immunization; lung and nasal cavity lavage fluids were collected after the last immunization. Nasal wash and lung lavage starting dilutions were 1 in 4 and serum starting dilutions were 1/100. ELISA was performed using whole virus as the detecting antigen. Briefly, 96 well round bottom microtiter plates (Immulon 2, Dynatech, Chantilly, Va.) were coated with antigen and incubated overnight. After aspiration of the antigen using a plate washer, plates were washed once with PBS containing Tween (PBS-T) and incubated with blocking solution containing PBS-T plus plus 2% powdered milk. After aspirating the blocking solution and washing with PBS-T, samples of sera, lung or nasal cavity lavage fluids, serially diluted 2-fold in blocking solution, were added and the plates were incubated for two hours at 37° C. After washing with PBS-T, affinity purified horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or IgA was added and plates were incubated at 37° C. for 30 min. After aspirating and washing twice with PBS-T, developing solution was added and plates were incubated for 15 min at r.t. prior to determining the absorbance values using a microtiter ELISA plate reader (Molecular Devices, Menlo Park, Calif.). Absorbances in the ELISA plate reader were determined at specified times. Antibody titers in the Figures are expressed as ng/ml of specific IgG or IgA determined from a standard curve produced using an ELISA capture assay using affinity purified mouse IgG and IgA standards (Sigma).
EXAMPLE 7 This Example Describes the in vitro Neutralization Assay to Measure Influenza Virus Neutralizing Antibodies in Serum and Lung Lavage Fluids
Neutralization of virus infectivity was determined by direct observation of cell lysis and cytopathic effect (CPE) in MDCK cells. The assay was performed in 96-well plates. Each sample was run in octuplicate. Serial dilutions of test samples (sera or lung lavage fluids) were incubated with 100 TCID50 of live influenza virus homologous to the vaccine strain, incubated for 90 minutes at room temperature and added to 2.4×105 MDCK cells/well. Plates were incubated at 32° C./5% CO2 for the remainder of the assay. Viral neutralization was determined during the virus growth phase (5-7 days of incubation) by evaluation of CPE using an inverted microscope. Neutralizing titers were determined by the Kärber formula (TCID50=Δ−δ(S−0.5)) where “Δ” is the log10 of the dilution with 100% positive cultures, “δ” is the log10 of the dilution factor and “S” is the sum of positive cultures per dilution including those at dilution with 100% infected cultures.
EXAMPLE 8 Evidence of Enhanced Immunogenicity and Immunity as Measured by Enhanced Serum HAI and Virus Specific IgG Titers Elicited by Proteosome-HA Vaccines
Serum IgG (ng/mL) * 188,956 6,006 43,885 50
HAI (GMT) ** 160 20 40 10
Lung IgA (ng/mL) *** 500 20 20 20
Serum IgG (ng/mL) * 373,400 *** 189,600 155,400 81,110
HAI (GMT) ** 320 320 320 320
EXAMPLE 9 This Example Describes the Mouse Immunization Live Virus Challenge Protocols and Results
As shown in FIG. 4 and Table 3, the IgG1/IgG2a ratio was shifted from 14-20 (for Flu antigen alone) down to the 1-2 range when the vaccine contained proteosomes for both nasal and injected vaccines for split flu antigens; and from 6-60 to 1.7 for the baculo HA antigen. This shift of immunity from a Th2 to Th1 response was confirmed for the recombinant HA antigen by measuring cytokines produced after re-stimulating spleen cells from immunized animals with inactivated purified influenza virus. Briefly, Balb/c mice were euthanized 14 days after the second immunization and the spleens from 5 mice from each group were harvested and cells teased into a single cell suspension using a 100-μm nylon cell strainer (Becton Dickinson, N.J.). Spleen cells were cultured at 2.0×106 cells/ml (200 μl/well) in RPMI 1640 medium (Gibco BRL, Life technologies, Burlington, ON) containing 8% fetal bovine serum (heat-inactivated for 1 hr at 56° C.; Gibco BRL), 2 mM glutamine (Gibco BRL), 50 μM 2-mercaptoethanol (Sigma Chemical Co., St-Louis, Mo.) and 50 μg/ml gentamycin (Gibco BRL) with or without UV-inactivated X-113 (A/Texas/36/94 (H1N1) and X-31 (H3N2) reassortant); influenza virus (NIBSC, Hertfordshire, UK) in 96-well cell culture cluster (Corning, N.Y.). Cells were incubated for 72 hrs at 37° C. and supernatants harvested and frozen at −80° C. Murine cytokines levels were measured using sandwich ELISAs (OptEIA set) purchased from Pharmingen (San Diego, Calif.). according to manufacturer's intructions. Recombinant cytokine were used as standards.
G1/ G1/ G1/
G2a* INFγ** IL-5** G2a INFγ IL-5 G2a INFγ IL-5
EXAMPLE 12 Induction of Serum HAI and Nasal Wash sIgA in Humans
Normalized KELISA rate=(specimen KELISA rate×150)÷specimen total sIgA conc.
≧4-fold ≧40 HAI
rises on or titer on or
before before HAI GMT
Treatment Group N day 42 (%) day 42 (%) day 42
15 μg A/Beijing 8 1 (13) 1 (13) 7.7
7.5 μg proteo-flu 8 2 (25) 2 (25) 10.9
15 μg proteo-flu 13 6 (46) 5 (38) 14.5
30 μg proteo-flu 13 7 (54) 6 (46) 21.1
EXAMPLE 13 SDS-PAGE Analysis for Proteosome-HA Vaccine Complexes Demonstrate Complexing of Proteosomes to Influenza-HA Antigen
EXAMPLE 14 Particle Size Analysis of Proteosome-HA Vaccine Complexes Demonstrate Complexing of Proteosomes to Influenza-HA Antigen
EXAMPLE 15 Demonstration of Complexing by Electron Microscopy
18. The method according to claim 1 wherein the influenza vaccine is prepared by a method comprising (a) providing a mixture of the influenza HA antigen with a proteosome preparation in the presence of detergent wherein the ratio of proteosomes to HA antigen is between 2:1 and 8:1; (b) removing the detergent from the mixture by diafiltration or ultrafiltration to obtain a proteosome-HA antigen composition; and (c) formulating the composition into an influenza vaccine.
US10771737 2000-02-15 2004-02-03 Proteosome influenza vaccine Active 2022-02-15 US7399840B2 (en)
US18247600 true 2000-02-15 2000-02-15
US09788280 US6743900B2 (en) 2000-02-15 2001-02-15 Proteosome influenza vaccine
US10771737 US7399840B2 (en) 2000-02-15 2004-02-03 Proteosome influenza vaccine
US12144722 US20080260781A1 (en) 2000-02-15 2008-06-24 Proteosome influenza vaccine
US09788280 Division US6743900B2 (en) 2000-02-15 2001-02-15 Proteosome influenza vaccine
US12144722 Continuation US20080260781A1 (en) 2000-02-15 2008-06-24 Proteosome influenza vaccine
US20040156867A1 true US20040156867A1 (en) 2004-08-12
US7399840B2 true US7399840B2 (en) 2008-07-15
US09788280 Active US6743900B2 (en) 2000-02-15 2001-02-15 Proteosome influenza vaccine
US10771737 Active 2022-02-15 US7399840B2 (en) 2000-02-15 2004-02-03 Proteosome influenza vaccine
US12144722 Abandoned US20080260781A1 (en) 2000-02-15 2008-06-24 Proteosome influenza vaccine
DE (2) DE60121136D1 (en)
WO (1) WO2001060402A3 (en)
US20090252762A1 (en) * 2001-03-09 2009-10-08 Id Biomedical Corporation Of Quebec novel proteosome-liposaccharide vaccine adjuvant
US8709447B2 (en) 2003-10-22 2014-04-29 Id Biomedical Corporation Of Quebec Compositions and methods for activating innate and allergic immunity
DE102014222605A1 (en) 2014-02-20 2015-08-20 Hyundai Motor Company Emergency button for vehicle
EP1578443B1 (en) 2002-11-20 2011-01-12 Bestewil Holding B.V. Compositions comprising antigen-complexes, method for making same as well as methods of using the antigen-complexes for vaccination
JP5274011B2 (en) * 2004-06-25 2013-08-28 アイディー バイオメディカル コーポレイション オブ ケベック Compositions and methods for treating neurological disorders
CA2719201A1 (en) * 2008-03-28 2009-10-01 Sea Lane Biotechnologies, Llc. Neutralizing molecules to viral antigens
WO1998001558A2 (en) 1996-07-10 1998-01-15 Intellivax, Inc. Protein and peptide vaccines for inducing mucosal immunity
Barchfield. et al. Vaccine (1999) 17:695-704.
Benyedidia, et al. Letters in Peptide Science (1998) 5(5-6):341-344.
Berstad, et al. Vaccine (2000) 18:1910-1919.
Crowe et al. Vaccine, 2006, vol. 24, p. 452-456. *
Dalseg, R., et al. Vaccines (1999) 17:2336-2345.
Fynan et al. International Journal of Immunopharmacology, 1995, vol. 17, p. 79-83. *
Gluck. et al. J. Infect. Dis. (2000) 181:1129-1132.
Hashigucci. et al. Vaccino (1996) 14.113-119.
Jahn-Schmidt, Beatrice, et al. "Toward selective elicitation of T<SUB>H</SUB>1-controlled responses: vaccine applications of bacterial surface layer proteins", Journal of Biotechnology (1996) 44:225-231.
Levi et al. vaccine , 1995, vol. 13, p. 1353-1359. *
Levi, R., et al. Vaccine (1995) 13:1353-1358.
Lowall, et al, Intec. & Imm.(1996) 64(11):4686-93.
Lowell, et al, Journal of Infectious diseases (1997) 175(2):292-301.
Lowell, et al. American Soc for microbio, 2nd Nat Conf. (1995) p. 81.
Lowell, et al. Infec. & Imm (1996) 64(5):1706-1713.
Lowell, et al. Journal of cell biochem (1995) S19A.
Lowell, G.H. in: New Generation Vaccines , G.C. Woodrow and M.M. Levine eds., Marcel Dekker, Inc., New York, (1990), p. 141-160; 325-348.
Lowell, G.H., et al. in: New Generation Vaccines . 2nd ed.. Marcel Dekker, Inc. New York, Basil, Hong Kong (1997) pp. 193-206.
Lowell, G.H., et al. J. Exp. Med (1998) 167:658-663.
Lowell, G.H., et al. Science (1955) 240:800-802.
Lynch, E.C., et al. Biophys. J. (1984) 45:104-107.
Mallett, C.P., et al. Infect. Immun. (1995) 63:2382-2388.
McGhee, et al. J. Immunol. (2000) 165:4778-4782.
Orr, N., et al. Infect. Immun. (1993) 61:2390.
Plante, et al. Abstracts of the General Meeting of the American Society for Microbiology. (2000) 100:297.
Slavik, et al. Acta Virologica (1993) 37(6):449-458.
Slavik, et al. Database accession No. 131.57769, XP002174983 (1998).
Tamura, et al. J. Immunol. (1992) 149:981-988.
Wetzler et al., "Characterization and Specificity of Antibodies to Protein I of Neisseria gonorrhoeae Produced by Injection with Various Protein I-Adjuvant Preparations," Journal of Experimental Medicine 168(5):1883-1897, Nov. 1988.
Wetzler et al., "Gonococcal Porin Vaccine Evaluation: Comparison of Por Proteosomes, Liposomes, and Blebs Isolated from rmp Deletion Mutants," Journal of Infectious Diseases 166(3):551-555, Sep. 1992.
Zollinger et al., "Complex of Meningococcal Group B Polysaccharide and Type 2 Outer Membrane Protein Immunogenic in Man," Journal of Clinical Investigation 63(5):836-848, May 1979.
WO2001060402A3 (en) 2002-03-21 application
US20080260781A1 (en) 2008-10-23 application
CA2400468C (en) 2012-12-18 grant
US20010053368A1 (en) 2001-12-20 application
DE60121136D1 (en) 2006-08-10 grant
US6743900B2 (en) 2004-06-01 grant
ES2267724T3 (en) 2007-03-16 grant
DE60121136T2 (en) 2007-06-06 grant
EP1255561B1 (en) 2006-06-28 grant
JP2003522802A (en) 2003-07-29 application
WO2001060402A2 (en) 2001-08-23 application
EP1255561A2 (en) 2002-11-13 application
CA2400468A1 (en) 2001-08-23 application
US20040156867A1 (en) 2004-08-12 application
JP2007051157A (en) 2007-03-01 application
JP3980884B2 (en) 2007-09-26 grant
US20040057962A1 (en) 2004-03-25 Immunogenic complex
Coulter et al. 2003 Intranasal vaccination with ISCOMATRIX® adjuvanted influenza vaccine
Jones et al. 1996 Orally administered microencapsulated Bordetella pertussis fimbriae protect mice from B. pertussis respiratory infection.
Sundquist et al. 1988 Influenza virus ISCOMs: antibody response in animals
WO2002072012A2 (en) 2002-09-19 A novel proteosome-liposaccharide vaccine adjuvant
US6136606A (en) 2000-10-24 Influenza vaccine compositions