(19) &
(11) EP 1 593 392 B1
(12) EUROPEAN PATENT SPECIFICATION
(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 39/205 (2006.01) A61K 39/42 (2006.01) 28.09.2011 Bulletin 2011/39 C07K 16/10 (2006.01)
(21) Application number: 04023168.0
(22) Date of filing: 29.09.2004
(54) Rabies vaccine Tollwut-Impfstoff Vaccin antirabique
(84) Designated Contracting States: • LODMELL D L ET AL: "Post-exposure DNA AT BE BG CH CY CZ DE DK EE ES FI FR GB GR vaccination protects mice against rabies virus" HU IE IT LI LU MC NL PL PT RO SE SI SK TR VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD,GB, vol. 19, no. 17-19, 21 March 2001 (30) Priority: 07.05.2004 US 569172 P (2001-03-21), pages 2468-2473, XP004231067 ISSN: 0264-410X (43) Date of publication of application: • SHIMAZAKI Y ET AL: "Immune response to 09.11.2005 Bulletin 2005/45 Japanese rabies vaccine in domestic dogs." JOURNAL OF VETERINARY MEDICINE SERIES (73) Proprietor: Novartis Vaccines and Diagnostics B, vol. 50, no. 2, March 2003 (2003-03), pages GmbH 95-98, XP002306648 ISSN: 0931-1793 35041 Marburg (DE) • ROOIJAKKERS E J M ET AL: "Potency of veterinary rabies vaccines in the Netherlands: A (72) Inventors: case for continued vigilance" VETERINARY • Banzhoff, Angelika, Dr. QUARTERLY, vol. 18, no. 4, 1996, pages 146-150, 35039 Marburg (DE) XP008038725 ISSN: 0165-2176 • Malerczyk, Claudius, Dr. • LODMELL D L ET AL: "Rabies cell culture 35039 Marburg (DE) vaccines reconstituted and stored at 4
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• ANONYMOUS: "Intradermal application of rabies • BRIGGS D J ET AL: "Antibody response of vaccines" REPORT OF A WHO CONSULTATION, patients after postexposure rabies vaccination [Online] 2000, XP002306653 Retrieved from the with small intradermal doses of purified chick Internet: URL:http://www.who.int/emc- embryo cell vaccine or purified Vero cell rabies documents/rabie s/docs/ vaccine" BULLETIN OF THE WORLD HEALTH whocdscsraph2005.pdf> [retrieved on 2004-11] ORGANIZATION, vol. 78, no. 5, 2000, pages • MADHUSUDANA S N ET AL: "Simulated post- 693-698, XP008038719 ISSN: 0042-9686 exposure rabies vaccination with purified chick • "IMOVAX Rabies"[Online] April 2000 (2000-04), embryo cell vaccine using a modified Thai Red XP002306655 Retrieved from the Internet: URL: Cross regimen." INTERNATIONAL JOURNAL OF http://www.aventispasteur.ca/avp_conte nt/ INFECTIOUS DISEASES : IJID : OFFICIAL docs/ca_products/IMOVAX_E.pdf> [retrieved on PUBLICATION OF THE INTERNATIONAL 2004-11] SOCIETY FOR INFECTIOUS DISEASES. MAY • JENKINSET AL.: "Compendium of Animal Rabies 2004, vol. 8, no. 3, May 2004 (2004-05), pages Prevention and Control, 2003" INTERNET 175-179, XP002306654 ISSN: 1201-9712 -& ARTICLE, [Online] 2004, XP002306656 Retrieved MADHUSUDANA S N: "Simulated post- exposure from the Internet: URL:http://www.avma.org/ rabies vaccination: authors’ response to Wilde et pubhlth/rabcont.as p> [retrieved on 2004-11] al" INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, INTERNATIONAL SOCIETY FOR INFECTIOUS DISEASES, HAMILTON, CA, vol. 8, no. 6, November 2004 (2004-11), page 376, XP004604576 ISSN: 1201-9712 -&WILDE H ET AL: "Simulated post-exposure rabies vaccination: comments on article by Madhusudana et al" INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, INTERNATIONAL SOCIETY FOR INFECTIOUS DISEASES, HAMILTON, CA, vol. 8, no. 6, November 2004 (2004-11)
2 1 EP 1 593 392 B1 2
Description BRIEF SUMMARY OF THE INVENTION
[0001] The invention provides for an immunogenic ra- [0004] Applicants have surprisingly discovered that bies vaccine comprising a reduced vaccine dose and sufficient neutralizing antibody titers can be achieved by methods of post-exposure immunization with a reduced 5 immunization with a substantially reduced rabies virus dose. The concentration of rabies vaccine antigen per vaccine antigen concentration. In particular, rabies virus dose is less than 2.5 IU/mL. For example, the concen- vaccine antigen concentrations of substantially less than tration of rabies vaccine antigen may be less than half, the current WHO recommended 2.5 IU/mL can provide less than one fourth, or less than one eighth of 2.5 IU/mL. neutralizing antibody titers similar to those generated Preferably, the concentration of rabies vaccine antigen 10 with the standard dose. is between 2.4 IU/mL and 0.25 IU/mL, more preferably [0005] The invention provides for a composition com- between 2.0 IU/mL and 1.0 IU/mL and even more pref- prising a rabies virus vaccine, wherein the concentration erably between 1.75 IU/mL and 1.25 IU/mL, wherein 1.5 of the rabies virus vaccine antigen is less than 2.5 IU/mL. IU/mL is most preferred. This means, the concentration For example, the concentration of the vaccine antigen of the rabies virus vaccine antigen in the composition can 15 may be less than half, less than one fourth, or less than be 2.2 IU/mL, 1.8 IU/mL, 1.6 IU/mL, 1.4 IU/mL, 1.2 IU/mL, one eighth of 2.5 IU/mL. In other words, the concentration 0.8 IU/mL, 0.6 IU/mL, 0.4 IU/mL, 0.3 IU/mL, or 0.25 of the rabies virus vaccine antigen in the composition is IU/mL. The composition is a single-dose unit having a preferably less than 1.25 IU/mL, more preferably less volume of 0.1 mL, the vaccines of the invention are ad- than 0.625 IU/mL, and even more preferably less than ministered intradermally as part of a Thai Red Cross20 0.3125 IU/mL. Preferably, the concentration of rabies (TRD) or the modified Thai Red Cross regimen. vaccine antigen is between 2.4 IU/mL and 0.25 IU/mL, more preferably between 2.0 IU/mL and 1.0 IU/mL and BRIEF DESCRIPTION OF THE FIGURES even more preferably between 1.75 IU/mL and 1.25 IU/mL, wherein 1.5 IU/mL is most preferred. Preferably, [0002] 25 the composition is a single-dose unit having a volume of 0.1 mL. This means, the concentration of the rabies virus Figure 1 shows a schematic depiction of the post- vaccine antigen in the composition can be 2.2 IU/mL, 1.8 exposure 2-site intradermal regimen (also known as IU/mL, 1.6 IU/mL, 1.4 IU/mL, 1.2 IU/mL, 0.8 IU/mL, 0.6 Thai Red Cross Regimen). Doses of 0.1 mL PCECV IU/mL, 0.4 IU/mL, 0.3 IU/mL, or 0.25 IU/mL. Preferably, are administered intradermally at two different sites 30 the rabies virus vaccine is produced in a continuous cell of the patient’s body. Optionally, rabies immunoglob- line such as a purified chicken embryo cell line, a purified ulin can be given together with the first vaccine dose. vero cell line, or a human diploid cell line. Processes and means for the production of rabies antigen which make BACKGROUND OF THE INVENTION use of these cell lines are described, for example, in "Vac- 35 cines", Plotkin and Orenstein (eds.), 2004, Chapter 37, [0003] Despite the availability of modern cell culture "Rabies Vaccine", by Plotkin, Rupprecht, Koprowski. rabies vaccines, human rabies deaths continue to occur [0006] The vaccines of the invention are given thera- in developing countries where canine rabies is endemic peutically as part of a complete post exposure regimen and the cost of post- exposure prophylaxis is unaffordable and include administration intradermally. for the majority of the population. Current WHO recom- 40 mendations recommend a concentration of rabies virus DETAILED DESCRIPTION OF THE INVENTION vaccine derived from cell culture vaccines of at least 2.5 IU/mL. To overcome the financial burden, the WHO has [0007] The practice of the present invention will em- recommended two intradermal (ID) post- exposure rabies ploy, unless otherwise indicated, conventional methods treatment regimens that have been successfully imple- 45 of chemistry, biochemistry, molecular biology, immunol- mented in several countries (cf. WHO recommendations ogy and pharmacology, within the skill of the art. Such on Rabies Post-Exposure Treatment and the Correct techniques are explained fully in the literature. See, e.g., Technique of Intradermal Immunization against Rabies Remington’s Pharmaceutical Sciences, Mack Publishing [WHO/ECM/ZOO/96.6], WHO Doc 1996.). The more Company, Easton, Pa., 19th Edition (1995); Methods In economic of these regimens is the Thai Red Cross ID 2- 50 Enzymology (S. Colowick and N. Kaplan, eds., Academic site (TRC) regimen. The TRC regimen, using 0.1 mL per Press, Inc.); and Handbook of Experimental Immunolo- ID dose, reduces the cost of vaccine by up to 80%. Pu- gy, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, rified chick embryo cell vaccine (PCECV, such as Rabi- Blackwell Scientific Publications); Sambrook, et al., Mo- pur®) has been proven to be highly immunogenic, well lecular Cloning: A Laboratory Manual (2nd Edition, tolerated and efficacious in this regimen. Despite the re- 55 1989); Handbook of Surface and Colloidal Chemistry duced cost for these ID regimens, a need for less expen- (Birdi, K.S. ed., CRC Press, 1997); Short Protocols in sive rabies immunization, particularly in developing Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, countries, persists. John Wiley & Sons); Molecular Biology Techniques: An
3 3 EP 1 593 392 B1 4
Intensive Laboratory Course, (Ream et al., eds., 1998, [0011] Alternatively, the IU of a rabies vaccine dose Academic Press); PCR (Introduction to Biotechniques may be determined using an antibody binding test Series), 2nd ed. (Newton & Graham eds., 1997, Springer (ABBT), which measurement is then converted into Verlag); Peters and Dalrymple, Fields Virology (2d ed), IU/mL bycomparison with theinternational standard. See Fields et al. (eds.), B.N. Raven Press, New York, NY. 5 Barth, "The modified antibody binding test for in vitro [0008] The rabies virus belongs to the family Rhab- qualification of rabies virus antigen in inactivated rabies doviridae, genus Lyssavirus and comprises an envel- vaccines", in Laboratory Techniques in Rabies (Meslin oped virion with a single, nonsegmented, negative- and Kaplan, eds.) WHO, Geneva 1998, p. 392 - 4. stranded RNA. The rabies virus is bullet-shaped with [0012] The rabies inactivated vaccine international trimers of a the rabies glycoprotein (G) projecting to the 10 standard may be obtained, for example, from the Inter- exterior of the virus. This G protein is thought to be the national Laboratory for Biological Standards, Statens Se- major antigen responsible for inducing production of neu- ruminstitute, Copenhagen, Denmark. tralizing antibodies and for conferring immunity against [0013] The vaccines of the invention may be derived lethal infection with rabies virus. from any of the known tissue or cell culture techniques [0009] As used herein, the term "International Units" 15 for rabies known in the art, including for example those or "IU" of rabies virus vaccine antigen refers to the inter- used with mammalian cells, baculoviruses, bacteria, or national potency concentration measurement used by, yeast. Suitable cell lines may be used to cultivate the for example, the WHO and other international health reg- rabies virus or to express polynucleotides encoding ra- ulatory agencies (see for example, European Pharma- bies viral antigens (such as the G protein). copoeia, 01/2005:0216, Rabies vaccine for human use 20 [0014] Preferably, the rabies virus is cultivated in cells prepared in cell cultures,. International Units indicate the of mammalian origin. Such cell lines include but are not predicted potency of a vaccine composition and can be limited to: human or non-human primate cells (e.g. MRC- measured using standard assays. For example, the ra- 5 (ATCC CCL-171), WI-38 (ATCC CCL-75), human em- bies vaccine antigen concentration of a sample inactivat- bryonic kidney cells (293 cells, typically transformed by ed rabies virus vaccine can be calculated using a stand- 25 sheared adenovirus type 5 DNA), VERO cells from mon- ard assay to measure the potency of the test vaccine in key kidneys), horse, cow (e.g. MDBK cells), sheep, dog mouse challenge experiments compared to the potency (e.g. MDCK cells from dog kidneys, ATCC CCL34 MDCK of a publicly available international reference standard. (NBL2) or MDCK 33016, deposit number DSM ACC 2219 These standard assays include the NIH mouse potency as described in WO97/37001), cat, and rodent (e.g. ham- test and the European Pharmacopoeia test see( WHO 30 ster cellssuch as BHK21- F,HKCC cells, or Chinese ham- Expert Committee on Biological Standardization. Thirty- ster ovary cells (CHO cells)), and may be obtained from seventh report. Geneva, World Health Organization, a wide variety of developmental stages, including for ex- WHO Technical Report Series, (1987) No. 760 and ample, adult, neonatal, fetal, and embryo. In certain em- (1994) No. 840. bodiments the cells are immortalized (e.g. PERC. 6 cells, [0010] For example, groups of at least ten mice, aged 35 as described in WO01/38362 and WO02/40665, and as 3 - 4 weeks, are inoculated with single, decreasing doses deposited under ECACC deposit number 96022940). of vaccine (in accordance with the European Pharmaco- [0015] In preferred embodiments, mammalian cells poeia) or with two doses, 1 week apart (in accordance are utilized, and may be selected from and/or derived with the NIH test). A sufficient number of dilutions of vac- from one or more of the following non- limiting cell types: cine are compared to estimate the dilution at which 50% 40 fibroblast cells (e.g. dermal, lung), endothelial cells (e.g. of the mice are protected against intracerebral challenge aortic, coronary, pulmonary, vascular, dermal microvas- 14 days later (50% protective dose = PD50). A WHO in- cular, umbilical), hepatocytes, keratinocytes, immune ternational standard vaccine is available to compare the cells (e.g. T cell, B cell, macrophage, NK, dendritic), titration curve of the test vaccine to that of the IU standard, mammary cells(e.g. epithelial), smooth muscle cells(e.g. 45 i.e., to ensure that the PD 50 of the test vaccine is at least vascular, aortic, coronary, arterial, uterine, bronchial, equivalent to the IU standard. The following parameters cervical, retinal pericytes), melanocytes, neural cells should be met in order for the titration curve calibration (e.g. astrocytes), prostate cells (e.g. epithelial, smooth to be valid: (i) for both the test vaccine and the interna- muscle), renal cells (e.g. epithelial, mesangial, proximal tional standard, the PD50 should lie between the largest tubule), skeletal cells (e.g. chondrocyte, osteoclast, os- and the smallest doses given to the mice; ( ii) the titration 50 teoblast), muscle cells (e.g. myoblast, skeletal, smooth, of the challenge virus suspension should show that 0.03 bronchial), liver cells, retinoblasts, and stromal cells. mL of the suspension contained at least 10 LD50 (the WO97/37000 and WO97/37001 describe production of LD50 should be in the range of 12 - 50 for a valid test); animal cells and cell lines that are capable of growth in (iii) the confidence interval (p = 0.95) for the test is not suspension and in serum free media and are useful in less than 25% and not more than 400% of the estimated 55 the production and replication of viruses. potency (i.e., statistical analysis shows a significant slope [0016] Preferred rabies virus vaccine cultivation sub- and no significant deviations from linearity or parallelism strates include purified chick embryo cells, purified vero of the dose/response lines). cells, human diploid cells, rhesus diploid cells, hamster
4 5 EP 1 593 392 B1 6 kidney cells, and duck embryo cells. Rabies virus vac- Pichia pastoris, Schizosaccharomyces pombe and Yar- cines for use in the invention include purified chicken rowia lipolytica. embryo cell vaccines (PCECV) such as Rabipur® (ob- [0020] Nucleic acid molecules comprising nucleotide tainable from Chiron-Behring, Marburg, Germany) and sequences of the viral antigens or antibodies of the in- RabAvert® obtainable from Chiron Corp., Emeryville, 5 vention can be stably integrated into a host cell genome USA; purified vero cell vaccine (PVRV) such as Vero- or maintained on a stable episomal element in a suitable rab™ (obtainable from Aventis Pasteur) and Immovax- host cell using various gene delivery techniques well Rabies vero™ (obtainable from Aventis Pasteur); chro- known in the art. See., e.g., US Patent No. 5,399,346. matographically purified Vero cell culture rabies vaccine [0021] Depending on the expression system and host (CPRV); human diploid cell vaccine (HDCV) such as Ra- 10 selected, the molecules are produced by growing host bivac® (obtainable from from Chiron-Behring, Marburg, cells transformed by an expression vector under condi- Germany); rhesus diploid cell culture vaccine (RVA) such tions whereby the protein is expressed. The expressed as fetal rhesus monkey lung fibroblast cultures form Bi- protein is then isolated from the host cells and purified. oPort; primary Hamster Kidney Cell Culture Vaccine If the expression system secretes the protein into growth (PHKCV); and purified duck embryo vaccine (PDEV)15 media, the product can be purified directly from the me- such as Lyssavax N™. Preferred rabies virus vaccines dia. If it is not secreted, it can be isolated from cell lysates. for use in the invention include PCECV, PVRV, and HD- The selection of the appropriate growth conditions and CV. Processes and means for the production of rabies recovery methods are within the skill of the art. antigen which make use of these cell lines are described, [0022] The rabies vaccines of the invention preferably for example, in "Vaccines", Plotkin and Orenstein (eds.), 20 comprise inactivated rabies virus cultivated in mamma- 2004, Chapter 37, "Rabies Vaccine", by Plotkin, Rup- lian cells. General methods of inactivating or killing virus- precht, Koprowski. es to destroy their ability to infect mammalian cells are [0017] In one embodiment, a PCECV inactivated ra- known in the art. Such methods include both chemical bies vaccine is prepared by cultivation of the Flury low and physical means. Chemical means for inactivating a egg passage (LEP) rabies strain in primary chick embryo 25 virus include treatment with an effective amount of one fibroblasts (see Example 1). The virus may be cultivated or more of the following agents: detergents, formalde- in a synthetic cell culture medium, optionally also includ- hyde, β-propiolactone, or UV light. Additional chemical ing human albumin, polygeline and antibiotics. The virus means for inactivation include treatment with methylene is then inactivated by approximately 0.025%β -propiol- blue, psoralen, carboxyfullerene (C60) or a combination actone, concentrated and purified by zonal centrifugation 30 of any thereof. Other methods of viral inactivation are in a sucrose density gradient, and lyophlizied. Further known in the art, such as for example binary ethylamine, discussion of commonly used cell cultures for rabies virus acetyl ethyleneimine, or gamma irradiation. The pre- cultivation can be found in Vaccines, Plotkin and Oren- ferred method of inactivation for rabies virus is treatment stein eds., 4th Ed. 2004, Chapter 37 (pp 1011 - 1038, with β-propiolactone. see in particular pp 1018 - 1022). 35 [0023] The rabies vaccines of the invention may com- [0018] Alternatively, the vaccines of the invention may prise further components such as human serum albumin, be cultivated in bacterial, insect, or plant cell culture sys- gelatin, phenolsulfonphthalein, or antibiotics such as ne- tems. Insect cell expression systems, such as baculovi- omycin, chlortetracycline, and amphotericin B. rus systems, are known to those of skill in the art and [0024] The vaccines of the invention may be described in, e.g., Summers and Smith, Texas Agricul- 40 lyophlilized to a powder form for storage or shipment, tural Experiment Station Bulletin No. 1555 (1987). Ma- and reconstituted in sterile water for immunization. terials and methods for baculovirus/ insert cell expression [0025] The vaccines of the invention are preferably systems are commercially available in kit form from, inter stored at cool temperatures, preferably 2° to 8° C. alia, Invitrogen, San Diego CA. Insect cells for use with [0026] The vaccines of the invention may be prepared baculovirus expression vectors include, inter alia, Aedes 45 using one or more concentration techniques such as zon- aegypti, Autographa californica, Bombyx mori, Dro- al centrifugation, density gradient centrifugation, ultrafil- sophila melanogaster, Spodoptera frugiperda, and Tri- tration, and chromatography. choplusia ni. [0027] The invention includes use of reduced concen- [0019] Similarly, bacterial and mammalian cell expres- tration rabies virus vaccines in post- exposure dosing reg- sion systems are also known in the art and described in, 50 imens by immunization via intradermal administration. e.g., Yeast Genetic Engineering (Barr et al., eds., 1989) The intradermal doses generally comprise a total volume Butterworths, London. Bacterial hosts such asE. coli, of 0.1 mL. Bacillus subtilis, and Streptococcus spp., could be used [0028] The invention provides for a method of immu- to express constructs encoding rabies viral antigens. nizing a subject with a rabies vaccine comprising injecting Yeast hosts useful in the present invention include, inter 55 the subject with one or more doses of rabies vaccine, alia, Saccharomyces cerevisiae, Candida albicans, wherein the concentration of the dose of rabies vaccine Candida maltosa, Hansenual polymorpha, Kluyveromy- antigen is less than 2.5 IU/mL. For example, the concen- ces fragilis, Kluyveromyces lactis, Pichia guillerimondii, tration of the vaccine antigen may be less than half, less
5 7 EP 1 593 392 B1 8 than one fourth, or less than one eighth of 2.5 IU/mL. costs of vaccinating can be considerably reduced by ad- Preferably, the concentration of rabies vaccine antigen ministering the vaccines of the invention. For example, is between 2.4 IU/mL and 0.25 IU/mL, more preferably only two 0.1 mL doses mL at two sites (such as the upper between 2.0 IU/mL and 1.0 IU/mL and even more pref- arm or over each deltoid) on days 0, 3 and 7 are sufficient erably between 1.75 IU/mL and 1.25 IU/mL, wherein 1.5 5 to produce a protective antibody titer of 0.5 IU/mL or IU/mL is most preferred. This means, the concentration more. Preferably, these doses can have an antigen con- of the rabies virus vaccine antigen in the composition can centration of about 1.5 IU/mL, 1.0 IU/mL or even 0.5 be 2.2 IU/mL, 1.8 IU/mL, 1.6 IU/mL, 1.4 IU/mL, 1.2 IU/mL, IU/mL or 0.25 IU/mL. Alternative regimens comprise two 0.8 IU/mL, 0.6 IU/mL, 0.4 IU/mL, 0.3 IU/mL, or 0.25 0.1 mL doses on days 0, 5 and 8, or single 0.1 mL doses IU/mL. 10 on days 0, 2, 4, 6 or on days 0, 3, 5, 7. [0029] Post-exposure dosing regimens are adminis- [0033] In the dosing regimens of the invention requiring tered intradermally. Preferred post- exposure rabies vac- intradermal administration, the intradermal dose volume cines include PCECV, HDCV or RVA vaccines. The in- is about 0.1 mL. The rabies virus vaccine antigen con- vention thus provides for a method of post-exposure im- centration per dose in intradermal dosing regimens is munization against rabies infection comprising immuniz- 15 consequently less than 2.5 IU/mL (i.e., less than 0.25 IU/ ing a subject who has been exposed to rabies infection 0.1mL). Preferably the rabies vaccine antigen concen- with a rabies vaccine, wherein the concentration of the tration in an intradermal dosing regimen is less than one dose of rabies vaccine antigen is less than 2.5 IU/mL. half the concentration of 2.5 IU/mL (i.e., less than 0.13 Preferably, the concentration of rabies vaccine antigen IU/0.1mL), less than one fourth the concentration of 2.5 is between 2.4 IU/mL and 0.25 IU/mL, more preferably 20 IU/mL (i.e., less than 0.06 IU/mL), or less than one eighth between 2.0 IU/mL and 1.0 IU/mL and even more pref- the concentration of 2.5 IU/mL (i.e., less than 0.03 IU/ erably between 1.75 IU/mL and 1.25 IU/mL, wherein 1.5 0.1mL). IU/mL is most preferred. This means, the concentration [0034] The reduced dose rabies vaccines of the inven- of the rabies virus vaccine antigen in the composition can tion generate neutralizing antibody titers in the subject be 2.2 IU/mL, 1.8 IU/mL, 1.6 IU/mL, 1.4 IU/mL, 1.2 IU/mL, 25 suitable for meeting international regulatory standards. 0.8 IU/mL, 0.6 IU/mL, 0.4 IU/mL, 0.3 IU/mL, or 0.25 Preferably, the vaccines of the invention provide for sub- IU/mL. The vaccine is administered in 0.1 mL doses. ject antibody titers greater than 0.5 IU/mL. Measurement [0030] Intradermal post-exposure dosing regimens for of neutralizing antibody titers in rabies vaccine immu- use in the invention include a two site intradermal method nized subjects is known in the art and may be accom- known as ("2-2-2-0-1-1"; the numerical abbreviation re- 30 plished by, for example, the rapid fluorescent focus inhi- fers to the numbers of doses administered on days bition test (RFFIT). 0,3,7,14,28, and 90; i.e. the number of doses adminis- [0035] Post-exposure immunization regimens should tered on the administration days scheduled in accord- be accompanied by administration of rabies immune ance with the standard WHO intramuscular regimen). globulin (RIG). Preferably, RIG is administered as a sin- The "2-2-2-0-1-1" regimen is also known as the Thai Red 35 gle dose of 20 IU per kg of body weight for human RIG Cross regimen or TRC-ID. This regimen comprises in- or as a single dose of 40 IU per kg of body weight for tradermal administration of 0.1 mL at two sites (such as heterologous (equine) RIG. This single dose of RIG is the upper arm or over each deltoid) at each of days 0, 3 preferably given at the same time as the first dose of and 7. This regimen further comprises intradermal ad- vaccine. If RIG is unavailable at day 0, it may be admin- ministration of 0.1 mL at one site (such as the upper arm) 40 istered up to day 7. on days 28 and 90. [0036] For subjects given pre-exposure vaccine regi- [0031] A modified version of the TRC- ID is designated mens, a post exposure boost of vaccine given on days ("2-2-2-0-2"). This regimen comprises intradermal immu- 0 and 3 is generally recommended. Such post exposure nization of 0.1 mL at two sites on days 0, 3, 7 and 28 boosts could be intramuscular or intradermal doses. (with the 90 day booster being omitted). 45 [0037] Subjects suitable for immunization with the vac- [0032] For example, the rabies vaccine according to cines of the invention are humans. the invention may be administered after contact with an [0038] Also disclosed herein is the use of a rabies virus animal which might be rabid. According to the WHO rec- antigen for the preparation of a medicament for the treat- ommendations for Rabies Postexposure Prophylaxis ment of rabies virus infection, wherein the concentration (WHO Expert Committee on Rabies; World Organisation 50 of rabies virus antigen is between 2.4 IU/mL and 0.25 Tech. Rep. Ser. 824: 1-84 (1992)), vaccination should be IU/mL, preferably between 2.0 IU/mL and 1.0 IU/mL, performed immediately after an animal suspected or con- more preferably between 1.75 IU/mL and 1.25 IU/mL, firmed of being rabid (upon contact with a human) caused and most preferably 1.5 IU/mL. Preferably, this treatment minor scratches or abrasions without bleeding, single or is a post-exposure treatment. Further, it is preferred that multiple transdermal bites or scratches, or even after nib- 55 the medicament is administered via intradermal admin- bling of uncovered skin. However, post-exposure treat- istration. The medicament preferably is to be adminis- ment can be stopped after a 10 days- observation period tered in at least two doses in a period of ten days after in which the animal remains healthy. In these cases, the contact with the animal. Preferably, the medicament is
6 9 EP 1 593 392 B1 10 to be administered in at least three, four, five, six or seven "MF59 -- Design and Evaluation of a Safe and Potent doses in a period of ten days after after contact with the Adjuvant for Human Vaccines" in Vaccine Design: The animal. Usally, the doses have a volume of 0.1 mL. Subunit and Adjuvant Approach (Powell, M.F. and New- [0039] While the use of adjuvants are not required to man, M.J. eds.) Plenum Press, New York, 1995, pp. obtain sufficient levels of neutralizing antibody titers, the 5 277-296). MF59 contains 4-5% w/v Squalene (e.g. reduced vaccine doses of the invention may be supple- 4.3%), 0.25-0.5% w/v Tween 80™, and 0.5% w/v Span mented with one or more adjuvants. Adjuvants for use 85™ and optionally contains various amounts of MTP- with the invention include, but are not limited to, one or PE, formulated into submicron particles using a micro- more of the following set forth below: fluidizer such as Model 110Y microfluidizer (Microfluid- 10 ics, Newton, MA). For example, MTP-PE may be present A. Mineral Containing Compositions in an amount of about 0-500 Pg/dose, more preferably 0-250 Pg/dose and most preferably, 0-100 Pg/dose. As [0040] Mineral containing compositions suitable for used herein, the term "MF59-0" refers to the above sub- use as adjuvants in the invention include mineral salts, micron oil-in-water emulsion lacking MTP-PE, while the such as aluminum salts and calcium salts. The invention 15 term MF59-MTP denotes a formulation that contains includes mineral salts such as hydroxides (e.g. oxyhy- MTP-PE. For instance, "MF59-100" contains 100P g droxides), phosphates (e.g. hydroxyphosphates, ortho- MTP-PE per dose, and so on. MF69, another submicron phosphates), sulfates, etc. (e.g. see chapters 8 & 9 of oil-in-water emulsion for use herein, contains 4.3% w/v Vaccine Design... (1995) eds. Powell & Newman. ISBN: squalene, 0.25% w/v Tween 80™, and 0.75% w/v Span 030644867X. Plenum.), or mixtures of different mineral 20 85™ and optionally MTP- PE. Yet another submicron oil- compounds (e.g. a mixture of a phosphate and a hydrox- in-water emulsion is MF75, also known as SAF, contain- ide adjuvant, optionally with an excess of the phosphate), ing 10% squalene, 0.4% Tween 80™, 5% pluronic- with the compounds taking any suitable form (e.g. gel, blocked polymer L121, and thr- MDP, also microfluidized crystalline, amorphous, etc.), and with adsorption to the into a submicron emulsion. MF75- MTP denotes an MF75 salt(s) being preferred. The mineral containing composi- 25 formulation that includes MTP, such as from 100-400 Pg tions may also be formulated as a particle of metal salt MTP-PE per dose. (WO00/23105). [0044] Submicron oil-in-water emulsions, methods of [0041] Aluminum salts may be included in vaccines of making the same and immunostimulating agents, such the invention such that the dose of Al3+ is between 0.2 as muramyl peptides, for use in the compositions, are and 1.0 mg per dose. 30 described in detail in International Publication No. WO90/14837 and US Patent Nos. 6,299,884 and 6,45 B. Oil-Emulsions 1,325,incorporated herein by reference in their entireties. [0045] Complete Freund’s adjuvant (CFA) and incom- [0042] Oil-emulsion compositions suitable for use as plete Freund’s adjuvant (IFA) may also be used as ad- adjuvants in the invention include squalene- water emul- 35 juvants in the invention. sions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles C. Saponin Formulations using a microfluidizer). See WO90/14837. See also, Podda, "The adjuvanted influenza vaccines with novel [0046] Saponin formulations, may also be used as ad- adjuvants: experience with the MF59-adjuvanted vac- 40 juvants in the invention. Saponins are a heterologous cine", Vaccine (2001) 19: 2673-2680. MF59 is used as group of sterol glycosides and triterpenoid glycosides the adjuvant in the FLUAD™ influenza virus trivalent sub- that are found in the bark, leaves, stems, roots and even unit vaccine. flowers of a wide range of plant species. Saponin from [0043] Particularly preferred adjuvants for use in the the bark of the Quillaia saponaria Molina tree have been compositions are submicron oil-in- water emulsions. Pre- 45 widely studied as adjuvants. Saponin can also be com- ferred submicron oil-in-water emulsions for use herein mercially obtained from Smilax ornata (sarsaprilla), Gyp- are squalene/water emulsions optionally containing var- sophilla paniculata (brides veil), and Saponaria officia- ying amounts of MTP-PE, such as a submicron oil-in- nalis (soap root). Saponin adjuvant formulations include water emulsion containing 4-5% w/v squalene,purified formulations, such as QS21, as well as lipid for- 0.25-1.0% w/v Tween 80™ (polyoxyelthylenesorbitan 50 mulations, such as ISCOMs. monooleate), and/or 0.25-1.0% Span 85™ (sorbitan tri- [0047] Saponin compositions have been purified using oleate), and, optionally, N-acetylmuramyl-L-alanyl-D- High Performance Thin Layer Chromatography (HP- LC) isogluatminyl-L-alanine-2-(1’-2’-dipalmitoyl-sn-glycero- and Reversed Phase High Performance Liquid Chroma- 3-huydroxyphosphophoryloxy)-ethylamine (MTP-PE), tography (RP-HPLC). Specific purified fractions using for example, the submicron oil-in- water emulsion known 55 these techniques have been identified, including QS7, as "MF59" (International Publication No. WO90/14837; QS17, QS18, QS21, QH- A, QH-B and QH- C. Preferably, US Patent Nos. 6,299,884 and 6,451,325, incorporated the saponin is QS21. A method of production of QS21 is herein by reference in their entireties; and Ott et al., disclosed in US Patent No. 5,057,540. Saponin formula-
7 11 EP 1 593 392 B1 12 tions may also comprise a sterol, such as cholesterol system in the intranasal trivalent INFLEXAL™ product (see WO96/33739). {Mischler & Metcalfe (2002) Vaccine 20 Suppl 5: B17-23} [0048] Combinations of saponins and cholesterols can and the INFLUVAC PLUS™ product. be used to form unique particles called Immunostimulat- ing Complexs (ISCOMs). ISCOMs typically also include 5 E. Bacterial or Microbial Derivatives a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in [0052] Adjuvants suitable for use in the invention in- ISCOMs. Preferably, the ISCOM includes one or more clude bacterial or microbial derivatives such as: of Quil A, QHA and QHC. ISCOMs are further described in EP0109942, WO96/11711 and WO96/33739. Option- 10 1. Non-toxic derivatives of enterobacterial lipopoly- ally, the ISCOMS may be devoid of additional detergent. saccharide (LPS) See WO00/07621. Such derivatives include Monophosphoryl lipid A [0049] A review of the development of saponin based (MPL) and 3- O-deacylated MPL (3dMPL). 3dMPL is adjuvants can be found at Barr, et al., "ISCOMs and other a mixture of 3 De-O-acylated monophosphoryl lipid saponin based adjuvants", Advanced Drug Delivery Re- 15 A with 4, 5 or 6 acylated chains. A preferred "small views (1998) 32: 247-271. See also Sjolander, et al., "Up- particle" form of 3 De-O-acylated monophosphoryl take and adjuvant activity of orally delivered saponin and lipid A is disclosed in EP 0 689 454. Such "small ISCOM vaccines", Advanced Drug Delivery Reviews particles" of 3dMPL are small enough to be sterile (1998) 32:321-338. filtered through a 0.22 micron membrane (see EP 0 20 689 454). Other non-toxic LPS derivatives include D. Virosomes and Virus Like Particles (VLPs) monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. [0050] Virosomes and Virus Like Particles (VLPs) can See Johnson et al. (1999) Bioorg Med Chem Lett 9: also be used as adjuvants in the invention. These struc- 2273-2278. tures generally contain one or more proteins from a virus 25 2. Lipid A Derivatives optionally combined or formulated with a phospholipid. Lipid A derivatives include derivatives of lipid A from They are generally non-pathogenic, non-replicating and Escherichia coli such as OM-174. OM-174 is de- generally do not contain any of the native viral genome. scribed for example in Meraldi et al., "OM- 174, a New The viral proteins may be recombinantly produced or iso- Adjuvant with a Potential for Human Use, Induces a lated from whole viruses. These viral proteins suitable 30 Protective Response with Administered with the for use in virosomes or VLPs include proteins derived Synthetic C-Terminal Fragment 242-310 from the from influenza virus (such as HA or NA), Hepatitis B virus circumsporozoite protein of Plasmodium berghei", (such as core or capsid proteins), Hepatitis E virus, mea- Vaccine (2003) 21:2485-2491; and Pajak, et al., sles virus, Sindbis virus, Rotavirus, Foot-and- Mouth Dis- "The Adjuvant OM-174 induces both the migration ease virus, Retrovirus, Norwalk virus, human Papilloma 35 and maturation of murine dendritic cells in vivo", Vac- virus, HIV, RNA-phages, Qβ-phage (such as coat pro- cine (2003) 21:836-842. teins), GA-phage, fr-phage, AP205 phage, and Ty (such 3. Immunostimulatory oligonucleotides as retrotransposon Ty protein p1). VLPs are discussed Immunostimulatory oligonucleotides suitable for use further in WO03/024480, WO03/024481, and Niikura et as adjuvants in the invention include nucleotide se- al., "Chimeric Recombinant Hepatitis E Virus- Like Parti- 40 quences containing a CpG motif (a sequence con- cles as an Oral Vaccine Vehicle Presenting Foreign taining an unmethylated cytosine followed by gua- Epitopes", Virology (2002) 293:273-280; Lenz et al., nosine and linked by a phosphate bond). Bacterial "Papillomarivurs-Like Particles Induce Acute Activation double stranded RNA or oligonucleotides containing of Dendritic Cells", Journal of Immunology (2001) palindromic or poly(dG) sequences have also been 5246-5355; Pinto, et al., "Cellular Immune Responses to 45 shown to be immunostimulatory. Human Papillomavirus (HPV)-16 L1 Healthy Volunteers The CpG’s can include nucleotide modifications/ Immunized with Recombinant HPV-16 L1 Virus- Like Par- analogs such as phosphorothioate modifications ticles", Journal of Infectious Diseases (2003) 188: and can be double- stranded or single-stranded. Op- 327-338; and Gerber et al., "Human Papillomavrisu Vi- tionally, the guanosine may be replaced with an an- rus-Like Particles Are Efficient Oral Immunogens when 50 alog such as 2’-deoxy-7-deazaguanosine. See Coadministered with Escherichia coli Heat- Labile Enter- Kandimalla, et al., "Divergent synthetic nucleotide toxin Mutant R192G or CpG", Journal of Virology (2001) motif recognition pattern: design and development 75(10):4752-4760. Virosomes are discussed further in, of potent immunomodulatory oligodeoxyribonucle- for example, Gluck et al., "New Technology Platforms in otide agents with distinct cytokine induction profiles", the Development of Vaccines for the Future", Vaccine 55 Nucleic Acids Research (2003) 31(9): 2393-2400; (2002) 20:B10 -B16. WO02/26757 and WO99/62923 for examples ofpos- [0051] Immunopotentiating reconstituted influenza vi- sible analog substitutions. The adjuvant effect of rosomes (IRIV) are used as the subunit antigen delivery CpG oligonucleotides is further discussed in Krieg,
8 13 EP 1 593 392 B1 14
"CpG motifs: the active ingredient in bacterial ex- Interferon after Coapplication onto Bare Skin", Infec- tracts?", Nature Medicine (2003) 9(7): 831-835; Mc- tion and Immunity (2002) 70(6):3012-3019; Pizza, Cluskie, et al., "Parenteral and mucosal prime-boost et al., "Mucosal vaccines: non toxic derivatives of LT immunization strategies in mice with hepatitis B sur- and CT as mucosal adjuvants", Vaccine (2001) 19: face antigen and CpG DNA", FEMS Immunology and 5 2534-2541; Pizza, et al., "LTK63 and LTR72, two Medical Microbiology (2002) 179-185; 32: mucosal adjuvants ready for clinical trials" Int. J. WO98/40100; US Patent No. 6,207,646; US Patent Med. Microbiol (2000) 290(4-5):455-461; Scharton- No. 6,239,116 and US Patent No. 6,429,199. Kersten et al., "Transcutaneous Immunization with The CpG sequence may be directed to TLR9, such Bacterial ADP-Ribosylating Exotoxins, Subunits and as the motif GTCGTT or TTCGTT. See Kandimalla, 10 Unrelated Adjuvants", Infection and Immunity (2000) et al., "Toll-like receptor 9: modulation of recognition 68(9):5306-5313; Ryan et al., "Mutants of Es- and cytokine induction by novel synthetic CpG cherichia coli Heat- Labile Toxin Act as Effective Mu- DNAs", Biochemical Society Transactions (2003) 31 cosal Adjuvants for Nasal Delivery of an Acellular (part 3): 654-658. The CpG sequence may be spe- Pertussis Vaccine: Differential Effects of the Nontox- cific for inducing a Th1 immune response, such as 15 ic AB Complex and Enzyme Activity on Th1 and Th2 a CpG- A ODN, or it may be more specific for inducing Cells" Infection and Immunity (1999)(12): 67 a B cell response, such a CpG-B ODN. CpG-A and 6270-6280; Partidos et al., "Heat-labile enterotoxin CpG-B ODNs are discussed in Blackwell, et al., of Escherichia coli and its site-directed mutant "CpG-A-Induced Monocyte IFN-gamma-Inducible LTK63 enhance the proliferative and cytotoxic T- cell Protein-10 Production is Regulated by Plasmacytoid 20 responses to intranasally co-immunized synthetic Dendritic Cell Derived IFN-alpha", J. Immunol. peptides", Immunol. Lett. (1999) (3): 67209-216; (2003) 170(8):4061-4068; Krieg, "From A to Z on Peppoloni et al., "Mutants of the Escherichia coli CpG", TRENDS in Immunology (2002) 23 (2): 64-65 heat-labile enterotoxin as safe and strong adjuvants and WO01/95935. Preferably, the CpG is a CpG-A for intranasal delivery of vaccines", Vaccines (2003) ODN. 25 2(2):285-293; and Pine et al., (2002) "Intranasal im- Preferably, the CpG oligonucleotide is constructed munization with influenza vaccine and a detoxified so that the 5’ end is accessible for receptor recogni- mutant of heat labile enterotoxin from Escherichia tion. Optionally, two CpG oligonucleotide sequences coli (LTK63)" J. Control Release (2002) (1-3): 85 may be attached at their 3’ ends to form "immuno- 263-270. Numerical reference for amino acid substi- mers". See, for example, Kandimalla, et al., "Sec- 30 tutions is preferably based on the alignments of the ondary structures in CpG oligonucleotides affect im- A and B subunits of ADP- ribosylating toxins set forth munostimulatory activity", BBRC (2003) 306: in Domenighini et al., Mol. Microbiol (1995) 15(6): 948-953; Kandimalla, et al., "Toll-like receptor 9: 1165-1167, specifically incorporated herein by ref- modulation of recognition and cytokine induction by erence in its entirety. novel synthetic GpG DNAs", Biochemical Society 35 Transactions (2003) 31(part 3):664-658; Bhagat et F. Bioadhesives and Mucoadhesives al., "CpG penta-and hexadeoxyribonucleotides as potent immunomodulatory agents" BBRC (2003) [0053] Bioadhesives and mucoadhesives may also be 300:853-861 and WO03/035836. used as adjuvants in the invention. Suitable bioadhesives 4. ADP-ribosylating toxins and detoxified deriva- 40 include esterified hyaluronic acid microspheres (Singh tives thereof. et al. (2001) J. Cont. Rele. 70:267-276) or mucoadhe- Bacterial ADP-ribosylating toxins and detoxified de- sives such as cross-linked derivatives of poly(acrylic ac- rivatives thereof may be used as adjuvants in the id), polyvinyl alcohol, polyvinyl pyrollidone, polysaccha- invention. Preferably, the protein is derived from E. rides and carboxymethylcellulose. Chitosan and deriva- coli (i.e., E. coli heat labile enterotoxin "LT), cholera 45 tives thereof may also be used as adjuvants in the inven- ("CT"), or pertussis ("PT"). The use of detoxified tion. E.g. WO99/27960. ADP-ribosylating toxins as mucosal adjuvants is de- scribed in WO95/17211 and as parenteral adjuvants G. Microparticles in WO98/42375. Preferably, the adjuvant is a detoxi- fied LT mutant such as K63, LT- LT-R72, and 50 [0054] Microparticles may also be used as adjuvants LTR192G. The use of ADP-ribosylating toxins and in the invention. Microparticles (i.e. a particle of ~100nm detoxified derivaties thereof, particularly LT-K63 and to ~150Pm in diameter, more preferably ~200nm to LT-R72, as adjuvants can be found in the following ~30Pm in diameter, and most preferably ~500nm to references, each of which is specifically incorporated -10pm in diameter) formed from materials that are bio- by reference herein in their entirety: Beignon, et55 degradable and non-toxic (e.g. a poly(α-hydroxy acid), al., "The LTR72 Mutant of Heat-Labile Enterotoxin a polyhydroxybutyric acid, a polyorthoester, a polyanhy- of Escherichia coli Enahnces the Ability of Peptide dride, a polycaprolactone, etc.), with poly (lactide-co-gly- Antigens to Elicit CD4+ T Cells and Secrete Gamma colide) are preferred, optionally treated to have a nega-
9 15 EP 1 593 392 B1 16 tively-charged surface (e.g. with SDS) or a positively- of aspects of one or more of the adjuvants identified charged surface (e.g. with a cationic detergent, such as above. For example, the following adjuvant compositions CTAB). may be used in the invention:
H. Liposomes 5 (1) a saponin and an water oil-in- emulsion (WO99/11241); [0055] Examples of liposome formulations suitable for (2) a saponin (e.g.., QS21) + a non- toxic LPS deriv- use as adjuvants are described in US Patent No. ative (e.g. 3dMPL) (see WO94/00153); 6,090,406, US Patent No. 5,916,588, and EP 0 626 169. (3) a saponin (e.g.., QS21) + a non- toxic LPS deriv- 10 ative (e.g. 3dMPL) + a cholesterol; I. Polyoxyethylene ether and Polyoxyethylene Ester For- (4) a saponin (e.g. QS21) + 3dMPL + IL-12 (option- mulations ally + a sterol) (WO98/57659); (5) combinations of 3dMPL with, for example, QS21 [0056] Adjuvants suitable for use in the invention in- and/or oil-in-water emulsions (See European patent clude polyoxyethylene ethers and polyoxyethylene es- 15 applications 0835318, 0735898 and 0761231); ters. WO99/52549. Such formulations further include (6) SAF, containing 10% Squalane, 0.4% Tween 80, polyoxyethylene sorbitan ester surfactants in combina- 5% pluronic-block polymer L121, and thr-MDP, ei- tion with an octoxynol (WO01/21207) as well as polyox- ther microfluidized into a submicron emulsion or vor- yethylene alkyl ethers or ester surfactants in combination texed to generate a larger particle size emulsion. with at least one additional non-ionic surfactant such as 20 (7) Ribi™ adjuvant system (RAS), (Ribi Immuno- an octoxynol (WO01/21152). chem) containing 2% Squalene, 0.2% Tween 80, [0057] Preferred polyoxyethylene ethers are selected and one or more bacterial cell wall components from from the following group: polyoxyethylene-9-lauryl ether the group consisting of monophosphorylipid A (laureth 9), polyoxyethylene-9-steoryl ether, polyox- (MPL), trehalose dimycolate (TDM), and cell wall ytheylene-8-steoryl ether, polyoxyethylene-4-lauryl25 skeleton (CWS), preferably MPL + CWS (Detox™); ether, polyoxyethylene-35-lauryl ether, and polyoxyeth- and ylene-23-lauryl ether. (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as J. Polyphosphazene (PCPP) 3dPML). 30 [0058] PCPP formulations are described, for example, M. Human Immunomodulators in Andrianov et al., "Preparation of hydrogel micro- spheres by coacervation of aqueous polyphophazene [0062] Human immunomodulators suitable for use as solutions", Biomaterials (1998) (1-3): 19 109-115 and adjuvants in the invention include cytokines, such as in- Payne et al., "Protein Release from Polyphosphazene 35 terleukins (e.g. IL- 1, IL-2, IL- 4, IL- 5, IL-6, IL- 7, IL-12, etc.), Matrices", Adv. Drug. Delivery Review (1998) (3): 31 interferons (e.g. interferon-γ), macrophage colony stim- 185-196. ulating factor, and tumor necrosis factor. [0063] Aluminum salts and MF59 are preferred adju- K. Muramyl peptides vants for use with injectable influenza vaccines. Bacterial 40 toxins and bioadhesives are preferred adjuvants for use [0059] Examples of muramyl peptides suitable for use with mucosally-delivered vaccines, such as nasal vac- as adjuvants in the invention include N-acetyl-muramyl- cines. L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normu- [0064] The following example illustrates generation of ramyl-1-alanyl-d-isoglutamine (nor-MDP), and N- acetyl- neutralizing antibodies by administration of low doses of muramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1’-2’-di- 45 rabies virus vaccine in a Thai Red Cross Indtradermal palmitoyl- sn- glycero- 3- hydroxyphosphoryloxy)-ethyl- immunization regimen. amine MTP-PE). EXAMPLES L. Imidazoquinolone Compounds 50 Generation of neutralizing antibodies by administra- [0060] Examples of imidazoquinolone compounds tion of low dose of PCECV in TRC ID regimen. suitable for use adjuvants in the invention include Imiq- uamod and its homologues, described further in Stanley, [0065] This study was conducted to evaluate the "Imiquimod and the imidazoquinolones: mechanism of amount of antigen required to elicit a satisfactory immune action and therapeutic potential" Clin Exp Dermatol55 response. As the administration of Human Rabies Immu- (2002) 27(7):571-577 and Jones, "Resiquimod 3M", Curr noglobulin (HRIG) has been reported to reduce the im- Opin Investig Drugs (2003) 4(2):214-218. mune response to vaccine in Post- Exposure Treatment, [0061] The invention may also comprise combinations HRIG was given concomitantly in this clinical trial to eval-
10 17 EP 1 593 392 B1 18 uate the worst case scenario. istered on days 0, 3, 7, 14, 28 and 90 of the vacci- [0066] The study which was performed at the Infec- nation regimen, or the modified Thai Red Cross reg- tious Deseases Department at the University Hospital of imen (2-2-2-0-2), with the 90 day booster being emit- Hradec Kralove (Czech Republic) from October 2003 to ted. January 2004 is a prospective, open-label, randomized, 5 controlled, single-center, clinical trial. All tests conducted 2. Composition for use according to claim 1, wherein in this trial were performed in accordance with the GCP- the concentration of rabies virus antigen in the vac- ICH guidelines after approval of the Ethics Committee cine is less than one half of 2.5 IU/ml. and the national authorities. [0067] PCECV was administered to healthy human 10 3. Composition for use according to claim 2, wherein subjects (n=165) using a simulated post-exposure TRC the concentration of rabies virus antigen in the vac- ID regimen in combination with human rabies immu- cine is less than one fourth of 2.5 IU/ml. noglobulin. Briefly, PCECV (potency 5.1 IU/ml) was ad- ministered in ID doses of 0.1 mL, either undiluted or in 4. Composition for use according to claim 3, wherein dilutions from 1:2 up to 1:16 (i.e. up to approximately15 the concentration of rabies virus antigen in the vac- 0.032 IU/dose). Specifically, the PCECV corresponded cine is less than one eighth of 2.5 IU/ml. to 0.51 IU/ID 0.1 mL dose (undiluted), 0.25/ID 0.1 mL dose (diluted 1:2; this potency corresponds to the mini- 5. Composition for use according to claim 1, comprising mal potency of 2.5 IU per IM dose recommended by the a rabies virus vaccine, wherein the concentration of WHO), 0.125 IU/ID 0.1 mL dose (diluted 1:4), 0.062/ID 20 rabies virus antigen in the vaccine is between 2.4 0.1 mL dose (diluted 1:8) and 0.031 IU/ID 0.1 mL dose IU/ml and 0.25 IU/ml. (diluted 1:16). [0068] The administration was performed in accord- 6. Composition for use according to claim 5, wherein ance with the 2-site intradermal regimen as depicted in the concentration of rabies virus antigen in the vac- figure 1 using seperate syringes and needles for each 25 cine is between 2.0 IU/ml and 1.0 IU/ml. dose. On day 0, human rabies immunoglobulin was ad- ministered intragluteally in a concentration of 20 IU/mL 7. Composition for use according to claim 6, wherein (for example, Berirab®, obtainable from Chiron Behring, the concentration of rabies virus antigen in the vac- Marburg, Germany). No serious adverse drug reactions cine is between 1.75 IU/ml and 1.25 IU/ml. were observed during the regimen. 30 [0069] Blood was withdrawn from subjects on Days 0, 8. Composition for use according to claim 7, wherein 7, 14, 30, 90 and 104 and rabies virus neutralizing anti- the concentration of rabies virus antigen in the vac- bodies were determined using the rapid fluorescent focus cine is 1.5 IU/ml. inhibition test (RFFIT). [0070] The results revealed an excellent and fast se- 35 9. Composition for useaccording to claims 1-8, wherein roprotection rate. The percentage of subjects having an the rabies virus vaccine is selected from the group adequate titer of neutralizing antibodies ≥ ( 0.5 IU/mL) consisting of purified chicken embryo cell vaccine were calculated. On Day 14, as expected, 100% of the (PCECV), purified vero cell vaccine (PVRV), and hu- subjects receiving undiluted vaccine had titers above 0.5 man diploid cell vaccine (HDCV). IU/ml. Also all subjects receiving 2 1: diluted vaccine 40 achieved protective titers. In the 1:4 and 1:8 groups all 10. Composition for useaccording to claims 1-9, wherein but one subjects had titers ≥ 0.5 IU/ml, whereas in the 1: post-exposure vaccination is accompanied by the 16 group again 100% seroprotection was achieved. Ex- administration of rabies immune globulin. actly the same seroprotection rates were found on Day 30 with 100% seroprotection in the 1:16 group. 45 Patentansprüche
Claims 1. Zusammensetzung zur Verwendung bei der postex- positionellen Impfung eines humanen Individuums, 1. Composition for use in the post-exposure vaccina- 50 das der Tollwut ausgesetzt war, wobei die Zusam- tion of a human subject who has been exposed to mensetzung einen Tollwutvirus-Impfstoff enthält, rabies, said composition comprising a rabies virus und wobei die Konzentration des Tollwutvirus-Anti- vaccine, wherein the concentration of rabies virus gens in dem Impfstoff weniger als 2,5 IU/ml beträgt, antigen in the vaccine is less than 2.5 IU/ml, and und wobei die Zusammensetzung für die intrader- wherein the composition is for intradermal adminis- 55 male Verabreichung in Dosen von 0,1 ml nach dem tration in doses of 0.1 ml according to the Thai Red Thai Red Cross-Verabreichungsschema (2-2-2- Cross regimen (2-2-2-0-1-1), wherein the numerical 0-1-1), wobei sich die numerischen Abkürzungen auf abbreviations refer to the numbers of doses admin- die Anzahl der zu verabreichenden Dosen an den
11 19 EP 1 593 392 B1 20
Tagen 0, 3, 7, 14, 28 und 90 des Impfschemas be- Revendications ziehen, oder nach dem modifizierten Thai Red Cross-Verabreichungsschema (2-2-2-0-2), bei dem 1. Composition destinée à être utilisée dans la vacci- die Auffrischungsimpfung am Tag 90 ausgelassen nation post-exposition d’un sujet humain qui a été wird, ist. 5 exposé à la rage, ladite composition comprenant un vaccin antirabique, dans laquelle la concentration 2. Zusammensetzung zur Verwendung nach Anspruch d’antigène viral rabique dans le vaccin est inférieure 1, wobei die Konzentration von Tollwutvirus- Antigen à 2,5 UI/ml, et dans laquelle la composition est des- in dem Impfstoff weniger als die Hälfte von 2,5 IU/ml tinée à être administrée par voie intradermique à des beträgt. 10 doses de 0,1 ml conformément au schéma d’admi- nistration de la Croix-Rouge thaïlandaise (2-2-2-0- 3. Zusammensetzung zur Verwendung nach Anspruch 1-1), dans lequel les abréviations numériques font 2, wobei die Konzentration von Tollwutvirus- Antigen référence au nombre de doses administrées aux in dem Impfstoff weniger als ein Viertel von 2,5 IU/ml jours 0, 3, 7, 14, 28 et 90 du schéma de vaccination beträgt. 15 ou selon le schéma d’administration modifié de la Croix-Rouge thaïlandaise (2-2-2-0-2) en omettant le 4. Zusammensetzung zur Verwendung nach Anspruch rappel à 90 jours. 3, wobei die Konzentration von Tollwutvirus- Antigen in dem Impfstoff weniger als ein Achtel von 2,5 IU/ml 2. Composition destinée à être utilisée selon la reven- beträgt. 20 dication 1, dans laquelle la concentration d’antigène viral rabique dans le vaccin est inférieure à la moitié 5. Zusammensetzung zur Verwendung nach Anspruch de 2,5 UI/ml. 1, die ein Tollwutvirus-Impfstoff umfasst, wobei die Konzentration des Tollwutvirus-Antigens in dem 3. Composition destinée à être utilisée selon la reven- Impfstoff zwischen 2,4 IU/ml und 0,25 IU/ml beträgt. 25 dication 2, dans laquelle la concentration d’antigène viral rabique dans le vaccin est inférieure à un quart 6. Zusammensetzung zur Verwendung nach Anspruch de 2,5 UI/ml. 5, wobei die Konzentration des Tollwutvirus-Anti- gens in dem Impfstoff zwischen 2,0 IU/ml und 1,0 4. Composition destinée à être utilisée selon la reven- IU/ml beträgt. 30 dication 3, dans laquelle la concentration d’antigène viral rabique dans le vaccin est inférieure à un hui- 7. Zusammensetzung zur Verwendung nach Anspruch tième de 2,5 UI/ml. 6, wobei die Konzentration des Tollwutvirus-Anti- gens in dem Impfstoff zwischen 1,75 IU/ml und 1,25 5. Composition destinée à être utilisée selon la reven- IU/ml beträgt. 35 dication 1, comprenant un vaccin antirabique, dans laquelle la concentration d’antigène viral rabique 8. Zusammensetzung zur Verwendung nach Anspruch dans le vaccin se situe entre 2,4 UI/ml et 0,25 UI/ml. 7, wobei die Konzentration des Tollwutvirus-Anti- gens in dem Impfstoff 1,5 IU/ml beträgt. 6. Composition destinée à être utilisée selon la reven- 40 dication 5, dans laquelle la concentration d’antigène 9. Zusammensetzung zur Verwendung nach den An- viral rabique dans le vaccin se situe entre 2,0 UI/ml sprüchen 1-8, wobei der Tollwutvirus-Impfstoff aus- et 1,0 UI/ml. gewählt ist aus der Gruppe bestehend aus einem Impfstoff aus gereinigten Hühnerembryozellen (pu- 7. Composition destinée à être utilisée selon la reven- rified chicken embryo cell vaccine, PCECV), einem 45 dication 6, dans laquelle la concentration d’antigène Impfstoff aus gereinigten Verozellen (purified vero viral rabique dans le vaccin se situe entre 1,75 UI/ml cell vaccine, PVRV) und einem Impfstoff aus huma- et 1,25 UI/ml. nen diploiden Zellen (human diploid cell vaccine, HDCV) 8. Composition destinée à être utilisée selon la reven- 50 dication 7, dans laquelle la concentration d’antigène 10. Zusammensetzung zurVerwendung gemäß den An- viral rabique dans le vaccin est de 1,5 UI/ml. sprüchen 1-9, wobei die postexpositionelle Impfung gemeinsam mit der Verabreichung von Tollwut-Im- 9. Composition destinée à être utilisée selon les reven- munglobulin erfolgt. dications 1 à 8, dans laquelle le vaccin antirabique 55 est choisi dans le groupe constitué par du vaccin purifié préparé sur cellules d’embryon de poulet (PCECV), du vaccin purifié préparé sur cellules Vero (PVRV) et du vaccin préparé sur cellules diploïdes
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humaines (HDCV).
10. Composition destinée à être utilisée selon les reven- dications 1 à 9, dans laquelle la vaccination post- exposition s’accompagne d’une administration d’im- 5 munoglobuline rabique.
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This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
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