HRP930517A2 - Complexes having adjuvant activity - Google Patents
Complexes having adjuvant activity Download PDFInfo
- Publication number
- HRP930517A2 HRP930517A2 HRP-1667/90A HRP930517A HRP930517A2 HR P930517 A2 HRP930517 A2 HR P930517A2 HR P930517 A HRP930517 A HR P930517A HR P930517 A2 HRP930517 A2 HR P930517A2
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- Prior art keywords
- antigen
- matrix
- glycosides
- phase
- vaccine
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Description
Sadašnji izum se odnosi na komplekse koji imaju pomoćnu aktivnost, na komp1ekse koji obuhvaćaju lipid i najmanje jedan glikozid i na nove g1ikozide za ubacivanje u spomenute komplekse. The present invention relates to complexes that have auxiliary activity, to complexes that include a lipid and at least one glycoside, and to new glycosides for inclusion in said complexes.
Zaštitna imunizacija životinja protiv mikroba obuhvaća davanje antigena koji je izveden iz mikroba zajedno sa materija1om koji povećava antitijelo i/i1i imunu reakciju posredovanu stanicama antigena u životinji, ovaj materijal je poznat kao pomoćno sredstvo. Jedini dodaci koji su trenutno autorizirani za korištenje na ljudima ili svinjama u mnogim zemljama su aluminij hidroksid i aluminij fosfat. Mada su ovi dodatci zadovoljavajući za mnoga cjepiva., proučavanja su pokaza1a da su Freundovo komp1etno pomoćno sredstvo (FCA) ili quill a (također poznati kao saponin) često efikasniji u polučivanju reakcije antitijela i stanično posredovanog imuniteta ali, na nesreću,, ova pomoćna sredstva mogu učiniti da životinje reagiraju štetno na cijepljenje. Protective immunization of animals against microbes involves the administration of an antigen derived from the microbe together with a material that increases the antibody and/or cell-mediated immune response to the antigen in the animal, this material being known as an adjuvant. The only additives currently authorized for use on humans or pigs in many countries are aluminum hydroxide and aluminum phosphate. Although these adjuvants are satisfactory for many vaccines, studies have shown that Freund's complete adjuvant (FCA) or quill a (also known as saponin) are often more effective in eliciting antibody responses and cell-mediated immunity but, unfortunately, these adjuvants can cause animals to react adversely to vaccination.
EP-A-O 109 942 i EP-A-O 180 564 opisuju imunogene komp1ekse koji su formirani između g1ikozida, takvih kao što su tritropenoidni saponini (naročito Qui1 A), i antigena koji sadrže hidrofobni region. Imuno-stimulacionim komp1eksima dato je ime "iscomi". Ko1ičina Quil-A u jednom iscomu može biti oko 10 do 100 puta niža nego kada se Quil A miješa s antigenom za proizvodnju istog antigenog efekta. Iscom ne stvara štetne reakcije koje prate Quil A. EP-A-0 109 942 and EP-A-0 180 564 describe immunogenic complexes which are formed between glycosides, such as tritropenoid saponins (especially Quil A), and antigens containing a hydrophobic region. Immuno-stimulating complexes are given the name "iscomi". The amount of Quil-A in a single iscom can be about 10 to 100 times lower than when Quil A is mixed with antigen to produce the same antigenic effect. Iscom does not produce the adverse reactions associated with Quil A.
EP-A-231 ukazuje da prisustvo antigena nije neophodno za formiranje bazne iscom strukture, koja se ovdje kasnije zove iscom "matrica", jer je moguće da se matrica formira iz nekog stero1a, kao što je ho1estero1, nekog fosfo1ipida kao što je fosfati-di1-etano1amin, i nekog g1ikozida kao što je Quil A. Međutim, nema opisa da su takve matrice korisne kao pomoćna sredstva; opisane su samo u odjeljku da se prikaže da se strukture slične iscomu mogu formirati bez prisustva antigena. Mi smo našli da se takve prazne matrice mogu koristiti za osiguranje pomoćne funkcije pomoć u dodavanja na antigene bakterija i mikrop1azme koji ne formiraju dio matrice iscoma. Da1je, u sva tri citirana dokumenta iz ranije tehnike, kada se koristi antigen je neotopiv u vodi, ovaj se otapa sa deterđentom i, tada, poslije miješanja sa glikozidom itd., deterđent se odvaja, na primjer, dijalizom, tako da se izaziva formiranje iscoma. Ml smo našli da će se iscomi formirati u prisustvu deterđenta i da ova faza odvajanja nije potrebna. Na s1ičan način mogu se formirati matrice iscoma. EP-A-231 indicates that the presence of antigen is not necessary for the formation of the basic iscom structure, hereafter called the iscom "matrix", because it is possible that the matrix is formed from some sterol, such as cholesterol, some phospholipid, such as phosphati- di1-ethanolamine, and a glycoside such as Quil A. However, there is no description of such matrices being useful as excipients; are described only in the section to show that iscoma-like structures can form in the absence of antigen. We have found that such empty matrices can be used to provide an auxiliary function by helping to add antigens to bacteria and microorganisms that do not form part of the iscoma matrix. Yes, in all three cited prior art documents, when an antigen is used that is insoluble in water, it is dissolved with a detergent and, then, after mixing with a glycoside, etc., the detergent is separated, for example by dialysis, so that the formation iscoma. We have found that iscomas will form in the presence of detergent and that this separation step is not necessary. Iscom matrices can be formed in a similar way.
Jedan aspekt pronalaska osigurava cjepivo za korištenje na čovjeku ili životinji koja obuhvaća (1) antigen udružen sa bakterijom ili mlkoplazmom i (2) matricu iscoma, pri čemu spomenuti antigen nije inkorporiran u iscom. One aspect of the invention provides a vaccine for use in a human or animal comprising (1) an antigen associated with a bacterium or a bacterium and (2) an iscoma matrix, wherein said antigen is not incorporated into the iscoma.
Pronalazak također obuhvaća postupak za spravljenje cjepiva za korištenje na čovjeku ili životinji, koji obuhvaća (1) formiranje kompleksa nekog gilkozida, sterola i, opciono, nekog fosfolipida, i (2) miješanje spomenutog kompleksa sa antigenom koji je udružen sa bakterijom ili mikop1azmom. The invention also includes a process for making a vaccine for human or animal use, which comprises (1) forming a complex of a gylcoside, a sterol and, optionally, a phospholipid, and (2) mixing said complex with an antigen associated with a bacterium or a mycoplasma.
Pod životinjom mi podrazumijevamo ne-humanog kralježnjaka, pože1jno sisavca kao što su krava, svinja, ovca, koza, konj, pas ili mačka. By animal we mean a non-human vertebrate, preferably a mammal such as a cow, pig, sheep, goat, horse, dog or cat.
Cjepivo može obuhvaćati jedani ili više razblaživača i nosača konvencionalne prirode. Ovi će se normalno dodati u ili poslije faze (2) postupka. The vaccine may comprise one or more diluents and carriers of a conventional nature. These will normally be added at or after stage (2) of the process.
Pod terminom "antigen udružen sa bakterijom ili mikop1azmom'' mi podrazuijevamo bilo koju jedinicu koja može proizvesti zaštitno antitijelo ili stanično posredovanu imuno1ošku reakciju protiv bakterije ili mikop1azme u kra1ješnju koji je iz1ožen antigenu. Antigen može biti sav ili dio proteina, glikoproteina, glikolipida, polisaharida ili lipopolisaharida koji je udružen sa bakterijom ili mikoplazmom, ili može biti neki polipeptid ili druga jedinica koja imitira cijeli ili dio takvog proteina, g1ikoproteina, gliko1ipida, polisaharida ili lipolisaharida. Zato što antigen nije inkorporiran u matricu iscoma za vrijeme formiranja zadnje, nije neophodno da on ima hidrofobni region ili da bude vezan za hidrofobnu grupu, kao u postupcima iz ranije tehnike. Sama bakterija ili mikoplazma ne mora biti prisutna u cjepivu, ali ona može smanjiti cijenu cjepiva, ako se koriste cijele stanice, na primjer Haemophi1us p1europneumoniae umjesto ekstrakata. By the term "antigen associated with a bacterium or a mycoplasma" we mean any unit that can produce a protective antibody or cell-mediated immune response against a bacterium or a mycoplasma in a cell exposed to the antigen. The antigen can be all or part of a protein, glycoprotein, glycolipid, polysaccharide. or lipopolysaccharide associated with a bacterium or mycoplasma, or may be a polypeptide or other unit that mimics all or part of such a protein, glycoprotein, glycolipid, polysaccharide or lipopolysaccharide. Because the antigen is not incorporated into the iscoma matrix during the formation of the latter, it is not necessary that it has a hydrophobic region or to be attached to a hydrophobic group, as in prior art methods.The bacterium or mycoplasma itself need not be present in the vaccine, but it can reduce the cost of the vaccine if whole cells are used, for example Haemophi1us p1europneumoniae instead of extracts.
Pod "mikoplazmama" mi uključujemo blisko srodne organizme koji su poznati kao ureaplazme i aholeplazme. By "mycoplasmas" we include the closely related organisms known as ureaplasmas and aholeplasmas.
Bakterijske bolesti uključuju one koje su izazvane Mycobacterium (npr., M. tubercu1osis & M. bovis), C1ostridium (npr., C .. we1wchii), Rickettsia , Spirochaetes (np r ., Treponema pal1idum ili Leptospira pomona), Eschericia (npr., E. coli), Staphylococci (npr., S. aureus), Hemophillius (npr., H. inf1uenzae i H. p1europneumoniae ), Bordetella (npr., B. pertussis), Vibrio (npr., V. cholerae), Salmonella (npr., S. typhi i S. paratyphi), Streptococci (npr., S.aga1actie, Neisseria (npr., N. gonorrhoea) Pasteurella (npr., P. multocida), Legione1la (npr., L. pneumoniae), Ch1amidia (npr., C. pistacci), Pseudomonas (npr., P. malliae), Actinobacillus (npr., A. pleuropneumafilae), Campylobacter (npr., C. jejuni), Listeria (npr., L. monocytogenes), Brucella (npr., B. Abortus), Corynebacterium (npr., C. diphteriae), Yersinia (npr., Y. pseudotuberculosis), Pneumococci (npr., Streptococcus pneumoniae), Bacillus (npr,. B. antracis), Klebsiella (npr., K. pneumoniae), Shigella (npr., S. dysenteriae) i Actinomycetes (npr., Nocardia asteroides). Bacterial diseases include those caused by Mycobacterium (eg, M. tubercu1osis & M. bovis), C1ostridium (eg, C .. we1wchii), Rickettsia , Spirochaetes (eg, Treponema pal1idum or Leptospira pomona), Eschericia (eg ., E. coli), Staphylococci (eg, S. aureus), Hemophillius (eg, H. inf1uenzae and H. p1europneumoniae ), Bordetella (eg, B. pertussis), Vibrio (eg, V. cholerae) , Salmonella (eg, S. typhi and S. paratyphi), Streptococci (eg, S.aga1actie, Neisseria (eg, N. gonorrhoea) Pasteurella (eg, P. multocida), Legionella (eg, L. pneumoniae), Ch1amidia (eg, C. pistacci), Pseudomonas (eg, P. malliae), Actinobacillus (eg, A. pleuropneumafilae), Campylobacter (eg, C. jejuni), Listeria (eg, L. monocytogenes), Brucella (eg, B. abortus), Corynebacterium (eg, C. diphteriae), Yersinia (eg, Y. pseudotuberculosis), Pneumococci (eg, Streptococcus pneumoniae), Bacillus (eg, B. anthracis ), Klebsiella (eg, K. pneumoniae), Shigella (eg, S. dysenteriae) and Actinomycetes (eg, Nocardia asteroides).
Poželjno je bakterija Mycobacterium (npr., M. tubercu1osisi), C1ostridium (npr., C. we1chii), Rickettsia, Spirochaetes (npr., Treponema pallidium), Escherichia (npr., E. coli), Staphy1ococci (npr., S. aureus), Hemophi1ius (npr. H. influenzae i H. pleuropneumoniae), Bordetella (npr., B. pertussis), Vibrio (npr., V. cholerae), Salmonella (npr., S. Typhi i S. paratyphi), Streptococci (npr., S. aga1actie), Neisseria (npr., N. gonorrhoea), Pasteurella (npr. P. mu1tocida), Legionella (npr., L. pneumoniae), Pseudomonas (npr., P. malliae), Actinobacillus (npr., A. pleuropneumoniae), Campylobacter (npr., C. jejuni), Listeria (npr., L. monocytogenes). Određeni antigeni uk1jučuju faktor adherencije u Colli, npr., pili K 88, proinski protein u npr,. Salmonella i druge membranske proteine iz B. pertussis i Neisseria maningitidis. Preferred bacteria are Mycobacterium (eg, M. tubercu1osisi), C1ostridium (eg, C. we1chii), Rickettsia, Spirochaetes (eg, Treponema pallidium), Escherichia (eg, E. coli), Staphy1ococci (eg, S . aureus), Hemophilus (eg, H. influenzae and H. pleuropneumoniae), Bordetella (eg, B. pertussis), Vibrio (eg, V. cholerae), Salmonella (eg, S. Typhi and S. paratyphi) , Streptococci (eg, S. aga1actie), Neisseria (eg, N. gonorrhoea), Pasteurella (eg, P. mu1tocida), Legionella (eg, L. pneumoniae), Pseudomonas (eg, P. malliae), Actinobacillus (eg, A. pleuropneumoniae), Campylobacter (eg, C. jejuni), Listeria (eg, L. monocytogenes). Certain antigens include the adhesion factor in Colla, eg, pili K 88, the proin protein in eg. Salmonella and other membrane proteins from B. pertussis and Neisseria meningitidis.
Pože1jno je antigen onaj koji ne formira konvencionalni iscom, tj. kao što je opisano u EP-A-109 i EP-A-180 564. Preferably, the antigen is one that does not form a conventional iscom, ie as described in EP-A-109 and EP-A-180 564.
Naročito je poželjno cijepljenje svinja protiv H. pleuropneumoniae. Vaccination of pigs against H. pleuropneumoniae is particularly desirable.
Mikoplazme od veterinarskog ili medicinskog interesa uk1jučuju Mycoplasma bovis, M. gallisepticum, M. agalactiae, M. hyopopneumoniae, M. synoviae, M. Arthriditis, M. capricolium, M. dispar, M. hominis. M. mycoides subs. capri, M.. ora1e, M. Oripneumoniae, M. pu1monis, M. cynos, M. hyorhinis, H. mycoides, M. salvarium, M. fermentas. Mycoplasmas of veterinary or medical interest include Mycoplasma bovis, M. gallisepticum, M. agalactiae, M. hyopopneumoniae, M. synoviae, M. Arthritis, M. capricolium, M. dispar, M. hominis. M. mycoides subs. capri, M.. ora1e, M. Oripneumoniae, M. pu1monis, M. cynos, M. hyorhinis, H. mycoides, M. salvarium, M. fermentas.
Mada antigen nije inkorporiran u matricu na raniji način, naime kao dio same strukture iscoma, može svejedno biti prisutan na vanjskom dijelu matrice iscoma ili se kasnije može integrirati u matricu iscoma. Alternativno, može biti prisutan potpuno odvojeno u otopini ili disperziji. Although the antigen is not incorporated into the matrix in an earlier way, namely as part of the iscoma structure itself, it may still be present on the outer part of the iscoma matrix or may later be integrated into the iscoma matrix. Alternatively, it may be present completely separately in solution or dispersion.
Matrica iscoma se može napraviti na način kao što je opisano u EP-A-0 231 0'39, naime zajedničkim mješanjern sterola, g1ikozida i (opcionalno) fosfo1ipida i tada odvajanjem sredstava za otapanje,. Alternativno se odvajanje sredstava za otapanje može izostaviti. Elektronski mikrograf iscoma dobivenog iz Qui1-a A, ho1estero1a i fosfatidi1etno1amina opisan je na slici 1 EP-A-0 231 039 (uvećanje 116.000 x). Ako se ne koriste fosfolipidi formiraju se dimenzionalne strukture. Primjer takvih struktura opisan je na slici 2 EP-A-0 231 039. Ova slika je e1ektronsk i mikrograf (uvećanje 146.000 x ) struktura dobivenih iz Quil-a A i ho1estero1a.Termin ''matrica iscoma'' koristi se i za 3-dimenzionalne i 2-dimenzionalne strukture. The iscoma matrix can be made in the manner described in EP-A-0 231 0'39, namely by mixing sterols, glycosides and (optionally) phospholipids together and then separating the solubilizing agents. Alternatively, the separation of the solubilizers can be omitted. An electron micrograph of iscoma obtained from Quil A, cholesterol and phosphatidylinosamine is described in Figure 1 of EP-A-0 231 039 (magnification 116,000x). If phospholipids are not used, dimensional structures are formed. An example of such structures is described in Figure 2 of EP-A-0 231 039. This image is an electron and micrograph (magnification 146,000 x ) of structures obtained from Quil A and ho1estero1a. The term ``iscoma matrix'' is also used for 3- dimensional and 2-dimensional structures.
Glikozidi koje treba koristiti u postupku prema pronalasku mogu biti isti kao oni koji su spomenuti u EP-A 0 109 942 i EP-A--0 231 039. Uglavnom su glikozidi, glikozidi koji pokazuju amfipatne osobine i obuhvaćaju hidrofobne i hidrofilne regione u molekulu. Pože1jno je koristiti saponine, naročito saponinski ekstrakt iz Quillaja saponaria M-olina na prvom mjestu DQ-ekstrakt napravljen prema K. Dalsgard: Saponin Adjuvants III Archiv fur die gesamte Virusforschung 44. 243-254 (1974). Drugi poželjni saponini su aescin iz Aesculus hippocastanum (T.Patt and W Winkler."Das terapeutische Wirsame Prinzip der Rosskatanie (Aesculus hippocastanum) Arzneimittelforschung 10 (4) 273-275 (1960) i sapoalbin iz Gypsophilla struthium (R. Vochten. P.Joos and R. Ruyssen. Physicochemlcal properties of sapoalbin and their relation to the foam stability, J. Pharm.Belg. 42 213.266 (1968). Naročito je poželjno korištenje Quil-a A. The glycosides to be used in the process according to the invention can be the same as those mentioned in EP-A 0 109 942 and EP-A--0 231 039. They are mainly glycosides, glycosides that show amphipathic properties and include hydrophobic and hydrophilic regions in the molecule . It is preferable to use saponins, especially the saponin extract from Quillaja saponaria M-olina in the first place DQ-extract made according to K. Dalsgard: Saponin Adjuvants III Archiv fur die gesamete Virusforschung 44. 243-254 (1974). Other preferred saponins are aescin from Aesculus hippocastanum (T.Patt and W Winkler."Das terapeutische Wirsame Prinzip der Rosskatanie (Aesculus hippocastanum) Arzneimittelforschung 10 (4) 273-275 (1960) and sapoalbin from Gypsophilla struthium (R. Vochten. P. Joos and R. Ruyssen. Physicochemical properties of sapoalbin and their relation to the foam stability, J. Pharm. Belg. 42 213.266 (1968). The use of Quil A is particularly preferred.
U postupku prema pronalasku glikozidi se koriste u najmanje kritičnoj koncentraciji za formiranje mmicela. U s1učaju Quli-a A ova koncentrac1ja je oko 0.03% mas. Uglavnom, rnolarni odnos glikozida (naročito kada je to Quil A) prema sterolu (naročito kada je to holesterol) prema fosfolipidu je 1:1:0 ± 20% (poželjno ne više od ± 10%) za svaku cifrz. Ovo je ekvivalentno sa masenim odnosom od oko 5:1 za Qui1 A : ho1estero1). In the process according to the invention, glycosides are used in the least critical concentration for the formation of micelles. In the case of Quli A, this concentration is about 0.03% by weight. Generally, the molar ratio of glycoside (especially when it is Quil A) to sterol (especially when it is cholesterol) to phospholipid is 1:1:0 ± 20% (preferably no more than ± 10%) for each digit. This is equivalent to a mass ratio of about 5:1 for Qui1 A : ho1estero1).
Steroli koji se koriste u postupku prema pronalasku mogu biti poznati steroli životinjskog ili biljnog porijekla, kao što su holesterol, lanosterol, 1umisterol, stigmasterol i sitosterol. Poželjno je holesterol sterol koji se koristi u postupku prema pronalasku. The sterols used in the process according to the invention can be known sterols of animal or vegetable origin, such as cholesterol, lanosterol, 1umisterol, stigmasterol and sitosterol. Cholesterol is preferably a sterol used in the process of the invention.
Poželjno je da se koristi fosfolipid tako da se formiraj u 3-dimenzionalni iscomi. Podesni fosfolipidi uključuju fosfatidilholin i fosfatidiletanolamin. Preferably, a phospholipid is used so that it forms into 3-dimensional iscomas. Suitable phospholipids include phosphatidylcholine and phosphatidylethanolamine.
Drugi aspekt pronalaska osigurava postupak za spravijanje imunogenog kompleksa koji obuhvaća u vodi netopiv antigen, pri čemu postupak obuhvaća otapanje antigena sa sredstvom za otapanje, mješanje otopljenog antigena, glikozida, sterola i opciono, fosfolipida i formiranje iscoma suštinski bez odvajanja sredstava za otapanje. Another aspect of the invention provides a method for preparing an immunogenic complex comprising a water-insoluble antigen, wherein the method comprises dissolving the antigen with a solubilizing agent, mixing the dissolved antigen, glycosides, sterols and optionally, phospholipids and forming an iscome essentially without separating the solubilizing agents.
Tako nema potrebe da se sredstvo za otapanje odvaja ultrafiltracijom, dijalizom, ultracentrifugiranjem ili hromatografskim postupcima radi formiranja iscoma. Thus, there is no need for the solvent to be separated by ultrafiltration, dialysis, ultracentrifugation or chromatographic procedures to form the iscoma.
Antigen je kao što je definirano gore, osim što može biti udružen s gljivicama, parazitima kao što su protozoe i g1iste ili virusi umjesto neoplazma ili bakterija. The antigen is as defined above, except that it may be associated with fungi, parasites such as protozoa and g1ists, or viruses instead of neoplasms or bacteria.
Gljivice koje su udružene sa bolešću uključuju: Candida spp., Cryptococcus spp., Aspergilus spp., Microsporum spp., Trichophyton spp., Epidermophyton spp., i Dermatophilus spp. Fungi associated with the disease include: Candida spp., Cryptococcus spp., Aspergilus spp., Microsporum spp., Trichophyton spp., Epidermophyton spp., and Dermatophilus spp.
Paraziti koji su praćeni bolešću uključuju one koji su navedeni u EP-A-109 942. Parasites associated with the disease include those listed in EP-A-109 942.
Virusi koji su praćeni bolešču ukiljučuju one koji su navedeni u EP-A-109 942. Mnogi od ovih imaju omote (kapsule) iz kojih se mogu ekstrahovati antigeni. Određeni virusi uk1jučuju virus konjske inf1uence, herpes virus konja, virus volovske virusne diaree, koronavirus, paravovirus, virus mačije leukemije, virus mačije insufijencije i virus konjskog virusnog arteritisa. Viruses that have been associated with the disease include those listed in EP-A-109 942. Many of these have envelopes (capsules) from which antigens can be extracted. Certain viruses include equine influenza virus, equine herpes virus, bovine viral diarrhea virus, coronavirus, paravovirus, feline leukemia virus, feline insufficiency virus, and equine viral arteritis virus.
Sredstvo za otapanje može biti bilo koje od onih koja su spomenuta u EP-A-O 109 942 ili EP-A-O 231 039, na primjer, neki deterđent, karbamid ili guanidin. The solubilizing agent can be any of those mentioned in EP-A-0 109 942 or EP-A-0 231 039, for example, a detergent, carbamide or guanidine.
Uglavnom se za ovu svrhu mogu koristiti ne-ionski, ionski ili cviter-ionski deterđent ili deterđent na bazi so1ne kiseline, kao što je natrij-dezoksiholat. Pože1jno je koristiti deterđent oktilglikoid, nonin-N-metilglukamid ili dekanoil-N-metrilglukamid, ali su također podesni alkilfenil-poliiksietilen etri, naročito polietilenglikolp-izooktilfeniletar koji ima 9 do 10 oksietilenskih grupa koji je komercijaliziran pod trgovačkim imenom Triton X-100R. In general, a non-ionic, ionic or zwitterionic detergent or a detergent based on hydrochloric acid, such as sodium deoxycholate, can be used for this purpose. It is preferred to use the detergent octylglycoid, nonine-N-methylglucamide or decanoyl-N-methylglucamide, but alkylphenyl-polyoxyethylene ethers are also suitable, especially polyethylene glycol p-isooctylphenylether having 9 to 10 oxyethylene groups and commercialized under the trade name Triton X-100R.
Postupak prema pronalasku se može koristiti za spravijanje imunogenskih kompleksa iz antigenih proteina ili peptida koji pokazuju amfipatme osobine, na primjer, onih koji su opisani u EP-A-109 942. The process according to the invention can be used to make immunogenic complexes from antigenic proteins or peptides showing amphipathic properties, for example, those described in EP-A-109 942.
U postupku prema drugom aspektu izuma opisani ili otopljeni antigen se uglavnom kontaktira sa otopinom koja sadrži glikozid u najmanje kritičnoj koncentraciji za formiranje micela, neki sterol i opciono neki fosfolipid. In the process according to the second aspect of the invention, the described or dissolved antigen is mainly contacted with a solution containing a glycoside in the least critical concentration for the formation of micelles, some sterol and optionally some phospholipid.
Ako se želi, dobivene otopine imunogenskih kompleksa mogu se liofilizirati. Liofilizirani preparati mogu se tada rekonstruirati za korištenje prije dodavanja vode. If desired, the obtained solutions of immunogenic complexes can be lyophilized. Lyophilized preparations can then be reconstituted for use before adding water.
Treći aspekt izuma osigurava postupak za spravljanje matrice iscoma pri čemu postupak obuhvaća otapanje strola, i opciono nekog fosfolipida, sa sredstvom za otapanje i miješanje sa otopinom glikozida, bez odvajanja sredstava za otapanje. A third aspect of the invention provides a process for preparing an iscoma matrix, wherein the process comprises dissolving a sterol, and optionally a phospholipid, with a solubilizing agent and mixing with a glycoside solution, without separating the solubilizing agents.
Sredstvo za otapanje, sterol, fosfolipid i glikozid su kao što ovdje definirani ranije. The solubilizing agent, sterol, phospholipid and glycoside are as previously defined herein.
Dalje pronalazak se odnosi na farmaceutski preparat koji sadrži imunogene komplekse koji su napravljeni pomoću prvog i drugog aspekta pronalaska. ove osobine mogu se dobiti spravljanjem imunogenog kompleksa u obliku koji je podesan za parenteral no davanje, poželjno za davan je injekcijom i, naročito za injekcije pomoću potkožnih ili intra-muskularnih načina. Ug1avnom farmaceutski preparati sadrže imuogenske komplekse u vodenoj, fiziološi prihvatljivoj sredini, koja, ako se želi, sadrži pufer i/ili takvu sol kao što je natrij-klorid za prilagođavanje osmotskom tlaku. Ovi postupci su dobro poznati u tehnici. Further, the invention relates to a pharmaceutical preparation containing immunogenic complexes made using the first and second aspects of the invention. these properties can be obtained by preparing the immunogenic complex in a form suitable for parenteral administration, preferably for administration by injection and, in particular, for injection by subcutaneous or intramuscular routes. Generally, pharmaceutical preparations contain immunogenic complexes in an aqueous, physiologically acceptable medium, which, if desired, contains a buffer and/or such a salt as sodium chloride to adjust the osmotic pressure. These procedures are well known in the art.
S1ijedeći neograničavajući primjeri ilustruraju raz1ičite poželjne aspekte izuma. The following non-limiting examples illustrate various preferred aspects of the invention.
Primjeri Examples
Primjeri 1-6: Spravljanje prazne matrice iscoma Examples 1-6: Creating an empty iscom matrix
Potrebni reagensi Required reagents
Fosfatni puferizitana slana otopina (Dulbeccdo A, Oxoid) Phosphate buffered saline (Dulbecco's A, Oxoid)
Lipidni miks (vidi niže) Lipid mix (see below)
10%-na w/v otopina Quil-a A (superfos) u sterilnoj desti1iranoj vodi. 10% w/v solution of Quil A (superfos) in sterile distilled water.
Spravljanje lipidnog miska Preparation of lipid membrane
Materijali: Kloroform (ana1itički reagens), holesterol(Kvaliteta I, iz svinjske jetre, Sigma). L-beta-fosfatidilholin )Sigina), Mega 9 ili 10 (Sigma) ili Triton (Mega 9 je nonil-N-metilglukamid, dok je Mega 10 dekanoil-N-metilg1ukamid) i steri1na desti1irana voda. Materials: Chloroform (analytical reagent), cholesterol (Quality I, from pig liver, Sigma). L-beta-phosphatidylcholine (Sigina), Mega 9 or 10 (Sigma) or Triton (Mega 9 is nonyl-N-methylglucamide, while Mega 10 is decanoyl-N-methylglucamide) and sterile distilled water.
Postupak Procedure
(a) Napraviti 20% w/v Mega 10 u sterilnoj destiliranoj vodi tj., izmjeriti 2g mega 10 i otopiti u 10 ml vode.,(b) izmjeriti 50 mg ho1esterola i dodati na 50 mg fosfatidilholina. (c) kada je holesterol otop1jen u fosfatidi1ho1inu dodati 2 ml k1oroforma (d) Dodati 10 m1 20% w/v Mega 10. Smjesa če biti tu mliječno-bije1a po izg1edu. Ovo će podići otopinu na 12 ml. Međutim, Kloromorf će ispariti poslije mješanja i postići će se finalni obujam 10 ml. (e) Staviti na magnetnu mješalicu u vrućoj prostoriji (37° C ), sa poklopcem kontejnera koji jeodvojen. Poslije nastavljenog miješanja, smjesa će postati mobilnija i bistra. (f)Spremati na sobnoj temperaturi (a) Make 20% w/v Mega 10 in sterile distilled water, ie, measure 2g of Mega 10 and dissolve in 10 ml of water., (b) Measure 50 mg of cholesterol and add to 50 mg of phosphatidylcholine. (c) when cholesterol is dissolved in phosphatidylcholine, add 2 ml of chloroform (d) Add 10 ml of 20% w/v Mega 10. The mixture will appear milky-white. This will bring the solution to 12 ml. However, Chloromorph will evaporate after mixing and the final volume will be 10 ml. (e) Place on a magnetic stirrer in a hot room (37° C), with the lid of the container detached. After continued mixing, the mixture will become more mobile and clear. (f) Store at room temperature
Primjer 1 Example 1
Protokol. Staviti 20 ml PBSA u sterilan univerzalan ba1on. Dodati 100µl lipidnog miksa. Vrtložno mješati otopinu punom brzinom 10 sekundi. Sterilizirati otopinu prolaženjem kroz filter 0.2, µm (Millipore). Dekantirati mali obujam za proučavanje elektronske mikroskopije. Protocol. Place 20 ml of PBSA in a sterile universal flask. Add 100 µl of lipid mix. Vortex the solution at full speed for 10 seconds. Sterilize the solution by passing it through a 0.2 µm filter (Millipore). Decant a small volume for electron microscopy study.
Primjer 2 Example 2
Prati se postupak iz primjera 1 ali sa slijedećim varijantama. Staviti 20 ml PBSA u sterilan univerzalan balon. Dodati 200 µl 10%-ne otopine Quil-a A. Dodati 800 µl lipidnog miksa. Vrtložno mješati otopinu punom brzinom 10n sekundi. Sterilzirati kroz 0.2 µm filter. Dekantirati mali obujam za proučavanje elektronske mikroskopije. Spremati na +4° C. The procedure from example 1 is followed, but with the following variants. Place 20 ml of PBSA in a sterile universal flask. Add 200 µl of 10% Quil A solution. Add 800 µl of lipid mix. Vortex the solution at full speed for 10 seconds. Sterilize through a 0.2 µm filter. Decant a small volume for electron microscopy study. Store at +4° C.
Primjer 3 Example 3
20 ml PBSA dodati Amicon mješanu stanicu sa 30K Mwt (molekulska težina) odsječenom membranom. U ovo dodati 800 µl lipidnog miksa plus 200 µl Quil-a A (10%- na otopina). Otopina se koncentrira na 5 ml i doda se 100 ml PBSA. Gornja faza se ponovi x2. Sadržaj od 20 ml se filtrira kroz markiran filter od 0.2µm i čuva se na +4°C. Add 20 ml of PBSA to an Amicon mixed cell with a 30K Mwt (molecular weight) membrane cut off. To this add 800 µl of lipid mix plus 200 µl of Quil A (10% solution). The solution was concentrated to 5 ml and 100 ml of PBSA was added. The above phase is repeated x2. The content of 20 ml is filtered through a marked filter of 0.2 µm and stored at +4°C.
Primjer 4 Example 4
20 ml PBSA dodaju se na Amicon mješanu stanicu sa odsječenom membranom od 30K na mjestu. Dodaju se 400µl lipidnog miks plus 100 µl 10% otopine Quil-a A. Gornja otopina se koncentrira na 5 ml i resuspendira na 100 ml sa PBSA. Gornja faza se ponovi x2. Finalni sadržaj od 20 ml stanice se sterilizira filtracijom kroz filter od 0.2 µm., markiraju i spramaju na +4°C. 20 ml of PBSA is added to the Amicon mixing cell with the cut-off 30K membrane in place. Add 400 µl lipid mix plus 100 µl 10% solution of Quil A. The above solution is concentrated to 5 ml and resuspended to 100 ml with PBSA. The above phase is repeated x2. The final contents of 20 ml of cells are sterilized by filtration through a 0.2 µm filter, labeled and stored at +4°C.
Primjer 5 Example 5
20 ml. PBSA doda se u Amicon mješanu stanicu, pri čemu je odsječena membrana od 30K Mwt.na mjestu. Dodaju se 200 µl lipodnog miksa plus 50 µl 10% otopine Quil-a A. Gornja otopina se koncentrira na 5 ml i resuspendira se u 100 ml PBSA Gornja faza se pomovi x2. Finalni preparat od 20 ml stanica se tada filtrira kroz filter od 0.2 µm, markira i sprema na +4°C. 20 ml. PBSA is added to the Amicon mixing cell, with the cut-off 30K Mwt membrane in place. Add 200 µl of lipid mix plus 50 µl of 10% Quil A solution. The above solution is concentrated to 5 ml and resuspended in 100 ml of PBSA. The upper phase is washed x2. The final preparation of 20 ml cells is then filtered through a 0.2 µm filter, labeled and stored at +4°C.
Primjer 6 Example 6
Dodaju se 20 ml PBSA u Amicon mješanu stanicu (odsječena membrana od 30 K Mwt). Dodaju se 100 µ1 lipidnog miksa plus 25 µl 10% otopine Quil-a A. Gornja otopina se koncentrira na 5 ml i resuspendira se sa 100 ml PBSA. Gornja faza se ponovi x2. Finalni sadržaj od 20 ml stanice se sterilizira filtracijom kroz filter od 0.2 µm, markiraju i spremaj u na +4° C. Add 20 ml of PBSA to an Amicon mixed cell (30 K Mwt cut-off membrane). Add 100 µl of the lipid mix plus 25 µl of a 10% solution of Quil A. The above solution is concentrated to 5 ml and resuspended with 100 ml of PBSA. The above phase is repeated x2. The final contents of 20 ml cells are sterilized by filtration through a 0.2 µm filter, labeled and stored at +4°C.
U proučavanju elektronske mikroskopije, izgleda da su matrice iscoma manje brojne nego u drugim primjerima. In electron microscopy studies, iscoma matrices appear to be less numerous than in other examples.
Primjer 7: Proučavanje na miševima: Efikasnost Example 7: Study on mice: Efficacy
Koriste se ženke miševa urođene vrste (Balb/c) iste starosti (8 tjedana). Miševi su dobiveni iz jedinice koja je održavana iza pregrade (SPF) Harlan-Olac (Bicester). U ovoj studiji korišten je spoj Mycoplasma hyopneumoniae iz National Cell Type Collection. Organizam se kultivira u modifikaciji Friis podloge. Spremljene kulture održavaju se na -70°C svaka suspenzija za inkorporiranje cjepiva napravljena je inokulacijom 40 ml juhe sa 4 ml spremljene kulture na pH 7.4 i tada inkubacijom na 37°C tokom 3 dana ili dok pH ne opadna na 6.8. Do ovog vremena organizam je dostigao log fazu rasta. Kulture se inaktiviraju korištenjem binarnog etilenimina, koncentriraju se korištenjem ultrafiltracije sa tangencijalnim protokom i isperu se sa fosfatnom puferovanom slanom otopinom (PBS). Koncentracija antigena podesi se tako da sva cjepiva sadrže 10 µg ukupnog proteina na dozu od 0.1 ml mišjeg cjepiva. Female mice of the inbred species (Balb/c) of the same age (8 weeks) are used. Mice were obtained from the Harlan-Olac maintained behind partition (SPF) unit (Bicester). The Mycoplasma hyopneumoniae compound from the National Cell Type Collection was used in this study. The organism is cultivated in a modified Friis medium. Stored cultures are maintained at -70°C each suspension for incorporating the vaccine is made by inoculating 40 ml of broth with 4 ml of the stored culture at pH 7.4 and then incubating at 37°C for 3 days or until the pH drops to 6.8. By this time, the organism has reached the log phase of growth. Cultures are inactivated using binary ethyleneimine, concentrated using tangential flow ultrafiltration, and washed with phosphate buffered saline (PBS). The antigen concentration is adjusted so that all vaccines contain 10 µg of total protein per dose of 0.1 ml of mouse vaccine.
Preparati iscoma iz primjera 3 do 6 korišteni su kako slijedi. Iscom preparations from examples 3 to 6 were used as follows.
Osam grupa od po šest miševa imuniziraju se u dvije prilike, i svaki miš prima po 0.1 ml. slijedećih preparata. Eight groups of six mice are immunized on two occasions, and each mouse receives 0.1 ml. the following preparations.
Grupa cijepiti Group to vaccinate
A samo 10 µg M. hyopneumoniae And only 10 µg of M. hyopneumoniae
B 10 µg M. hyopneumoniae dopunjeni sa Freundovim kompletom B 10 µg M. hyopneumoniae supplemented with Freund's kit
C 10 µg M. hyopneumoniae sa matricom iscoma koja sadrži 10 µg QA miš u dozi C 10 µg M. hyopneumoniae with iscoma matrix containing 10 µg QA mouse in dose
D 10 µg M. hyopneumoniae sa matricom iscoma koja sadrži 5 µg QA miš u dozi D 10 µg M. hyopneumoniae with iscoma matrix containing 5 µg QA mouse in dose
E 10 µg M. Hyopnemoniae sa matricom iscoma koja sadrži 2.5 µg QA miš u dozi E 10 µg M. Hyopnemoniae with iscoma matrix containing 2.5 µg QA mouse in dose
F 10 µg M. hyopneumoniae sa matricom iscoma koja sadrži 1.25 µg QA miš u dozi F 10 µg M. hyopneumoniae with iscoma matrix containing 1.25 µg QA mouse in dose
G 0 µg M. hyopneumoniae sa matricom iscoma koja sadrži 5 µg QA miš u dozi G 0 µg M. hyopneumoniae with iscoma matrix containing 5 µg QA mouse in dose
H 10 µg M. hyopneumoniae + 107 cfu H. pleurpneumoniae sa matricom iscoma koja sadrži 5 µg QA miš u dozi H 10 µg M. hyopneumoniae + 107 cfu H. pleurpneumoniae with iscoma matrix containing 5 µg QA mouse in dose
QA=Quil A QA=Quil A
Grupe miševa se pred-iskrvare kroz repnu venu prije primarne imuni zacije i tada 14 dana kasnije. 28 dana poslije primarne imunizacije životinje su primile daljnje cijepljenje i iskrvavljene su 7 dana kasnije i finalno 28 dana poslije sekundarnog cijepljenja. Serum je napravljen iz krvi na dan skupljanja i spreman je na -70°C za analizu pomoću ELISA Miševi su promatrani poslije cijep1jenja na bilo kakve štetne lokalne i sistematske reakcije. Groups of mice are prebled through the tail vein before primary immunization and then 14 days later. 28 days after the primary immunization, the animals received a further vaccination and were bled 7 days later and finally 28 days after the secondary vaccination. Serum was made from blood on the day of collection and was stored at -70°C for analysis by ELISA. Mice were observed after vaccination for any adverse local and systemic reactions.
Rezultati the results
Rezultati eksperimenata planiranih za procjenu pomoćnog efekta Quil-a A a u preparatu iscomma, gdje je antigen vanjski za preparat, prikazani su ispod u tablici 1 i 2. The results of the experiments planned to evaluate the adjuvant effect of Quil A in the iscomma preparation, where the antigen is external to the preparation, are shown below in Tables 1 and 2.
Rezultati jasno demonstriraju da ISCOM preparat sa najvišim proteinom Quil A odnos od 10 µg: 10 µg/ miš doza, proizvodi najveću reakciju na cjepivo, dajuči isto tako dobru reakciju., ako ne i bolju, kao cjepivo sa Freund-ovim pomoćnim sredstvom. The results clearly demonstrate that the ISCOM preparation with the highest protein Quil A ratio of 10 µg:10 µg/mouse dose, produces the highest vaccine response, giving as good a response, if not better, than the Freund's adjuvant vaccine.
Grupe miševa koje su primile iscom cjepiva koja sadrže manje od 10 µg Qull-a A po mišu u dozi, nisu bile bolje, u proizvodnji reakcije na antigen, samo od nespomenutog antigena. Nisu zapažene reakcije na cjepivo niti lokalne niti sistematske na bilo kojim miševima koji su primili iscom cjepivo ili samo antigen. Na miševima koji su imunizirani sa Freund-om, zapaženi su mali granulomi približe veličine 3 mm na točki imunizacije, ali kao i s a svim drugim miševima nisu zapaženi znaci sistematskih poremećaja. Groups of mice that received iscom vaccines containing less than 10 µg of Qull A per mouse dose were no better, in producing a response to the antigen, than the blank antigen alone. No local or systemic reactions to the vaccine were observed in any mice that received iscom vaccine or antigen alone. In mice immunized with Freund, small granulomas of approximately 3 mm in size were observed at the point of immunization, but as with all other mice, no signs of systemic disorders were observed.
Tablica 1: Anti M. hyopneumoniae na miševima kao što je izmjereno indirektnim ELSA testom Table 1: Anti M. hyopneumoniae in mice as measured by the indirect ELSA assay
[image] [image]
Izostavljena su standardna odstupanja DP2°C - dani poslije sekundarnog cijepljenja. Standard deviations of DP2°C - days after secondary vaccination are omitted.
Primjer 8: Proučavanja na miševima: Procjena prazne iscom matrice kao pomoćnog sredstva za Hemophilius pleuropneumoniae cjepiva na miševima. Example 8: Studies in mice: Evaluation of blank iscom matrix as an adjuvant for Hemophilius pleuropneumoniae vaccines in mice.
Miševi se imuniziraju na cjepivima koja sadrže cijele inaktivirane H. pleuropneumoniae bakterije i iscom matricu koja je formulirana sa različitim nivoima Quil-a A. Miševi koji su imunizirani dva puta sa cjepivom koje sadrži 10 µg Quil-a A/miš dala je veću antitijelo reakciju od miša koji su imunizirani sa ekvivalentnim brojevima bakterija u Freund-ovom pomoćnom sredstvu. Niže doze Quil-a A su imale vidljivi pomoćni efekat. Nisu zapaženi štetni lokalni ili sistematski efekti poslije davanja bilo kojeg cjepiva koje sadrži matricu iscoma. Mice are immunized with vaccines containing whole inactivated H. pleuropneumoniae bacteria and an iscom matrix formulated with different levels of Quil A. Mice immunized twice with a vaccine containing 10 µg of Quil A/mouse gave a greater antibody response. from mice immunized with equivalent numbers of bacteria in Freund's adjuvant. Lower doses of Quil A had a visible auxiliary effect. No adverse local or systemic effects have been observed following administration of any vaccine containing iscoma matrix.
NCTC referentni sloj H. pleuropneumoniea serotip 3 (sloj 1421) kultiviran je do log faze rasta u podlozi sa kvascevim ekstraktom koja je dopunjena sa B NAD. Kultura se inaktivira sa 0.2% formalina i ispere se tri puta sa fosfatnim puferiziranom slanom otopinom (PBS) tako da se dobiva isprani preparat inaktiviranog antigena iz cijele stanice. Antigen se standardizira enumeracijom bakterija korištenjem Neubaumer komore sa modificiranim Thoma pravilima i sprema se u PBS na 4°C do korištenja. NCTC reference strain H. pleuropneumoniea serotype 3 (strain 1421) was cultured to log phase growth in yeast extract medium supplemented with B NAD. The culture is inactivated with 0.2% formalin and washed three times with phosphate buffered saline (PBS) so that a washed preparation of the inactivated antigen from the whole cell is obtained. Antigen is standardized by bacterial enumeration using a Neubaumer chamber with modified Thoma rules and stored in PBS at 4°C until use.
Grupe miševa, šest po grupi, imuniziraju se samo sa H. pleuropneumoniae, sa H. pleuromoniae u FIA ili H. pleuropneumoniae ili H. pleuropneumoniae zajedno sa matricom iscorna koja sadrži različite nivoe Quil-a A, kao što je detaljno dato niže. Finalna grupa primila je i H. pleuropneumoniae i H. hypopneumoniae antigene zajedno sa matricom iscoma tako da se istraži moguća interakcija između dva antigena. Miševi su primili dvije potkožne injekcije od po 0.1 ml odgovarajućeg cjepiva u dvije prilike sa 3-tjednim intervalom između imunizacija. Groups of mice, six per group, are immunized with H. pleuropneumoniae alone, with H. pleuromoniae in FIA or H. pleuropneumoniae or H. pleuropneumoniae together with iscorna matrix containing different levels of Quil A, as detailed below. The final group received both H. pleuropneumoniae and H. hypopneumoniae antigens together with the iscoma matrix to investigate a possible interaction between the two antigens. Mice received two subcutaneous injections of 0.1 ml of the respective vaccine on two occasions with a 3-week interval between immunizations.
Imunizacija miševa Immunization of mice
Grupa Cijepljenje Group Vaccination
1 ništa 1 nothing
2 samo Hpl 2 only Hpl
3 Hpl u FIA 3 Hpl in FIA
4 Hpl sa iscom matricom koja sadrži 10 µg QA/po mišu u dozi cjepiva 4 Hpl with iscom matrix containing 10 µg QA/per mouse at vaccine dose
5 Hpl sa iscom matricom koja sadrži 5 µg QA/po mišu u dozi cjepiva 5 Hpl with iscom matrix containing 5 µg QA/per mouse at the vaccine dose
6 Hpl sa iscom matricom ko a sadrži 2.5 µ gQA/po mišu u dozi cjepiva 6 Hpl with isca matrix containing 2.5 µ gQA/per mouse in the vaccine dose
7 Hpl sa iscom matricom koja sadrži 1.2 µg QA/po mišu u dozi cjepiva 7 Hpl with iscom matrix containing 1.2 µg QA/per mouse at the vaccine dose
8 Hpl sa iscom matricom koja sadrži 5 µg Q A / po mišu u dozi cjepiva i 10 µg/po mišu u dozi Mycoplasma hyopneumoniae antigena. 8 Hpl with iscom matrix containing 5 µg Q A / per mouse in the dose of vaccine and 10 µg / per mouse in the dose of Mycoplasma hyopneumoniae antigen.
Poslije primarnog cijep1jenja bilo je ma1o antitijela reakcije u bilo kojoj grupi. Poslije sekundarnog cijepljenja nestalo je brzo i naglašeno povećanje u nivoima antitijela u 2 od 8 imuniziranih grupa. Najveća reakcija viđena je na miševima koji su imunizirani sa bakterijama zajedno sa iscom matricom koja sadrži 10 µg/miš Quil-a A koja je imala prosječan pik od 1.24 (± 0.17). Jedinica optičke gustoće; (UOD ) kada je mjereno 1 tjedan poslije sekundarnog cjepljenja. Miševi imunizirani sa bakterijama i FIA imali su sličnu, ali neznatno manju antitijelo reakciju sa prosječnim pikom od 1.11 (± 0.08) UOD istom prilikom. Miševi imunizirani samo sa bakterijama, sa bakterijama sa nižim dozama Quil-a A, ili sa bakterijama zajedno sa M. hyopneumoniae antigenom su svi imali slične, niže, reakcije antitijela sa prosječnim vrijednostim između 0.43 i 0.63 UOD kada se mjeri jedan mjesec poslije sekundarne imunizacije. After primary vaccination, there were few antibody reactions in any group. After the secondary vaccination, the rapid and pronounced increase in antibody levels disappeared in 2 of the 8 immunized groups. The largest response was seen in mice immunized with the bacteria together with isca matrix containing 10 µg/mouse Quil A which had an average peak of 1.24 (± 0.17). Unit of optical density; (UOD ) when measured 1 week after secondary vaccination. Mice immunized with bacteria and FIA had a similar but slightly lower antibody response with an average peak of 1.11 (± 0.08) UOD on the same occasion. Mice immunized with bacteria alone, bacteria with lower doses of Quil A, or bacteria together with M. hyopneumoniae antigen all had similar, lower, antibody responses with an average value between 0.43 and 0.63 UOD when measured one month after the secondary immunization .
Tablica 2: Anti-H. pleuropneumoniae reakcija, kao što je izmjerena indirektnim ELSA testom Table 2: Anti-H. pleuropneumoniae reaction, as measured by the indirect ELSA test
[image] [image]
Primjer 9: Proučavanje na svinjama Example 9: Study on pigs
30 landrace-ukrštenih praščića starosti 3 tjedna dobiveni su iz krda za koj se zna da je slobodno do svih kliničkih i seroloških dokaza infekcije sa H. pleuropneumoniae i M. Hyopneumoniae. Prasad se održava izolirano od cijelog ostalog krda i hrane se po volj i sa standardnom hranom za rast odojaka odbijenih od sise. 30 3-week-old landrace-cross piglets were obtained from a herd known to be free of all clinical and serological evidence of infection with H. pleuropneumoniae and M. Hyopneumoniae. Piglets are kept in isolation from the rest of the herd and are fed ad libitum and standard weanling feed.
Kultivira se poljsi izoiat sa malo prolaza (manje od 10) (SVA3) M. hyopneumoniae do log faze rasta u Friis podlozi. A low passage (less than 10) field isoiate (SVA3) of M. hyopneumoniae is cultured to the log phase of growth in Friis medium.
Na 20 ml sterilnog PBSA doda se 400 µl 10% Quil-a A otopine i 1600 µl lipidnog miksa. Smjesa se tada vrtložno mješa 20 sekundi sa maksimalnom brzinom i onda se filtrira kroz filter od 0.45 µm. Na 10 ml ovog ISCOM miksa doda se 10 ml 4 mg/ml M. hyopneumoniae antigena tako da se dobiva ISCOM miks koji sadrži 2 mg/ml antigena i 1 mg/ml Quil-a A. 400 µl of 10% Quil A solution and 1600 µl of lipid mix are added to 20 ml of sterile PBSA. The mixture is then vortexed for 20 seconds at maximum speed and then filtered through a 0.45 µm filter. To 10 ml of this ISCOM mix, 10 ml of 4 mg/ml M. hyopneumoniae antigen is added so that an ISCOM mix containing 2 mg/ml antigen and 1 mg/ml Quil A is obtained.
Grupa od 5 praščića imunizira se sa 1 ml ISCOM matrice koja sadrži 2 mg/ml antigena cijele juhe M. Hyopneurnoniae. Daljih 5 primili su 1 ml 2 mg/ml M. hyopneumoniae sa Freundovim pomoćnim sredstvom. Finalnih 5 praščića primili su samo PBSA. A group of 5 piglets is immunized with 1 ml of ISCOM matrix containing 2 mg/ml of M. Hyopneurnoniae whole broth antigen. Another 5 received 1 ml of 2 mg/ml M. hyopneumoniae with Freund's adjuvant. The final 5 piglets received only PBSA.
Praščići su primili njihovo prvo cijep1jenje u 3 tjedna starosti dubokom intramuskularnom injekcijom 1 ml odgovarajućeg cjepiva u duboke mišiće lijeve strane vrata. Druga slična injekcija data je 6 tjedana kasnije u desnu stranu vrata. 2 tjedna poslije druge imunizacije, kada su bili stari 11 tjedana, praščići su eksperimentalno zaraženi. Prasčići su ubijen i priblžno 2 tjedna poslije zaraze za posmrtno ispitivanje patologije pluća. Piglets received their first vaccination at 3 weeks of age by deep intramuscular injection of 1 ml of the appropriate vaccine into the deep muscles of the left side of the neck. A second similar injection was given 6 weeks later in the right side of the neck. 2 weeks after the second immunization, when they were 11 weeks old, the piglets were experimentally infected. Piglets were killed approximately 2 weeks after infection for postmortem examination of lung pathology.
a) Kliničke reakcije na cijepljenje a) Clinical reactions to vaccination
Nisu zapaženi štetni klinički znaci na bilo kojim praščićima poslije primarnog ili sekundarnog cijepljenja. Nema značajnih povećanja temperature poslije primarne ili sekundarne imunizacije u bilo kojoj grupi. Najveće povećanje temperature od 1.1 ± 0.23°C, zapaženo je na praščićima koji su imunizirani sa M. hyopneumoniae antigenom u Freundovom nekompletnom pomoćnom sredstvu poslije sekundarne imunizacije. Uglavnom, prosječno se temperatura u grupi praščića penjala za približno 0.3 °C što je bilo nedovoljno za izazivanje kliničkih znakova pireksije. Cjepiva koja su sadržavala M. hyopneumoniae nisu bila značajno pirogenija od onih koja nisu. No adverse clinical signs were observed in any piglets after primary or secondary vaccination. There were no significant increases in temperature after primary or secondary immunization in any group. The highest temperature increase of 1.1 ± 0.23°C was observed in piglets immunized with M. hyopneumoniae antigen in Freund's incomplete adjuvant after secondary immunization. Basically, the average temperature in the group of piglets rose by approximately 0.3 °C, which was insufficient to cause clinical signs of pyrexia. Vaccines that contained M. hyopneumoniae were not significantly more pyrogenic than those that did not.
Nisu bile vidljive lokalne reakcije niti su se mogla palpirat mjesta injekcija bilo kojeg cjepiva u bilo koje ispitivano vrijeme. No local reactions were visible nor could the injection sites of any vaccine be palpated at any time examined.
Nema razlike između prirasta težine kod praščića. There is no difference between weight gain in piglets.
b) Kliničke reakcije na cjepiva b) Clinical reactions to vaccines
Kliničke reakcije koje prate injekcije nisu bile vidljive. Eksperimentalna zaraza nije izazvala pireksiju, praščići su ostali klinički normalni 2 tjedna poslije zaraze. Prirast težine tijekom ovog perioda i unošenje hrane nisu pokazali značajne razlike između grupa. Clinical reactions accompanying the injections were not visible. The experimental infection did not cause pyrexia, the piglets remained clinically normal 2 weeks after the infection. Weight gain during this period and food intake did not show significant differences between groups.
c) Mjerenje antitijela u serumu c) Measurement of antibodies in serum
Nakon cijepljenja samo grupa koja je primila M. hyopneumoniae sa Freundovim pomoćnim sredstvom demonstrirala je antitijelo reakciju. Međutim, nakon druge imunizacije nestalo je naglašeno povećanje nivoa antitijela u svim i životinjama koje su primile cjepivo M. hyopneumoniae. Reakcije serumskog M. hyopneumoniae antitijela mjerene su sa ELSA OD (na 492 nm) prije zaraze i bile su kako slijedi: After vaccination, only the group that received M. hyopneumoniae with Freund's adjuvant demonstrated an antibody response. However, after the second immunization, the pronounced increase in antibody levels disappeared in all animals that received the M. hyopneumoniae vaccine. Serum M. hyopneumoniae antibody responses were measured by ELSA OD (at 492 nm) before challenge and were as follows:
za životinje koje su primile ISCOM cjepivo 0.6±0.156 for animals that received ISCOM vaccine 0.6±0.156
za životinje koje su primile cjepivo sa Freundovim sredstvom 1.7±0.089 for animals that received a vaccine with Freund's agent 1.7±0.089
Reakcija anti M. hyopneumoniae antitijela (OD na 492 nm= 0.6±0.156) također je zapažena na životinjama koje su primile cjepivo sa ISCOM matricom. An anti M. hyopneumoniae antibody reaction (OD at 492 nm= 0.6±0.156) was also observed in animals that received the ISCOM matrix vaccine.
Nije bi1o daljnjeg značajnijeg povećanja antitijela u bilo kojoj grupi infekcije. There was no further significant increase in antibodies in any infection group.
d) Lokalne reakcije na cijepljenje d) Local reactions to vaccination
Najjače reakcije zapažene su na praščićima koji su imunizirani sa M. hyopneumoniae antigenom u Freundovom dodatku. Reakcije su se sastojale od višestruko disperziranih uzročnih granulomata i bile su karakteristične reakcije na Freudovo pomoćno sredstvo na praščićima. Nisu zapažena oštećenja na praščićima koji su primili ISCOM cjepivo. The strongest reactions were observed in piglets immunized with M. hyopneumoniae antigen in Freund's adjunct. The reactions consisted of multiple dispersed causative granulomas and were characteristic reactions to Freud's adjuvant in piglets. No damage was observed on the piglets that received the ISCOM vaccine.
Primjer 10: Proučavanje na ovcama Example 10: Study on sheep
Blackface x Swledale ovce dobivene su sa farme za koju se zna da nije bilo OEA najmanje 4 godine. 5-6 godina stare životinje su prethodno ispitane prije korištenja na antitijela na Chlamydia psittaci i Toxoplasma gondii. Ovce se drže u izolaciji od drugih krda i hrane se sijenom i koncentratima pro odgovarajućim nivoima za njihov fiziološki i nutricioni status. Blackface x Swledale ewes were obtained from a farm known to have been free of OEA for at least 4 years. 5-6 year old animals were previously tested for antibodies to Chlamydia psittaci and Toxoplasma gondii before use. Sheep are kept in isolation from other herds and are fed hay and concentrates at appropriate levels for their physiological and nutritional status.
1. Cjepiva 1. Vaccines
OEA sojevi A22 i S26/3 Chlamydia psittaci se kultiviraju i kasnije se polupročiste. Početni obujam kulture za svaki sloj od 10L koncentrira sa na 1L u ovom postupku tako da se dobiva 10X Antigen. OEA strains A22 and S26/3 Chlamydia psittaci are cultured and later semi-purified. The initial culture volume for each 10L layer is concentrated to 1L in this procedure so that 10X Antigen is obtained.
Iscom cjepivo napravljeno je kako slijedi (IV): 50 m1 10X antigena tretira se sa 100 µl stoka (50%) glutaraldehida tokom 15 min na 4°C. (b) Doda se 50 ml 0.15M glicerina steriliziranog na filteru PBS i uspenzija se inkubira na 5 min 4°C. (c) Suspenzija se centrifugira na 17000g tijekom 45 min na 4°C , tada se granula ispere dva puta steriliziranim PBS pH 7.2. (d) Granula se resuspendira u 50 ml PBS. (e) Doda se tiomersal (1%; steriliziran filtracijom), 0.5 ml, tako da se dobija finalna koncentracija 0.1%. Iscom vaccine was made as follows (IV): 50 m1 of 10X antigen is treated with 100 µl of stock (50%) glutaraldehyde for 15 min at 4°C. (b) 50 ml of 0.15 M glycerin sterilized on a PBS filter is added and the suspension is incubated for 5 min at 4°C. (c) The suspension is centrifuged at 17,000 g for 45 min at 4°C, then the granule is washed twice with sterilized PBS pH 7.2. (d) The pellet is resuspended in 50 ml of PBS. (e) Add thiomersal (1%; sterilized by filtration), 0.5 ml, so that a final concentration of 0.1% is obtained.
Na 100 ml steriliziranog PBSA dodoaje se 50 µl 10% Quil-a A otopine i 2 ml lipidnog miksa. Smjesa se mješa pod tlakom od 62 kN.m2 i koncentrira se na obujam 20 ml Dodaje se sterilni PBSA da se otopina dopuni do 100 ml i obujam se smanji na 20 ml. Ovo se ponovi dva puta. Finalni obujam se filtrira kroz filter od 0.45µm. 50 µl of 10% Quil A solution and 2 ml of lipid mix are added to 100 ml of sterilized PBSA. The mixture is mixed under a pressure of 62 kN.m2 and concentrated to a volume of 20 ml. Sterile PBSA is added to make the solution up to 100 ml and the volume is reduced to 20 ml. This is repeated twice. The final volume is filtered through a 0.45 µm filter.
Preparat antigena se dodaje Iscom miksu tako da se dobija koncentracija antigena 0.25 X antigen/ml. The antigen preparation is added to the Iscom mix so that the antigen concentration is 0.25 X antigen/ml.
Sačini se jedno cjepivo na bazi ulja (OBV) i jedno vodeno cjepivo sa adsorbiranim Alhydrogelom (AAV) kao kontrole tako da se dobivaju 1X antigen/ml, odnosno 0.25 X antigen/ml. One oil-based vaccine (OBV) and one aqueous vaccine with adsorbed Alhydrogel (AAV) are prepared as controls so that 1X antigen/ml and 0.25 X antigen/ml are obtained.
Eksperiment je obuhvaćao 5 grupa i ukupno 106 ovaca. Grupe 1, 2 i 3 cijepljene su sa OBV, AAV, odnosno sa IV. Grupe 4 i 5 činile su kontrole, i bile su inficirane/necijepljene, odnosno neinficirane/necijepljene. The experiment included 5 groups and a total of 106 sheep. Groups 1, 2 and 3 were vaccinated with OBV, AAV and IV. Groups 4 and 5 were controls, and were infected/unvaccinated and uninfected/unvaccinated, respectively.
OBV cjepivo je davano jednom dnevno kao doza od 1 m1 davano 9 tjedana prije sazrijevanja. Druga cjepiva davana su dva puta, 9 tjedana i 3 tjedna prije sazrijevanja, i svaka injekcija obuhvaća dozo od 2 ml (sva cjepiva isporučivala su količinu od 4 µg Chlamydial proteina). The OBV vaccine was given once daily as a dose of 1 m1 given 9 weeks before maturation. The other vaccines were given twice, 9 weeks and 3 weeks before maturation, and each injection comprised a dose of 2 ml (all vaccines delivered an amount of 4 µg of Chlamydial protein).
Sva cjepiva su injektirana potkožno na identificiranom ogoljelom mjestu na desnoj strani vrata. All vaccines were injected subcutaneously at an identified exposed site on the right side of the neck.
Ovce iz grupa 1, 2, 3 i 4 zaraze se sa živim chlamydia organizmima pri 85-90 d.g. Grupa 5 je ostala netretirana. Sheep from groups 1, 2, 3 and 4 are infected with live chlamydia organisms at 85-90 d.g. Group 5 remained untreated.
Prilikom janjenja ili abortusa uzimane su placente i ako su pristupačne, za uzorkovanje. Bilježeno je stanje janjeta u ovo vrijeme (živo, mrtvorođeno ili mrtvo). Nije rađen pokušaj da se drže živim bolesni janjci, osim osiguranja kolostruma. During lambing or abortion, placentas were taken and, if accessible, for sampling. The state of the lamb at this time was recorded (alive, stillborn or dead). No attempt was made to keep sick lambs alive, other than providing colostrum.
Zapaženo je prisustvo ili drukčijih OEA oštećenja u placentama i procjenjen je njihov sadržaj. Placente (ili vaginalni brisevi) su ispitani na chlamydial tijela bojenjem pomoću Ziehl-Neelsen-ovog postupka, i kultivirane su u BHK21 stanicama koje su tretirane sa 5-jododeoksiridinom. The presence or other OEA damage in the placentas was noted and their content was evaluated. Placentas (or vaginal swabs) were examined for chlamydial bodies by staining using the Ziehl-Neelsen method, and were cultured in BHK21 cells treated with 5-iododeoxyridine.
Rezultati the results
Rezultati su pokazali da je ISCOM cjepivo osiguralo.zaštitu protiv Chlamydial infekcije koja je bila najmanje isto tako dobi a kao što je osiguraju konvencionalna cjepiva korištena u eksperimentu. The results showed that the ISCOM vaccine provided protection against Chlamydial infection that was at least as old as that provided by the conventional vaccines used in the experiment.
Primjer 11: Iscom preparat za virus konjske influence Example 11: Iscom preparation for equine influenza virus
Protokol: Staviti 20 ml PBSA u sterilni univerzalni balon. Dodati 100 µl 10%-ne otopine Quil-a A. Dodati 400 µg lipidnog miksa. Dodati ERIK-14 antigensku komponentu konjskr gripe u finalnoj koncentraciji 5 µg/ml. Vrtložno mješati punom brzinom tijekom 20 sekundi. Sterilizirati otopinu prolaženjem kroz 0.2µm filter (Milipore). Dekantirati mali obujam za. proučavanja elektronske mikroskopije (zapažene iscom strukture) i provjeru bakterijske kontaminacije. Spremati na +4°C. Protocol: Place 20 ml of PBSA in a sterile universal flask. Add 100 µl of 10% Quil A solution. Add 400 µg of lipid mix. Add ERIK-14 antigenic component of equine influenza at a final concentration of 5 µg/ml. Vortex at full speed for 20 seconds. Sterilize the solution by passing it through a 0.2 µm filter (Millipore). Decant a small volume for. electron microscopy studies (observed by iscom structures) and verification of bacterial contamination. Store at +4°C.
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