CA1120855A - Chagas vaccine and process for its manufacture, killed trypanosoma and their use for the manufacture of immunologically active cell constituents - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to a process for the manufac-ture of a vaccine against the Chagas disease in which an aqueous suspension of 1 x 106 to 6 x 109 per ml of Try-panosoma cruzi organisms obtained from culture media is incubated with at least 0.5% (g/v) of an acylating agent or of an aliphatic mono- or dialdehyde having a chain length of 1 to 6 carbon atoms, at 4 - 40° C, until all organisms are killed, the killed organisms are separated from the incubation medium, suspended in a physiologi-cally tolerated medium and further worked up in known manner to yield a vaccine. Further objects of the in-vention are a Chagas vaccine prepared according to the processes of the invention, killed Trypanosoma and their use for the preparation of immunologically active cell constituents.

Description

-`-" liZ~355 The invention relates to a vaccine against the Chagas disease,t:~e e~sential aotive oo~ponent of which ~BiS~8 of killed trypanosoma and to a process for the manufacture of lcilled trypanosoma.
The ger~ of the Chagas disease which is affecting millions of humans is the parasite Trypanosoma cruzi. There have been made se~eral attempts to control this disease by immunization.
Thus, in several reports it has been alleged that the ~arasite could be killed and that a vaccine could be prepared therefrom.
However, intensi~e investigations of the processes used in this respect have shown that the immunizing activity of such vaccines is caused by small proportions of not killed germs. HoweverJ
objections remained regarding the infectiosity of such vaccines.
At present, those skilled in the art are of the opinion that 2 vaccine which is free from living Trypanosoma is inefficient, DUt thatJ on the other hand-, livin~ Try~ano~oma include tho risk o~
&:n~~inrection o~ the vacclnated person.
Now, we have found that, sùrprisingly, killed Trypanosoma retain their immunogenicityJ if a relatively high concentration of inactivating agents is used for killin~ the Tr~panoso~a.
additionJ these concentrations make sure that liYing Try-panosoma are no longer present in the final product.
Accordi~glyJ the object of the invention is a process for the preparation of a vaccine against th~ Chagas di~easeJ which is characterized by incubating an aqueous suspension of 1 x 1o6 to 6 x 109J preferably 1 x 109 to 3 x lOgJ per ml of Trypanoso~a cruzi or~anisms obtai~ed from culture media uith at least 0.
~ w/~J) g'v) of an acylating agent or of an aliphatic mo~o- or dialdeh~-de having a chain length of 1 to 6 C-atoms, at 4 - 40 CJ un'~il all organisms are ~illedJ separating the killed organisms from the

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incubation snedium, su~pending them ln a physiologically tolerated medium and ~vorking u~ in known manner to a vaccine.
When combined w~th a protein-stabilizing agent, the vaccine can be lyophylized without loss o~ its im~unogenicity.
Preferred agents for killin~ the Try~anosoma in the sense of the invention are acetic acid anhydridc in a concentration range of irom 10 to 30%, sulfosalicylic acid in a concentration range o~ ~rom 10 to 30%, maleic acid anhydride in a concentration range o~ ~rom 1 to 20%, pyrocarbonic acid ethyl ester in a concentration range oi from 10 to 3070, each time ~ , ln order to mention by way of example so~e o~ the acylating agents.
Fro~ the aldehydes, it is preferred to use ~or~aldehyde in a concentration range o~ from 1 to 870, glutardialdehyde in a concentration range of from 0.5 to 10%, propionaldehyde in a concentration range of from 1 to 20~, benzaldehyde in z concen-tration range of irom 1 to 30%, butyraldehyde in a concentration ran~e o~ from 10 to 30%, isobu~yraldehyde in a concentra~ion range of fro~ 0.5 to 1%, each ti~e ~
It is obvlous from the values given by way o~ example that the acti~ity ~naximum of the individual agents used for ~illing the Trypanoso~a is different. It may be said in general tkat . ~ , a~ minlmum concentration oi 0.5% o~ the inactivating agent is requlred for obt~ning the desired effect. ~atis~actory results are obtained with at leaæt 1%, good resu}ts gencrally with at least 5% of the ~nactivating agent in thc solution. The pre--ferred inactivatlon range is ~etween 8 and ~0%.
The agents used ~or kllling are dissolYed in a suitabl~
801ve~t, preferably w~ter or ethyl alcohol, and the Trypanosoma to be killed are suspended in this s~lution~ This is followed by the ~cubation.

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The substances used accord~Dg to the invention are suf~i-ciently active in the preferably used concentratio~ range in a time of 1 to 5 hours,-pre~erab~y about 2 hours, to obtain ~illed Trypanosoma which are suitable for a vaccine. With lower con-centrations, the activity can be iDcreased by USiDg a prolonged incubation time, with higher concéntrations the in~u~ation time can be shortened.
The killing of the Trypanosoma cruzi organisms within the scope of the invention can be examined by inoculating the suspension into 1 to 2 days old mice of a inbred strain. I~
the inoculation does not cause parasitemia within 10 ~o 30 days, i.e. no occurrenc0 of living Trypanosoma in the blood of the animals, the killing of the total population of the Trypanosoma suspension by the process of the lnvention can be considered as sure.
For this reason, it is adviaable, prior to each larger ~atch, to carry out test batches which are advantageously tested for the success~ul termination in the afore-described baby mouse odel.

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The trypanosoma killed according to the invention are immuno-genio~,~i.e. they lead ln the test person immunized with them, to~a protection agai~st a subsequent infection with Trypanosoma cruzi. The immunogenicity is ~ur~iciently strong: -that adJuvants which are not toler~ted by hu~ans need not ~e used. However, in order to increase tLta immunological activity, it is suitable to add i~munolo6ical adjuvants usual in human medicine to increase the~humoral or cellular immunity. These are, ~or example, alumiDium hydroxide or quartz powder in a very fine distribution, but alsQ ~ater-soluble adjuvants~ lor example neuraminidase.

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~ he fact i8 particularly surprising that the vaccine pre-pared accordln~ to the inventlon leads at a very early time after the injection to a test person to its protection and that the vaccine also has a prophylactic e~fect if it i8 administered ln due time, l.e. a few days after infection of the patient.
The mea~ures according to the invention cau~e a stabilization of the immunologlcal component. ~or ~torage the suspension con~
taininOE the killed Trypano~oma for the vaccine preparcd there~rom, can be lyophylized, suitably after addition of a stabilizer, for e~ample 0.01 to 10~ of carbohydrates, preferably 2 - 3~ of lactose or 0.02~ of mannit, wlthout considerably impairing the immunologioal activity.
The stability of the immunological components i8 al~o evidenced by the fact that it is possible to treat the killed Trypanosoma with mechanical mean~ which destroy the cell structure and to obtain there~rom the immunologically active products.
The cell structure Or the Trypanosoma organisms can be destructed not only by sound vibration, but also by shaking in the presence o~ geometric&l bodieq of solid material, or with the aid of pressure ulth following pressure relea~e. However, even chemical agent~ can be used, for example deterBents at elevated temperatures.
Separat~ion of the immunologically active components of the or-gani~ms can be carried out by electrophores~s, but also ~y gradi~nt centrifugation at bigh ~peed. Some ~ractions which can be isolated therefrom give, after inooulation to te~t per~ons, a protection again~t the Chagas disoa~e. Thus, another ob~ect of the invention i- the use of the Trypanosoma cruzi killed accordlng to ths invention for the pre~paratlon of immunogenically active component~
of the Trypano~oma or~anl~ms.

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- ~2Vt35~i The invention is illustrated by the following example~
EXAMPLE I:
1) Cultivation _~ Trypanosoma Trypanosoma cruzi of the strain D 1 are transferred, after a pre-cultiYation in a biphasic medium containing blood agar into a fermenter ~hich contains 18 liters of the medium CMRL 1066.
The medium contains:
mg/liter Amino-acids:
~-Alanine HCl 25.0 1-Arginine 70.0 1-A9paragine 30.0 1-Cysteine HCl-H20 .260.0 l-Cystinic acid 20.0 I-Glutamic acid 75.0 1-Glutamine 100.0 1-Glycine 50.o 1-Histidine HCl . H20 20~0 Hydroxyproli.ne 10.0 Isoleucine 20.0 ~: :
Leucine 60.0 ~ 1-Lysine-HCl 70,0 ; ~ 1-Methionine ~ 15.0 Phenylalanine 25 0 .
l-Proline 40.0 : 1-Serine. 25.0 - 1-T~hreonine 30.0 1-Tryptophan 10.0 l-Tyrosine 40.0 1-Valine 25,0 S
Vitamine~:
p-Aminobenzoic acid 0.050 Ascorbic acid 50.000 D-Biotin 0.010 D-Ca-Panthotenate 0.010 Choline chloride 0,500 Cocarboxylase 1.000 Folic acid 0.010 i-Inoæite 0.500 Nicotine-a~ide 0.025 Nlcotinic acid 0.025 Peridoxal HCl 0.025 Peridoxine HCl 0.025 Ribo~lavine 0.010 Thiamine HCl 0.010 Inorganic saltæ and other components:
Tween 80 5.0 Cholesterin 0.2 Coenzyme A 2.5 Desoxyadenosine 10.0 De~oxycytidine HCl 10.0 Deso~cyguanosine 10.0 Diphosphopyridine-uucleotide- tetrahydrates DPN~H20?
Fl:avine adeni`ne-dinucleotide (FAD) 1.0 Glutathic-ne ~ 10~0 NaH2PO~ . H20 NaHC03 2200 ' .

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' -` 112(~8~;5 Sodium acetat~ 3 H20 83.o Na Olucuronates 4.8 Thymidine 10,0 Triphosphopyridine-nucleotlde mono --Na-salt (TPN) 1.0 Urldlne-5'-tr~phosphate, tetra-Na-tetrahydrate (UTP) 1.0 . NaCl 6800 KCl 4 MgS~4 7 H20 200 : CaC12 (anhydr.) 200 aluoose 1000 ~: Phenol red 17 : .
The harvest was er~eoted at a~germ density of' 20 x 10 o~
; Trypanosoma/ml.
The Try~anosoma f'ermenter culture was centrl~uged at about 40,000 x g in a centrif'uge which was provided with a through-rotor and the resldue was suspended in a 0.~85~ strength ~ ~ :
NaCl solution. The suspenslon was again oentrlruged ~or 15~minutes at about~3;,000 x g.
The: sediment was resuspended in a 0.85~ strength NaCl-solut~on to~a~germ density of 3 x 10 Trypanosoma/ml, centrifuged and suspended in A phosphate-bu~iered sodium chloride solution oi pH 7.2 agail~ in a concentration o~ 3 x 109~ml.
2. Klllin~ Q~ ~ panosoma The suspension was again centr:i~uged and resuspended in the , ~

-rigin~l volume Or a phosphate bur~er Or p~ 7.2 which contained 8~ ~ Or ~ormaldehyde. ~ 37% strength acid-rree rormaldehyde solutlon was used, 8 ml o~ the ~7% strength solution were added to 29 ml o~ phosphate-burfered NaCl-solutlon. The suspension was kept ror 2 hours at 28 C while stirring, then lt was centrl-~uged, washed twlce with a 0.9% strength NaCl-solutlon and rlnally suspended at the original volume in a pAosphate-bu~ered NaCl-solutlon having a pH-value Or 7.2.

3. Preparatlon o~ the vaccfne The suspension obtained accord~ng to 2) was mlxed with the equal volume o~ a 0.2~ strenæth ~ suspension Or gamma-~l(OH)3 and stlrred ~or 30 mlnutes.

4) Test o~ the vaccine ror lnocoussness .
--0.05 ml or-the vacclne suspension was-admin~stered subcu-- taneously to two-days ol~mlce Or the straln NMRI. None Or thc inocculated mlce showed parasltemia arter 20 days.

5) Test ror the activity o~ the vacclne -- 0.5 ml Or the vacclne was administered subcutaneously per mouse. ~ter 18 days, the mice were inrected subcutaneously with 1 x 105 ortrypomastigotlc ~ormes o~ Trypanosoma cruzl suspended in 0.5 ml o~ rermentatlon medlum. 80~ o~ the immunized ~ ln-ected mice survived the ln~eotion.
With the same success as wlth 8% rormaldehyde, correspondin2 vaccines could be prepared uslng the rollowing substances:

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`- llZU855 1 % of glutaraldehyde (w/v) in water 20 % of sulfosalicylic acid (w/v) in water ',15 ~ of propionaldehyde (w/v~ in water 20 % of maleic acid ~w/v) in water 30 % of acetic acid anhydride (w/v) in ethanol (96 4 strength) 0.1 % of isobutyraldehyde (w/v) in ethanol (96 % strength) 30 % of pyrocarbonic acid ethyl ester (w/v) 'in ethanol ~ ' 20 % of maleic acid anhydride (g/v) in water ', 5 % of benzaldehyde ~g/v) in ethanol, Instead of~the 0.2~%~of aluminium hydroxide~used as adjuvant in Example I,,also 0.1% of aluminium hydroxide, 15~ 0~.~2~of the finé;1y~dispered quartz powder AEROSIL or 0.5 ,to~S unlts of~néuramlnldase:~yL~Ld;~comparable proteotlve ~i values;~qf tbe~vacc;l~e.
EXAMPLE II~
Disin~tegration~with~ultra-sound of the Trypanosoma ,20~ druzl killed açc'ord1ng to~Example I) ~
he~,s~uspens~ion~o;f Trypanosoma killed according to bxample I 2) or by a;corresponding treatment aacording to~the invent~ion~was treated 4 x 30 seconds with an ultra- , sound~apparatus~of ~.essrs.~Bronson~Desintegrator Sonifier 25~;Model B-l2~on the stage 4 with an~interruption time of each time l~minute.~

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The Trypanosoma treated with ultrasound were then centrifuged with 30,000 x g and the sediment was isolated.
A vaccine was prepared from the sediment according to Example 1. It showed a protection of 65~ of the immunized animals.

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~lZ~ 5 2. ~lslnte~rat~ion~o~ the ~ater~al treated with ultra-sound and ~il,led with ror~aldehyde ~ rter the ultra-sound treatment, the suspension was subJected to a gradlent centrifugation with 50 60 % Or saccharose as the medlum. The most effective rraction showed a protection o~ 80%' o'~ tne animals immunized th0rewith.

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Claims (4)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS: `

1. A process for the preparation of a vaccine against Chagas' disease, in which an aqueous suspension of 1 x 106 to 6 x 109 per ml of Trypanosoma cruzi organisms obtained from culture media is incubated with at least 0.5% (w/v) of an acylating agent or with more than 0.526% (w/v) of an aliphatic mono- or di-aldehyde having a chain length of 1 to 6 carbon atoms, at 4 to 40°C until all organisms are killed, the killed organisms are separated from the incubation medium, and suspended in a physiologically tolerated medium and a vaccine is subsequently formed.

2. A vaccine against Chagas' disease, whenever obtained according to a process as claimed in claim 1 or by an obvious chemical equivalent thereof.

3. A process as claimed in claim 1 in which the vaccine is combined with a protein-stabilizing agent and lyophylized.

4. A vaccine against Chagas' disease, whenever obtained according to a process as claimed in claim 3 or by an obvious chemical equivalent thereof.