NZ177422A - Anti-pregnancy vaccine - Google Patents
Anti-pregnancy vaccineInfo
- Publication number
- NZ177422A NZ177422A NZ177422A NZ17742275A NZ177422A NZ 177422 A NZ177422 A NZ 177422A NZ 177422 A NZ177422 A NZ 177422A NZ 17742275 A NZ17742275 A NZ 17742275A NZ 177422 A NZ177422 A NZ 177422A
- Authority
- NZ
- New Zealand
- Prior art keywords
- vaccine
- unit
- hcg
- sera
- beta sub
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
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Description
r
A
177 42"
Priority Date(s): i ;7?. < J?.'!?; 7?., Complete G?9ci«joct:cn Fi'sd: 7*C Class: j ?&:. .
PubSicatkm Date:
P.O. Journal Via:
2.4. AUG j984
'2-(s<
Patents Form No. 5
PATENTS ACT 1953
COMPLETE
SPECIFICATION
"ANTIPREGNANCY VACCINE"
I/WE, ALL INDIA INSTITUTE OF MEDICAL SCIENCES of New Delhi, 110016, India f s^nblt s htj under ab Act of Pa r I,-aw -t a f (A[l htjfa /hs/i'/k^ of S ot * t* cc s Ac-t , HS6 (tfo-15 °f HSC ) )
hereby declare the invention, for which l/we pray tha a patent may be granted to i}©/us, and the method by which it is to be performed, to be particularly described in and by the following statement
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(The present invention relates to a vaccine for preventing pregnancy, to a method of preparing the vaccine and to the method of using the vaccine for preventing pregnancy.
A variety of methods for controlling fertility and for preventing and terminating pregnancy are known. A variety of approaches such as condomes, ITJCD's, mechanical devices, spermicidal creams and jellies, foam tablets,
oral pills, etc. are currently available as a prevention against pregnancy. These methods, though effective to a variable extent, also have limitations. Kost of them require constant motivation on the part of the user.
Some require specialized medical attention.
The development of immunological methods of controlling fertility has been the subject of experimentation going back at least to the beginning of this century.
A number of approaches are described by Albert Tyler in "Approaches to the Control of Fertility Based on Immunological Phenomena," Journal of Reproduction and Fertility, (1961)2, pages 473-506."
Among the approaches followed in recent years by investigators have been immunization with placental,
fetal material, and hormones. To date, however, to Applicant's knowledge, there has been no effective anti-fertility vaccine developed. The present approach is based upon eliciting antibody response by active immunization to a hormone critical to establishment and maintenance of pregnancy.
It is an object of the present invention to - provide an antipregnancy vaccine.
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It is a further object of the present invention to provide a process for preventing pregnancy in mammals utilizing vaccines which are free of significant adverse side-reactions.
It is a further object of the present invention to provide a method for preparing an antipregnancy vaccine which produces antibodies with minimal cross-reaction with other endogenous hormones at physiological levels.
It is a further object of the present invention to provide a process which prevents pregnancy using a vaccine that can be administered by paramedical auxiliary personnel and which requires only periodic supplemental treatment.
It has now been found that a vaccine for the prevention of pregnancy is provided by forming a conjugate of a subject-compatible immunogenic carrier with a preparation of beta sub-unit of hCG, the preparation being free of determinants capable of reacting with high affinity with anti .LH sera. Specifically, antipregnancy vaccines are obtained with conjugates of said carriers with a substance selected from the group consisting of
(i) C-terminal fragments of the beta sub-unit of human chorionic gonadotropin, having from 30 to 38 amino acid residues,
(ii) the nitro derivative of the beta sub-unit of hCG, and
(iii) the chemically purified beta sub-unit of hCG, all substances free of determinants capable of reacting with high affinity with anti LH sera. The term immunochemically purified, in this context, includes treatment
1 77422
which, removes components of the beta sub-unit preparation which, if retained, would cause formation of antibodies that would cross-react with endogenous LH. The vaccine of the present invention is substantially free of cross reaction with other endogenous hormones, such as the pituitary hormones required for normal function of reproductive organs.
In accordance with one preferred embodiment of the invention, the beta sub-unit is separated from the alpha sub-unit of human chorionic gonadotropin in accordance with known procedures, is thereafter chemically purified by known means, and then immunochemically purified using a heterologous anti LH immunosorbent. The vaccine in a preferred form is a conjugate of a chemically and immuno-chemically purified form of beta sub-unit of human chorionic gonadotropin with tetanus toxoid in molecular proportions of about 1:5 to about 1:20. The conjugation takes place in the presence of a condensing agent such as glutaraldehyde or ethyl aminopropyl carbodiimide to obtain a covalently linked conjugate. The conjugate is thereafter subjected to the conventional steps of dialyzing, sterility control .andprecipitation to form the vaccine.
As known in the art, placenta is a tissue which develops only in pregnancy; it is not a normal part of the nonpregnant female. The vaccine of this invention is capable of counteracting the establishment and. maintenance of pregnancy. In humans, for example, a critical hormone, human chorionic gonadotropin is produced by the trophoblasts on about the eighth day after fertilization. This hormone supports, at this stage, the corpus luteum and ensures the
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receptivity of the uterus. The hormone may also protect the blastocyst by other means. Its neutralization results in the onset of menstruation, thus preventing the maintenance of pregnancy,
Human chorionic gonadotropin (hereinafter sometimes referred to as hCG) is a glyco-proteinic hormone composed of 2 sub-units: alpha and 'oeta. The alpha sub-unit is nearly identical with'the respective alpha sub-units of the hormones Thyroid Stimulating Hormone (TSH), Follicle Stimulating Hormone (FSH), and Luteinizing Hormone (LH). The beta sub-unit of the hormone, in each case, ascribes principally the hormonal specificity.
The beta sub-unit of hCG is a polypeptide of 146-147 amino acids with 3 out of 5 carbohydrate residues in the C-terminal end. A portion of the beta sub-unit bears amino acid homologies with the beta sub-unit of LH. The beta hCG prepared by dissociation of highly purified hCG has a fair degree of microheterogeneity. The protein resolves into 8 bands by electrophoresis on analytical polyacrylamide gels. This microheterogeneity is observed in commercially available hCG-, which is prepared from pregnancy urine» The microheterogeneity may be due to a variable degree of cleavage and/or modification of the molecule during its passage through various tissues.
hGG as a whole molecule or the beta sub-unit of hCG, when injected into women, does not elicit the formation of antibodies capable of neutralizing the activity of hCG. It can, however,-be made antigenic by coupling it with strongly antigenic haptens. Dia?,otized sulfanilic acid derivatives have been tried and it has been demonstrated
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that anti hCG antibodies ars induced in postmenopausal women. This is described by Vernon G. Stevens and 0.
Beans Grystle in "Effects of Immunization with Hapten-Coupled HCG on the Human rlenstrual Cycle", Obstetrics and Gynecology, Vol., 42, No. 4, October, 1973- However,
subjects had a depression or obliteration of raid-cycle LH peats, suggesting to the investigator that the antibodies cross-reacted in vivo with indogenous LH. Such a cross-reaction with a pituitary hormone required for normal function of reproductive organs is undesirable and potentially hazardous.
It is important according to the present invention that the beta sub-unit of hCG used in the reaction mixture be highly purified both chemically and immunochemically. The chemical purification of the beta sub-unit may be effected in any manner known in the art, such as the procedure described in Morgan, P. J. and E. E. Canfield, Endocrinology 88, 1045, 197/and 3?. J. Korgan, E. E. Canfield, J. L. Vaitukaitis, and G.. T. Eoss, Endocrinology 94, pages 1601-1605, 19?4. Another suitable method of obtaining a chemically pure beta sub-unit of hCG is described by Swaminathan, K. and Bahl, O.P. in "Dissociation and Recombination of Subunits of Human Chorionic Gonadotropin", Biochem, Biophys. Res. Commun,, Vol. 40, page 422, (1970),, However, these chemical purification procedures are not sufficient to remove determinants capable of reacting with high affinity with anti LH sera.
The immunochemical 'purification step consists of dissolving the chemically purified beta sub-unit of human chorionic gonadotropin in a physiological solvent, such as
177422
phosphate "buffered salios, and stirring the solution at room temperature for a controlled length of time with an immunosorbent, such as rabbit antiovine L5, The treatment with a heterologous anti LH immunosorbent, under carefully controlled conditions, is designed to remove those parts of the preparation which have determinants capable of reacting with high affinity with anti LH sera. The immunosorbent used in the process of this invention may be selected from any of several heterologous LH antisera such as rabbit antiovine LH, monkey antiovine or anti bovine LH. The stirring is effected at a temperature of, for example, 25°C, for periods of from 1 to 60 minutes or more, depending upon the temperature, the nature of the particular batch of beta sub-unit of hCG- and the potency of immunosorbent used. The optimum condition should be determined for each sample of beta sub-unit preparation and for each immunosorbent. The preparation is thereafter subjected to the step of centrifugation; the precipitate is discarded and the filtrate contains chemically and immunochemically purified beta sub-unit of human chorionic gonadotropin.
The C-terminal fragments of the beta sub-unit may be prepared by enzyme cleavage of beta sub-units of hCG or by synthesis of the C-terminal peptides by the standard procedures of Nerrifield, E. B., Journal of Am. Chem. Soc♦, Vol. 86, 304, 19S4-. Suitable methods utilizing enzyme cleavage are described in Babi et al., Biochem.
Biophys. Bes. Coutran. 48, 416, 1972, and Morgan et al., Mol. Cell. Biochem.2, 97, 1973.
The nitro derivative of beta sub-unit of hGG may
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"be prepared by the process dascribed in bokolousky ot al., Biochemistry 5 ■ pages 5582-2539 (1966) .
These preparations are subjected to the immunochemical purification step to exclude determinants capable of reacting with high•affinity with anti LH sera.
The term "high affinity" as used according to this invention describes a characteristic of those moieties which combine with the anti L11 sera at a rapid rate at the temperature used. The anti LH sera for this purpose is preferably raised to heterologous LH.
The preparation of beta sub-unit of human chorionic gonadotropin free of determinants capable of reacting with high affinity with anti LH sera is then reacted with a subject-compatible, immunogenic carrier such as tetanus toxoid in desired proportions using a condensing agent,
thus conjugating the beta sub-unit with the carrier.
Suitable carriers for use in preparing the vaccine of the •present invention include tetanus toxoid, polymerized flagellin, ELH (Keyhole Lympet Ilem'ocyanin) , dextran, heat or irradiation inactivated polio and yellow fever viruses,
heat or irradiation inactivated typhoid and para-typhoid
( BacHtu-l of C&fme.ti'e and vaccines, cholera toxoid, BOG/, antiplasmodium vaccine,
pertussis and diphtheria toxoid and biodegradable polymers, such as pOlylactic acid, polyglycolic acid, collagen, synthetic polypeptides, and polynucleotides.
±n accordance with one of the preferred embodiments of the present invention, the reaction product is a complex polymer of tetanus toxoid and the chemically and immuno-chemically ourifiea beta sub—unit of human chorionic gonadotropin conjugated covalently in the molecular
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(
proportions from about 1:5 to about 1:20 in the presence :
of a condensing or coupling agent such as glutaraldehyde \
I
or l-ethyl-3-(3~<Jimethyl aminopropyl) carbodiimide. j
When the condensing agent consists of l-ethyl~3-( j
. . . . ' . ?■ 2-dimethyl-aminopropyl) carbodiimide, purified tetanus toxoid and beta sub-unit of human chorionic- gonadotropin is admixed in molecular proportions within the range given above together with the condensing agent and the mixture is then incubated at a temperature not exceeding 25°C, and for a period not exceeding six hours depending upon the temperature. It is preferred that the reaction is carried out at a temperature not exceeding 10°0 and for a period of 4 to 6 hours.
When the condensing agent consists of glutaraldehyde, purified tetanus toxoid and beta sub-unit of human chorionic gonadotropin is admixed in the desired molecular proportions together with the condensing agent and the mixture is then incubated at a temperature not exceeding 37°C and £ov a period not greater than six hours. Preferably, the step of incubation is carried out at a temperature of about 20 to about 30°0 and for a period of 2 to 4 hours.
In the embodiment using the C-terminal fragments of B-hCG, it is combined with the tetanus toxoid in the molecular proportions of toxoid to fragments ranging from 1:10 to 1:100, preferably 1:40. When the nitro derivative is used in the conjugate, it is used in the molecular proportion, toxoid to derivative, of about 1:5 to about 1:20.
The compound so obtained is thereafter dialyzed thoroughly against phosphate buffer saline pll 7.2, 0.005 II , in cold, e.g. 5°C, and collected in a sterile tube using a
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1
j millipore filter and syringe assembly, using saline for transfer. .The preparation is then subjected to the step of sterility testing, in a manner'known as such in the art, to ensure the absence of micro-organisms.
Subsequent to the step of sterilization, the preparation is precipitated on alumiwn, using sterile alum, A£|< C • 13-H-lOJ
potash^and sterile sodium carbonate solutions. Instead of alumh*4 any other suitable adjuvants may also be used.
« However, it has been found that precipitation on alumiwfc i
■j 10 protects the active substance from adsorption on glass l<; containers and also provides a long lasting response. The
■ vaccine is preferably used as an emulsion with vegetable
"j - oils at the time of injection, and this further improves
1
■ antigenicity.
Further objects and advantages of this invention
.J
i will be more apparent from the ensuing description relating to Examples for the preparation and use of the vaccine.
EXAI-iPIE I
An antipregnancy vaccine was prepared by the 20 following procedure:
A.
Chemical Purification of Beta Bub-unit of human
Chorionic Gonadotropin
I Purified human chorionic gonadotropin was prepared
, EVpq _ Res. Z2-> :/2v-, /97/
from crude hCG- by the method of Canfield et ajj.: It was incubated at 40°C for 1 hour with 8 M'freshly prepared urea solution that had been purified on. a mixed cationanion exchange resin column, specifically a bed of equal quantities j 33owex-l X-8 and Dowex-50 X-4, 200 mesh. The column sifce
, was 2 x 40 cm. In the presence of urea, the beta sub-units
1
™ -10-
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are dissociated and then separated by the method described by Morgan and Canfield, Endocrino 1 ofzy 88, 104,5, 1971- In accordance with this process, the mixture is passed through ion exchange columns and Sephadex columns repeatedly to obtain the dissociated beta sub-unit with minimal contamination of the whole human chorionic gonadotropin molecule, or its alpha sub-unit.
B.
Immunochemical purification of beta sub-unit of human chorionic gonadotropin
From the beta sub-unit of hCG prepared in Step A. above, micro samples were taken. A series of 100 ug samples, 5 in number, of the beta sub-unit of hCG were each dissolved in 0.1 ml of 100 mil sodium phosphate (rali^PO^ and Jl^HFO^) buffered saline (0.85% KaCl) (PBS) (pH 7-2). Each sample was stirred at room temperature (25°C) with an immunosorbent of rs.bbit antiovine LH prepared in the manner described in
C. infra. Stirring times "varied from 1, 5, 10, 20 and 30 minutes. After the mixing, the tubes were centrifuged for 10 minutes at 0-4°C. The supernatent from each tube was tested for reactivity against anti hCG and anti LH. By this test the appropriate conditions for purifying the entire batch of beta sub-unit of hCG prepared in A, were determined to be 30 minutes of stirring at 25°C The selection of 20 minutes was based on the observation that the sample provided a supernatent with minimum level of reactivity with LH.
The characteristics of the beta hCG of the purified beta sub-unit of hCG prepared in A. above was compared with the iimnunochemically purified beta sub-unit preparation
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prepared above in E. figures .1 and 2 of the attached drawings show the reactivity of the respective preparations with anti hCG and anti LH serum before (Figure 1) and after
(Figure 2) the immunesorption purification step. The absorption step $lso improves the microheterogeneity of the preparation. Referring to Figures 1 and 2, the beta hCG preparations were subjected to electrophoresis in analytical polyaerylamide gels at pH 9.0. A set of the.gel was stained for densimetric measurements and another identical gel was transversely sliced into about 2.5 mm thick segments. Proteins from each segment were eluted in 0.9% sodium chloride solution. In each of Figux^es 1
and 2, the curve ( ——— ) represents proteins measures by densitometry; the curve (o o) represents proteins
125
competing with beta hCG VI for binding with anti beta hGG
sertim; and the curve o o represents proteins competing
125
with hLH ^1 for binding with anti hLH serum. It will be noted that the immunochemically purified preparation shows a substantial reduction in reactivity with, anti hLH. sex'um.
It may be noted that the optimum conditions of time of stirring and temperature are determined by the nature- of the particular batch of beta sub-unit and the potency of the immunosorbent used. It is recognized that the latter varies with the titres of the antiserum, used and with storage period of the immunosorbent.
0.
PPJEPAj&i'PIOg QP Immunosorbent
ml of antiserum (rabbit antiovine LH) obtained . by immunisation of rabbit antiovine LH is dialyzed thoroughly
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L
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1
against PBS at 0-4-°C and pH is adjusted, to pH 5 with'lH sodium acetate buffer. 3 ml of a 2.5% aqueous solution of glutaraldehyde is added gently with stirring. The reaction mixture is allowed to stand at room temperature (25°) for 3 hours. The polymerized material is homogenized in. a Potter ElveJhem homogenizer with Teflon pestle.. It is washed with PBS repeatedly till free of 280 absorbing material (non-polymerized free protein). It is then washed with 200 mM glycine - HC1 buffer pII2.2 and neutralized with 10 2 M K^HPO^ solution and finally equilibrated with PBS. The j immunosorbent is stored at 4°C with 0.1% Na azide as a
| preservative in glycine HC1 buffer pending the follow-up.
« ' ' i.,
A 1
j PREPARATION OF vaccine using l-ethyl-3~(3-dimethyl-
;«j aminopropyl) carbodiimide as condensing agent
i Preparation (for 4-0 doses)
3-2 mg beta sub-unit of hCG, as prepared in B. above was mixed with 400 Lf tetanus toxoid (1500 Lf/rag protein N) and 16 mg of l-ethyl-3-(3-dimethyl-aminopropyl) 20 . ' carbodiimide in 6.5 ml of PBS, and the solution was ^ incubated at 10°C for 5 hours with occasional shaking, The molecular proportion of tetanus toxoid to ^-sub-unit of hCG is about 1:10. The reaction.product was dialyzed ^ thoroughly against phosphate buffer saline pH ?«2, 0.005 M
in cold. The product was collected in a sterile tube using a millipore filter and syringe assembly, and using saline for transfer. The product v;as tested for sterility on thioglycolate'medium for seven days. It was then.
adsorbed on alumiawr using sterile 10% potash alumina and 30 sterile 10% sodium carbonate solutions. The vaccine was
\*
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then distributed, sealed into ampules, and sampled for pyrogens and sterility.
D2
PREPARATION OF vaccine using Glutaraldehyde as condensing agent - Preparation of 1 dose 80 ug of the beta sub-unit of hOG, as prepared in C. above was mixed with 1 dose tetanus toxoid, 10 Lf units and 50 ul of 1% aqueous glutaraldehyde, The mixture was incubated at 30°C with, occasional iaixing for 2 hours, and then dialyzed thoroughly against PBS pH 7.2, 0.005 The rest of the procedure is the same as that described in preparation .
PROPERTIES:
The vaccine prepared in accordance with described above, hereinafter designated Beta-hCG-TT was found to be immunogenic in mice, rabbits, goats, monkeys; as well as in the human female. The vaccine under d.escribed above was found to have similar immunogenic characteristics in monkeys. Both gave rise to a fairly long lasting antibody response.
A single injection of the vaccine Di given with Freund's adjuvant at multiple sites in the goat induced rising antibody titers for about 9 months after which a plateau is reached. This was followed by s. declining trend. A second booster injection in the declining phase lead to a sharp rise in antibody titers. This is shown in figure J.
The pattern of response in 10 experimental rhesus monkeys is found to be similar, although there are individual variations. This is also shown .in Figure 3.
In examining the immunological px-operties of anti
-1A-
1 774
(Be ta-hCG-TT) serum, it was found that such serum raised in the goat or monkey reacts immunologically with whole hGG molecules. The antiserum does not give a cross-rea-ction with human growth hormone (hGH), human prolactin, and human placental lactogen (hPL), F3H, TSH, or LH, as tested at the physiological and surge levels. Table 1 below summarizes the results.
TABLE I .
SPECIFICITY OF AMEI g HCG-T. T. SERA FROM MONKEY AND.GOAT
Hormone
Concentration
%30UED to Antibody
Monkey (1:5000) Goat (1:4000)
^Completion . Monkey Goat
Maximum conc. where competition appears.
' B-HCG
ng
13
9
87
91
-
2 ng
72
68
28
32
-
HCG
ng
50
70
70
-
2 ng
70
'85
,
-
HLH
1.2 ng**5
100
95
0
4
ng
0.4 ng*
100
98
0
2
-
HFSH
0.7 ng**
100
100
0
0
50 ng
0.3 ng*
100
100
0
0
-
HTSE
2.5 ng**
100
100
0
0
7>1C0 ng
HPL
12.5 ng**
100
100
0
0
>>1 v.g
HGE
0.2p ng**
100
100
0
0
»1 "<iS
*Basic or tonic concentration *^Maximum physiological concentration
177 4 22
The biological properties of the anti (Beta-hCG-TT) serum produced in the goat and monkey and the ability of the serum to neutralize hCG was tested in a number of systems. It was found effective .for inhibition of the hCG induced increase of the ventral prostate weight in immature rats. Experimental data summarising this work is shown in Figure 4. It also counteracted the hGG indiiced rise in production of progesterone in corpus luteum slices in vitro. This is shown in Table II.
TABLE II
EFFECT OF ANTI £-HCG-TT ON PROGESTERONE SYNTHESIS BY CORPUS LUTEUK Additions . P,, u^/g tissue*
1. None 79.4- _+ 15.2
2. + HCG (0.1 ug/ial) 183.2 _+ 30-6
3. + HCG + anti /ff-TT 105.4- + 23.4
4. + anti /9-TH. 96.7 + 23.2
*Kean _+ S.E. of 6 experiments
It also prevents the binding of hCG to receptors in membrane preparations from the testes and ovaries. Experimental data showing this is summarized in Figure 5« All these tests show that the antibodies raised against the compound Beta-hCG-TT has the ability to not only react immunologically with the whole hCG molecules but also to neutralise its biological activity.
The (Beta--hCG-TT) vaccine preparation was tested for safety as per British Pharmacopoeia requirements, and was found to give rise to no death in guinea pigs over the 4 weeks of observation period. It was also devoid of
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acute and subacute toxicity as tested in two species,
namely, the rodents and the monkey. Its tolerance has also been tested in 65 mice, 7 rabbits, and 15 monkeys for periods ranging up to 1 year and beyond. The survival of the immunized animals was 100%. Other parameters such as hematology (hemoglobin, TLC/DLC, PCV, peripheral smear examination for platelets, reticulocytes) hepatic functions (serum bilirubin, alkaline phosphatase, the transaminases GOT and GFT, LDH isoenzymes, cholinesterase), Renal function tests, blood glucose, serum lipids, cholesterol, triglyceiaes free fatty acids, and thyroid function tests were normal in immunized monkeys. The immunized monkeys did not develop immediate (Arthus) or delayed hypersensitivity to. hCG Clinical Studies:
Six human femal volunteers in the reproductive age group of 20-32 years were injected with either the vaccine preparation made according to above, or with altej^nate placebos in blind trials. Since the object of these tests was to establish the'immunogeneity of the vaccine of the present invention, the six women selected for this stage of the testing were those which had previously undergone tubal ligation. The. placebos, in all cases, gave no response. Each of the three subjects receiving the vaccine elicited antibody response against both tetanus toxoid and human chorionic gonadotropin. Following a lag period which lasted from 4-6 weeks from subject to subject, a progressive rise in antibody titers followed. Antibodies were also elicited simultaneously to tetanus.
Figures 6, 7» and 8 of the attached drawings show the response of the three huiiian volunteers that received the
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vaccine. Krs. E. V., S'igure 8 of. the drawings, was 30 years old vuth 5 pregnancies and 4- living children. The rise of antibodies reacting against hCG- is shown by the (c_™__—0) curve, and the anti tetanus antibody by the (o ...i ... o) curve. The anti hCG titres attained a plateau and have remained at this level at about 9 months after injection. The tests are continuing. The date of menses are indicated by an X mark at the top of the graph. "The subject has been menstruating regularly. The endometrium biopsy which was taken at 150 days, showed evidence of ovulation, which was confirmed by vaginal cytology and by progesterone levels in the secretory phase of the cycle.
The second subject Firs. A. K., Figure 7j was 32' years old, with 3 pregnancies and 2 live children. Figure 7 shows a similar type of immune response kinetics for Mrs. A. M. The endometrium biopsy in her case, as also the vaginal cytology and progesterone levels, indicate the patency of ovulation.
The third subject, Mrs. N.B. (Figure 6) was 30 years old with 5 pregnancies and 3 live children. Mrs. H. D. had been feeding a child during the first about 150 days of the study, which explains the lack of menses during this period. Immunization with the vaccine preparation did not produce any disturbance in lactation in spite of a fairly good immune response. She has since started menstruating and has had normal menstrual periods.
libido and other reproductive functions in all 3 subjects have been completely normal. Thorough clinical examination has shown no detectable abnormality of any type. Other parameters such as hematology, (hemoglobin, TLC/'DLC-
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1 77 4 22
POV, peripheral smear examination for platelets, reticulocytes) , hepatic functions (serum bilirubin, alkaline phosphatase, the transaminase s GOT and GPT, LDil isoenzymes, chclinesterase), Henal function tests, blood glucose,
serum lipids, cholesterol, triglyceides, free fatty acids, and thyroid function tests were normal and immunization has produced no alteration in organ functions and metabolism. The subjects have also shown normal response to insulin hypoglycaemic test in terms of production of Cortisol, and growth hormone.
The sera of these immunized women as well as of monkeys and goats hyperimmunized with the B-hCG-TT preparation has been examined by various tests to exclude possible autoimmune reactions against body organs. They were found negative for antinuclear antibodies, antimicro-somal antibodies and were devoid of rheumatoid factor reactivity. This information is summarized below in Table 2.
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TABLE 2
Immunopathology Investigations on Immunized Subjects
Sera from
Investigations
Observations
1. N.D. 50 Fig. 6 23.8.74
2. U.D. 50 20.9.74'
3. N.D. 30 2.12.74
4. A.M., 32 Pig. 7 . 11.10.74
. K.W., 30 Fig. 8 28.10.7^
6. E.W., 30 2.12.74
7. Eon-immunized control
1. Antinuclear antibodies.
2. Antimicrosomal antibodies.
3. Latex agglutination for Rheumatoid factor.
No reaction . detectable with any of the sera tested.
17742
They were also found to be negative in their reactivity against human thyroid, parathyroid, adrenals, kidney, testes, and ovaries. The data establishing these facts are summarized in Table 3 below.
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TABLE 3
Reactivity of Anti g-TT sera against Human organs
.■ vi
Sera from
Human Organs Tested
Observations i
ro i
Hormal human female Immunized human (U.P.) Fig. 6
Normal monkey Immunized monkey
Thyroid
Parathyroid
Adrenal
Kidney
Testis
Ovary
No reaction of any sera seen by inimunoflores-cence with any of the organs
NJ
S|
K) NJ
1 77422
They were also negative ixx their reactivity against tissue substrates from mice, rabbit and baboon as summarized in Table 4.
3
TABLE 4
Reactivity of Anti >g-hCC--TT sera with Tissue substrates from various snecies
Sera tested
Tissue substrates from
Tests performed
Observations i
ro vn i
Normal human
Immunized female (N.D.) Fig. 6
Normal mo like y Immunized monkey
Mice
Rabbit
Baboon a) Indirect Immunofluorescence b) Anti DNA antibodies
1) No tissue reacting antibodies detected
2) No anti DNA antibodies found
N) IS>
177422
The antibodies generated .in the human volunteers were found competent- for neutralizing the biological activity of the whole hGG. .Figure 9 of the attached drawings shows the ability of these antibodies from volunteer Krs. 35.D- to block binding of hCG to corpus luteum preparations in a radioreceptors assay. The ability of these antibodies to bind with hCG in vivo is demonstrated by the determinations in Figure 10 of the attached drawings.
The total duration of immune response to hCG in humans following primary immunization with four fortnightly injections of the vaccine is expected to be beyond one year, although individual variations may occur. Active immunization has not evoked undesirable reactions. The subjects.
have had normal menstrual cycles. There is evidence of ovulation. The kidney, liver, pituitary, cardiovascular, thyroid and other organ functions are normal and undisturbed.
Example II
A vaccine was prepared using the Beta-hCG end fragment (BEF) containing the las't 32-33 amino acid residxies, , in place of the immunologically purified beta-hCG used in and following the procedure outlined in Example I. In this Example, beta-hCG end fragment (BEF) comprising the last 32-33 amino acid residues, is covalently linked to tetanus toxoid in'a ratio of 40:1 using l-ethyl--3-(3-dimethyl aminopropyl) carbodiimide., The BEF is obtained from beta-hCG by papain cleavage, as described according to the method described above. Alternatively, the BEF may be synthesized chemically, according to known procedures»
Sufficient vaccine for 30 doses is prepared by ^mixing 2.5 mg B.SF with 300 Lf of tetanus toxoid and 15 mg
JV
1774 22
of l-ethyI-5~(3-dimethyl aminopropyl) carbodiimide, in phosphate "buffer saline pH 7-2 to final volume .5*0 ml.
This is incubated at 4-°C for 6 hours with occasional mixing. The product is dialyzed against PBS 7»2 in the cold for '+8 hours with 5-6 changes of 1 litre buffer each.
The balance of the procedure, .including sterilization and preparation of the vaccine, is the same as described in Example I.
Example III
A vaccine was prepared using a nitro derivative of the beta sub-unit of hCG, following the procedure outlined in Example I, . The molecular ratio of the derivative to tetanus toxoid was 10:1. The balance of the procedure, including sterilization and preparation of the vaccine, is the same as described in Example I.
JV • —27—
Claims (17)
1. A vaccine comprising & conjugate of a subject--compatible immunogenic carrier and a preparation of beta sub-unit of human chorionic gonadotropin* the preparation being free of determinants capable of reacting with high affinity with anti LH, sera.
2. A vaccine comprising a conjugate of a subject-compatible immunogenic carrier and a substance selected from the group consisting of (a) C-terminal fragments of the beta sub-unit of human chorionic gonadotropin, having; from 20 to 58 amino acid residues, (b) the nitro derivative of the beta sub-unit of hCG-, and (c) the iiuaiunochemically purified beta sub-unit of hCG, each substance being free of determinants capable of S<2 fa reacting with high affinity with anti LH/. J.
A vaccine comprising a conjugate of a subject- compatible immunogenic carrier and C-terminal fragments of the beta sub-unit of hCG having from 50 to 58 amino acid residues, the C-terminal fragment of beta sub-unit of hCG- being free of determinants capable of reacting with high affinity with anti LH sera.
4. The vaccine of claim 3, wherein the carrier is tetanus toxoid. ■.. . , A vaccine comprising a conjugate of a subject-compatible immunogenic carrier and the nitro derivative of the beta sub-unit of hCG, the nitro derivative being-free of determinants capable of reacting with high affinii N.Z. PATENT QF^Cg' "
5 AUG S977 'j 177422 with anti LH sera.
6. The vaccine of claim 5, wherein the carrier is tetanus toxoid.
7. A vaccine comprising a.conjugate of chemically purified beta sub-units of hCG and a subject-compatible immunogenic carrier, the beta sub-unit of hCG being free of determinants capable of reacting with high affinity with anti LH sera.
8. The vaccine of claim 7, wherein the carrier is tetanus toxoid.
9. A vaccine comprising a conjugate of tetanus toxoid and a beta sub-unit of hCG, the beta sub-unit having been immunochemically purified by reaction with polymerized heterologous LH anti sera prior to conjugation to remove determinants capable of reacting with high affinity with anti LH sera.
10. A process for preparing a vaccine which comprises the steps of (a) obtaining a preparation of chemically pure beta sub-unit of hCG free of determinants capable of reacting with high affinity with anti LH sera, and (b) condensing the substance in (a) with a subject-compatible immunogenic carrier to form a conjugate.
11. The process of claim 10, wherein the conjugate is precipitated on alum to form a vaccine.
12. A process for obtaining a vaccine for the prevention of pregnancy which comprises the steps of (a) obtaining a substance selected from the group consisting of (i) C-terminal fragments of the beta sub- -I 1# 177422 ■ fro* 2o -ft 3% ctw° acidrei'dufd unit of human chorionic aon^doti-epi (ii) the nitro derivative of the beta .il:.;— unit of hCG, and . (iii) the •••(■■•chemically purified beta -ub-unit of hGG , the substances being free of determinants capable of reacting with high affinity with anti L1I sera) (b) condensing at least one substance fro:;1 (&) uifL a subject-compatible immunogenic carrier. j 10
13. She process of claim 12, wherein the carrier in tetanus toxoid. , A
14. *i»rprocess for preparing a vaccine 1'or the ■ prevention of pregnancy which comprises the steps of. (a) obtaining a chemically pure beta sub-unit J. of hCG, •1 . , . . ' • (b) reacting said chemically pure beta sub-unit if 1 with an LH immunosorbent to free the preparation of detexnninants capable of reacting with high affinity with anti LH sera, and 20 (c) condensing the thus treated beta sub-unit preparation with a subject-compatible immunogenic carrier ~j to obtain a vaccine. i
15. The process of claim 14, wherein the LH immuno- sorbent is rabbit antiovine LH. ~
16. The process of claim 15, -wherein the carrier <3 1 is tetanus toxoid. j
17. The process of claim 14, wherein the LH immuno-. sorbent is prepared by J (ft) thoroughly dialyaing rabbit antiovine LH £ 30 ^sera against 100 mil phosphate-buffered saline at pH 7.2, JV -30- 177422 (t>) polymerizing the antis^ra by st.vnOing at room temperature for 5 hours .with the addifcioi; of glut;-. r-Ai-.dehyde, (c) homogenizing the polyinorised material followed by washing with phosphate-"buffered saline until, the washings are free of non-polymerized free protein, (d) washing the homogenate with a 200 inl'i glycine - HC1 buffer at pH 2.2, •(e) neutralizing the homogenate with' K^HPO^. solution, and (f) equilibrating the immunosorbent thus produced with phosphate-buffered saline.
18. The process of claim 14, -wherein the treated beta sub-unit is condensed with tetanus toxoid in tbe molecular proportions of 5:1 to 20:1 in the presence of a condensing agent and the resxilting product is subjected to the steps of dialyzation and precipitation on alum'', to form a vaccine. 19- The process of claim 18, wherein the condensing agent is selected from the group consisting of glutaraldehyde, and a carbodiimide.
20. The process of claim 18, wherein the condensing agent is l-ethyl-5-(3-aminoprogyl) carbodiimide and the condensing step is conducted at a temperature not in excess of room temperature and for a period not exceeding wbuufc 6 hours.
21. The process of claim 20, wherein the temperature does not exceed 10°0.
22. The process of claim 19, wherein the condensing agent is glutaraldehyde and the reaction is conducted.at a 177422 temperature not exceeding 37_°C for,, a period not exceeding 6 hours.
23. The process of claim 22, wherein the reaction temperature is within the range of 20-30°C.
24. A process for preventing pregnancy which comprises administering to a fonale non-human mammal a vaccine comprising a conjugate of a subject-compatible immunogenic carrier with a preparation of beta sub-unit of hCG, the preparation being free of determinants capable of reacting with high affinity with anti LH sera, the vaccine being administered at a level non-human sufficient to raise antibodies in the female/mammal to neutralize endogenous chorionic gonadotropin normally produced by the , trophoblasts.
25. A process for treating female non-human mammals to prevent pregnancy which comprises administering a vaccine comprising a conjugate of a subject-compatible immunogenic carrier with a substance selected from the group consisting of (i) C-terminal fragments of the beta sub-unit of human chorionic gonadotropin, having from 30 to 38 amino acid residues, (ii) the nitro derivative of the beta sub-unit of hCG, and (iii) the immunochemically purified beta sub-unit of hCG, the preparation being free of determinants capable of reacting with high affinity with anti LH sera, the vaccine being administered at a level sufficient to raise antibodies in the subject to neutralize endogenous chorionic gonadotropin normally produced by the trophoblasts.
26. A.process for-preventing pregnancy in the non-human mammalian • : > female which comprises administering to a subject a vaccine comprising a conjugate of tetanus toxoid and a ■inraunochemically purified beta sub-unit of bCG free of determinants capable cf reacting with high affinity with anti I.-H sera, the vaccine being administered to raise antibodies to^endogenous chorionic gonadotropin normally produced by the trophoblasts. JV 177422 27* A prooeBB for raising antibodies capable of neutralising endogenous chorionic gonadotropin normally non-human produced by the trophoblasts in female/mammals comprising non-human administering to a/<mammal a sufficient amount of a conjugate of (a) chemically end imnunoohemioally purified /5 -eutninit of human chorionic gonadotropin which 1b free of determinants capable of reacting with high affinity with anti-LH sera, and {b) a Bubjeot-compatible immunogenic carrier. 28* A prooesB for raising antibodies oapable of neu trail ting endogenous chorionic gonadotropin normally fion-human produced by the trophoblasts In female/mammal b comprising non-human administering to a female/mammal a sufficient amount of a conjugate of (a) a ohemloally and lmmunoohemically purified eubstanoe whioh 1b free of determinants capable cf reaction with high affinity with anti-LH sera, said substance being selected from the group consisting of $-hCG, and the C-termlnal fragment of /B-hOG having y> to 58 amino acid residues, and (b) a subject-compatible Immunogenic carrier selected from the group consisting of tetanus toxoid, polymerised flagellin, keyhole limpet hemocyanin, dextran, Inactivated polio virus, inactivated yellow fever virus, inactivated typhoid and paratyphoid vaccines, cholera toxoid, Bacillus cf Calmette and Ouerln, antiplasmodium vaoclne, pertussis and diphtheria toxoid, and biodegradeable polymers.
29. The prooeBB according to claim 28 wherein the carrier Is tetanus toxoid. 177422 30^ The prooese according to claim 28 wherein the carrier is polymerized flagellin.
31. The process according to claim 28 wherein the carrier is keyhole limpet hemocyanin.
32. The process according to claim 28 wherein the carrier is inactivated polio virus.
33. The procesB according to claim 28 wherein the conjugate comprises 0-hCO and tetanus toxoid. 34* The process according to any one of claimB 28 to 33 wherein the molecular proportions of (a) to (b) range from mliput 5e 1 to abou* 20i1. 35* A prooess for preparing a vaccine for the prevention of pregnancy which comprises: (a) obtaining chemically pure fi-hCO; (b) reacting said chemically pure /S-hOG with rabbit antiovine LH immunosorbent to obtain a yg-hCG preparation which is free of determinants capable of reacting with high affinity with anti-LH sera; and (o) condensing the thus treated £-hGG preparation with a subject-compatible immunogenic carrier to obtain a vaccine.
36. The process of claim 35» wherein the carrier is tetanus toxoid.' 37» The process of claim 35, wherein the immunosorbent Is prepared by (a) thoroughly dialyzing rabbit antiovine LH sera against 100 mM phosphate buffered saline at pH 7*2, (b) polymerizing the antisera by standing at room temperature for 3 hours with the addition of glutaraldehyde, (c) homogenizing the polymerized material followed . '••• " ' o; 1984 j * ij - IV 17 7422 by washing with ffhoaphafeb4e^buffered saline until the washings are free of non-polymerized free protein, (d) washing the homogenate with u 200 mM glycine -HC1 buffer at pH 2,2, (e) neutralizing the homogenate with KgHPO,^ solution, and (f) equilibrating the immunosorbent thus produced with phoophatido/buffared saline,
38. The process of claim 35, wherein the treated fi-hQCr is condensed with tetanus toxoid in molecular proportions of 5:1 to 20:1 in the presence of a condensing agent and the resulting product is subjected to the steps of dialyzation and precipitation on alum to form a vaccine.
39. The process of claim 38, wherein the condens- /15 /agent is selected from the group consisting of glutaraldehyde and a carbodiimide .
40. The process of claim 30, wherein the condensing agent is 1-ethyl-3-(3-aminopropyl) carbodiimide and the condensing step i3 conducted at a temperature not in excess 'of room temperature and for a period not exceeding about 6 hours .
41. The process of claim 40, wherein the temperature does not exceed 10°G.
42. The process of claim 39, wherein the condensing agent is glutaraldehyde and the reaction is conduoted at a temperature not exceeding 37°0. for a period not exceeding 6 hours .
43. The process of claim 42, wherein the reaction temperature is within the range of uubou^i 20-30°C. 4-
44. The vaccine of c3.aim ^wherein the molecular 177422 proportion}? of carrier to fragments is in the range of 1:10 to 1 :100.
45. The vaccine of claimwherein the molecular proportion of carrier to nitro derivative is in the range of 1;5 to 1:20.
46. The vaccine of claim Pf, wherein the molecular proportion of carrier to beta sub-unit is in the range of 1:5 to 1:20. /-//•/ CkCtQr&fTCf ft> C*fOlirv\ (
47. A vaccin<^ substantially as herein described in any one of the Examples. , &C-COrd,s\g~f' o c!q tnn j
48. A process for preparing a vaccine/ substantially as herein described in any one of the liixamples. ALL INDIA INSTITUTE OF MEDICAL SCIENCES By their attorneys BALDWIN, SON & CAREY.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN2287/CAL/74A IN140168B (en) | 1974-10-14 | 1974-10-14 | |
US54467775A | 1975-01-28 | 1975-01-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ177422A true NZ177422A (en) | 1984-08-24 |
Family
ID=26324459
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NZ177422A NZ177422A (en) | 1974-10-14 | 1975-05-05 | Anti-pregnancy vaccine |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU503647B2 (en) |
NZ (1) | NZ177422A (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0282501A4 (en) * | 1986-08-18 | 1989-11-14 | Bunge Australia | Regulating animal reproduction. |
AU602187B2 (en) * | 1987-01-14 | 1990-10-04 | Commonwealth Scientific And Industrial Research Organisation | Anti-reproductive hormone |
-
1975
- 1975-05-05 NZ NZ177422A patent/NZ177422A/en unknown
- 1975-05-05 AU AU80826/75A patent/AU503647B2/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
AU503647B2 (en) | 1979-09-13 |
AU8082675A (en) | 1976-11-11 |
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