IL44779A

IL44779A - Chemically modified polypeptide antigens their production and pharmaceutical compositions containing them

Info

Publication number: IL44779A
Application number: IL44779A
Authority: IL
Original assignee: Univ Ohio State
Priority date: 1973-05-07
Filing date: 1974-05-07
Publication date: 1977-10-31
Also published as: DD113919A5; FR2228477B1; AT356263B; NZ174192A; FR2228477A1; HK41280A; NL7406098A; PL97318B1; IL44779A0; AU6869574A; SE7406096L; GB1473601A; CA1057742A; DE2421943A1; IE42002L; DE2421943C2; CH622426A5; AU524925B2; IE42002B1; JPS5069221A

Abstract

The composition has an active immunising action against reproduction in humans and mammals. It contains as active component a biologically determinant peptide of one of the protein-reproducing hormones follicle-stimulating hormone (FSH), luteinising hormone (LH), human placental lactogen (HPL), human prolactin or human chorionic gonadotropin (HCG). The peptide is modified to an artificial antigen in an immunochemically known way. [GB1473601A]

Description

, o» a ?9 ay© O» »¾S)S T9 ; a *xa *oJK .. onis Ghemicaiiy' modified antigens, their produc ion and pharmaceutical compositions containing them THE OHIO STATE UNIVERSITY C. 42863 118-3329 ANTIGENIC MODIFICATION OF POLYPEPTIDES ^ ^ This invention relates to the chemical modification of polypeptides to provide antigens which may,be used in active immunisation for the purposes of the control or treatment of various physiological processes, particularly reproduction.

■ It is well known that polypeptides, particularly proteins, are responsible for, contribute , to or influence many physiological processes. For example, certain protein hormones and non-hormonal proteins are knov/n to be essential for the normal events of the reproductive process. Unusual excesses of certain polypeptides, such as gastrin, angiotensin II or somatomedin, are also known to cause or influence various disease states and maladies .

The present invention is concerned with the control or treatment of such physiological processes by active immunisation (i.e. administration of antigens) causing formation of antibodies which neutralise (render biologically ineffectual) the endogenous polypeptides which are responsible for, contribute to or influence the' physiological process in question.

- - - Immunology, as a means of controlling reproduction, has been the subject of much recent investigatioft-r-' R.G. Edwards, in a paper entitled "Immunology of Conception and Pregnancy (British Medical Journal, 26 , 72-78 [1970]), surveys the literature on this topic, for example that concerning the production and use of antibodies against testes or sperm. Hormone antibodies have also been studied for many years and the effects of specific antisera have been recorded. Most of the past approaches to contraception by immunological means have, however, used passive immunisation, that is injection of antibodies produced elsewhere. There are serious limitations to the use of passive immunisation for human therapy since the repeated injection of animal antibodies into humans is known to produce undesirable reaction in many individuals.

It has now been found that by chemically modifying natural, endogenous polypeptides which play a role in the physiological process to be treated or controlled, antigens are obtained which are capable of inducing formation of antibodies which neutralise not only the anti-genically modified polypeptide but also its unmodified endogenous counterpart. Thus, immunisation apparently takes place because of the inability of the antibodies produced to distinguish between the antigenically modified polypeptides and their unmodified endogenous counterparts. -, She modified polypeptides are produced from the natural, endogenous polypeptides in the species involved or are immunologically equivalent to the modified polypeptides so produced. In practice, the polypeptides to be modified are derived from the species involved or from a closely related species.

As used herein, the term "fragment" in relation to an endogenous polypeptide means a sequence of amino acids in the complete amino acid chain of the endogenous polypeptide. These fragments must be sufficiently large and distinctive in chemical and physical characte to enable them to be recognised as a specific part of the whole. As used herein, the term "polypeptide which is immunologically equivalent to" in relation to an endogenous polypeptide or fragment thereof, means a polypeptide which, although not identical to the polypeptide or fragmen in question, elicits, when modified in accordance with the invention, essentially the same antibody response as the endogenous polypeptide or fragment concerned.

Such polypeptides may be naturally occurring or syntheticI ; T e invention accordingly provides an antigen for use in active immunisation for the control or treatment of a physiological process in a particular species, comprising a chemically modified polypeptide, the unmodified polypeptide being a) an endogenous polypeptide which influences such from protein reproductive hormones, non-hormonal protein antigens isolated from placental tissue, gastrin, Angiotensi^ix II, growth hormone, soma- tomedi^n, parathyroid hormone, glucagon, thyroid stimulating hormone or secretin; b) a fragment (as herein defined) of such endogenous polypeptide; or c) a polypeptide which is immunologically equivalent (as herein defined) to such endogenous polypeptide or fragment thereof, and the chemical modification being such as to permit the chemically modified polypeptide to induce, in that species, formation of antibodies which biologically neutralise the endogenous polypeptide as well as the chemically modified polypeptide, ϋίαβ invention also provides a process for the production of such an antigen comprising chemically modifying an appropriate polypeptide to the necessary extent.

The invention also provides the antibodies generated in th species in response to the action of the antigens of the invention, and antlsera containing such antibodies.

The invention also provides a method of controlling or influencing a physiological process in a species comprising injecting into that species an immunologically effective amount of the antigens or antibodies of the invention. The invention particularly provides such a method of contraception.

U.K. Patent Specification 1,058,828 discloses broadly the chemical modification of protein peptides, by coupling to a carrier protein, to obtain antigens. There is, however, no -suggestion whatsoever in that specification of modifying ¾e specific polypeptides of the present, invention and, indeed the. only proteins which are specifically mentioned in that U.K. Specification, namely polio virus and tobacco mosaic virus, are totally unrelated to the polypeptides modified in accordance with the present invention.

Eur. J. Biochem. 18, 561-572, (1972), describes the coupling of alanine peptides, to proteins. Again, however, there is no suggestion in that article of chemically modifying the specific polypeptides of the present invention, nor does the article suggest any use for. the modified proteins. - 5 - 118-3329 Polypeptides which may be chemically modified to provide the antigens of the invention include protein reproductive hormones, such as Follicle Stimulating Hormone (FSH) , L^ut&nising Hormone (LH) , Human Placental Lactogen (HPL) , Human Prolactin and Human Chorionic Gonadotropin, and specific fragments thereof.

Specific fragments of protein reproductive hormones v.'hich may be modified in accordance with the invention include the β-subunit of FSH and specific unique fragments of natural HPL or Human Prolactin, which fragments may bear little resemblance to analogous portions of other protein hormones. Preferred fragments include the β-subunit of HCG which, according to the two authoritative views:, has either structure I or II as follows (""indicates site locations of carbohydrate moieties) :- 10 « Ser~Lys~Glu--Pro-~Lsu~Arg-Pro-/-g~C¾'s^ ■ 20 « Val~Glu..Lys~Glu-G3y«Cys~Pro-Val-Ctys~^^ hO ' 50 Ala-Gly~Tiy»(^s~Pro-Thr-Met-Tlir"Arg-Val--Leu-Gln-Gly-Yal.-Leu-Pro-Ala- 60 Lsii»Pro~Gln~Vra~Val~C s~/iSn~a ^ 70 80 Leu-Pro-Gly-CVs-Pro-Are-GIy Val-Asn-Pro-Val-Val-Ser-Iyr-Ala-Yal-i a- 90 100 Leu~Ser~Cys»Gln-.Cys-Ala-Leu-Cys~/^ 110 Pi'0"I^s-Asp~His-Pro-Leu--T}ir-Cys-Asp»A3p-Pro-Ar6~Phe-Gln-A5p~Ser-S£r- .20 « ' * no * Ber-Scr"Lys-/0.a-Pro-Pro»Pi-'o-Ser--Leu»Pro~Ser-Pro-fjei*« 'g-JJeu-Pr -Gly~ * ihO Pro-Ser-Aap-Thr-Pro-Ile-Lsu-Pro-Gln (Structure I) - 6 - 118-3329 10 * " Ser»Lys-Gln-Pro-Leu- g-.Pro-to^ 20 29 * AJ-a-Val-Glu-Iyo-Glu-Gly-Cys-Pro- ^ 1)0 ne~Cys^Ala~Gly~ yr..CyB~Pro-Thr- et^ hr-Arg^al~Le -Gln-Gly»Val~ 50 60 keu~Pro~ u~Leu-Pro-Glx-^eu-Val~CyG 70 80 Glu-Ser-Ile-Ai-g-Leu-Pi'O Gly-Cj's-Pro~Arg-Gly-Val-Asn-Pro«Val-Val^ (Structure II) For specificity of antibody action, it is desirable that polypeptides be modified which comprise molecular structures completely or substantially completely different to those of other protein hormones. In this connection, the β-subunit of HCG ' ossesses a chain or chains of amino acids which differ greatly from those of LH and such chains may also be modified in accordance with the invention. Such chains include 20-30 or 30-39 amino acid peptides consisting of the C-terminal residues of the β-subunit of HCG. More particul rly, suitable such chains include those of the following formulae III and IV (C-terminal portions of structure I, above) and V and VI (C-terminal portions of Structure II, above) : - - - * Asp~Asp-Pro-Arg-Phe-Gln-Asp-Ser--Ser-Ser-Ser-Lys~Ala-Pro-- Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro- Ser-Asp-Thr-Pro-Ile-Le.u-Pro-Gln . (Structure III) •k it Gln~Asp~Ser~Ser-Ser-Ser-Lys~Ala-Pro-Pro-Pro-Ser-Leu--Pro- * * Ser~Pro~Ser-Arg-Leu-Pro--Gly-Pro-Ser-Asp-Thr-Pro-Ile- Leu-Pro-Gln.

(Structure IV) Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys" * * Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr~Pro-Ile-Leu-Pro-Gln~Ser-Leu-Pro.

(Structure V) * Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu- * * Pro-Ser-Pro-Ser~Arg-Leu-Pro~Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro .

(Structure VI) Such structures may be obtained by purely synthetic methods or by enzymatic degradation of the parent polypeptide [Carlson et. al.f J. Biological Chemistry, 284 (19) / 6810 (1973)]. A polytyrosine chain may also be added to the structures III to VI and the resulting polypeptide modified in accordance with the invention. ~ 8 - 118-3329 Another group of polypeptides which may be modified according to the invention are specific non-hormonal protein antigens isolated from placental tissue.

Other proteins which may be modified according to the invention and used for active immunisation in the treatment of various disease states and maladies, include gastrin, the hormonal polypeptide known as Angiotensin II, growth hormone, Somatcmedi n, parathyroid hormone, insulin, glucagon, thyroid stimulating hormone (TSH) and secretin.

The degree of chemical modification of the polypeptides, in accordance with the invention, is, as indicated, such that the resulting antigens will induce generation of antibodies which will neutralise not only the antigens but also some, at least, of their natural endogenous counterparts and this as well as the type of modification v/ill depend on the specific problem being treated as well as the nature of the polypeptide involved If too little modification is effected, the body may not recognise the modified polypeptide as a foreign intruder and may therefore not generate antibodies against it. If, on the other hand, too much modification is effected, the body will generate antibodies specific to the injected antigen which v/ill not neutral- - 9 - 118-3329 ilse the natural endogenous protein involved. · In general, the chemical modification involves addition of foreign modifying groups to the polypeptide involved (hapten-coupling) . The number of foreign modifying groups to be added will, of course, vary depending on the circumstances but, generally , it is preferred that 1 to 40, preferably 2 to 40, more preferably 5 to 30, and most suitably 10 to 26 modifying groups per molecule of polypeptide be attached.

Given modifying groups will attach to particular amino acid sites in the peptide molecule, so that the maximum possible number of a given modifying group which illi attach to a given polypeptide can readily be calculated. The nature of the modifying group may be selected accordingly. It is also possible that several modifying groups may attach to each other and then attach to a single amino acid moiety but, for the purposes of this invention, such a substitution is regarded as attachment of a single modifying group.

The modifying groups may as indicated vary in chemistry depending on the circumstances. Suitable modifying groups include diazo groups . These may suitcibly be introduced by reaction v/ith the appropriate number of - 10 - 118-3329 moles of diazosulfanilic acid. Introduction of diazo groups into proteins is a well-known technique and may , for example be effected as described, by .

Cinader et al., J. Am. Chem. Soc. 78_, 746 (1955); by Phillips et al. , J. Biol. Chem. 24£, 575(1969); by.

Tabachnick et al. , J. Biol. Chem. 234(7), 1726(1959) or by Crampton et al., Proc. Soc. Ex. Biol. & Med. 8_0, 448(1952) . In general, the methods of Cinader et al. and Phillips et al. are preferred.

Additional modifying groups include those introduced by reaction of the polypeptides with dinitro-phenol, trinitrophenol , ε-acetomercaptosuccinic ahhydrid' polytyrosine or polyalanines ( n either straight or branched chains) , biodegradable polydextran or, rather less preferably, natural proteins r.uch as thyro-globulin. Generally, synthetic modifiers are preferred to natural modifiers.

The above reactions, as well as many other suitable hapten coupling reactionsr are well known in protein chemistry. The following references may, for example, be- cited in this connection:- 1. Klotz et al., Arch. Biochem. & Biophys, 96, 605-612 (1966) ; ' 2. Khorana, Chem. Rev. 53, 145(1953); 3. Sela et al., Biochem. J. 85, 223(1962); 4. Eisen et al., J. Am. Chem. Soc. 75, 4583 (195'3) 5. Certano et al., Fed. Proc. (ABSTR--. ) 2S_, 729 (1966) 6. Sokolowski et al., J. Am. Chem. Soc. 86_, 1212 (1964) ; 7. Goodfriend et al. , Science 144, 1344(1964); 8. Sela et al., J. Am. Chem, Soc. 78_, 746 (1955), and 9. Bah , J. Biol. Chem. 2 , 575 (1969).

The chemical modification may alternatively or additionally comprise removal of moieties from the polypeptides. Thus, for example where certain of the natural proteins have carbohydrate moieties, these may be removed in conventional manner, using, for example,.

N-acetyJ.neurami.nidase or N-acetylglucosidase , materials useful for removing specific carbohydrate moieties.

As indicated, the modified polypeptides of the invention are indicated for use as antigens in active immunisation for the purposes of control or treatment of various physiological processes.

More particularly, the modified protein reproductive hormones or fragments thereof, of the invention are indicated for use as antigens in active immunisation for the purposes of controlling reproduction. Thus, these materials induce formation of antibodies which neutralise not only the antigens but also the natural endogenous protein reproductive hormones which are essential for the normal events of the reproductive process, thus disrupting the natural processes of conception and/or gestation. These antigens are therefore indicated for use in contraception and for terminating pregnancies soon after conception. The specific non-hormonal protein antigens isolated from the placental tissue and modified according to this invention are also indicated for such use. There is direct evidence that inhibition of substances which are specific to the placental tissue and do not have similar antigenic properties to antigens from other organs, can result in disruption of pregnancies by passive immunisation. Such specific placental substances when modified in accordance with the invention can be injected into the body of the same, species as a fertility control means by active immunisation. The advantage of these substances is that placental antigens are foreign to the nonpregnant female humans and are. therefore unlikely to cause any cross-reaction or disruption of normal body functions in the non-pregnant female.

It will be understood while the methods of controlling reproduction described are mainly applic- r^ able to females/ certain antigens, in particular FSH, its β-subunit and fragments thereof, when modified in accordance with the invention, may be applicable to males.

Gastrin, when modified in accordance with the invention is indicated for use in the treatment by active immunisation of the digestive disorder knovm as the Zollinger-Ellison Syndrome. This state is generally described as a condition in which there is hyper-secretion of the polypeptide gastrin, which, is produced in the pancreas and brings about a state of hyperacidity in the stomach resulting in a chronic digestive disorder:.

Injections of modified gastrin will enhance formation of antibodies against the hypersecretion of gastrin and thereby alleviate the disease without the need for the surgical treatment which is the only effective treatment presently knovm.

Angiotensin II, when modified in accordance with the invention, is an antigen for use in the treatment of hypertension. In general terms, the state of hypertension is the abnormal level or fluctuation of ones blood pressure. The blood pressure in an individual - 14 - 118-3329 is controlled by many physiological processes in the body However, one major substance affecting the regulation of such pressure is the hormonal polypeptide known as Angiotensin II. In certain states of high blood pressure (hypertension) it is difficult to medically control the secretion and therefore the level of Angiotension II in the circulatory system. By the appropriate modification of this hormone and subsequent immunisation with the resulting antigen, it is possible to reduce the secretion of angiotension II in patients, with 'chronically elevated hormone levels. The predictable and controlled reduction of this substance is beneficial to certain patients with chronic problems of hypertension.

Growth Hormone and Somatomedin , when modified according to the invention are indicated for use as antigens in immunological treatment of diabetes and associated micro and macro-vascular disorders. Currently the treatment of diabetes is limited to dietary and/or drug treatment to regulate blood glucose levels.

Recent scientific data support the concept that Growth Hormone and Somatomediin (both polypeptides) are intimately involved in the disease syndrome.

Antigens of the invention are suitable admixed - - with a pharmaceutically acceptable liquid carrier and administered parenterally.

Suitable liquid carriers are e.g. mannlde mono-oleate, saline or an oil. The dosage to be administered will, of course, vary depending on various factors, including the condition being treated and its severity. However, i general, unit doses of 0.1 to 50 mg are indicated, suitably administered one to five times at intervals of one to three weeks.

As indicated, the theory leading to the present invention was that the chemical modification of a protein essential for reproduction or influencing a particular disease state would render it antigenic such that it would cause formation of antibodies which would, at least partially, neutralise the endogenous protein in addition to the antigen. With this in mind, reproductive hormones of various species were modified and tested i baboons as described in Example 1, hereinafter. The results demonstrated that modified hormones of unrelated species do not produce the desired result whereas modified hormones of the same species or closely related species do. She remaining Examples 2 to 9 also illustrate the invention, Example I_ Adult female baboons vere studied for at least one menstrual -C cycle for patterns of urinary estrogens, plasma, progestin, and in some cases urinary LH. Onl those animals displaying normal patterns of these hormones vere immunized. The criteria for normality and the procedures for housing animals are veil knovn and will hot be described.

Gonadotropin 'Prepara ions Human Luteinizing Hormone (HLII) «» partially purified preparation from human pituitaries vith a biological potency of 2„5 units per mg.

(NIH-LH-Sl)..

Human Follicle Stimulating Hormone (HFSH) - a partially purified preparation from human pituitaries with a biological potency of 86 units per mg. ( IH~FSH~SI).

Human. Chorionic Gonadotropin (UCG) - a highly purified preparation from human pregnancy urine vith biological potency of 13*200 IU/mg. ('2nd IRP-HCG ) .

Monkey Luteinizing Hormone (MLH) ~ a crude preparation from rhesus monkey .pituitaries vith a biological potency of 0.75 units per mg„ (KIH-LH-SI).

• Ovine Luteinizing Hormone (OLH) (NIH-LH-S5 ) .

Baboon Luteinizing Hormone (BLH) - partially purified baboon pituitary preparation vith a biological potency of 1.1 units per mg. ' (NIH-Lli-Sl)..

All preparations, excepting the OLH, vere prepared in the inventor's laboratory. LH and HCG biological activity vas determined by the ovarian ascorbic acid depletion test and the FSH preparation assayed by the ovarian augmentation assay.

Hormones vere altered as antigens by coupling vith a hapten in varyin ratios of hapten to hormone as described by Cinader et al. , supra. tnipra, may provide a more stable bond under certain circumstances. In this procedure, the. protein hormone serves as a carrier and the hapten is coupled to it by diazo bonds. Although a variety of hapten groups vere coupled to different hormones, the same basic procedure vas used for any combination. Fifteen to thirty-five hapteni'c groups per hormone molecule vere found most useful for preparing immunizing antigens. The basic reaction consisted of diazotizing the hapten (suJfanilic acid) by adding it to a solution of 0.1.1 N HC1 and then slowly adding this solution drop-vise to a 1 percent solution of NaNOg with constant stirring at k°C.

Diazotization was considered complete when free HKOp was detected in the reaction mixture. Although the above reaction was accomplished at U°C,t optimum temperatures for the reaction normally are about 0-6 C. , although °C. is preferred.

The hapten-protein coupling vas performed by dissolving the protein hormone in an alkaline buffer, pll 8.0. The diazotized hapten was added slowly to the hormone solution with continuous stirring at h°C, The pH of the reaction was constantly monitored and kept near 8.0. After all the hapten vas added, the pll vas finally adjusted to 8.0, stirred for 1-2 hours and allowed to stand at k° overnight. 'The mixture was thoroughly dialyzed for 6-8 days against distilled water to remove unreacted hapten.

Although the number of diazo groups per hormone molecule could be regulated by the number of moles of hapten and hormone reacted, a-parallel control experiment with 8 35 labelled sulfan lic acid to evaluate the precise composition of the hapten-protein .samples vras performed vrith each diazotization. The same hormone preparation to be used for immunization was used in the control experiment. After the reaction vras completed, an aliquot vras taken from the reaction mixture and the remainder thoroughly dialyzed. Equal volumes of the dialyzed and undialyzed solutions were and undialyzed samples, the moles of hapten coupled to each mole of hormone was calculated since the unreacted hapten vas removed by dialysis. For this calculation, a molecular weight of 30,000 vas assumed for all Gonadotropin preparations.

Following dialysis, hapten-honnones vere lyophilized and stored at h°C . D azo-HCG (35 groups/molecule) and HLH (26 groups/molecule) were bioassayed by the ovarian ascorbic acid depletion method and found to retain 62 and 85 percent respectively of the activity of the unaltered hormones from which they .were derived. Kone of the other hormones were assayed for biological activity.

Immunisation Procedures Female baboons received their initial immunization on days 3-5 of the menstrual cycle and the second and third injections one week apart. The fourth injection was given 2-3 weeks after the third. Λ few animals received a fifth injection at γο-80-days after the' irst injections. All antigens were administered subcutaneously' in a suspension of mannide nan-oleate or peanut oil. Dose3 of antigens for each injection varied between 3 and 5 mg. Injection sites were inspected daily for 5 days after each immunization for local reactions.

Monitoring 3Sffects of Immunization.

Daily 2h hour urine specimens and frequent serum samples were collected during at least one menstrual cycle prior to immunizations and following immunizations until the effects of treatment vere assessedo Urinary LH, urinary estrogens and plasma progestins were measured. Antibodies were detected in post-imrcun zation serum samples by reacting 0.2 ml. of a 1 : 1000 dilution of serum in phosphate-buffered saline (pH 7 · Ό 0.5 percent normal baboon serum with 50 pg of 1 131 labelled hormone.

Sera vere reacted vith both the unaltered immunizing hormone and unaltered baboon LH for antibod detection. Λ urified baboon LH re aration 1. vere precipitrvted with ovine anti-baboon gamma globulin after a 2h hr. incubation at k°C. Antibody levels vere expressed as pg of labelled hormone bound. Significant antibody levels vere considered to be those 131 that would bind 5.0 pg or more of the Γ labelled antigen.

Antisera vere fractionated .by gel filtration of Sephadex G-200 according to the procedure of Fahey and Terry (at p. 36, Experimental Immunology, F.A, Davis Co.- Philadelphia, Pa., 106?, incorporated by reference to the extent necessary to understand the invention) to determine the proportion of IgM and-IgG antibodies in the baboon sera. Since the IgG fraction in this procedure contained a portion of IgA and IgD antibodies, onl IgM and total titers vere determined. The IgM fraction from 131 the column was reacted with 1 * hormones and the binding capacity determined. The volumes of the fractionated sera vere adjusted so that antibody levels vould be comparable to those of vhole serum.

No significant reactions vere observed at the site of injection following any immunization. On h occasions s a slight induration (2-3 cm in diameter) vas seen vhem mannide manoleate was used as 0. vehicle but the redness and swelling disappeared within H-5 days. Antibodies vere detected against the immunizing antigen within 3-5 weeks in all animals. The extent, duration and cross reactivity of these antibodies is recorded. Generally speaking, higher levels vere observed to heterologous gonadotropin immunization than to homologous ones.

The cross~reat:tivity of induced antibodies vith baboon LH vas studied on each animal. Cross-reactivity of antisera at peak levels vas recorded. Although relatively high antibody activity against human LH and HCG vere seen, relatively little reaction vith baboon LH occurred. An intermediate cross-reaction was noted vith anti-ovine LH and a high degree of cross-reactivit vas seen with anti-monke LH. Diazo-huraan FSH waa veakly antigenic in the baboon. The duration of antibody production wai _^ generally longer vith the human and sheep gonadotropin immun nation than with those of monkey or" baboon origin.

Peak antibody levels usually occurred at the time when the antibodies had shifted to principally the IgG type. Early antibodies had. a larger proportion of IgM type and were generally more cross-reactive with baboon LH. The change in the proportion of the total antobody propitiation that was IgM was recorded from the time antibodies were first detected. Significant cross-reactivity to baboon LH was observed in anti-huraan gonadotropins when IgM was abundant but dropped sharply as the antisera shifted to nearly all IgG. This drop in cross-reactivity did not occur vith monkey and baboon immunizations. A a n, the ovine LH immunizations produced an intermediate change in reactivity vith the shift from IgM to IgG.

Effects on the Menstrual Cycle The effects of immunization upon the events of the menstrual cycle were determined by observing changes in sex skin turgescence and levels of pituitary and/or ovarian hormones. Based on these parameters, the delay or retardation of ovulation from the expected- time,, as judged by the control cycle was calculated. One animal immunized vith HCG had no interruption in ovulation arid another immunized vith HFSH was delayed for only one cycle. Two animals injected with HLH and two injected with HCG had ovulation delays equivalent to two menstrual cycles. A third animal immunized vith HLH was delayed a calculated 86 days. Ovine LH immuniza ions produced an 88 day delay in ovulation.

Immun zations with diazo-monkey or baboon LH resulted in longer disruption of the menstrual cycle. Calculated delays in ovulation for the two animals receiving monkey LH was II6 and 122 days whereas the animal3 ' receiving altered baboon LH were retarded from ovulation 2?.h and 210 days.

Effects on cpecific hormone patterns following immunization considered to represent a "cycle". Urinary estrogens and plasma progestin patterns indicated that no ovulation occurred during the cycle of immunization vhich was 85 days in duration. Urinary estrogens were elevated during treatment but did not reflect a typical pattern. Plasma progestins were not elevated until about day 19 of the first post-treatinent cycle. Patterns of both estrogens and progestins were within normal limits during the second post-treatment cycle. Antibody levels were elevated from about day 35 of the treatment cycle until 289 days from the first detection of antibodies. An LH assay was not available when this animal was studied and no data on plasma or urinary levels of this hormone was obtained.

Hormonal patterns following an immunization with diazo-baboon LH were recorded. In this animal, antibody levels were lower and persisted, in general, for a shorter period than- did immunizations with human gonadotropins. During the treatment cycle, levels of urinary estrogens and plasma progestins followed a normal pattern but were qualitatively lower than normal. Urinary LH patterns fluctuated markedly due to the injections of diazo-LH during this period. No conclusive evidence of ovulation was obtained for the treatment cycle. The first post-treatment cycle lasted 2k6 days. During this cycle urinary LH and estrogens were elevated on days 35~'<1 but there was no subsequent elevation in plasma progestins that would indicate ovulation had occurred. Following day h2 of this cycle „ there was no significant elevation in any of the three hormone levels until day 231 when significant elevations of urinary . estrogens and LH occurred. These rises were followed 3 days later by an elevation in plasma progestins indicating the presence of a functioning cox"pus luteum. , A second post-treatinent menstrual cycle was of normal duration and. the endocrine patterns were normal.

Antibodies to unaltered baboon LH attained maximum levels by about day TO of the post-treatment cycle and remained relatively constant timee a peak of LH commensurate with a normal inidcycle elevation vas observed. From tliis point the animal appeared to liave the normal function of the pituitary-ovarian axis. Hormonal patterns in animals with other heterologous gonadotropin immun zations were similar to animal receiving HLH and other animals receiving monkey or baboon L3I were similar in response to animal receiving baboon LH.

These results in baboons indicated that the modificatio of a reproductive hormone, by the procedures outlined, did render it antigenic and the antibodies thus formed did neutralize natural end genous hormones if the natural hormone was obtained from the species receiving the immuni~ zations with modified hormone.

EXAMPLE^- 2 HCG is a hormone naturally present only in pregnant women. HCG is. also commercially available. LH hormone is immunologically and bio-logically identical to HCG hormone, even though there are chemical differences. Since they are biologically identical and HCG is readily available from commercial sources it was presumed that the ef ectiveness of this immunological procedure could be evaluated by injecting modified HCG into non-pregnant women and monitoring the blood levels of LH.

Antibodies formed will neutralize both the LH and the modified HCG.

Women have a pattern of LH levels; the level is substantially constant until the middle period betwe cycles, immediately prior to ovulation; ~at that point the LH level rises greatly and. helps induce the ovulation. Monitoring the LH level and the antibody level vill show that the procedure used did or did not cause the production of antibodies capable of neutralizing the endogenous reproductive hormone, namely LH.

A woman aged 27 years was selected for study. Hormone was injected into the subject. It is veil known that antibodies to HCG . react identically to LH as veil as HCG. The effect of the immunization was elevated, principally by monitoring blood levels of LH hormone.

Finally the results were evaluated.

Preparation of Hormone, Clinical grade HCG derived from pregnanc urine vas obtained from the Vitaraerican Corp., Little Falls, New Jersey. This material has an immunological potency pf 2600 IU/mg. Contaminants were detected in this preparation. Purification consisted of chromatography and elut on. Fractions were dialyzsd and lyophylized. ' The most potent fraction contains approximately "600 IU/iog., however, it was heterogenous on oloyaerylamide gel electrophoresis.

The fraction was further purified by gel filtration. The elution profile revealed two major protein peaks. The most potent HCG. was found in the first peak and had on immunological potency of 13,670 IU per mgo This fraction was subjected to polyacrylamide gel electrophoresis.

Further purification by gel filtration showed no evidence of heterogenit of the HCG at this stage. Consequently, materials for. study were processed according to the above■ procedure. 131 The contamination of this purified HCG vas tested with I used for identification and a sample was reacted with antisera against several proteins offering potential contamination. Those proteins were follicle stimulating hormone, human growth hormone, whole human serum, human albumin, transferin, alpha one globulin, alpha two globulin and orosomucoid. Ho detectable binding of the puri ed HCG vas observed with any antisera at a dilution of 1:^0 of each. These negative results, calculated against potential binding of the respective proteins, indicated that contamination with, any was less than 0„005 percent.

■Alteratio.n of Hormone f^j- Homione vas altered by coupling vith a hapten (sulfanilazo).

This method couples the hapten molecules to the protein via the amino group of. the aliphatic or aromatic portion of the hapten. The number of hapten molecules coupled to each HCG molecule (Ha-HCG) can be regulated and for this study, forty haptenic groups per HCG molecule were used for preparing the immunizing antigen.

Followin the hapten-coupling process, the Ha-HCG vas sterilized and testede Subject The subject was multiparous and had terminated her reproductive capabilities by prior elective bilateral salpingectomy,, She vas in good health and had regular cyclic menstruation. She underwent complete histroy, physical examination and laboratory evaluation including blood count, urinalysis, latex fixation and Papanicolau smear. She had no history of allergy.

To demonstrate normal functioning of the pituitary-ovarian axis prior to immunization, blood samples were obtained every other day from the first day of menses for 10 days, then daily for 10 days and finall » every other day until the next menses. Serum determinations of FSH, LH, estrone, estradiol and progesterone vere performed. These studies indicated an ovulatory pattern.

^Immunization Procedures .

Ten mg. of the Ha-HCG antigen vas dissolved in 1.0 ml. of saline and emulsified vith an equal volume of oil. Prior to injection, scratch tests to antigen and vehicle vere performed. Immunizations vere begun in the luteal phase of the treatment cycle to prevent superovulation from the administered HCG. Four injections at two veek intervals vere given to cu ~ in saline only via the intradermal route. Folloving each injection, blood pressure readings were taken and the subject observed for allergic reactions.

Monitoring E ects o_f Immunizations Blood samples were collected at weekly intervals beginning two veeks after the initial injection to test for the presence of humoral . and cel.lu3.ar antibodies. Following completion of the immunization schedules, blood samples were collected in the same manner as in the control cycle to assess effects of immunization on hormonal patterns of the menstrual cycle. Since antibodies to HCG react identically to LH as with HCG, LH was monitored as an index of e ectiveness of the procedure. A third cycle was similarly studied six months after initial inununiza ion. Upon completion of the study, physical and pelvic examinations and laboratory evaluations w.ere repeated,, Serum samples from the control and post- • . The subject was tested for delayed hyperteneivity before immunisation and at two veek intervals \intil the injection schedule vas completed by an ivi v o lymphocyte transformation test,* 'Results Temporal relationships of serum pituitary and gonadal hormones in the control cycles of the subject vere recorded. Antibody titers to HCG vere detected in the subject after two injections. Menses occurred at regular intervals during the immunizations .

Following the initial injection in mannide manoleate, some itching and swelling at the injection site occurred. Subsequent intradermal injections in saline produced no reactions and it was concluded that the local reactions were induced by the mannide manoleate. Lympho=- cyte transformation tests on plasma samples were negative.

In the post-treatment cycle, baseline follicular and luteal phase LH levels were not noticeably changed in the subject. Very small midcycle elecations in LH levels vere observed as compared to the normal large increases . FSH patterns in the post-treatment cycle vrere normal. This indicated that the antibodies vere neutralizing the action of endogenous LH.

The subject shoved an ovulatory progesterone pattern but attained relatively high antibody titers to LH and JICG after only tvo injections of Ha-JICG. ;.

The subject vas studied during another cycle approximately six months from tJie first immun sation. Significant antibody titers vrere found. LH patterns indicated a small midcycle elevation.- FSH patterns vere essentially normal. Thus, the specificity of anti-HCG antibodies to LH vas shown bu not to FSH.

EXAMPLE 3 Another woman aged 29 years vas selected for further study.

Hormone vas obtained, purified, and modified as in Example 2. . This modified hormone vas injected into tlris subject in the same way as in Example II. The subject was monitored and tested as in Example 2.

The results vere similar to the results found in Example 2 except that (l) the levels of estrone and estradiol vas substant ally norroalj, (2) the subject acquired significant antibody titers late in the post-immunization cycle, end (3) in the cycle studied after six months this subject shoved no significant midcycle elevation in LH patterns.

EXAMPLE jft." Another woman aged 29 years was selected for further study.

Hormone vafj obtained and purified and modified as in Example 2. This The results vere similar to the results found in Example 2 except that (1) baseline follicular and luteal phase LH levels vere ... r noticeably depressed in the post-treatment cycle, (2) no midcycle elevations vere observed in LH, (3) estrone levels vere elevated' during the follicular phase of the post-immun nation cycle, and {k) during the six months study there vas no significant midcycle elevation in LH patterns.

' EXAMPLE J> Another voman aged 35 years vas selected for further study.

Hormone vas obtained, purified, and modified as in Example 2. This modified hormone vas injected into this subject in the same vay as in Example 2. The subject vas monitored and tested as in Example 2.

The results vere similar to the results found in Example 2 except that (l) baseline follicular and luteal phase LH levels vere noticeably depressed in the post-treatment cycle, (2) a very email mid-cycle elevations of LH vere observed, (3) levels of FSH patterns in the post-treatment cycle vere depressed, and (h) levels of both estrone and estradiol vere reduced, during the follicular phase of the post-immunization.

■ EXAMPLE Another voman aged 28 years vas selected for further study.

Hormone vas obtained,: purified, and modified as in Example 2. This modified hormone vas injected into this subject in the same vay as in Example 2· Th subject vas monitored and tested as in Example 2.

The results vere similar to results found in Example 2 except that (l) baseline follicular and luteal phase LH levels were depressed in the post-treatment cycle., (2) no peaks vere observed in midcycle levels of LH, (3) estrone levels appeal'ed elevated in the follicular phase of the post immunization cycle, and (Ό LH patterns indicated no significant midcycle elevation in the six month post«immuni¾ation cycle.

EXAMPLE 1 ' Another woman aged 28 was selected for further study. Hormone was obtained, purified, and modified as in Example 2. This modified hormone was injected into this subject in the s me vay as in Example 2. The subject was monitored and tested as in Example 2.

The results were similar to results found in Example 2 except that (l) antibody titers to HCG were not detected until after three injections,. (2) baseline follicular and luteal phase LH levels were depress in the post-treatment cycle, (3) no peaks nor midcycle elevation in the LH were observed, (h) estrone levels were elevated during the follicular, phase, and (5) no significant antibody titers were found in the six month cycle.

All the above examples show the practicality of injecting modified hormones for the purpose of neutralising, an endogenous reproductive hormone and thereby offering a procedure for the prevention of conception or the disruption of gestation.

EXAffLE. 8_ Data obtained in earlier experiments and discussed in Examples 1 to 7 showed that a modified natural reproductive hormone, when injected into an animal of species from which it was derived, would produce antibodies, that would neutralize the action of the unmodified endogenous natural hormone, in the body of the animal. Hormones used in experiments 1 to 7 were FSH, LH and HCG. New experiments were performed, based on this knowledge, to identify another reproductive hormone (placental' lactogen) that could be used in a similar fashion.

Prepa ation 'o Hormone lactogen to immunize baboons* Placentae were extracted and purified on column chromatograph according to previously published procedures. The purity was tested by polyacrylamide gel, electrophoresis and by radio» immunoassa . The material obtained shoved a high degree of purity on electrophoresis and ■ adioimmunoassay shoved no contamination with other placental hormones.

Hormone Mod ica ion 'and Immu isations -The baboon placental lactogen (BPL) vas altered by coupling with the diazohium salt of sulfanilic acid as outlined for other hormones in Example- I. The number of diaso molecules per BPL molecule in this instance was 15« Immun zat on jDrocedures vere also similar to those described in Example I for other hormones » l^suJ-ts^ Within i-!-6 weeks after the first injection of diazo-BPL, anti-body levels to natural unmodif ed BPL in, yjitro vere detected in 6 female baboons. Levels rose to a plateau within 8-10 weeks and remained there for several months. Hormonal measurements indicated that there were no effects on the normal e^/ents of the menstrual cycle due to the immuniza tions. Since BPL is normally secreted only in pregnanc 5 this was not a surprising observation.

All six females were mated with a male of proven fertility thre times (once each in three different cycles during the fertile period). Pregnancy diagnosis by hormonal measurement vas performed after each mating. From the 18 matings, there were .13 conceptions as judged. by pregnancy tests. The animals that were pregnant bad menstrual bleeding 7-12 days later than vas expected for their normal menstrual cycles.

Subsequent hormonal measurementrjconfirmed that these 13 pregnancies were terminated by abortions approximately one week after the tine of expected menses » These findings suggest that the antibodies formed in the animals body after immunization had no effect on the non-pregnant menstrual cycle but when pregnancy was established, they neutralized the baboon placental lactogen in the baboon placenta and the result was abortion very early after conception. . · When, in Exs.mp.les 1. to 8 above, structure's 1 to 7 are modified by use of diazosul anilic acid, dinitrophenol, or S-aceto mercaptosuccinic anhydride or structures III. «VI -modified by addition of polytyrosine or polyalanines, are used, similar results may be obtained.

Similarly, when FSH, somatomedin, growth hornone or angio-tension II modified by use of diazosulfanilic acid or trinitrophenol, the results obtainable upon administration of the purified modified poly» peptide into a male or female human or animal indicate the stimulation of anti bodies which neutralise all or some of the modified polypeptide as well as corresponding endogenous polypeptide. ■ 'Example 9 The subjects used in the studies reported in the example are female baboons. All baboons were adults of reproductive age. A description of subjects and the conditions of experimentation have been described in Example 1. The. animals were studied using highly purified beta -cubunits of HCG using a preparation with a biological activity of less than 1,0 IU/rng_ -Animals were immunized with l¾-26 moles/mole of .polypeptide^ of diazasulfanilic acid coupled subunits in mannide lnanoleate .

Antibody levels were assessed by determining the binding of 125 serum dilutions with I labelled antigens. Cross-reactivxty of anta.sera was measured by direct binding of labelled antigens and by displacement radioimmunoassays- Antifertility effects in actively immunized animals anti-B-HCG vere detei-inined by monitoring serum levels of gonadotropins -aland sex steroid hormones before and after immunizations.

Eight female baboons vrere immunized with the modified beta eubunit of HCG. Significant.'.antibody levels vere attained in all animals , Baboon immun zations with modified beta subunit of HCG resulted in high antibody levels reacting to HCG, human LH and baboon CG but not to baboon LH. * All animals remained ovulatory, however, no pregnancies resulted from numerous matings vith males of proven fertility. Passive immunization of non-immunized pregnant baboons vith sheep anti-3-K G serum produced abortions within 3 —U hours.

Claims

44779/2 118-3329 CLAIMS ;

1. An antigen for use in active immunisation for the control or treatment of a physiological process in a particular species, comprising a chemically modified polypeptide, the unmodified polypeptide being a) an endogenous polypeptide which influences such physiological process in that species and selected from protein reproductive hormones, non-hormonal protein antigens isolated from placental tissue, gastrin, Angiotensin II, growth hormone, soma- tomedifln, parathyroid hormone, glucagon, thyroid stimulating hormone or secretin? b) a fragment (as herein defined) of such endogenous polypeptide; or c) a polypeptide which is immunologically equivalent .(as herein defined) to such endogenous polypeptide or fragment thereof, and the chemical modification, being such that as to v permit the chemically modified polypeptide to induce in that species, formation of antibodies which biologically neutralise the endogenous polypeptide as well as the chemically modified polypeptide. 44779/2 118-3329

2. An antigen according to Claim 1, in which the chemical modification comprises a) the attachment of one or more modifying groups to the the unmodified polypeptide? or b) the removal of one or more moieties from the unmodified polypeptide.

3. An antigen according to Claim 1 or 2, for use in active immunisation for the control of fertility, in which the unmodified polypeptide is a) a protein reproductive hormone; b) a fragment (as herein defined) thereof; c) a polypeptide which is immunologically equivalent (as herein defined) to such protein reproductive hormone or fragment thereof.

4. An antigen according to Claim 3, in which the protein reproductive hormone is Follicle Stimulating Hormone, L^ut¾nising Hormone, Human Placental Lactogen, Human Prolactin or Human Chorionic Gonadotropin.

5. An antigen according to Claim 4, in which the protein reproductive hormone is Follicle Stimulating Hormone .

6. An antigen according to Claim 5, in which the unmodified polypeptide is Follicle Stimulating Hor-. mone or the β-subunit thereof. - 33 - 44779/2 118-3329

7. An antigen according to Claim 4, in which the protein reproductive hormone is Human Placental Lactogen or Human Prolactin.

8. An antigen according to Claim 7, in which the protein reproductive hormone is Human Chorionic Gonadotropin.

9. An antigen according to Claim 8, in which the unmodified polypeptide is Human Chorionic Gonadotropin .

10. An antigen according to Claim 8, in which the unmodified polypeptide is the β-subunit of Human Chorionic Gonadotropin.

11. An antigen according to Claim 8, in which the unmodified polypeptide is a 20 to 30 amino acid C-terminal fragment of the β-subunit of Human Chorionic Gonadotropin.

12. An antigen according to Claim 8, in which the unmodified polypeptide is a 30 to 39 amino acid C-terminal fragment of the β-subunit of Human Chorionic Gonadotropin... 44779/2 118-3329

13. An antigen according to Claim 8, in which the unmodified polypeptide is of structure III, * Asp-Asp-Pro-Zirg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala- * * Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly- * Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln.

14. , An antigen according to Claim 8, in which the unmodified polypeptide is of structure IV, * * Gln-Ar.p-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Prc- * * Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile- Lcu-P-ro-Gln .

15. An antigen according to Claim 8, in which the unmodified polypeptide is of structure V, * Thr-Cys~Asp-Asp~Pro-Arg-Phe-Gln-Asp-Ser~Ser~Ser~Lys--Ala- * * Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-AKX-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro.,

16. An antigen according to Claim 8 , in v.'hich the unmodified polypeptide is of ture VI, Phe~Gln-Asp-Scr-£er-Ser-Lys-Ala-Pro-Pro-Pro-Ser-LeU" * * Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Se -Levi-Pro. - 35 - 44779/2 118-3329

17. An antigen according to Claim 1 or 2 , in which the unmodified polypeptide is a) a non-hormonal protein antigen isolated from placental tissue; b) a fragment (as herein described) thereof; or c) a polypeptide which is immunologically equivalent (as herein defined) to such antigen or fragment thereof.

18. An antigen according to Claim 1 or.2 , for use in active immunisation for the treatment of the Zollinger-Ellison syndrome, in which the unmodified polypeptide is a) gastrin; b) a fragment (as herein defined) thereof; or c) a polypeptide which is immunolpgically equivalent (as herein defined) to such gastrin or fragment thereof.

19. An antigen according to Claim 1. or 2, for use in active immunisation for the treatment of hypertension, in which the unmodified polypeptide is a) Angiotensin II; b) a fragment (as herein defined) thereof; or c) a polypeptide which is immunologically equivalent (as herein defined) to such Angiotensin II or fragment thereof. - 36 - 44779/2 118-3329

20. An antigen according to Claim 1 or 2, for use in active immunisation for the treatment of diabetes and associated micro- and macro- vascular disorders, in which the unmodified polypeptide is a) Growth Hormone or Somatomedin; b) a fragment (as herein defined) of such Growth Hormone or Somatomedin ; or c) a polypeptide which is immunologically equivalent (as herein defined) to such Growth Hormone or Somatomedin or fragment thereof.

21. An antigen according to any one of the preceding Claims, in which the chemical modification comprises the attachment of one or more modifying groups to the unmodified polypeptide.

22. An antigen according to Claim 21, in which 1 to 40 modifying groups are attached per mole of the unmodified polypeptide.

23. An antigen according to Claim 22, in which 2 to 40 modifying groups are attached per mole of the unmodified polypeptide.

24. An antigen according to Claim 23, in which 5 to 30 modifying groups are attached per mole of the unmodified polypeptide. - 37 - 44779/2 118-3329

25. An antigen according to Claim 24, in which 10 to 26 modifying groups are attached per mole of the unmodified polypeptide.

26. An antigen according to any one of Claims 2 to 25, in which the modifying groups are diazo groups.

27. An antigen according to Claim 26, in which the diazo groups are introduced by reaction of the polypeptide with diazo sulfanilic acid.

28. An antigen according to any one of Claims- 2 to 25, in which the modifying groups are derived from dinotrophenol, trinitrophenol, S-aceto-mercaptosuccinic anhydride, a polytyrosine, a polyalanine biodegradable polydextran, or thyroglobulin .

29. Human Chorionic Gonadotropin modified by introduction of 35 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

30. Human Leutinising Hormone modified by introduction of 26 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

31. Human Chorionic Gonadotropin modified by introduction of 40 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

32. Baboon Placental Lactogen modified by introduction of 15 diazo groups per molecule, the diazc - 38 - 44779/2 118-3329 A groups being derived from diazotised sulfanilic acid.

33. The p-subunit of Human Chorionic Gonadotropin modified by introduction of 14 to 26 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

34. An antigen according to Claim 1, substantially as herein described with reference to any one of the Examples .

35. A pharmaceutical composition comprising an antigen according to any one of Claims 1 to 33, in association with a pharmaceutically acceptable liquid carrier, and in a form suitable for parenteral administration.

36. A pharmaceutical composition according to Claim 35, in which the liquid carrier is mannide mono-oleate, saline or an oil.

37. A pharmaceutical composition according to Claim 35 or 36 in unit dosage form and containing 0.1 to 50 mg of the antigen.

38. A pharmaceutical composition according to Claim 35, substantially as herein described with reference to any one of the Examples. - 39 - t 44779/2 118-3329

39. A method of contraception comprising injection of an immunologically effective amount of an antigen according to any one of Claims 3 to 17 , 21 to 28 (as dependent on Claims 3 to 17) and 29 to 33. 5

40. A method according to Claim 39, comprising injection of 0.1 mg to 50 mg of the antigen.

41. A method according to Claim 39 or 40, in which the antigen is injected in the form of a solution or suspension in a pharmaceutically . acceptable 10 liquid carrier.

42. A method according to Claim 41, in which the liquid carrier is mannide monooleate, saline or an oil.

43. A method according to Claim 39, substantially as herein described with reference to any one of 15 the Examples. - 40 -