the incorporation of hcg peptide antigens into the inorganic salt / biopolymer matrix is accomplished by methods described by royer ( u . s . pat . nos . 6 , 391 , 336 , 6 , 497 , 901 and 6 , 630 , 486 , by their entirety , incorporated by reference herewith ). the inorganic salt employed for use in the current invention is calcium sulfate hemi - hydrate and the biopolymer used is dextran sulfate , the preferred form having an average molecular weight of 8000 daltons . two of the hcg peptide antigens entrapped represent either hcg beta subunit sequences 109 - 145 and 38 - 57 or analogues of these peptides conjugated to dt in a specified ratio of peptide vs . dt or combinations of these conjugates . tt can be substituted for dt . each conjugate antigen , preferably in a dry state , is mixed separately with calcium sulfate hemi - hydrate in a proportion by weight of 1 - 10 parts salt to 1 part antigen with the preferred ratio being 4 : 1 . after thorough mixing of the dry components , a volume of aqueous 10 percent dextran sulfate ( wt / volume ) is added to the appropriate weight of dry conjugate / salt premix with continual stirring to form a slurry with a uniform dispersion of the conjugate within . the weight of the mixture in the wet state is recorded . the mixture is allowed to dry at room temperature , with ventilation , for 2 - 5 days until no further change in the weight of the mixture is observed . a final weight is recorded before the solid matrix is ground to a fine powder using a mortar and pestle , or mechanical grinder , and the water content of the powder determined . water content plus loss of water on drying is used to calculate the concentration of antigen in the solid matrix ( mass balance ). the desired micro - particle sizes for use are obtained by sieving through a series of standard mesh sieves . particles used for vaccine formulation range from 10 - 800 micrometers with a preferred size range of 45 - 150 micrometers . the dried antigen - containing particles are typically stable to long - term storage at ambient temperature , but may be stored under refrigeration if desired or necessitated by properties of particular antigens . to formulate a vaccine , the antigen - containing matrix particles are suspended in a liquid medium with sufficient viscosity to maintain a uniform distribution of particles throughout the suspension . alternately a less viscous liquid medium may be used so long as the particles can be easily resuspended prior to injection . various liquid vehicles can be used , such as water - in - oil emulsions , oil - in - water emulsions , 0 . 1 - 2 . 0 percent carboxymethyl cellulose , hyaluronic acid solutions , lecithin solutions or other acceptable pharmaceutical vehicles . one preferred vehicle for hcg conjugate antigens is a water - in - oil emulsion of squalene and phosphate - buffered saline ( pbs ) in a ratio of 3 parts oil phase to 2 parts pbs ( v / v ). an acceptable oil is provided by a mixture of 4 parts squalene to 1 part mm , which acts as an emulsifier and stabilizes the emulsion . the emulsion is prepared using a mechanical mixer or by hand using hypodermic syringes connected by a three - way stopcock . other ratios of oil phase vs . pbs are useful for other antigens depending upon their solubility and / or dose required in a vaccine . other oils , such as squalane , may also be used . most weak antigens , including the hcg conjugates used in the current invention , require an adjuvant compound to be administered together with the antigen to stimulate the desired immune response . such compounds can be added to the matrix and mixed with antigen at the time the matrix is prepared or added to the aqueous phase of the emulsion before the emulsion is formed . adjuvant compounds that have been shown to be effective for this purpose include mycobacteria , synthetic fragments of bacteria membranes or synthetic analogues of these , aluminum salts , magnesium salts , lipids and numerous other compounds ( adams a . synthetic adjuvants . new york : john wiley & amp ; sons , 1985 ). a preferred adjuvant for the invention disclosed herein is a synthetic analogue of a bacteria membrane , nor muramyl dipeptide ( nor mdp ), which is chemically defined as n - acetyl - d - glucosamine - 3 - y - l - ala - d - isogln sodium salt . this compound is highly water - soluble and is added to the aqueous phase of the squalene / pbs emulsion in concentrations such that the final emulsion has a concentration of 5 - 100 micrograms per milliliter . for final formulation of a vaccine , a pbs and oil emulsion is first formed in a double syringe system , by forcing the oil / aqueous mixture back and forth between the syringes until a smooth , milky emulsion is achieved . next , the syringe containing the emulsion is detached and reconnected to a new syringe containing dry antigen particles . the selected dose of matrix particles ( determined by antigen concentration in the matrix and the weight of antigen needed ) is then suspended in the delivery vehicle by mixing back and forth until an even suspension of particles in the vehicle is achieved . the dry matrix particles may be mixed with the vehicle at the time of immunization or by batch mixing of particles with the vehicle in bulk volumes for dispensing unit doses into sterile vials , ampoules , syringes , or other suitable delivery means . vaccines formulated in the emulsion preferably are stored at temperatures of 2 - 8 degrees c . antigen concentrations in the matrix can vary from 12 - 24 percent by weight with a preferred concentration of 16 - 18 percent . doses of hcg conjugate for human immunizations can range from 10 - 500 micrograms and can be administered in 0 . 1 - 1 . 5 milliliters . selected antigen and adjuvant doses , in the appropriate volume , are injected intramuscularly to recipients at a medically acceptable site . in humans , this normally is in the upper arm , thigh or buttocks . the release of entrapped antigens is much slower when particles are suspended in the emulsion than when suspended in an aqueous vehicle ( fig1 ). fig1 illustrates the comparison of in vitro release rates of the conjugate dt / beta subunit peptide 109 - 145 ( ctp / dt ) entrapped in the inorganic / biopolymer matrix , either suspended directly in extracting medium ( pbs ) or after first emulsifying with squalene / mannide monooleate / pbs emulsion . particles or emulsified particles were placed in 13 × 100 mm disposable tubes with aliquots of 1 . 0 ml pbs , mixed by vortexing and rotated in an incubator at 37 degrees c . at the indicated times , the tubes were centrifuged at low speed ( 900 × g ) for 5 - 7 minutes to settle the particles or partition the emulsion / oil / pbs . the supernatant pbs was drawn off with a micropipet and tested for the presence of extracted conjugate by a lowry protein assay procedure . another 1 . 0 ml of pbs was added back to each tube , and samples were mixed by vortexing and then rotated at 37 degrees c . as before . curves indicate the total amount of conjugate extracted from particles directly suspended in pbs ( diamonds ) and particles suspended in emulsion and then mixed with pbs ( squares ). fig2 illustrates the mean antibody levels obtained in four rabbits following a single injection of matrix particles containing 1 . 0 mg of the conjugate dt / hcg beta subunit peptide 109 - 145 ( ctp / dt ) in 1 . 0 ml of a squalene / pbs emulsion containing 0 . 025 mg of the nor - mdp adjuvant . fig3 illustrates the mean antibody levels obtained in four rabbits following a single injection of matrix particles containing 1 . 0 mg of the conjugate dt / beta subunit peptide 38 - 57 analogue ( loop / dt ) in 1 . 0 ml of a squalene / pbs emulsion containing 0 . 025 mg of the nor - mdp adjuvant . fig4 illustrates the mean antibody levels obtained in two groups of rabbits ( 4 - 8 rabbits / group ) following injections of either ( a ) three inoculations of vaccine without the conjugate being entrapped in matrix particles ( diamonds ), or ( b ) a single inoculation of vaccine with conjugate entrapped in matrix particles ( squares ), where each formulation contains equal amounts of the conjugates dt / beta subunit peptides 109 - 145 ( ctp / dt ) and 38 - 57 analogue ( loop / dt ). all doses were given in squalene / pbs emulsion containing 0 . 025 mg of the nor - mpd adjuvant . each inoculation involved 0 . 05 mg of conjugates and was administered to rabbits . for the group receiving matrix - containing vaccine , the single dose of vaccine in 0 . 5 ml of emulsion was given on day one ( left arrow ) and no further injections were given . for the group receiving vaccine without matrix particles , doses were given in 1 . 0 ml of emulsion , on day 1 ( left arrow ) and again at weeks 3 and 6 ( middle and right arrows ). fig5 illustrates the mean antibody levels in three groups of rabbits ( 4 - 8 rabbits / group ) following injections of matrix particles containing equal amounts of the conjugates dt / beta subunit peptides 109 - 145 ( ctp / dt ) and 38 - 57 analogue ( loop / dt ) using combined doses of 0 . 045 , 0 . 090 or 0 . 135 mg administered to each rabbit on day 1 and at 24 weeks . all doses were given in 0 . 5 ml of a squalene / pbs emulsion containing 0 . 025 mg of the nor - mpd adjuvant . in order to investigate release of immunogen from the inorganic / biopolymer matrix , an in vitro method was developed to determine the release rate . the conjugate dt / beta subunit peptide 109 - 145 ( ctp / dt ) was entrapped in the inorganic / biopolymer matrix , and then either suspended directly in extracting medium ( pbs ) or suspended in pbs after first suspending the particles in a preformed squalene / mm / pbs emulsion . particles or emulsified particles were then placed in 13 × 100 mm disposable tubes with aliquots of 1 . 0 ml pbs , mixed by vortexing and then rotated in an incubator at 37 degrees c . at specified times , the tubes were centrifuged at low speed ( 900 × g ) for 5 - 7 minutes to settle the particles or partition the emulsion / oil / pbs . the supernatant pbs was drawn off with a micropipet and tested for the presence of extracted conjugate by a lowry protein assay procedure . another 1 . 0 ml of pbs was then added back to each tube , samples were mixed by vortexing and then rotated at 37 degrees c . as before . the extraction curves obtained are shown in fig1 . in both cases , the inorganic / biopolymer matrix retained significant amounts of the conjugate and released it over a number of days . however , the conjugate extracted much more quickly from particles directly suspended in pbs ( diamonds ) than from particles suspended in emulsion and then mixed with pbs ( squares ). these slower release rates of conjugate are likely to be responsible for high titers and long duration of titers seen in immunization studies ( examples ii and iii , below ), because slow release from the injection site is known to augment immune responses to vaccines . based on the superior retention of conjugate by the emulsified particles , further studies focused on this formulation of the conjugate . a test of the immunogenicity of the antigen beta hcg peptide 109 - 145 conjugated to dt ( ctp / dt ) incorporated in the inorganic / biopolymer matrix was conducted using rabbits as vaccine recipients . four rabbits were each injected with a formulation containing 1 . 0 mg of the conjugate in 1 . 0 ml of an emulsion of squalene / pbs ( 60 %/ 40 % v / v ) which contained 0 . 025 mg of nor mdp . antibody levels were measured weekly using a radioimmunoassay and expressed as nm / l binding capacity . a single intramuscular injection resulted in the production of elevated antibodies by two weeks after the injection . peak antibody levels of over 1 , 500 nm / l were attained by 7 weeks , and were sustained at levels above 100 nm / l for several months . these levels are 40 - 100 times higher than required in colon cancer patients to provide benefit from therapy ( fig2 ). this study showed that the formulation was highly effective . compared to results previously obtained in inoculations with identical amounts of conjugate but where the conjugate was not entrapped in matrix particles , the data indicated that a lower dose of antigen entrapped in the matrix could be used to obtain the necessary level of antibodies for therapy than with the non matrix - entrapped formulation . another test was performed using identical conditions as in example ii except a beta hcg 38 - 57 analogue peptide conjugated to dt ( loop / dt ) was used as the antigen . very similar results were obtained ( fig3 ). compared to results previously obtained in inoculations with identical amounts of conjugate but where the conjugate was not entrapped in matrix particles , the data indicated that lower doses of antigen entrapped in the matrix were more effective for use in a vaccine than the non matrix - entrapped formulation . a comparison of the efficacy of vaccines identical except for the presence or absence of the inorganic / biopolymer matrix was conducted using rabbits as vaccine recipients . four rabbits were each injected intramuscularly with a formulation containing 0 . 5 mg of each of the two conjugates ( ctp / dt and loop / dt as in examples ii and iii above ) in 0 . 5 ml of an emulsion of squalene / pbs ( 60 %/ 40 %) which contained 0 . 025 mg of nor mdp . antibody levels were measured weekly using a radioimmunoassay and expressed as nm / l binding capacity . a single intramuscular injection of inorganic / biopolymer - matrix - containing vaccine was compared to three injections of vaccine lacking the inorganic / biopolymer matrix , in order to achieve approximate parity of effects . fig4 shows that both vaccination regimens resulted in the production of elevated antibodies by two weeks after the injection . however , titers obtained from the injections of inorganic / biopolymer - matrix - containing vaccine reached significantly higher levels and remained higher than those from vaccine lacking the inorganic / biopolymer matrix , despite the latter having been injected three times vs . one time for the inorganic / biopolymer - matrix - containing vaccine . table 1 compares the tissue reactions obtained for the immunizations . for the vaccine not entrapped in the inorganic / biopolymer matrix , the tissue reactions at the injection sites , measured as in example vi , were minimal to moderate in all animals , but the average reaction ( lesion score ) was 1 . 4 , substantially above the clinically acceptable level of 1 . 0 . in contrast , the tissue reactions were negligible for the inorganic / biopolymer - matrix - containing vaccine , and well within clinically acceptable limits . a series of tests using individual conjugates indicated that safe and effective doses of each hcg conjugate incorporated into the matrix was between 0 . 010 and 0 . 10 mg . a study was then initiated in which a dose response was obtained for a combination of both ctp / dt and loop / dt antigens in equal proportions . doses containing a total of 0 . 045 - 0 . 135 mg of the combined conjugates ( 50 / 50 ratio , w / w ) were tested for their ability to induce hcg - specific antibody production in rabbits . injections were given on day 1 and at 24 weeks from the first injection . the antibody profiles from this study are shown in fig5 . antibody levels , even at the lowest dose studied , were more than adequate for clinical use . the tissue reactions measured as in example vi were minimal at the lowest doses and moderate , but acceptable , at the highest doses used . in order to evaluate the gross ( macroscopic ) appearance of the thigh muscle after injection of test materials , the skin of euthanized animals was peeled off the examined thigh by making a transverse and longitudinal incision and then peeling off the skin . care is taken to make a clean separation from muscle tissue , without damaging the latter . using a sharp lancet , each injection site was incised to expose the interior of the thigh muscle . additional incisions were made if necessary to ensure complete viewing and assessment of pathology . biopsy specimens were preserved in 10 % buffered formalin . no visible pathology . at times yellow fatty / fibrous tissue appeared after complete resolution of inflammation in the muscle tissue . such a change was not rated as pathological . a typical appearance included small (& lt ; 3 mm diameter ) hard nodules representing encapsulated and resolving sterile abscesses or inflammatory sites . the combined volume of such lesions was required to be less than 5 % of the total thigh muscle volume . nodules were larger ( 3 - 10 mm diameter ) and included hard to the touch ( old fibrosis ) or soft ( more recently encapsulated ) lesions . on squeezing such lesions , pus or injection material sometimes was expressed . free ( unencapsulated ) material was occasionally seen . in that case , the longitudinal diameter of the lesion was required to be no larger than 10 mm . the combined volume of the lesions was required to be between 5 - 10 % of the total thigh muscle volume . large , encapsulated or unencapsulated lesions , greater than 10 mm in longitudinal diameter . typically , lesions contained pus ( sterile abscesses ) or injection material ( emulsion ). total volume of the lesions was & gt ; 10 % of total thigh muscle volume . when lesions did not fall unequivocally within the definition of a certain grade , intermediate grades were designated as 0 - 1 , 1 - 2 , or 2 - 3 . to calculate group means , intermediate grades were assigned half values ( for example , 0 - 1 was assigned a value of 0 . 5 ).
this invention comprises a method of immunization in which human chorionic gonadotropin vaccine antigens are incorporated into an inorganic salt / biopolymer complex using a simple manufacturing process . the resulting solid matrix is administered to human subjects in the form of microparticles . the method comprises suspending microparticles in an emulsion of a natural oil and water containing an adjuvant compound acceptable for human use and injecting a pharmaceutical dose of the suspension intramuscularly . the vaccine antigens are conjugates of peptide fragments of the beta subunit of hcg with a carrier protein , diphtheria toxoid . vaccine antigens delivered in this formulation are effective for eliciting antibodies in recipients for the treatment of cancer or hormone - related diseases .