LV13128B - Multivalent meningococcal polysaccharide-protein conjugate vaccine - Google Patents

Multivalent meningococcal polysaccharide-protein conjugate vaccine Download PDF

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Publication number
LV13128B
LV13128B LVP-03-79A LV030079A LV13128B LV 13128 B LV13128 B LV 13128B LV 030079 A LV030079 A LV 030079A LV 13128 B LV13128 B LV 13128B
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polysaccharide
vaccine
conjugate
vvas
protein
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LVP-03-79A
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Robert P Ryall
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Aventis Pasteur
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Description

MULT1VALENT MENINGOCOCCAL POLĪSACCHARIDE-PROTEIN CONJUGATE VACCINE [OOO1] This application claims the benefit of U.S. provisional appiication no. 60/263,435, filed January 23, 2001.
BACKGROUND OF THE INVENTION
Fieid of the Invention [0002] The present invention relates to the fieid of medicine generally, and more specificaliy ' to microbiology, immunology, vaccines and the prevention of infection by a bacterial pathogen by immunization.
Summary of the Related Art [0003] Neisseria meningitidis is a Ieading cause of bacterial meningitis and sepsis throughout the world. The incidence of endemic meningococcai disease during the last thirty years ranges from 1 to 5 per 100,000 in the developed world, and from 10 to 25 per 100,000 in developing countries (Reido, F.X., et. al. 1995). During epidemics the incidence of meningococcai disease approaches 1000 per 1000,000. There are approximately 2,600 cases of bacterial meningitis per year in the United States, and on average 330,000 cases in developing countries. The case fatality rāte ranges betvveen 10 and 20%.
[0004] Pathogenic meningococci are enveloped by a polysaccharide capsuie that is attached to the outer membrane surface of the organism. Thirteen different serogroups of meningococci have been identified on the basis of the immunological specificrty of the capsular polysaccharide (Frasch, C.E., et. al. 1985). Of these thirteen serogroups, five cause the majority of meningococcai disease; these include serogroups A, B, C, W135, and Y. Serogroup A is responsible for most epidemic disease. Serogroups B, C, and Y cause the majority of endemic disease and localized outbreaks.
[0005] The human naso-oropharyngeal mucosa is the only knovvn natūrai reservoir of Neisseria meningitidis. Colonization takes place both at the exterior surface of the mucosal celi and the subepithelial tissue of the nasopharynx. Carriage of meningococci can last for months. Spreading of meningococci occurs by direct contact or via air droplets. Meningococci become invasive by passing through the mucosal epithelium via phagocytic vacuoles as a result of endocytosis. Host defense of invasive meningococci is dependent upon complement-mediated bacteriolysis. The serum antibodies that are responsible for complement-mediated bacteriolysis are directed in large part against the outer capsular polysaccharide.
[0006] Vaccines based on meningococcai poiysaccharide have been described vvhich elicit an immune response against the capsular polysaccharide. These antibodies are capable of complement-mediated bacteriolysis of the serogroup specific meningococci. The meningococcal polysaccharide vaccines vvere shovvn to be efficacious in children and adults (Peltola, H., et. al. 1977 and Artenstein, M.S., et. al. 1970), but the efficacy vvas limited in infants and young children (Reingold, A.L., et. al. 1985). Subsequent doses of the polysaccharide in younger populations elicrted a weak or no booster response (Goldschneider, I., et. al. 1973 and Gold, R., et. al. 1977). The duration of protection elicited by the meningococcal polysaccharide vaccines is not long lasting, and has been estimated to be betvveen 3 to 5 years in adults and children above four years of age (Brandt, B., et. al. 1975, Kāyhty, H., et. al. 1980, and Ceesay, S. J., et. al. 1993). For children from one to four years old the duration of protection is less than three years (Reingold, A.L., et. al. 1985).
[0007] Polysaccharides are incapable of binding to the major histocompatibility complex molecules, a prerequisite for antigen presentation to and stimulation of T-helper lymphocytes, i.e., they are T-cell independent antigens. Polysaccharides are able to stimulate B lymphocytes for antibody production vvithout the help of T-helper lymphocytes. As a result of the T-independent stimulation of the B lymphocytes, there is a lack of memory induction follovving immunization by these antigens. The polysaccharide antigens are capable of eiiciting very effective T-independent responses in adults, but these T-independent responses are weak in the immature immune system of infants and young children.
[0008] T-independent polysaccharide antigens can be converted to T-dependent antigens by covaient attachment of the polysaccharides to protein molecules (“carriers or “carrier proteīns”). B celis that bind the polysaccharide component of the conjugate vaccine can be activated by helper T ceils specific for peptides that are a part of the conjugated carrier protein. The T-helper response to the carrier protein serves to augment the antibody production to the polysaccharide. [0009] The serogroup B polysaccharide has been shovvn to be poorly to non-immunogenic in the human population (Wyle, F.A., et. al. 1972). Chemical attachment of this serogroup polysaccharide to proteīns has not significantly altered the immune response in laboratory animals (Jennings, H. J., et. al. 1981). The reason for the iack of immune response to this serogroup poiysaccharide is thought to arise from structural similarities betvveen the serogroup B polysaccharide and polysialylated host glycoproteins, such as the neural celi adhesion molecules.
[0010] A meningococcal conjugate vaccine based on serogroup C polysaccharide has been described. This monovalent vaccine elicits a strong functional antibody response to the capsular polysaccharide present on strains of N. meningitidis corresponding to serogroup C. Such a vaccine is only capable of protecting against disease caused by serogroup C bacteria.
[0011] Existing vaccines based on meningococcal polysaccharide are of limited use in young children and do not provide long-lasting protection in adults. The only meningococcal vaccine vvhich as been shovvn to be capable of eliciting iong-iasting protection in ali groups, including children, at risk for meningococcal infection is based on a polysaccharide from a single serogroup of N. meningitidis and provides no protection against infection by other serogroups. Thus, a need exists for a meningococcal conjugate vaccine capable of conferring broad, long-lived protection against meningococcal disease in children and adults at risk for meningococcal infection. The multivalent meningococcal polysaccharides of the present invention solve this need by providing vaccine formulations in vvhich immunogenic polysaccharides from the major pathogenic serogroups of N. meningitidis have been converted to T-dependent antigens through conjugations to carrier proteins.
SUMMARY OF THE INVENTION [0012] The present invention provides immunological compositions for treatment of meningococcal polysaccharide-protein conjugates caused by pathogenic Neisseria meningitidis.
[0013] The present invention provides immunological compositions comprising two or more protein-polysaccharide conjugates, vvherein each of the conjugates comprises a capsular polysaccharide from N. meningitidis conjugated to a carrier protein.
[0014] The present invention provides immunological compositions comprising two or more distinct protein-polysaccharide conjugates, vvherein each of the conjugates comprises a capsular polysaccharide from a different serogroup of N. meningitidis conjugated to a carrier protein. [0015] The present invention provides vaccines for meningococcal polysaccharide-protein conjugates caused by pathogenic Neisseria meningitidis. The present invention provides multivalent meningococcal vaccines comprised of immunologically effective amounts of from two to four distinct protein-po(ysaccharide conjugates, vvherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and vvherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W135 andY.
[0016] The present invention also provides methods of manufacture of a multivalent meningococcal polysaccharide-protein compos’rtion comprising purifying two or more capsular polysaccharides from pathogenic Neisseria meningitidis; conjugating the purified polysaccharides to one or more carrier proteins and-combining the conjugates to make the multivalent meningococcal polysaccharide-protein composition.
[0017] The present invention further provides a method of inducing an immunological response to capsular polysaccharide of N. meningitidis comprising administering an immunologically effective amount of the immunological composition of the invention to a human or animal. [0018] The present invention provides a multivalent meningococcal vaccine comprised of immunologically effective amounts of from tvvo to four distinct protein-polysaccharide conjugates, vvherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and vvherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W-135 and Y.
[0019] The present invention provides a method of protecting a human or animal susceptible to infection from N. meningitidis comprising administering an immunologically effective dose of the vaccine of the invention to the human or animal.
[0020] Ali patents, patent applications, and other publications recited herein are hereby incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE INVENTION [0021] The present invention comprises an immunological composition of tvvo or more distinct protein-polysaccharide conjugates, vvherein each of the conjugates comprises a capsular polysaccharide conjugated to a carrier protein. Thus, the present invention inciudes compositions that comprise tvvo or more different capsular poiysaccharides conjugated to one or more carrier protein(s).
[0022] Capsular po!ysaccharides can be prepared by Standard techniques knovvn to those of skill in the art (ref). In the present invention capsular polysaccharides prepared from serogroups A, C, W-135 and Y of N. meningitidis are preferred.
[0023] In a preferred embodiment, these meningococcal serogroup conjugates are prepared by separate processes and formulated into a single dosage formulation. For example, capsular polysaccharides from serogroups A, C, W-135 and Y of N. meningitidis are separately purified. [0024] In a preferred embodiment of the present invention the purified polysaccharide is depolymerized and activated prior to conjugation to a carrier protein. In a preferred embodiment of the present invention capsular polysaccharides of serogroups A, C, W-135, and Y from N. meningitidis are partially depolymerized using rrtild oxidative conditions.
[0025] The depolymerization or partiai depolymerization of the polysaccahrides may then be follovved by an activation step. By “activation is meant Chemical treatment of the polysaccharide to provide Chemical groups capabie of reacting vvith the carrier protein. A preferred activation method involves treatment vvith adipic acid dihyrazide in physiological saline atpH 5.0±0.1 for approximately two hours at 15 to 30°C. One process for activation is described in U.S. Patent 5,965,714.
[0026] Once activated, the capsular polysaccharides may then be conjugated to one or more carrier proteīns. In a preferred embodiment of the present invention each capsular polysaccharide is separately conjugated to a single carrier protein species. In a preferred embodiment the capsular poiysaccharides from serogroups A, C, W-135 and Y of N. meningitidis are each separately conjugated to the same carrier protein species.
[0027] Carrier proteīns may include inactivated bacterial toxins such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteīns such as, outer membrane complex c (OMPC), porins, transferrin binding proteīns, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesin protein (PsaA), could also be used. Other proteīns, such as ovalbumin, keyhole limpit hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) may also be used as carrier proteīns. Carrier proteīns are preferably proteīns that are non-toxic and non-reactogenic and obtainable in sufficient amount and purity. Carrier proteīns should be amenable to Standard conjugation procedures. In a preferred embodiment of the present invention diphtheria toxin purified from cultures of Corynebacteria diphtheriae and chemically detoxified using formaldehyde is used as the carrier protein.
[0028] After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates may be purified (enriched vvith respect to the amount of polysaccharide-protein conjugate) by a variety of techniques. One goal of the purification step is to remove the unbound polysaccharide from the polysaccharide-protein conjugate. One method for purification, involving ultrafiltration in the presence of ammonium sulfate, is described in U.S. Patent 6,146,902. Alternatively, conjugates can be purified awayfrom unreacted protein and polysaccharide by any number of Standard techniques including, inter alia, size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography or ammonium sulfate fractionation. See, e.g., P.W, Anderson, et. al. (1986). J. Immunol. 137:1181-1186. See also H. J. Jennings and C. Lugovvski (1981) J. Immunol. 127:1011-1018.
[0029] After conjugation of the polysaccharide and carrier protein the immunological compositions of the present invention are made by combining the various polysaccharide-protein conjugates. The immunological compositions of the present invention comprise two or more different capsular polysaccharides conjugated to one or more carrier protein(s). A preferred embodiment of the present invention is a bivalent immunological čomposition comprising capsular polysaccharides from serogroups A and C of N. meningitidis separately conjugated to diptheria toxoid. More preferably the present invention is a tetravalent immunological čomposition comprising capsular polysaccharides from serogroups A, C, W-135 and Y of N. meningitidis $eparately conjugated to diptheria toxoid.
[0030] Preparation and use of carrier proteīns, and a variety of potential conjugation procedures, are vvell known to those skilled in the art. Conjugates of the present invention can be prepared by such skilled persons using the teachings contained in the present invention as vvell as Information readily available in the general literature. Guidance can also be obtained from any one or ali of the follovving U.S. patents, the teachings of which are hereby incorporated in their entirety by reference: U.S. 4,356,170; U.S. 4,619,828; U.S. 5,153,312; U.S. 5,422,427 and U.S. 5,445,817.
[0031] The immunological compositions of the present invention are made by separately preparing polysaccharide-protein conjugates from different meningococcal serogroups and then combining the conjugates. The immunological compositions of the present invention can be used as vaccines. Formulation of the vaccines of the present invention can be accomplished using art recognized methods. The vaccine compositions of the present invention may aiso contain one or more adjuvants. Adjuvants include, by way of example and not limitation, aluminum adjuvants, Freund’s Adjuvant, ΒΑΥ, DC-chol, pcpp, monophoshoryl lipid A, CpG, QS-21, cholera toxin and formyl methionyl peptide. See, e.g., Vaccine Design, the Subunit and Adjuvant Approach, 1995 (M.F. Powell and M. J. Nevvman, eds., Plenum Press, NY). The adjuvant is preferabiy an aluminum adjuvant, such as aluminum hydroxide or aluminum phosphate.
[0032] As demonstrated belovv, the vaccines and immunological compositions according to the invention elicit a T-dependent-like immune response in various animal models, vvhereas the polysaccharide vaccine elicits a T-independent-like immune response. Thus, the compositions of the invention are also useful research tools for studying the biological pathways and processes involved in T-dependent-Iike immune responses to N. meningitidis antigens.
[0033] The amount of vaccine of the invention to be administered a human or animal and the regime of administration can be determined in accordance vvith Standard techniques vvell knovvn to those of ordinary skill in the pharmaceutical and veterinary arts taking into consideration such factors as the particular antigen, the adjuvant (if present), the age, sex, vveight, species and condition of the particular animal or patient, and the route of administration. In the present invention, the amount of polysaccharide-protein carrier to provide an efficacious dose for vaccination against N. meningitidis can be from betvveen about 0.02 gg to about 5 gg per kg body vveight. In a preferred composition and method of the present invention the dosage is betvveen about 0.1 pg to 3 pg per kg of body vveight. For example, an efficacious dosage vvill require less antibody if the post-infection time elapsed is less since there is less time for the bacteria to proliferate. In like manner an efficacious dosage vvill depend on the bacterial load at the time of diagnosis. Multiple injections administered over a period of days could be considered for therapeutic usage. [0034] The multivalent conjugates of the present invention can be administered as a single dose or in a series (i.e., vvith a “booster” or boosters”). For example, a child could receive a single dose early in life, then be administered a booster dose up to ten years later, as is currently recommended for otfier vaccines to prevent childhood diseases.
[0035] The booster dose vvill generate antibodies from primed B-cells, i.e., an anamnestic response. That is, the multivalent conjugate vaccine elicits a high primary (i.e., follovving a single administration of vaccine) functional antibody response in younger populations vvhen compared to the licensed polysaccharide vaccine, and is capable of eliciting an anamnestic response (i.e., follovving a booster administration), demonstrating that the protective immune response elicited by the multivalent conjugate vaccine of the present invention is long-lived.
[0036] Compositions of the invention can include liquid preparations for orifice, e.g., oral, nasal, anal, vaginai, peroral, intragastric, mucosal (e.g., perlinqual, alveolar, gingival, olfactory or respiratory mucosa) etc„ administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneious, intradermai, intramuscular, intraperitoneal or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Intravenous and parenteral administration are preferred. Such compositions may be in admixture vvith a suitable carrier, diluent, or excipient such as sterile vvater, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as vvetting or emulsifying aģents, pH buffering aģents, gelling or viscosrty enhancing additives, preservatives, flavoring aģents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON’S PHARMACEUTICAL SCIENCE”, 17tt edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, vvithout undue experimentation.
[0037] Compositions of the invention are conveniently provided as liquid preparations, e.g., isotonic aqueous Solutions, suspensions, emulsions or viscous compositions that may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention can be in the “solid form of pilis, tablets, capsules, caplets and the like, including “solid” prepara7 tions vvhich are time-reieased or vvhich have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosoi dispenser. Aerosols are usualiy under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size.
[0038] Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somevvhat more convenient to administer, especially by injection or orally, to animals, children, parti'cularly smali children, and others who may have difficulty swallowing a pili, tabiet, capsule or the like, or in multi-dose situations. Viscous compositions, on the other hand, can be formulated vvithin the appropriate viscosity range to provide longer contact periods vvith mucosa, such as the lining of the stomach or nasal mucosa.
[0039] Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., Iiquid dosage for (e.g., vvhether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., vvhether the composition is to be formulated into a pili, tablet, capsule, caplet, time release form or liquid-filled form).
[0040] Solutions, suspensions and gels, normally contain a major amount of vvater (preferably purified vvater) in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing aģents, buffering aģents, preservatives, vvetting aģents, jeiling aģents, (e.g., methyicellulose), colors and/or fiavors may also be present The compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal fluid.
[0041] The desired isotonicity of the compositions of this invention may be accomplished using sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
[0042] Viscosity of the compositions may be maintained at the selected Ievel using a pharmaceutically acceptable thickening aģent. Methylcel!uiose is preferred because it is readily and economically available and is easy to work vvith. Other suitable thickening aģents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the aģent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normāli;/ prepared from Solutions by the addition of such thickening aģents.
[0043] A pharmaceutically acceptable preservative can be employed to increase the shelf life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the aģent selected.
[0044] Those skilled in the art will recognize that the components of the compositions must be selected to be chemically inert vvith respect to the N. meningitidis polysaccharide-protein carrier conjugates.
[0045] The invention will be further deseribed by reference to the follovving illustrative, nonlimiting examples setting forth in detaii several preferred embodiments of the inventive concept. Other examples of this invention wili be apparent to those skilled in the art vvithout departing from the spirit of the invention.
EXAMPLES Example 1
Preparation of Neisseria meningitidis serogroups A, C, W-135, and Y purified capsular polysaccharides powders Crude paste preparation [0046] Separately, Neisseria meningitidis serogroup A, C, W135, and Y vvet frozen seed cultures were thavved and recovered vvith the aid of liquid Watson Scherp medium and planted in Blake botiJes containing Mueller Hinton agar medium. The Biake were incubated at 35 to 37°C in a C02 atmosphere for 15 to 19 hours. Follovving the incubation period, the grovvth from the Blake bottles were dislodged and added to 4L flasks containing Watson Scherp medium. The flasks were incubated at 35 to 37°C for 3 to 7 hours on a platform shaker. The contents of the 4L flasks were transferred to a fermenter vessel containing Watson Scherp medium. The fermenter vessel was incubated at 35 to 37°C for 7 to 12 hours controlling dissolved oxygen content and pH vvith supplement feed and antifoam additions. After the incubation period, the contents of the fermentor vessel were transferred to a 500L tank, Cetavlon was added, and the material mixed for 1 hours. The Cetavlon treated grovvth was centrifuged at approximately 15,000 to 17,000xg at a flow rāte of approximately 30 to 70 liters per hours. The crude poiysaccharide was precipitated from the supernatant vvith a second Cetavlon precipitation. Cetavlon was added to the supernatant and the material mixed for at least 1 hour at room temperature. The material vvas stored at 1 to 5°C for 8 to 12 hours. The precipitated poiysaccharide was collected centrifugation at approximateiy 45,000 to 50,000xg at a flow rāte of 300 to 400nil per minūte. The collected paste was stored at -60°C or lower until further processed.
Purified polvsaccharide powder preparation [0047] The inactivated paste was thawed and transferred to a blender. The paste was blended vvith 0.9M calcium chloride to yield a homogeneous suspension. The suspension was centrifuged at approximately 10,000xg for 15 minūtes. The supernatant was decanted through a iint free pad into a Container as the first extract. A second volume of 0.9M calcium chloride was added to the paste, and blended to yield a homogeneous suspension. The suspension was centrifuged as above, and the supernatant combined vvith the supernatant from the first extraction. A total of four extractions were performed, and the supernatants pooled. The pooled extracts were concentrated by ultrifiitration using 10-30kDA MWC0 spiral vvould ultrafiltration units.
[0048] Magnesium chloride was added to the concentrated, and the pH adjusted to 7.2 to 7,5 using sodium hydroxide. DNase and RNase were added to the concentrate, and incubated at 25 to 28°C vvith mixing for 4 hours. Ethanol was added to a concentration of 30 to 50%. Precipitated nucleic acid and protein vvere removed by centrifugation at 10,000xg for 2 hours. The supernatant was recovered and the polysaccharide precipitated by adding ethanol to 80% and ailovving it to stand ovemight at 1 to 5°C. The alcohol was siphoned off, and the precipitated polysaccharide was centrifuged for 5 minūtes at 10,000xg. The precipitated polysaccharide was vvashed vvith alcohol. The polysaccharide was vvashed vvith acetone, centrifuged at 15 to 20 minūtes at 10,000xg. The polysaccharide was dried under vacuum. The initial polysaccharide povvder was dissolved into sodium acetate solution. Magnesium chloride was added and the pH adjusted to 7.2 to 7.5 using sodium hydroxide solution. DNase and RNase vvere added to the solution and incubated at 25 to 28°C vvith mixing for 4 hours to remove residual nucleic acids. After incubation vvith these enzymes, an equal volume of sodium acetate-phenol solution was added to the polysaccharide-enzyme mixture, and placed on a platform shaker at 1 to 5°C for approximately 30 minūtes. The mixture was centrifuged at 10,000xg for 15 to 20 minūtes. The upper aqueous layer was recovered and saved. An equal volume of sodium acetate-phenol solution was added to the aqueous layer, and extracted as above. A totai of four extractions vvere performed to remove protein and endotoxin from the polysaccharide solution. The combined aqueous extracts vvere diluted up to ten fold vvith vvater for injection, and diafiltered against 10 volumes of vvater for injection. Calcium chloride was added to the diafiltered poiysaccharide. The poiysaccharide vvas precipitated ovemight at 1 to 5°C by adding ethanol to 80%. The alcohol super10 natant vvas withdrawn, and die polysaccharide collected by centrifugation at 10,000xg for 15 minūtes. The purified polysaccharide was vvashed tvvo times vvith ethanol, and once vvith acetone. The vvashed povvder was dried under vacuum in a desiccator. The dried povvder was stored at 30°C or lovver until processed onto conjugate.
Example 2
Depo}ymerization ofNeisseria meningitidis serogroups A,C, W135, and Y purified capsular polysaccharide povvder [0049] Materials used in the preparation include purified capsular polysaccharide povvders from Neisseria meningitidis serogroups A, C, W-135, and Y (prepared in accordance vvith Example 1), sterile 50mM sodium acetate buffer, pH 6.0, sterile IN hydrocholoric acid, sterile IN sodium hydroxide, 30% hydrogen peroxide, and sterile physiological saline (0.85% sodium chloride). [0050] Each serogroup polysaccharide was depolymerized in a separate reaction. A stainless steel tank was charged vvith up to 60g of purified capsular polysaccharide povvder. Sterile 50mM sodium acetate buffer, pH 6.0 was added to the polysaccharide to yield a concentration of 2.5g poiysaccharide per liter. The poiysaccharide solution was allovved to mix at 1 to 5°C for 12 to 24 hours to effect solution. The reaction tank was connected to a heat exchanger unit. Additional 50mM sodium acetate buffer, pH 6.0, was added to dilute the polysaccharide to reaction concentration of 1.25g per liter. The polysaccharide solution was heated to 55°C±0.1. An aliquot of 30% hydrogen peroxide was added to the reaction mixture to yield a reaction concentration of 1% hydrogen peroxide.
[0051] The course of the reaction was monitored by follovving the change in the molecular size of the polysaccharide over time. Every 15 to 20 minūtes, aliquots were removed from the reaction mixture and injected onto a HPSEC column to measure the molecular size of the polysaccharide. When the molecular size of the polysaccharide reached the targeted molecular size, the heating unit vvas tumed off and the polysaccharide solution rapidiy cooled to 5°C by circulation through an ice vvater bath. The depolymerized polysaccharide solution vvas concentrated to 15g per iiters by connecting the reaction tank to an ultrafiitration unit equipped vvith 3000 MWC0 regenerated cellulose cartridges. The concentrated depolymerized polysaccharide solution vvas diafiitered against 10 volumes of sterile physiological saline (0.85% sodium chloride). The depolymerized polysaccharide vvas stored at 1 to 5°C until the next process step.
[0052] The molecular size of the depolymerized polysaccharide vvas determined by passage through a gei filtration chromatography column sold under the tradename “Ultahydrogel™250’' that vvas calibrated using dextran molecular size standards and by muiti-angle laser light scatter11 ing. The quantity of polysaccharide vvas determined by phosphorus content for serogroup A using the method of Bartlet, G.R.J. (1959) Journal of Biological Chemistry, 234, pp-466-468, and by the sialic acid content for serogroups C, W135 and Y using the method of Svennerhoim, L.
(1955) Biochimica Biophysica Acta 24, pp604-611. The 0-acetyl content vvas determined by the method of Hesterin, S. (1949) Journal of Biological Chemistry 180, p249, Reducing activity vvas determined by the method of Park, J.T. and Johnson, M.J. (1949 Journal of Biological Chemistry 181, ppl49-l51. The structural integrity of the depolymerized polysaccharide vvas determined by protein *Η and 13C NMR. The purity of the depolymerized polysaccharide vvas determined by measuring the LAL (endotoxin) content and the residual hydrogen peroxide content.
Example 3
Derivatization ofNeisseria meningitidis serogroups A, C,W-135r and Y depolymerized polysaccharide [0053] Materials used in this preparation inciude hydrogen peroxide depolymerized capsular polysaccharide serogroups A, C, W-135, and Y from Neisseria meningitidis (prepared in accordance vvith Example 2), adipic acid dihydrazide, l-ethyl-3-(3-dimethylaminopropy!) carbodiimide (EDAC) for serogroup A only, sodium cyanborohydride, steriie IN hydrocholoric acid, sterile IN sodium hydroxide, steriie IM sodium chloride, and sterile physio)ogical saline (0.85% sodium chloride).
[0054] Each serogroup poiysaccharide vvas derivatized in a separate reaction. A stainless steel tank vvas charged vvith the purified depolymerized polysaccharide, and diluted vvith sterile 0.85% physiological saline to achieve a final reaction concentration of 6g polysaccharide per liter. To this solution vvas added a concentrated aliquot of adipic acid dihydrazide dissolved in sterile 0.85% physiological saline, in order to achieve a reaction concentration of lg per liter. For serogroup A oniy, EDAC vvas added as a concentrated aliquot dissolved in sterile 0.85% physiological saline, to achieve a reaction concentration of lg per liter. The pH vvas adjusted to 5.0+0.1, and this pH vvas maintained for 2 hours using sterile IN hydrochloric acid and sterile IN sodium hydroxide at room temperature (15 to 30°C). After tvvo hours, a concentrated aliquot of sodium cyanoborohydride, dissolved in 0.85% physiological saline, vvas added to the reaction mixture to achieve a reaction concentration of 2g per liter. The reaction vvas stirred at room temperature (15 to 30°C) for 44 hours ±4 hours vvhile maintaining the pH at 5.5±0.5. Follovving this reaction period, the pH vvas adjusted to 6.0±0.1, and the derivatized polysaccharide vvas concentrated to 12g poiysaccharide per liter by connecting the reaction tank to a ultrafiitration unit equipped vvith a 3000 MWC0 regenerated cellulose cartridges. The concentrated derivatized polysaccharide vvas diafiltered against 30 volumes of IM sodium chloride, follovved by 10 voiumes of 0.15M sodium chloride. The tank vvas disconnected from the ultrafiltration unit and stored at 1 to 5°C for 7 days. The tank vvas reconnected to an ultrafiltration unit equipped vvith 3000 MWC0 regenerated cellulose cartridges, and diafiltered against 30 volumes of IM sodium chloride, follovved by 10 volumes of 0.15M sodium chloride.
[0055] The molecular size of the derivatized polysaccharide, the quantity of polysaccharide, and the O-acetyl content vvere measured by the same methods used on the depolymerized polysaccharide. The hydrazide content vvas measured by the 2,4, 6-trinitrobenzensulfonic acid method of Snyder, S.L. and Sobocinski, P.Z. (1975) Analytical Biochemistry 64, pp282-288. The structural integrity of the derivatized polysaccharide vvas determined by proton :H and 13C NMR. The purity of the derivatized polysaccharide vvas determined by measuring the Ievel of unbound hydrazide, the LAL (endotoxin) content, and the residual cyanoborohydride content.
Example 4
Preparation of carrier protein Preparation of crude diphtheria toxoid protein [0056] Lyophilized seed cultures were reconstituted and incubated for 16 to 18 hours. An aliquot from the culture vvas transferred to a 0.5-liter flask containing grovvth medium, and the culture flask vvas incubated at 34.5 to 36.5°C on a rotary shaker for 7 to 9 hours. An aliquot from the culture flask vvas transferred to a 4-liter flask containing grovvth medium, and the culture flask vvas incubated at 34.5 to 36.5°C on a rotary shaker for 14 to 22 hours. The cultures from the 4-liter flask vvere used to inoculate a fermenter containing grovvth media. The fermenter vvas incubated at 34.5 to 36.5°C for 70 to 144 hours. The contents of the fermenter vvere filtered through depth filters into a collection vessel. An aliquot of formaldehyde solution, 37% vvas added to the harvest to achieve a concentration of 0.2%. The pH vvas adjusted to 7.4 to 7.6.
The harvest vvas filtered through a 0.2 micron filter cartridge into sterile 20 liter bottles. The bottles vvere incubated at 34.5 to 36.5°C for 7 days. An aliquot of formaldehyde solution, 37%, vvas added to each 20 liter bottle to achieve a concentration of 0.4%. The pH of the mixtures vvas adjusted to 7.4 to 7.6. The bottles vvere incubated at 34.5 to 36.5°C for 7 days on a shaker. An aliquot of formaldehyde solution, 37%, vvas added to each 20 liter bottle to achieve a concentration of 0.5%. The pH of the mixtures vvas adjusted to 7.4 to 7.6. The bottles vvere incubated at 34.5 to 36.5°C for 8 vveeks. The crude toxoid vvas tested for detoxification. The bottles vvere stored at 1 to 5°C during the testing period.
Purification of the crude diphtheria toxoid protein [0057] The crude toxoid vvas allovved to warm to room temperature, and the contents of the 20-liter bottles vvere combined into a purification tank. The pH of the toxoid vvas adjusted to 7.2 to 7.4, and charcoal vvas added to the crude toxoid and mixed for 2 minūtes. The charcoal toxoid mixture vvas allovved to stand for 1 hours, and vvas then filtered through a depth filter cartridge into a second purification tank. Solid ammonium sulfate vvas added to the filtrate to achieve 70% of saturation. The pH vvas adjusted to 6.8 to 7.2, and the solution vvas allovved to stand for 16 hours. The precipitated protein vvas collected by filtration and vvashed vvith 70% of saturation ammonium sulfate solution, pH 7.0. The precipitate vvas dissolved into steriie distilied vvater, and the protein solution vvas filtered into a stainless Steel collection vessel. The pH vvas adjusted to 6.8 to 7.2, and ammonium sulfate vvas added to 40% of saturation. The pH of the solution vvas adjusted to 7.0 to 7.2, and the solution vvas allovved to stand for 16 hours. The precipitate vvas removed by filtration and discarded. Ammonium sulfate vvas added to the filtrate to 60% of saturation, and the pH adjusted to 7.0 to 7.2. The mixture vvas allovved to stand for 16 hours, and the precipitated protein vvas collected by filtration. The precipitate vvas dissolved into steriie distilied vvater, filtered to remove undissolved protein, and diafiltered against 0.85% physiological saline.
Concentration and steriie filtration of the purified diphtheria toxoid protein [0058] The protein solution vvas concentrated to 15g per liter and diafiltered against 10 volumes of 0.85% physiological saline suing a 10,000 MWC0 regenerated cellulose filter cartridge. The concentrated protein solution vvas steriiized by filtration through a 0.2 micron membrane.
The protein solution vvas stored at 1 to 5°C until processed onto conjugate.
[0059] The protein concentration vvas determined by the method of Lowry, O.H. et. al (1951) Journal of Biological Chemistry 193, p265-275. The purity of the protein vvas measured by steriiity, LAL (endotoxin) content, and residual formaidehyde content.
Example 5
Preparation ofmonovalent Conjugates ofNeisseria meningitidis serogroups A, C, W-135, and Y polysaccharide to diphtheria toxoid protein [0060] Materials used in this preparation include adipic acid derivatized polysaccharide from
Neisseria meningitidis serogroups A, C, W-135, and Y (prepared in accordance vvith Example 3), steriie diphtheria toxoid protein (prepared in accordance vvith Example 4), EDAC, ammonium sulfate, steriie IN hydrochloric acid, steriie IN sodium hydroxide, and steriie physiologicai saline (0.85%).
[0061] Each serogroup polysaccharide conjugate was prepared by a separate reaction. Ali four conjugates vvere prepared by the follovving process. A stainless Steel tank vvas charged vvith the purified adipic acid derivatized poiysaccharide at a reaction concentration of 700 to lOOOpmoies of reactive hydrazide per liter and purified diphtheria toxoid protein at a reaction concentration of 3.8 to 4.0g protein per liter. Physiological saline 0.85%, vvas used to dilute the starting materiāls to the target reaction concentrations and the pH vvas adjusted to 5.0±0.1. An aliquot of EDAC vvas added to the polysaccharide protein mixture to achieve a reaction concentration of 2.28 to 2.4g per liter. The pH of the reaction vvas ķept at 5.0±0.1 for 2 hours at 15 to 30°C. After tvvo hours, the pH vvas adjusted to 7.0±0.1 using sterile IN sodium hydroxide, and the reaction vvas stored at 1 to 5°C for 16 to 20 hours.
[0062] The reaction mixture vvas allovved to vvarm to 15 to 30°C and the reaction vessel vvas connected to an ultrafiltration unit equipped vvith a 30,000 MWC0 regenerated cellulose cartridge. Solid ammonium sulfate vvas added to 60% of saturation (for serogroups A, W-135 and Y) and 50% of saturation (for serogroup C). The conjugate reaction mixture vvas diafiltered against 20 volumes of 60% of saturated ammonium sulfate solution (for serogroups A, W-135 and Y) and 50% of saturated ammonium sulfate solution (for serogroup 0, follovved by 20 volumes of physiological saline, 0.85%. The diafiltered conjugate vvas first filtered through a filter capsule containing a 1.2 micron and a 0.45 micron filter, and then through a second filter capsule containing a 0.22 micron filter.
. [0063] The quantity of polysaccharide and 0-acetyl content vvere measured by the same methods used on the depolymerized and derivatized polysaccharide. The quantity of protein vvas determined by the Lowry method. The molecular size of the conjugate vvas determined by passage through a gel filtration chromatography column sold under the tradename “TSK6000PW” that used DNA as the void volume marker, ATP as the total volume marker, and bovine thyroglobulin as a reference marker. In addition, the molecular size of the conjugate eluted from the TKS6000PW column vvas measured by multi-angle laser light scattering. The antigenic character of the conjugate vvas measured by binding to anti-polysaccharide serogroup specific antibody using double-sandvvich ELISA method. The purrty of the conjugates vvas determined by measuring the amount of unbound (unconjugated) polysaccharide by elution though a hydrophobic interaction chromatography column, unconjugated protein by capillary electrophoresis, steriiity, LAL (endotoxin) content, residual EDAC content, and residual ammonium ion content.
Example 6
Formulation of a multivalent meningococcal A, C, W-135, and Y polysaccharide diphtheria toxoid conjugate vaccine [0064] Materials used in this preparation include, serogroups A, C, W-135, and Y polysaccharide-diphtheria toxoid conjugates (prepared in accordance vvith Example 5), steriie lOOmM sodium phosphate buffered physiological saline (0.85% sodium chloride).
[0065] An aliquot of steriie 100-500mM sodium phosphate buffered physiological saline was added to physiological saline (0.85%) in a stainless steel bulking tank to yield a final vaccine concentration of lOmM sodium phosphate. An aliquot of each of from two to four of the steriie monovalent meningococcal polysaccharide-diphtheria toxoid conjugates vvas added to the bulking tank containing 10mM steriie sodium phosphate physiological saline to yield a final concentration of 8pg of each serogroup polysaccharide per milliliter of buffer. The formulated tetravalent conjugate vvas mixed and filtered through a 0.2gm fiiter into a second bulking tank.
[0066] The quantity of each serogroup poiysaccharide present in the multivalent formulation vvas determined by component saccharide analysis using high pH anion-exchange chromatography vvith puised amperometric detection. The quantity of protein vvas measured by the method of Lowry. Th pH of the vaccine vvas measured using a combination electrode connected to a pH meter. The antigenic character of the multivaient conjugate vaccine vvas measured by binding to anti-polysaccharide serogroup specific antibody using a doubie-sandvvich ELISA method. Immunogenicity of the multivalent conjugate vaccine vvas measured the ability of each conjugate present in the vaccine to elicit both a primary and booster anti-polysaccharide IgG immune response in an anima! modei. The purity of the multivalent conjugate vaccine vvas determined by measuring the amount of unbound (unconjugated) polysaccharide using high pH anion-exchange chromatography vvith puised amperometric detection, sterility, LAL (endotoxin) content, pyrogenic content, and general safety.
Example 7
Preparation of aluminum-hydroxide adjuvanted multivalent meningococcal polysaccharide diphtheria toxoid protein conjugate [0067] Preparation of conjugate adsorbed to aluminum hydroxide. Materials used in this preparation include serogroups A, C, W-135, and Y polysaccharide-diphtheria toxoid conjugates preparation described in Example 5, steriie physiological saline (0.85% sodium chloride), and steriie aluminum hydroxide in physiologicai saline (0.85% sodium chloride).
[0068] An aliquot of each of the sterile monovalent meningococcal polysaccharide diphtheria toxoid conjugates vvas added to the bulking tank containing physiological saline to yield a final concentration of 8pg of each serogroup poiysaccharide per millilfter of buffer. An aliquot of sterile aluminum hydroxide in physiological saline {0.85% sodium chloride) vvas added to the muitivalent conjugate vaccine to achieve a final concentration of 0.44mg aluminum ion per mīlliliter vaccine.
Example 8
Preparation of aluminum phosphate-adjuvanted conjugate [3389] Materials used in this preparation include serogroups A, C, W-135, and Y polysaccharide-diphtheria toxoid conjugates preparation described in Example 5, sterile physiological saline (0.85% sodium chloride), and sterile aluminum phosphate in physiological saline (0.85% sodium chloride).
[0070] An aliquot of each of the sterile monovalent meningococcal polysaccharide-diphtheria toxoid conjugates vvas added to the bulking tank containing physioiogical saline to yield a final concentration of 8pg of each serogroup polysaccharide per miililiter of buffer. An aliquot of sterile aluminum phosphate in physiological saline (0.85% sodium chloride) vvas added to the multivalent conjugate vaccine to achieve a final concentration of 0.44mg aluminum ion per miililiter vaccine.
Example 9 lmmunogenicity of the tetravalent conjugate vaccine [0071] The tetravalent conjugate vaccine vvas studied for its abiiity to elicit an immune response in smail laboratory animals prior to evaluation in the clinic. Mice, rats and rabbits have been used to study the immunogenicity of conjugate vaccines reiative to the polysaccharide vaccines. These animal modeis are useful, because they are capabie of distinguishing the conjugate vaccine from the corresponding polysaccharide by their immune response pattern. The conjugate vaccine elicits a T-dependent-like immune response in these modeis, vvhereas the po(ysaccharide vaccine elicits a T-independent-like immune response.
[0072] In the mouse immunogenicity studies, the conjugate vvas diluted vvith physiological saline (0.85% sodium chloride) to administer betvveen one-quarter to one-sixteenth of a human dose. The mice were administered one or tvvo doses of vaccine, either conjugate or polysaccharide, and blood specimens were taken tvvo vveeks post vaccination. One group of mice served as an unimmunized control group. Antibodies to each of the serogroup polysaccharides were measured by an ELISA method. The serum samples were incubated vvith excess of each capsular polysaccharide that vvas bound to a ELISA microtiter plate well. Methylated human serum albumin vvas used to bind each serogroup polysaccharide to the microtiter vvell. Follovving incubation the microtiter vvell vvas vvashed vvith buffer, and a secondary antibody-enzyme conjugate vvas added to the antibody-poiysaccharide complex vvhich binds to the anti-meningococcal polysaccharide antibody. The microtiter plate vvas vvashed, and a Chemical substrate vvas added to the polysaccharide-meningococcal antibody-secondary antibody-enzyme conjugate. The enzyme hydrolyzes a portion of the Chemical substrate that results in color formation. The amount of color formation is proportional to the amount of polysaccharide-meningococcal antibodysecondary antibody-enzyme conjugate that is bound to the microtiter vvell. The potency of the vaccine vvas determined by comparison to reference antisera for each serogroup, vvhich is measured in the same microtiter plate, by a parallel line calculation using a four-parameter fit. The mouse reference antisera vvas generated in the same strain of mice that vvere individually immunized vvith three doses of each serogroup conjugate vaccine. The mouse reference antisera vvere assigned titers based on the inverse of dilution yielding an optical density of 1.0.
[0073] Presented in Table 1 is a summary of anti-polysaccharide IgG titers for each serogroup achieved in Swiss-Webster mice who vvere vaccinated vvith two doses of either the tetravalent conjugate vaccine, both liquid and aluminum hydroxide formulation, or the corresponding tetravalent polysaccharide vaccine. The IgG titers vvere measured on pooled sera from a set of ten mice. Two sets of 10 mice vvere used to measure the immune response to each vaccine formulation. Both sets vvere vaccinated on day 1. On day 15 (2 vveeks post vaccination) blood specimens vvere taken from one set of 10 mice, and the second set of ten mice vvere vaccinated vvith a second dose of vaccine on day 15. Two vveeks late on day 29, blood specimens vvere taken from the second set of 10 mice, and from the unimmunized control group. Ali antibodies vvere titrated at the same time, that is, both the day 15 and day 29 blood specimens vvere assayed at the same time along vvith the unimmunized Controls and the mouse reference sera.
Table 1
Anti-polysaccharide IgG titers on pooled sera from Swiss-Webster mice ' vaccinated with either tetravalent conjugate or polysaccharide.
Vaccine Group Dosage μδ PS Anti-Men A Anti-Men C Anti-Men W135 Anti-Men Y
D15 D29 D15 D29 D15 D29 D15 D29
Conjugate (no adjuvant) 0.25 131 2640 250 1510 1350 6100 5660 4830
SUBSTITUTE SHEET (RULE 26)
Vaccine Group Dos- age ugps Anti-Men A Anti-Men C Anti-Men W135 Anti-Men Y
D15 D29 D15 D29 D15 D29 D15 D29
Conjugate (no adjuvant) 0.50 171 6220 416 2050 849 26000 5980 112000
Conjugate (no adjuvant) 1.0 249 4500 525 2740 1450 16600 11300 59100
Conjugate (Alum. Hyd.) 0.25 2920 4500 1010 2980 2300 33700 11600 124000
Conjugate (Alum. Hyd.) 0.50 5800 9550 2280 1010 4810 71900 26400 330000
Conjugate (Alum. Hyd.) 1.0 6210 9350 2630 12800 7870 94000 32700 302000
Polysaccharide (no adjuvant) 1.0 136 173 184 205 612 608 4470 3910
Unimmu- nized n.a. - 110 - 145 - 623 - 777
[0074] The tetravalent conjugate vaccine, both unadjuvanted and adjuvanted vvith aiuminum hydroxide, is capable of eliciting a strong anti-polysaccharide IgG immune response in this mouse modei. The aiuminum hydroxide adjuvant serves to improve both the primary and booster response to each of the four serogroup polysačcharide conjugates. The tetravalent polysaccharide vaccine elicits a negligible immune response to serogroups A, C, and W135 in this mouse modei relative to the unimmunized control, vvhereas serogroup Y does elicit a respectable immune response, but not a booster response. The tetravalent polysaccharide vaccine fails to elicit a booster response to ali four serogroup polysaccharides in this modei. This modei can readi!y differentiate betvveen the polysaccharide vaccine and the conjugate vaccine both by the magnitude of the immune response and booster response pattern to each of the serogroup conjugate vaccines.
[0075] The unadjuvanted form of the tetravalent conjugate vaccine has been studied in the clinic for safety and immunogenicity in young healthy aduits and in younģ healthy children. In the adult study, subjects vvere vaccinated vvith a single dose of vaccine, formulated to contain 4pg of each of the four conjugates, as polysaccharide. Blood specimens vvere taken immediately prior to vaccination and 28-days post vaccination. Antibodies to each of the serogroup conjugates vvere measured by an ELISA measurement that quantified the amount of anti-polysaccharide IgG. The ELISA method is very similar to the method used to measure the amount if IgG antibody present in mouse sera.
[0076] Briefly, the serum samples vvere incubated in ELISA microtiter vvelis that vvere coated vvith excess meningococcal poiysaccharide that vvas bound to the plate vvith methylated human
SUBST1TUTE SHEET (RULE 26) serum albumin. The amount of bound antibody was determined by a reaction vvith peroxidaselabeled mouse anti-human IgG specific monoclonal antibody. A subsequent reaction using peroxidase substrate generates a chromogenic product that vvas measured spectrophotometrically. The resulting optical density of the chromophore correlates vvith the amount of IgG antibody in the serum that is bound to the meningococcal polysaccharide on the microtiter plate. The amount of antibody vvas calculated by comparison to a human reference sera (CDC 1922) vvith an assigned value using a 4-parameter logistic curve method. In addition, the antibodies vvere measured for their ability to lyse serogroup specific bacteria. The serum samples are first heat-inactivated to destroy complement. The serum samples are diluted by two-fold dilutions in a sterile 96-well microtiter plate. Serogroup specific bacteria along vvith baby rabbit complement vvere added to the serum dilutions and allovved to incubate. After an incubation period, an agar overlay medium vvas added to the serum/complemeni/bacteria mixture, The agar overlay vvas allovved to harden, and then incubated overnight at 37°C vvith 5% carbon dioxide. The next day, bacterial colonies present in the vvells vvere counted. The endpoint titer vvas determined by the reciprocal serum dilution yielding greater than 50% killing as compared to the mean of the complement control vvells. [0077] Presented in Table 2 is a summary of the anti-polysaccharide mean IgG concentrations for each serogroup and the mean serum bactericidal antibody (SBA) titer in adult sera pre and post vaccination vvith the tetravalent conjugate vaccine formulated at 4pg polysaccharide per dose. The immune response to ali four serogroup conjugates vvere satisfactory, that is comparable to the immune response achieved by the licensed polysaccharide vaccine in terms of both IgG antibody and functional bactericidal antibody responses. The vaccine vvas found to be safe for this age group and the safety profilē vvas found to be similar to that of the licensed polysaccharide vaccine.
Table 2
Anti-polysaccharide IgG GMC (group mean concentration and Serum Bactericidal Antibody GMTs (group mean titers) foryoung health adults vaccinated with a tetravalent meningococcal conjugate vaccine formulated at 4pg per dose by polysaccharide.
Immune Re- sponse by Serogroup Npre/Npost IgG GMC (gg/ml) [95% Cl] SBA GMT [95%CI]
Pre Post Pre Post
A 28/28 3.3 38.4 487 6720
[2.34.8] [22.2-66.4] [231-1027] [4666-15428]
C 28/28 0.4 [0.2-0.7] [3.0-10.1] 16.4 [7.1-37.7] 1560 [8004042]
W-135 28/28 0.6 [0.3-1.0] 5.8 [2.9-11,7] 10.0 [5.9-16.9] 609 [250-1481]
Immune Re- sponse by Serogroup Npre/Npost IgG GMC (pg/ml) [95% Cl] SBA GMT [95%CI]
Pre Post Pre Post
Y 28/28 1.3 [0.7-2.5] 6.8 [3.2-14.6] 19.0 [8.0-41.2] 390 [143-1061]
[0078] In younger age groups, children less than 2 years of age, the immune response to the polysaccharide vaccine is weak and the immunity has been estimated to wane after one year. Children 12 to 15 months of age vvere administered a single dose of tetravalent conjugate vaccine formulated at 4pg of each serogroup polysaccharide per dose, and they vvere administered a second dose of tetravalent conjugate vaccine tvvo months follovving the first dose. Blood specimens vvere taken prior to the first and second vaccination, and one month post the second vaccination. The antibody responses to the four serogroup conjugates are summarized in Table 3. For each serogroup a booster response for IgG antibody and for functional-bactericidal antibody vvas observed follovving a second dose of tetravalent conjugate. The Ievel of IgG antibody elicited by the conjugate vaccine is comparable to that elicited by the licensed polysaccharide for this age group; a post 6 week response of 3.64yg/ml (2.96-4.49) IgG antibody response to serogroup C polysaccharide. Hovvever, the Ievel of bactericidal antibody elicited by the conjugate vaccine is much higher than what is normally elicited by the licensed polysaccharide vaccine for this age group; a post 6 week SBA titer of 7.2 (5.0-10.4). The reason for this discordance betvveen IgG antibody and bactericidal antibody in the younger populations is thought to resuit from the polysaccharide eliciting a high proportion of low avidity antibody in the younger populations. Conversely, the conjugate appears to elicit a much higher proportion of high avidity antibody.
High avidity antibody is thought to be responsible for the bactericidal activity.
Table 3
Anti-polysaccharide IgG GMC (group mean concentration) and Serum Bactericidal Antibody GMTs (group mean titers) for young healthy children (1 to 2 years of age) vaccinated vvith two doses of tetravalent meningococcal conjugate vaccine formulated at 4pg per dose by polysaccharide
Immune Response By Serogroup Νχ/Νζ/Ν 3 IgG GMC (pg/ml) [95% Cl] SBA GMT [95% Cl]
Pre dose 1 Pre dose 2 Post dose 2 Pre dose 1 Pre dose 2 Post dose 2
A 8/8/8 0.2 [0.1-0.4] 2.1 [0.94.8] 4.4 [2.1-9.1] 8.7 [1.4-55.1] 1328 [179- 9871] 3158 [1857- 5371]
C 8/8/8 0.2 [0.0-0.7] 1.0 [0.3-3.1] 1.5 [0.6-3.6] 6.7 [2.0-23.0] 117 [37.7- 365] 304 [128- 721]
W-135 8/8/8 0.1 [0.1-0.2] 0.6 [0.2-1.9] 1.5 [0.8-3.1] 6.2 [2.2-17.2] 22.6 [2.8-185] 430 [172- 1076]
Y 8/8/8 0.3 [0.2-0.4] 1.2 [0.5-2.8] 4.5 [2.7-7.6] 5.7 [3.7-8.8] 98.7 [20.4- 478] 304 [101- 920]
[0079] In addition to the ability of the tetravalent conjugate vaccine to elicit a high functional antibody response in younger populations compared to the licensed polysaccharide vaccine, the tetravalent conjugate vaccine is capable of eliciting an anamnestic response, demonstrating that protection elicited by the tetravalent conjugate vaccine of the present invention is long-lived. In the development of the tetravalent conjugate vaccine, studies vvere first conducted on a bivalent AC conjugate formulation. The vaccine offers vvider coverage than the current licensed monovalent C conjugate, but does not protect against disease caused by serogroups W135 and Y. [0080] A clinical study vvas performed vvith infant subjects that compared the immune response to the bivalent AC polysaccharide vaccine versus the bivalent AC conjugate vaccine. In this study, a third group of infants vvere enrolled to serve as a control group and they received a Haemophilus.influenzae type b conjugate. Ali three vaccine groups receive the same pediatric vaccines. The bivalent AC conjugate group received three doses of conjugate vaccine (4pg polysaccharide per dose) at 6,10, and 14 vveeks of age. The bivalent AC poiysaccharide group received tvvo doses of a bivalent AC poiysaccharide vaccine (50pg polysaccharide per dose) at 10 and 14 vveeks of age. The Haemophilus influenzae type b conjugate group received three doses of conjugate vaccine at 6,10, and 14 vveeks of age. Blood specimens vvere taken at 6 vveeks, pre-vaccination, and at 18 vveeks, 4 vveeks post vaccination. When the children vvere 11 to 12 months of age, blood specimens vvere taken and the children who had received e'rther the bivalent AC conjugate or the bivalent AC polysaccharide vaccine received a booster dose of AC polysaccharide. The reason for the booster dose of po!ysaccharide vvas to evaluate vvhether or not the subjects would elicit an anemestic response.
[0081] The results of this study, both the primary and polysaccharide booster immune responses are presented in Table 4 for the IgG antibody response and Table 5 for the SBA antibody response. The IgG antibody response post primary series vvas approximately the same for both the polysaccharide and conjugate vaccine. Hovvever, the bactericidal antibody response in the conjugate vaccinated subjects vvas much higher than that for the polysaccharide vaccinated sub22 jects. As observed vvith the one year old subjects, vaccination of infants vvith the polysaccharide elicits very little functional-bactericidai antibody. The antibody elicited by the infants to the polysaccharide vaccine is presumably low avidity antibody, vvhereas, the conjugate vaccine appears to elicit high avidity antibody, thereby accounting for the much higher titer of bactericidal antibody. The high Ievel of functional antibody elicited by the booster dose of polysaccharide vaccine in the subjects who had received the conjugate vaccine in the primary vaccination series, indicates that these subjects have been primed for a memory or T-cell dependent antibody response. The subjects who received the polysaccharide vaccine in the primary vaccination series elicited a modest response to the polysaccharide booster dose, that is indicative of a T-cell independent response.
Table 4
Anti-polysaccharide IgG GMC (group mean concentration) in infants against serogroups A and C before and after both the primary series immunization (6,10 and 14 vveeks ofage) and the booster vaccination vvith bivalent AC polysaccharide given at 11 to 12 months of age.
Immune Response by Vaccine Group Primary Vaccination GMC [95% Cl] PS Booster Vaccination GMC [95% Cl]
N Pre Post N Pre Post
Serogroup A:
AC Conjugate 34 3.4 [2.2-5.4] 5.8 [4.3-8.0] 31 0.2 [0.1-0.3] 7.0 [4.0-12.0]
AC Polysaccharide 35 3.0 [1.7-5.31 5.5 [4.1-7.31 30 0.9 [0.5-1.4] 3.1 [2.04.71
HIB Conjugate 36 3.2 [2.2-4.51 0.6 [0.40.8] NA NA NA
Serogroup C:
AC Conjugate 31 1.6 [0.9-2.81 2.8 [2.0-3.91 31 0.1 [0.1-0.21 8.1 [4.5-14.5]
AC Polysaccharide 35 2.3 [1.4-3.9] 5.3 [3.8-7.41 30 0.6 [0.3-1.01 2.8 [1.7-4.71
HIB Conjugate 36 2.0 [1.2-3.5] 0.5 [0.3-0.73 NA NA NA
Table 5
SBA antibody GMT (group mean titer) in infants against serogroups A and C before and after both the primary series immunization (6,10 and 14weeks ofage) and booster vaccination vvith bivalent AC polysaccharide given at 11 to 12 months of age.
Immune Response By Vaccine Group Primary Vaccination GMT [95% Cl] PS Booster Vaccination GMT [95% Cl]
N Pre Post N Pre Post
Serogroup A:
AC Conjugate 34 11.8 [7.2-19.3] 177 [101-312] 24 10.1 [5.6-18.0] 373 [162-853]
AC Polysaccharide 32 14.7 [8.5-25.4] 7.0 [4.7-10.5] 26 6.1 [3.9-9.5] 24.1 [11-53]
HIB Conjugate 35 11.2 [6.8-18.3] 6.7 [4.3-10.5] NA NA NA
Serogroup C;
AC Conjugate 34 50.8 [24-107] 189 [128-278] 27 4.6 [3.6-5.6] 287 [96.2-858]
AC Polysaccharide 32 62.7 [29-131] 25.4 [14.444.6] 26 4.1 [3.9-4.3] 14.4 [7.9-26.1]
HIB Conjugate 36 45.3 [21.9-133] 7.3 [4.7-11.3] NA NA NA
[0082] In addition to the benefits that this invention offers to the improved protection against meningococcai disease in young populations and the wider protection against serogroups A, C, W-135 and Y, the tetravalent conjugate may provide protection to other pathogens by inducing an antibody response to the carrier protein. When the tetravalent conjugate vaccine, using diphtheria toxoid conjugate, was administered to infants, these subjects also received the routine pediatric immunizations, vvhich included diphtheria toxoid. Therefore, in these subjects there was no apparent improvement in the antibody response to diphtheria toxoid. Hovvever, when the diphtheria toxoid conjugate was administered to subjects that did not receive concomitant diphtheria toxoid containing vaccines, a strong booster response to diphtheria toxoid was observed. These subjects had received a three dose regiment of DTP at 2, 3, and 4 months of age. In this study, the subjects received either single dose of a bivalent AC conjugate or a single dose of bivalent AC polysaccharide vaccine betvveen 2 and 3 year of age. Blood specimens were taken at the time of vaccination and 30-days post vaccination. The bivalent AC conjugate used diphtheria toxoid as the carrier protein.
[0083] The immune response of diphtheria toxoid in the tvvo vaccine groups is presented in Table 6. The polysaccharide did not serve to stimulate an anti-diphtheria immune response in these subjects as expected, hovvever a strong anti-diphtheria immune response was observed for the subjects receiving the AC conjugate. Therefore, the meningococcai conjugate vaccine may provide an added benefit of stimuiating an immune response to carrier protein thereby providing protection against diseases caused by Corynebacteria diphtheriae when diphtheria toxoid is used as a carrier protein.
Table6
Anti-diphtheria antibody by ELISA GMT (group mean titer) in lU/ml in young healthy children vaccinated with either a bivalentAC diphtheria toxoid conjugate vaccine formulated at 4pg as polysaccharide per dose or a bivalentAC polysaccharide vaccine formulated at 50pg as polysaccharide per dose
Immune Response by Vaccine Group Npre/Npost Anti-Diphtheria Antibody (ELISA - lU/ml) [95%CI]
Pre Post
AC Conjugate 104/103 0.047 [0.036-0.060] 21.2 [11.6-38.6]
AC Polysaccharide 103/102 0.059 [0.045-0.076] 0.059 [0.045-0.077]
References:
[0084] Frasch, C.E., Zollenger, W.D. and Poolman, J.T. (1985) Review of Infectious Diseases 7, pp. 504-510.
[0085] Reido, F.X., Plikaytis, B.D. and Broome, C. V. (1995) Pediatric Infectious Disease Journal 14, pp.643-657.
[0086] Artenstein, M.S., Gold, R., Zimmerly, J.G., Wyie, F.A., Schneider, H. and Harkins,C. (1970) The Nevv Engiand Journal of Medicine 282, pp. 417-420.
[0087] Peltola, H., Mākelā, H., Kāyhty, H., Jousimies, H., Herva, E., Hāilstrom, K., Sivonen,
A., Renkonen, O.V., Pettay, 0., Karanko, V., Ahvonen, P., and Sarna, S. (1997) The Nevv Engiand Journal of Medicine 297, pp. 686-691.
[0088] Reingold, A.L., Broome, C.V., Hightovver, A.W., Ajello, G.W., Bolan, G.A., Adamsbaum, C., Jones, E.E., Phillips, C., Tiendrebeogo, H., and Yada, A. (1985) The Lancet 2, pp. 114-118. [0089] Goldschneider, I., Lepovv, M.L., Gotschlich, E.C., Mauck, F.T., Bachl, F., and Randolph, M. (1973) The Journal of Infectious Diseases 128, pp. 769-776.
[0090] Gold, R., Lepovv, M.L., Goldschneider, I., and Gotschlich, E.C. (1977) The Journal of Infectious Diseases 136, S31-S35.
[0091] Brandt, B.L. and Artenstein, M.S. (1975) The Journal of Infectious Diseases 131, pp. S69-S72.
[0092] Kāyhty, H., Karanko, V., Peltola, H., Sarna, S, and Mākelā, H. (1980) The Journal of Infectious Diseases 142, pp. 861-868.
[0093] Cessey, S.J., Allen, S.J., Menon, A., Todd, J.E., Cham, K., Carlone, G.M., Turner,
S.H., Gheesling, L.L., DeWitt, W., Plikaytis, B.D., and Greenvvood, B. (1993) The Journal of Infectious Diseases 167, pp 1212-1216.
[0094] Wyle, F.A., Artenstein, M.S., Brandt, G.L, Tramont, E.C., Kasper, D.L., Altieri, P.L., Berman, S.L., and Lovventhal, J.P. (1972) The Journal of Infectious Diseases, 126, pp. 514-522. [0095] Jennings, H.J. and Lugowski, C. (1981) The Journal of lmmunology 127, pp. 10111018.

Claims (17)

1. An immunological composition comprising tvvo, three, or four distinct proteinpolysaccharide conjugates, vvherein each of the conjugates comprises a capsular polysaccharide from tvvo or more serogroup of N. meningitidis conjugated to one or more of carrier protein(s).
2. The immunological composition of claim 1, vvherein the capsular polysaccharides are se lected from the group consisting of capsular polysaccharides from serogroups A, C, W135 and Y of N. meningitidis.
3. The immunological composition of claim 1, vvherein the capsular polysaccharides are from serogroups A and C of N. meningitidis.
4. The immunological composition of claim 1, vvherein the capsular polysaccharides are from serogroups A, C, W-135 and Y of N. meningitidis.
5. The immunological composition of claim 1, vvherein the carrier protein in diphtheria toxoid.
6. The immunological composition of claim 1, further comprising an adjuvant.
i
7. The immunological composition of claim 6, vvherein the adjuvant is aluminum hydroxide.
8. The immunological composition of claim 6, vvherein the adjuvant is aluminum phosphate.
9. Use of the immunological composition of claim 1 for inducing an immunological response to capsular polysaccharide of N. meningitidis by administering an immunologically effective amount of the composition.
10. A multivalent meningococcal vaccine comprised of immunologically effective amounts of from tvvo to four distinct protein-polysaccharide conjugates, vvherein each of the conju gates contains a different capsular polysaccharide conjugated to a carrier protein, and vvherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W-135 and Y.
11. The multivalent meningococcal vaccine of claim 10, vvherein the capsular polysaccharides are prepared from serogroups A and C of N. meningitidis.
12. The multivalent meningococcal vaccine of ciaim 10, vvherein the capsular polysaccharides are prepared from serogroups A, C, W-135 and Y of N. meningitidis.
13. The multivalent meningococcal vaccine of claim 10, vvherein the carrier protein is diphthe ria toxoid.
14. The multivalent meningococcal vaccine of claim 10, further comprising an adjuvant.
15. The multivalent meningococcal vaccine of claim 14, vvherein the adjuvant is aluminum hy droxide.
16. The multivalent meningococcal vaccine of claim 14, vvherein the adjuvant is aluminum phosphate.
17. Use of the vaccine of claim 10 for protecting a human or animal susceptible to infection from N. meningitidis by administering to the human or animal an immunologicaily effective amount of the vaccine.
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