AU2011226941A1 - Immunization method against neisseria meningitidis serogroups A and C - Google Patents

Abstract

IMMUNIZATION METHOD AGAINST NEISSERA MENINGITIDIS SEROGROUPS A AND C Abstract The present invention describes methods of immunizing a patient with a combined s vaccine that offers protection against meningococcal disease caused by pathogenic bacteria Neisseria meningitidis serogroups A and C. The vaccine comprises at least two distinct polysaccharide-protein conjugates that are formulated as a single dose of vaccine. The purified capsular polysaccharides of Neisseria meningitidis serogroups A and C are chemically activated and selectively attached to a carrier protein by means of a covalent io chemical bond, forming polysaccharide-protein conjugates capable of eliciting long lasting immunity to a variety of N. meningitidis strains in infants.

Description

S&F Ref: 747930D2 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address Sanofi Pasteur, Inc., of Discovery Drive, Swiftwater, of Applicant: Pennsylvania, 18370, United States of America Actual Inventor(s): Robert P. Ryall Address for Service: Spruson & Ferguson St Martins Tower Level 35 31 Market Street Sydney NSW 2000 (CCN 3710000177) Invention Title: Immunization method against neisseria meningitidis serogroups A and C The following statement is a full description of this invention, including the best method of performing it known to me/us: 5845c(5631461_1) IMMUNIZATION METHOD AGAINST NEISSERIA MENINGITIDIS SEROGROUPS A AND C The present application claims priority to U.S. provisional application No.: 60/480,925 filed 5 on June 23, 2003, the entire disclosure of which is herein incorporated by reference in its entirety. Field of the Invention The present invention relates to the field of medicine generally, and more specifically to mi crobiology, immunology, vaccines and the prevention of infection by a bacterial pathogen by immu 10 nization. Background of the Invention Neisseria neningiuidis is a leading cause of bacterial meningitis and sepsis throughout the world. The incidence of endemic meningococcal disease during the last thirty years ranges from I to 15 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 meningococcal 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 rate ranges between 10 and 20%. 20 Pathogenic meningococci are enveloped by a polysaccharide capsule that is attached to the outer membrane surface of the organism. Thirteen different serogroups of meningococci have been identified on the basis of the inmunological specificity of the capsular polysaccharide (Frasch, C.E., et al. 1985). Of these thirteen serogroups, five cause the majority of meningococcal disease; these include serogroups A, B, C, W- 135, and Y. Serogroup A is responsible for most epidemic disease. 25 Serogroups B, C, and Y cause the majority of endemic disease and localized outbreaks. The human naso-oropharyngeal mucosa is the only known natural reservoir of Neisseria meningitidis. Colonization takes place both at the exterior surface of the mucosal cell and the subepithe lial tissue of the nasopharynx. Carriage of meningococci can last for months. Spreading of meningo cocci occurs by direct contact or via air droplets. Meningococci become invasive by passing through 30 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. Vaccines based on meningococcal polysaccharide have been described which elicit an im 35 mune response against the capsular polysaccharide. These antibodies are capable of complement mediated bacteriolysis of the serogroup specific meningococci. The meningococcal polysaccharide vaccines are shown to be efficacious in children and adults (Peltola, H., et al. 1977 and Artenstein, M.S., et al. 1970), but the efficacy is limited in infants and young children (Reingold, A.L., et al. 1985). Subsequent doses of the polysaccharide in younger populations elicited 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 be 5 tween 3 to 5 years in adults and children above four years of age (Brandt, B., ei al. 1975, Ksyhty, H., et al. 1980, and Ceesay, S. J., et a. 1993). For children from one to four years old the duration of protection is less than three years (Reingold, A.L., et al. 1985). 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 10 independent antigens. Polysaccharides are able to stimulate B lymphocytes for antibody production without the help of T-helper lymphocytes. As a result of the T-independent stimulation of the B lym phocytes, there is a lack of memory induction following immunization by these antigens. The poly saccharide antigens are capable of eliciting very effective T-independent responses in adults, but these T-independent responses are weak in the immature immune system of infants and young children. 15 T-independent polysaccharide antigens can be converted to T-dependent antigens by covalent attachment of the polysaccharides to protein molecules ("carriers" or "carrier proteins"). B cells that bind the polysaccharide component of the conjugate vaccine can be activated by helper T cells spe cific 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. Conjugation to a carrier 20 protein has not always resulted in a vaccine capable of inducing memory against the polysaccbaride. MacLennan et al. describe a study of a meningococcal A/C adjuvanted conjugate vaccine given to infants, less than six months old. MacLennan, J. et al., J. [nfect.Dis. 2001;183:97-104. The conjugate vaccine contained I I pg of each polysaccharide and 49 pg of CRM 197 adjuvanted with I mg of aluminum hydroxide. The children are boosted with either a mengococcal A/C polysaccharide 25 vaccine containing 50 sg of each polysaccharide or the conjugate when the children are between 18 and 24 months, and revaccinated at about 5 years of age with a single meningococcal A/C vaccine containing 10 pg of each polysaccharide. Blood samples are drawn at pre-vaccination and ten days post-vaccination. The authors noted that prevaccination Group A antibody concentrations are high in all groups, and concluded that they did not believe that immunologic memory to the group A compo 30 nent of this vaccine is conclusively proven. The serogroup B polysaccharide has been shown to be poorly to non-immunogenic in the human population (Wyle, F.A., et al. 1972). Chemical attachment of this serogroup polysaccharide to proteins has not significantly altered the immune response in laboratory animals (Jennings, H. J., et al. 1981). The reason for the lack of immune response to this serogroup polysaccharide is thought to 35 arise from structural similarities between the serogroup B polysaccharide and polysialylated host gly coproteins, such as the neural cell adhesion molecules. 2.

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. neningitidis corresponding to serogroup C. Such a vaccine is only capable of protecting against disease caused by serogroup C bacteria. 5 USP 5,425,946 describes an immunogenic conjugate comprising a modified group C meningococcal polysaccharide (GCMP) coupled to a carrier molecule. The GCMP is modified by 0 deacetylation to a varying extent. The patent describes selectively removing the 0-acetyl groups on positions 7 and/or 8 of the sialyl moieties in the group C polysaccharide from OAc+ strains are to a varying extent from the meningococcal group C polysaccharide by treatment with an appropriate re 10 agent. Methods for making polysaccharide-protein conjugates using an adipic dihydrazide spacer is described by Schneerson, R., et al, Preparation, Characterization and inmunogenicity of Haemophilus Influenzae Type b Polysaccharide-Protein Conjugates, J. Exp. Med., 1952, 361-476 (1980), and in U.S. Pat. No. 4,644,059 to Lance K. Gordon. Other linker methods, such as a binary spacer technol 15 ogy as described by Marburg, S., et al, "Biomolecular Chemistry of Macromolecules: Synthesis of Bacterial Polysaccharide Conjugates with Neisseria meningitidus Membrane Protein", J. Am. Chem. Soc., 108, 5282-5287 (1986) and a reducing ends methodology, as referred to by Anderson in U.S. Pat. No. 4,673,574 are known. Existing vaccines based on meningococcal polysaccharide are of limited use in young chil 20 dren and do not provide long-lasting protection in adults. The only meningococcal vaccine which as been shown to be capable of eliciting long-lasting protection in all groups, including children, at risk for meningococcal infection is based on a polysaccharide from'a single serogroup of N. ineningitidis and provides no protection against infection by other serogroups. Thus, a need exists for a meningo coccal conjugate vaccine capable of conferring broad, long-lived protection against meningococcal 25 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 which immunogenic polysaccharides from the major pathogenic serogroups of N. ieningitidis have been converted to T-dependent antigens through conjugations to carrier proteins. SUMMARY OF THE INVENTION 30 The present invention provides a method for prevention of diseases caused by pathogenic Neisseria neningitidis serogroups A and C by administration of a composition comprising aluminum free meningococcal polysaccharide-protein conjugates. The present invention provides a method of inducing an immunological response to capsular polysaccharide serogroups A and C of N. meningitidis by administering an immunologically effective 35 amount of the iununological composition to a human. The immunological composition is a multiva lent meningococcal vaccine comprising at least two distinct protein-polysaccharide conjugates, one conjugate comprising a capsular polysaccharide of serogroup A conjugated, either directly or by a linker, to a carrier protein, and a second conjugate comprising a capsular polysaccharide of serogroup C conjugated, either directly or by a linker, to a carrier protein. The immunological composition is aluminum-free. The immunological composition may contain other compounds, such as aluminum 5 free adjuvants, or preservatives. All patents, patent applications, and other publications recited herein are hereby incorporated by reference in their entirety. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method of inducing an immunological response to capsular 10 polysaccharides of serogroups A and C of N. meningifidis by administering to a human an aluminum free immunologically effective amount of the inununological composition comprising capsular poly saccharides of serogroups A and C each conjugated to a carrier protein. The capsular polysaccharides of serogroups A and C are preferably individually conjugated to a carrier protein. Conjugation may be a direct chemical linkage between the polysaccharide and the carrier protein, or an indirect linkage 15 whereby the polysaccharide and carrier protein are each chemically via a chemical linker molecule. The polysaccharide may be first covalently attached to the linker molecule, then the carrier protein covalently attached to the linker molecule. Alternatively, the carrier protein may be first covalently attached to the linker molecule, then the polysaccharide attached to the linker molecule. The immu nological composition may contain other compounds, such as aluminum-free adjuvants, or preserva 20 tives. Methods to prepare capsular polysaccharides of N. meningitidis serogroups A and C arc well known in the art, as vaccines containing N. neningitidis polysaccharides have been licensed for many years. For example, methods for obtaining capsular polysaccharides from serogroup A of N. meningi tidis are described in Moreau U.S. Patent 6,045,805, using a method described in Gotschlich et al., 25 Prog. Iminunobiol. Standard. (1972) 5: 485. USP 6,045,805 describes preparing an oligosaccharide from a larger, native polysaccharide by depolymerizing the polysaccharide and eluting the smaller oligosaccharide from a chromatography column. The oligosaccharide may be isolated using a number of conventional techniques, for example, by precipitation using an appropriate precipitating agent such as acetone or alcohol, by filtration on a membrane having an appropriate separation threshold, by 30 exclusion-diffusion or by ion-exchange chromatography. Subsequently, oligosaccharide fractions con taining molecules having an elution constant equal to, or in the vicinity of, the mean elution constant may be obtained. The polysaccharide according to the invention may be coupled, via covalent bonding, with a compound of peptide or protein nature or with another organic polymer such as for example polyacr 35 late in order to form a conjugate capable of promoting the immunogenicity of the polysaccharide es pecially in a mammal. It is preferred that the polysaccharide is conjugated to a bacterial protein, more 4 preferably, a bacterial toxin, the corresponding anatoxin or a subunit of a multimeric toxin as well as a membrane protein, a subunit of a multimeric membrane protein or a cytoplasmic protein. Preferred toxins include, pertussis toxin, cholera toxin, tetanus toxin and diphtheria toxin. These proteins can be extracted from the original bacteria or alternatively can be made recombinantly. 5 Chemical methods for preparing polysaccharide-protein conjugates are well known. For example, a functional group may be created on the oligosaccharide which is capable of reacting with a functional group of the carrier protein. A bifunctional coupling agent may also be reacted with the oligosaccharide and then with a carrier protein, or vice versa. W. E. Dick and M. Beurret in Conju gates Vaccines, J. M. Cruse, R. E. Lewis Jr Eds, Contrib. Microbiol. Immunol. Basel, Karger (1989) 10 10 : 48 provides a review of these various coupling methods. Furthermore, the oxidation-reduction fragmentation process introduces reducing groups, especially into the oligosaccharide derived from a polysaccharide of N. meningitidis group A. In a preferred embodiment, these meningococcal serogroup conjugates are prepared by sepa rate processes and formulated into a single dosage formulation. For example, capsular polysaccha 15 rides from serogroups A and C of N. meningitidis are separately purified. In a preferred embodiment of the present invention, the purified A and C polysaccharides are separately depolymerized and separately activated prior to conjugation to a carrier protein. Prefera bly, the capsular polysaccharides of serogroups A an'd C of N. meningitidis are partially depolymer ized separately using mild oxidative conditions. 20 The depolymerization or partial depolymerization of the polysaccahrides may then be fol lowed by an activation step. By "activation" is meant chemical treatment of the polysaccharide to provide chemical groups capable of reacting with the carrier protein. A preferred activation method involves treatment with adipic acid dihyrazide in physiological saline at pH 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. 25 Once activated, the capsular polysaccharides may then be conjugated to one or more carrier proteins. In a preferred embodiment of the present invention, each A and C capsular polysaccharide is separately conjugated to a single carrier protein, more preferably, each is conjugated to the same carrier protein. Carrier proteins may include inactivated bacterial toxins such as diphtheria toxoid, CRM 97 , 30 tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aerugi nosa. Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, trans ferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneuniococcal adhe sin protein (PsaA), could also be used. Other proteins, such as ovalbumin, keyhole limpit hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) may also be 35 used as carrier proteins. Carrier proteins are preferably proteins that are non-toxic and non-reactogenic and obtainable in sufficient amount and purity. Carrier proteins should be amenable to standard con jugation procedures. In a preferred embodiment of the present invention diphtheria toxin purified 5 from cultures of Corynebacteria diplitheriae and chemically detoxified using formaldehyde is used as the carrier protein. After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide protein conjugates may be purified (enriched with respect to the amount of polysaccharide-protein 5 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. Alterna tively, conjugates can be purified away from unreacted protein and polysaccharide by any number of standard techniques including, inter alia, size exclusion chromatography, density gradient centrifuga 10 tion, hydrophobic interaction chromatography or ammonium sulfate fractionation. See, e.g., P.W. Anderson, et a/. (1986). J. Immunol. 137: 1181-1186. See also H. J. Jennings and C. Lugowski (1981) J. Immunol. 127:1011-1018. After conjugation of the polysaccharide and carrier protein, the immunological compositions of the present invention are made by combining the various polysaccharide-protein conjugates, pref 15 erably in about equal amounts. The immunological compositions of the present invention comprise two or more different capsular polysaccharides conjugated to one or more carrier protein(s). A pre ferred embodiment of the present invention is a bivalent immunological composition comprising cap sular polysaccharides from serogroups A and C of N. meningitidis each separately conjugated to dip theria toxoid. 20 The total amount of polysaccharide in the composition contains about 0.5 to about 50 pg polysaccharide, more preferably, about 2 to about 30 Rg polysaccharide, and more preferably, about 5 to about 20 pg polysaccharide. The relative amounts of A and C polysaccharide in a given composi tion may vary, but preferably, are present in equal amounts within about 25% difference, more pref erably, within about 15% difference, or alternatively in a range of A:C polysaccharide ratio of 1:3 to 25 3:1, more preferably, of a range of 1:2 to 2:1. Preparation and use of carrier proteins, and a variety of potential conjugation procedures, are well 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 well as information readily available in the general literature. Guidance can also be obtained from any one or all of the following 30 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. The total amount of carrier protein in the composition contains about 20 to about 75 pg carrier protein, and more preferably, about 30 to about 50 pg carrier protein. The immunological compositions of the present invention are made by separately preparing 35 polysaccharide-protein conjugates from different meningococcal serogroups and then combining the conjugates. The immunological compositions of the present invention can be used as vaccines. For mulation of the vaccines of the present invention can be accomplished using art recognized methods. 6 The vaccine compositions of the present invention may also contain one or more aluminum-free adju vants. Adjuvants include, by way of example and not limitation, Freund's Adjuvant, BAY, 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. Newman, eds., 5 Plenum Press, NY). The present invention is directed to a method of inducing an immunological response in a pa tient, preferably a human patient. As demonstrated below, the vaccines and immunological compositions according to the in vention elicit a T-dependent-like immune response in various animal models, whereas the polysaccha 0 ride 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-like immune responses to N. ineningitidis antigens. The amount of vaccine of the invention to be administered a human or animal and the regime of administration can be determined in accordance with standard techniques well known to those of 5 ordinary skill in the pharmaceutical and veterinary arts taking into consideration such factors as the particular antigen, the adjuvant (if present), the age, sex, weight, species and condition of the particu lar animal or patient, and the route of administration. In the present invention, the amount of polysac charide-protein carrier to provide an efficacious dose for vaccination against N. imeningitidis can be from between about 0.02 pg to about 5 ltg per kg body weight. In a preferred composition and !0 method of the present invention the dosage is between about 0.1 pg to 3 pg per kg of body weight. For example, an efficacious dosage will 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 will de pend on the bacterial load at the time of diagnosis. Multiple injections administered over a period of days could be considered for therapeutic usage. 25 The present invention provides a method for boosting in a human subject an anti meningococcal immune response against a meningococcal capsular polysaccharides A and C. The method generally entails a primary vaccination using an aluminum-free polysaccharide-protein conju gate vaccine composition comprising meningococcal capsular polysaccharides A and C conjugated to a carrier protein e.g., A/C conjugate vaccine. In a preferred embodiment, a single primary vaccination 30 is sufficient to elicit an anti-meningococcal immune response in the vaccinated subject which is spe cific for meningococcal serogroups A and C. After the immune response elicited by the primary vac cination has declined to sub-protective levels, a boosting vaccination is performed in order to provide a boosted anti-meningococcal immune response. The boosting vaccination may be a meningococcal A and C polysaccharide vaccine, or a meningococcal A and C conjugated to a carrier protein, e.g., A/C 35 conjugate vaccine. 7 The multivalent conjugates of the present invention can be administered as a single dose or in a series (i.e., with 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 other vaccines to prevent childhood diseases. Preferably, the patient is immunized in a single dose before 5 one year of age. The present invention demonstrates that immunization with the A/C conjugate vac cine of the invention may be safely administered concomitantly with other childhood vaccines, such as DTP and OPV. The booster dose will generate antibodies from primed B-cells, i.e., an anamnestic response. That is, the multivalent conjugate vaccine elicits a high primary (i.e., following a single administra 10 tion of vaccine) functional antibody response in younger populations when compared to the licensed polysaccharide vaccine, and is capable of eliciting an anamnestic response (i.e., following a booster administration), demonstrating that the protective immune response elicited by the multivalent conju gate vaccine of the present invention is long-lived. Compositions of the invention can include liquid preparations for orifice, e.g., oral, nasal, 15 anal, vaginal, peroral, intragastric, mucosal (e.g., perlinqual, alveolar, gingival, olfactory or respira tory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for par enteral, subcutaneious, intradermal, intramuscular, intraperitoneal or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Intravenous and parenteral ad ministration are preferred. Such compositions may be in admixture with a suitable carrier, diluent, or 20 excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pI buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17*h edition, 1985, incorporated 25 herein by reference, may be consulted to prepare suitable preparations, without undue experimenta tion. 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 30 of pills, tablets, capsules, caplets and the like, including "solid" preparations which are time-released or which 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, composi tions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dis penser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can pref 35 er""ly dispense a metered dose or a dose having a particular particle size. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, 8 especially by injection or orally, to animals, children, particularly small children, and others who may have difficulty swallowing a pill, tablet, capsule or the like, or in multi-dose situations. Viscous com positions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa. 5 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., liquid dosage for (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage fonn (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form). 10 Solutions, suspensions and gels, nonnally contain a major amount of water (preferably puri fied water) in addition to the active ingredient. Minor amounts of other ingredients such as pH ad justers (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and/or flavors may also be present. The compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal fluid. 15 The desired isotonicity of the compositions of this invention may be accomplished using so dium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions. Viscosity of the compositions may be maintained at the selected level using a pharmaceuti cally acceptable thickening agent. Methylcellulose is preferred because it is readily and economically 20 available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred con centration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents. 25 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 ex ample, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suit able 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 agent selected. 30 Those skilled in the art will recognize that the components of the compositions must be se lected to be chemically inert with respect to the N. meningitidis polysaccharide-protein carrier conju gates. The invention will be further described by reference to the following illustrative, non-limiting examples setting forth in detail several preferred embodiments of the inventive concept. Other exam 35 ples of this invention will be apparent to those skilled in the art without departing from the spirit of the invention.

EXAMPLES Example 1 Preparation of Neisseria Meningitidis Serogroups A and C Purified Capsular Polysaccharide Powders 5 Crude Paste Preparation Separately, Neisseria meningitidis serogroup A and C wet frozen seed cultures are thawed and re covered with the aid of liquid Watson Scherp medium and planted in Blake bottles containing Mueller Hinton agar medium. The Blake are incubated at 35 to 37 deg. C. in a CO 2 atmosphere for 15 to 19 hours. Following the incubation period, the growth from the Blake bottles are dislodged and added to 10 4 L flasks containing Watson Scherp medium. The flasks are incubated at 35 to 37 deg. C. for 3 to 7 hours on a platform shaker. The contents of the 4 L flasks are transferred to a fermenter vessel con taining Watson Scherp medium. The fermenter vessel is incubated at 35 to 37 deg. C. for 7 to 12 hours controlling dissolved oxygen content and pH with supplement feed and antifoam additions. Af ter the incubation period, the contents of the fermentor vessel are transferred to a 500 L tank, Cetav 15 lonTm is added, and the material mixed for I hours. The Cetavlon treated growth is centrifuged at ap proximately 15,000 to 17,000 x g at a flow rate of approximately 30 to 70 liters per hours. The crude polysaccharide is precipitated from the supernatant with a second CetavonTM precipitation. Cetav lonTm is added to the supernatant and the material mixed for at least I hour at room temperature. The material is stored at I to 5 deg. C. for 8 to 12 hours. The precipitated polysaccharide is collected cen 20 trifugation at approximately 45,000 to 50,000 x g at a flow rate of 300 to 400 ml per minute. The col lected inactivated paste is stored at -60 deg. C. or lower until further processed. The inactivated paste may be prepared in several batches and combined. Purified Polysaccharide Powder Preparation 25 The inactivated paste is thawed and transferred to a blender. The paste is blended with 0.9 M calcium chloride to yield a homogeneous suspension. The suspension is centrifuged at approximately 10,000 x g for 15 minutes. The supernatant is decanted through a lint free pad into a container as the first ex tract. A second volume of 0.9 M calcium chloride is added to the paste, and blended to yield a homo 30 geneous suspension. The suspension is centrifuged as above, and the supernatant combined with the supernatant from the first extraction. A total of four extractions are performed, and the supernatants pooled. The pooled extracts are concentrated by ultrifiltration using 10-30 kDA MWCO spiral would ultrafiltration units. 35 Magnesium chloride is added to the concentrated, and the pH adjusted to 7.2 to 7.5 using sodium hy droxide. DNase and RNase are added to the concentrate, and incubated at 25 to 28 deg. C. with mix ing for 4 hours. Ethanol is added to a concentration of 30 to 50%. Precipitated nucleic acid and pro 10 tein are removed by centrifugation at 10,000 x g for 2 hours. The supernatant is recovered and the polysaccharide precipitated by adding ethanol to 80% and allowing it to stand overnight at I to 5 deg. C. The alcohol is siphoned off, and the precipitated polysaccharide is centrifuged for 5 minutes at 10,000 x g. The precipitated polysaccharide is ished with alcohol. The polysaccharide is ished with 5 acetone, centrifuged at 15 to 20 minutes at 10,000 x g. The polysaccharide is dried under vacuum. The initial polysaccharide powder is dissolved into sodium acetate solution. Magnesium chloride is added and the pH adjusted to 7.2 to 7.5 using sodium hydroxide solution. DNase and RNase are added to the solution and incubated at 25 to 28 deg. C. with mixing for 4 hours to remove residual nucleic acids. After incubation with these enzymes, an equal volume of sodium acetate-phenol solution is added to 10 the polysaccharide-enzyne mixture, and placed on a platform shaker at I to 5 deg. C. for approxi mately 30 minutes. The mixture is centrifuged at 10,000 x g for 15 to 20 minutes. The upper aqueous layer is recovered and saved. An equal volume of sodium acetate-phenol solution is added to the aqueous layer, and extracted as above. A total of four extractions are perfonned to remove protein and endotoxin from the polysaccharide solution. The combined aqueous extracts are diluted up to ten fold 15 with water for injection, and diatiltered against 10 volumes of water for injection. Calcium chloride is added to the diafiltered polysaccharide. The polysaccharide is precipitated overnight at I to 5 deg. C. by adding ethanol to 80%. The alcohol supernatant is withdrawn, and the polysaccharide collected by centrifugation at 10,000 x g for 15 minutes. The purified polysaccharide is ished two times with etha nol., and once with acetone. The ished powder is dried under vacuum in a desiccator. The dried pow 20 der is stored at -30 deg. C. or lower until processed onto conjugate. Example 2 Depolymerization of Neisseria Meningitidis scrogroups A and C Purified Capsu lar Polysaccharide Powder 25 Materials used in the preparation include purified capsular polysaccharide powders from Neisseria meningitidis serogroups A and C prepared in accordance with the above Example, sterile 50 mM so dium acetate buffer, pH 6.0, sterile I N hydrocholoric acid, sterile IN sodium hydroxide, 30% hydro gen peroxide, and sterile physiological saline (0.S5% sodium chloride). Alternatively, citrate buffer may be substituted for sodium acetate buffer. 30 Each serogroup polysaccharide is depolymerized in a separate reaction. A stainless steel tank is charged with up to 60 g of purified capsular polysaccharide powder. Sterile 50 mM sodium acetate buffer, pH 6.0 is added to the polysaccharide to yield a concentration of 2.5 g polysaccharide per liter. The polysaccharide solution is allowed to mix at I to 5 deg. C. for 12 to 24 hours to effect solution. 35 The reaction tank is connected to a heat exchanger unit. Additional 50 mM sodium acetate buffer, pH 6.0, is added to dilute the polysaccharide to reaction concentration of 1.25 g per liter. The polysaccha ride solution is heated to 55 deg. C..+-.0. 1. An aliquot of 30% hydrogen peroxide is added to the reac I I tion mixture to yield a reaction concentration of 1% hydrogen peroxide. The course of the reaction is monitored by following the change in the molecular size of the polysac charide over time. Every 15 to 20 minutes, aliquots are removed from the reaction mixture and in 5 jected onto a HPSEC column to measure the molecular size of the polysaccharide. When the molecu lar size of the polysaccharide reached the targeted molecular size, the heating unit is turned off and the polysaccharide solution rapidly cooled to 5 deg. C. by circulation through an ice water bath. The de polymerized polysaccharide solution is concentrated to 15 g per liters by connecting the reaction tank to an ultrafiltration unit equipped with 3000 MWCO regenerated cellulose cartridges. The concen 10 trated depolymerized polysaccharide solution is diafiltered against about 5 to 15 volumes, preferably about 6 to 10 volumes, or more preferably, 10 volumes of sterile physiological saline (0.85% sodium chloride). The depolymerized polysaccharide is stored at I to 5 deg. C. until the next process step. The depolymerized polysaccharide may be prepared in batches and combined. The preferred targeted size for the depolymerized polysaccharide is between about 5 and 75 kDa, 15 preferably, between about 5 and 40 kDa, and more preferably, between about 10 and 25 kDa. The molecular size of the depolymerized polysaccharide is determined by passage through a gel filtra tion chromatography column sold under the tradename "Ultahydrogel.TM.250" that is calibrated us ing dextran molecular size standards and by multi-angle laser light scattering. The quantity of poly 20 saccharide is determined by phosphorus content for serogroup A using the method of Bartlet, G. R. J. (1959) Journal of Biological Chemistry, 234, pp- 4 6 6

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4 6 8 , and by the sialic acid content for sero groups C, W1 35 and Y using the method of Svennerholm, L. (1955) Biochimica Biophysica Acta 24, pp604-611. The O-acetyl content is determined by the method of Hesterin, S. (1949) Journal of Bio logical Chemistry 180, p 2 4 9 . Reducing activity is determined by the method of Park, J. T. and John 25 son, M. J. (1949 Journal of Biological Chemistry 181, pp 14 9

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1 5 1 . The structural integrity of the de polymerized polysaccharide is determined by protein .sup. I H and sup. I 3C NMR. The purity of the depolymerized polysaccharide is determined by measuring the LAL (endotoxin) content and the re sidual hydrogen peroxide content. 30 Example 3 Derivatization of Neisseria Meningitidis Serogroups A, C, W-135, and Y Depolymerized Polysaccharide Materials used in this preparation include hydrogen peroxide, depolymerized capsular polysaccharide serogroups A and C from Neisseria meningitidis, prepared in accordance with the above Example 2, 35 adipic acid dihydrazide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) for serogroup A only, sodium cyanborohydride, sterile IN hydrocholoric acid, sterile IN sodium hydroxide, sterile 1 12 M sodium chloride, and sterile physiological saline (0.85% sodium chloride). Each serogroup polysaccharide is derivatized in a separate reaction. A stainless steel tank is charged with the purified depolymerized polysaccharide, and diluted with sterile 0.85% physiological saline to 5 achieve a final reaction concentration of 6 g polysaccharide'per liter. To this solution is added a con centrated aliquot of adipic acid dihydrazide dissolved in sterile 0.85% physiological saline, in order to achieve a reaction concentration of Ig per liter. For serogroup A only, EDAC is added as a concen trated aliquot dissolved in sterile 0.85% physiological saline, to achieve a reaction concentration of Ig per liter. The pH is adjusted to 5.0.+-.0. 1, and this pH is maintained for 2 hours using sterile IN hy 10 drochloric acid and sterile IN sodium hydroxide at room temperature (15 to 30 deg. C.). After two hours, a concentrated aliquot of sodium cyanoborohydride, dissolved in 0.85% physiological saline, is added to the reaction mixture to achieve a reaction concentration of 2 g per liter. The reaction is stirred at room temperature (15 to 30 deg. C.) for 44 hours.+-.4 hours while maintaining the pH at 5.5.+-.0.5. Following this reaction period, the pH is adjusted to 6.0.+-.0.1, and the derivatized poly 15 saccharide is concentrated to 12 g polysaccharide per liter by connecting the reaction tank to a ultrafil tration unit equipped with a 3000 MWCO regenerated cellulose cartridges. The concentrated derivat ized polysaccharide is diafiltered against 30 volumes of I M sodium chloride, followed by 10 vol umes of 0.15 M sodium chloride. The tank is disconnected from the ultrafiltration unit and stored at I to 5 deg. C. for 7 days. The tank is reconnected to an ultrafiltration unit equipped with 3000 MWCO 20 regenerated cellulose cartridges, and diafiltered against 30 volumes of I M sodium chloride, followed by 10 volumes of 0.15 M sodium chloride. Alternatively, the concentrated derivatized polysaccharide is dialyzed against about 10 to about 30 volumes IM sodium chloride and then against 10 to about 30 volumes physiological saline. 25 The molecular size of the derivatized polysaccharide, the quantity of polysaccharide, and the O-acetyl content are measured by the same methods used on the depolyrnerized polysaccharide. The hydrazide content is 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 poly saccharide is determined by proton 'H and 3 C NMR. The purity of the dcrivatized polysaccharide is 30 determined by measuring the level of unbound hydrazide, the LAL (endotoxin) content, and the resid ual cyanoborohydride content. Example 4 Preparation of Carrier Protein 35 Preparation of Crude Diphtheria Toxoid Protein Lyophilized seed cultures are reconstituted and incubated for 16 to 18 hours. An aliquot from the cul '3 ture is transferred to a 0.5-liter flask containing growth medium, and the culture flask is incubated at 34.5 to 36.5 deg. C. on a rotary shaker for 7 to 9 hours. An aliquot from the culture flask is transferred to a 4-liter flask containing growth medium, and the culture flask is incubated at 34.5 to 36.5 deg. C. on a rotary shaker for 14 to 22 hours. The cultures from the 4-liter flask are used to inoculate a fer 5 nienter containing growth media. The fermenter is incubated at 34.5 to 36.5 deg. C. for 70 to 144 hours. The contents of the fermenter are filtered through depth filters into a collection vessel. An ali quot of formaldehyde solution, 37% is added to the harvest to achieve a concentration of 0.2%. The pH is adjusted to 7.4 to 7.6. The harvest is filtered through a 0.2 micron filter cartridge into sterile 20 liter bottles. The bottles are incubated at 34.5 to 36.5 deg. C. for 7 days. An aliquot of formaldehyde 10 solution, 37%, is added to each 20 liter bottle to achieve a concentration of 0.4%. The pH1 of the mix tures is adjusted to 7.4 to 7.6. The bottles are incubated at 34.5 to 36.5 deg. C. for 7 days on a shaker. An aliquot of formaldehyde solution, 37%., is added to each 20 liter bottle to achieve a concentration of 0.5%. The pH of the mixtures is adjusted to 7.4 to 7.6. The bottles are incubated at 34.5 to 36.5 deg. C. for 8 weeks. The crude toxoid is tested for detoxification. The bottles are stored at I to 5 deg. 15 C. during the testing period. Purification of the Crude Diphtheria Toxoid Protein The crude toxoid is allowed to warm to room temperature, and the contents of the 20-liter bottles are 20 combined into a purification tank. The pH of the toxoid is adjusted to 7.2 to 7.4, and charcoal is added to the crude toxoid and mixed for 2 minutes. The charcoal toxoid mixture is allowed to stand for I hours, and is then filtered through a depth filter cartridge into a second purification tank. Solid ammo nium sulfate is added to the filtrate to achieve 70% of saturation. The pH is adjusted to 6.8 to 7.2, and the solution is allowed to stand for 16 hours. The precipitated protein is collected by filtration and 25 ished with 70% of saturation ammonium sulfate solution, pH 7.0. The precipitate is dissolved into sterile distilled water, and the protein solution is filtered into a stainless steel collection vessel. The pH is adjusted to 6.8 to 7.2, and ammonium sulfate is added to 40% of saturation. The pH of the solu tion is adjusted to 7.0 to 7.2, and the solution is allowed to stand for 16 hours. The precipitate is re moved by filtration and discarded. Ammonium sulfate is added to the filtrate to 60% of saturation, 30 and the pH adjusted to 7.0 to 7.2. The mixture is allowed to stand for 16 hours, and the precipitated protein is collected by filtration. The precipitate is dissolved into sterile distilled water, filtered to re move undissolved protein, and diafiltered against 0.85% physiological saline. Concentration and Sterile Filtration of the Purified Diphtheria Toxoid Protein 35 The protein solution is concentrated to 15 g per liter and diafiltered against 10 volumes of 0.85% physiological saline suing a 10,000 MWCO regenerated cellulose filter cartridge. The concentrated 14 protein solution is sterilized by filtration through a 0.2 micron membrane. The protein solution is stored at 1 to 5 deg. C. until processed onto conjugate. The protein concentration is determined by the method of Lowry, 0. H. et. al (195 1) Journal of Bio 5 logical Chemistry 193, p 2 6 5

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2 7 5 . The purity of the protein is measured by sterility, LAL (endotoxin) content, and residual fonnaldehyde content. Example 5 Preparation of Monovalent Conjugates of Neisseria Meningitidis Serogroups A and C Polysaccharide to Diphtheria Toxoid Protein 10 Materials used in this preparation include adipic acid derivatized polysaccharide from Neisseria men ingitidis serogroups A and C, prepared in accordance with the above Example, sterile diphtheria toxoid protein, prepared in accordance with the above Example, EDAC, ammonium sulfate, sterile IN hydrochloric acid, sterile IN sodium hydroxide, and sterile physiological saline (0.85%). 15 Each serogroup polysaccharide conjugate is prepared by a separate reaction. All four conjugates are prepared by the following process. A stainless steel tank is charged with the purified adipic acid deri vatized polysaccharide at a reaction concentration of 700 to 1000 .nu.moles of reactive hydrazide per liter and purified diphtheria toxoid protein at a reaction concentration of 3.8 to 4.0 g protein per liter. 20 Physiological saline 0.85%, is used to dilute the starting materials to the target reaction concentrations and the pH is adjusted to 5.0.+-.0.1. An aliquot of EDAC is added to the polysaccharide protein mix ture to achieve a reaction concentration of 2.28 to 2.4 g per liter. The pH of the reaction is kept at 5.0.+-.0.1 for 2 hours at 15 to 30 deg. C. After two hours, the pH is adjusted to 7.0.+-.0.1 using sterile IN sodium hydroxide, and the reaction is stored at I to 5 deg. C. for 16 to 20 hours. 25 The reaction mixture is allowed to warm to 15 to 30 deg. C. and the reaction vessel is connected to an ultrafiltration unit equipped with a 30,000 MWCO regenerated cellulose cartridge. For serogroup A, solid ammonium sulfate is added to 60% of saturation, and for serogroup C, solid ammonium sulfate is added to 50% of saturation. For serogroups A, the conjugate reaction mixture is diafiltered against 30 20 volumes of 60% of saturated ammoniurn sulfate solution, and for serogroup C, the conjugate reac tion mixture is diafiltered against 20 volumes of 50% of saturated ammonium sulfate solution, fol lowed by 20 volumes of physiological saline, 0.85%. The diafiltered conjugate is first filtered through a filter capsule containing a 1.2 micron and a 0.45 micron filter, and then through a second filter cap sule containing a 0.22 micron filter. Alternatively, the conjugate reaction mixture may be purified by 35 several, preferably about three, ammonium sulfate precipitations.

The quantity of polysaccharide and O-acetyl content are measured by the same methods used on the depolymerized and derivatized polysaccharide. The quantity of protein is determined by the Lowry method. The molecular size of the conjugate is dctennined by passage through a gel filtration chroma tography column sold under the tradename "TSK6000PW" that used DNA as the void volume marker, 5 ATP as the total volume marker, and bovine thyroglobulin as a reference marker. In addition, the mo lecular size of the conjugate eluted from the TKS6000PW column is measured by multi-angle laser light scattering. The antigenic character of the conjugate is measured by binding to anti polysaccharide serogroup specific antibody using double-sandwich ELISA method. The purity of the conjugates is determined by measuring the amount of unbound (unconjugated) polysaccharide by elu 10 tion though a hydrophobic interaction chromatography column, unconjugated protein by capillary electrophoresis, sterility, LAL (endotoxin) content, residual EDAC content, and residual ammonium ion content. Example 6 Formulation of an aluminun-free multivalent meningococcal A and C polysac 15 charide diphtheria toxoid conjugate vaccine Materials used in preparing a meningococcal A and C conjugates may be prepared in accordance with the above methods. Preferably, the vaccine composition is formulated in sterile pyrogen-free, phos phate buffered physiological saline. The saline concentration may be achieved by 0.9% of 15 mM 20 sodium chloride and 10 mM sodium phosphate. Preferably, the vaccine composition does not contain aluminum. Example 7 Imniunogenicity of an aluminum-free multivalent nicningococcal A and C poly saccharide diphtheria toxoid conjugate vaccine in Human Patients 25 A clinical study is performed with infant subjects that compared the immune response to the bivalent A/C polysaccharide vaccine versus the bivalent A/C conjugate vaccine. In this study, a third group of infants are enrolled to serve as a control group and they received a Haenophilus influenzae type b conjugate. All three vaccine groups receive the same pediatric vaccines. The bivalent A/C conjugate group received three doses of diptheria conjugate vaccine (4 ig polysaccharide per dose) at 6, 10, and 30 14 weeks of age. The bivalent A/C polysaccharide group received two doses of a bivalent AC poly saccharide vaccine (50 ig polysaccharide per dose) at 10 and 14 weeks of age. The Haemophilus influenzae type b conjugate group received three doses of conjugate vaccine at 6, 10, and 14 weeks of age. Blood specimens are taken at 6 weeks, pre-vaccination, and at 18 weeks, 4 weeks post vaccina tion. When the children are I Ito 12 months of age, blood specimens are taken and the children who 35 had received either the bivalent AC conjugate or the bivalent AC polysaccharide vaccine received a 16 booster dose of AC polysaccharide. The reason for the booster dose of polysaccharide is to evaluate whether or not the subjects would elicit an anemestic response. The results of this study, both the primary and polysaccharide booster immune responses are pre 5 sented in Table I for the gG antibody response and Table 2 for the SBA antibody response. The IgG antibody response post primary series is approximately the same for both the polysaccharide and con jugate vaccine. However, the bactericidal antibody response in the conjugate vaccinated subjects is much higher than that for the polysaccharide vaccinated subjects. As observed with the one year old subjects, vaccination of infants with the polysaccharide elicits very little functional-bactericidal anti 10 body. The antibody elicited by the infants to the polysaccharide vaccine is presumably low avidity antibody, whereas, the conjugate vaccine appears to elicit high avidity antibody, thereby accounting for the much higher titer of bactericidal antibody. The high level 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 1 5 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 I shows anti-polysaccharide IgG GMC (group mean concentration) in infants against sero 20 groups A and C before and after both the primary series immunization (6, 10 and 14 weeks of age) and the booster vaccination with bivalent AC polysaccharide given at 11 to 12 months of age. Table 1 Primary Vaccination PS Booster Vaccination GMC Immune Response GMC [95% CI) 195% cI] by Vaccine Group N Pre Post N Pre Post Serogroup A: AC Conjugate 34 5.8 31 0.2 7.0 [2.2-5.41 [4.3-8.0] 31 (0.1-0.3] [4.0-12.0] AC Polysaccha- 3.0 5.5 30 0.9 3.1 ride [1.7-5.3] [4.1-7.3) [0.5-1.4] [2.0-4.7) 1IB Conjugate 36 32.25] .4-0.8] NA NA NA Serogroup C: I__ 1.6 2.8 0.1 8.1 AC Conjugate 31 [2.0-3.9 1 0.1-0.2] (4.5-14.5} AC 2.3 5.3 0.6 2.8 Polysaccharide [1.4-3.9] [3.8-7.4 0 [0.3-1.0] (1.7-4.7] HIB Conjugate 36 [2-3.5) [0. 7) NA NA NA [ . - . 1 1 03-.

Table 2 shows SBA antibody GMT (group mean titer) in infants against serogroups A and C before and after both the primary series immunization (6, 10 and 14 weeks of age) and booster vaccination with bivalent AC polysaccharide given at 11 to 12 months of age. Table 2 Immune Response Primary Vaccination GMT PS Booster Vaccination GMT By Vaccine Group1 CI Post 195% C11 N Pre N Post N Pe Serogroup A: ACConjugate 3 11.8 177 24 10.1 373 ACConjugate 34_ [7.2-19.3] [101-312] [ 5.6-18.01 [162-853] AC 32 14.7 7.0 26 6.1 24.1 Polysaccharide [8.5-25.4] [4.7-10.51 [3.9-9.5] [11-53] HIB Conjugate 35 11.2 6.7 NA NA NA ___[6.8-18.31 [4.3-10.5)_____ Serogroup C: 50.S 189 4.6 287 AC Conjugate 34 [24-107] [128-278] 27 [3.6-5.6] [96.2-858] AC 32 62.7 25.4 26 4.1 14.4 Polysaccharide 32 [29-131] [14.4-44.6] 26 [3.9-4.3] [7.9-26.1] HIB Conjugate 36 5.9-1331 [47-11.3] NA NA NA 5 In addition to the benefits that this invention offers to the improved protection against meningococcal disease in young populations and the wider protection against serogroups A, C, W-135 and Y, the tet ravalent 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, is admin 10 istered to infants, these subjects also received the routine pediatric immunizations, which included diphtheria toxoid. Therefore, in these subjects there is no apparent improvement in the antibody re sponse to diphtheria toxoid. However, when the diphtheria toxoid conjugate is administered to sub jects that did not receive concomitant diphtheria toxoid containing vaccines, a strong booster response to diphtheria toxoid is observed. These subjects had received a three dose regiment of DTP at 2, 3, 15 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 between 2 and 3 year of age. Blood speci mens are taken at the time of vaccination and 30-days post vaccination. The bivalent AC conjugate used diphtheria toxoid as the carrier protein. 20 The immune response of diphtheria toxoid in the two vaccine groups is presented in Table 3. The polysaccharide did not serve to stimulate an anti-diphtheria immune response in these subjects as ex pected, however a strong anti-diphtheria immune response is observed for the subjects receiving the AC conjugate. Therefore, the meningococcal conjugate vaccine may provide an added benefit of 18 stimulating an immune response to carrier protein thereby providing protection against diseases caused by Corynebacteria diphtheriae when diphtheria toxoid is used as a carrier protein. Table 3 shows anti-diphtheria antibody by ELISA GMT (group mean titer) in IU/mI 5 in young healthy children vaccinated with either a bivalent AC diphtheria toxoid conjugate vaccine fonnulated at 4 pg as polysaccharide per dose or a bivalent AC polysaccharide vaccine formulated at 50 jig as polysaccharide per dose Table 3 I Anti-Diphtheria Antibody (ELISA Imune Response by Vaccine Group NpreINpost lU/m) 195%C1I ________ ____Pre JPost 0.047 21.2 AC Conjugate 104/103 [0.036 - 0.060] [11.6- 38.6] 0.059 0.059 AC Polysaccharide 103/102 [0.045 - 0.076] [0.045 - 0.077] 10 Example 8 Immunogenicity, safety, and memory of different schedules of an unadjuvanted Neisse-ia mcningiridis A/C-diphtheria toxoid conjugate vaccine in infants A clinical study in an open-label, randomized controlled trial of 618 infants in Niger receiving one to four doses of a vaccine of polysaccharides A and C conjugated to diphtheria toxoid, (A/C Conjugate) 15 or a standard A/C polysaccharide (A/C PS) vaccine simultaneous with routine infant immunizations is presented. At 24 months, A/C PS vaccine is given and memory response measured one week later. Serum bactericidal activity (SBA) and IgG antibody by ELISA are measured. The vaccine comprised capsular polysaccharides of N. ineningitidis serogroups A and C conjugated to. 20 diphtheria toxoid. The vaccine is in a 0.5 ml disposable syringe containing 4 pg of each of the two polysaccharides conjugated to 48 Ig of diphtheria toxoid. The unadjuvanted A/C conjugate vaccine is formulated into a dose of 0.5 mL of pyrogen-free, phosphate buffered physiological saline with no preservative, specifically, 0.9% of 15 mM sodium chloride and 10 mM sodium phosphate. 25 The 618 infants enrolled in the study are randomized into 6 groups of equal size. Inclusion criteria are: 1) infant in good health with rectal temperature <380C; 2) between 5 and 11 weeks of age; 3) delivered at >36 weeks gestation; 4) family resided permanently in Niamey and 5) parents providing written consent. Exclusion criteria are: severe chronic illness; enrolled in another clinical trial; previ ously vaccinated with DTP vaccine, Meningococcal PS, or Haemophilus influenzae b (Hib) conjugate '9q vaccine; preceding meningitis; administration of BCG or corticosteroid therapy within the past 3 weeks; or a contraindication to vaccination. Children randomized to the control groups received either Meningococcal A/C polysaccharide 5 (Men.PS, Aventis Pasteur) that contained 50 gg of each polysaccharide, or Hib conjugate vaccine (Act-Hib, Aventis Pasteur). Intramuscular injections of MenD, MenPS and Act-Hib are given in the anterolateral right thigh. Children had received BCG and oral polio vaccine (OPV) at birth. In accor dance with the Expanded Program on Inmunization (EPI) schedule, they received DTP and OPV at 6, 10, and 14 weeks, with boosters at 15 months. Measles and yellow fever vaccines are given at age 9 10 months. EPI injections are given intramuscularly in the left deltoid muscle. There are 6 groups of 103 infants who received four (Group 1), three (group 2), two (group 3), or one dose (groups 4 and 5) of A/C Conjugate or one dose of A/C PS (group 6) during the first 9 months of life concomitant with routine EPI vaccines, see Table 4 below: 15 20 Table 4 Group assignments, trial schedule, and number of subjects with evaluable blood specimens Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8 Visit 9 6 weeks 10 14 weeks 18 9 months 10 15 months 24 24 weeks weeks months months months + 1 week -- MenPS Group I MenD MenD MenD MenD N=104 N=98 N=99 N=84 N=77 - MenPS Group 2 MenD MenD MenD N=103 N=97 N=92 N=85 N=74 MenPS Group 3 MenD MenD N=103 _ N=94 N=89 N=73 N=66 MenPS Group 4 MenD N= 103 N=97 N=93 N=83 N=70 - MenPS Group 5 Act-Hib Act- Act-Hib MenD N=103 Hib N=101 N=97 N=87 N=76 MenPS Group 6 Act-Hib Act- Act-Hib MenPS N=102 Hib N=97 N=88 N=82 N=73 Other DTP DTP DTP Yellow DTP vaccines OPV OPV OPV fever OPV Measles Specie Blood Blood Blood Blood ns all sample sample sample sample At 24 months of age, subjects received a dose of A/C PS, in order to evaluate the anamnestic response 5 and simulate immune response on encountering N. meningilidis bacteria. Four 3 mL of blood speci mens are collected, at 18 weeks of age, 10 months, 24 months, and one week later. Children are monitored for 30 minutes after each injection for immediate reactions that might repre sent hypersensitivity reactions. Follow up evaluations are made during home visits 24 and 72 hours 10 after study injections. Serum bactericidal activity is measured for both A and C serogroups by the standard methods using baby rabbit complement, Maslanka SE, el al., Clin Diagn Lab !lnnmnol 1997; 4: 155-67. Bactericidal activity is defined as the reciprocal serum dilution yielding 2 50% of bacterial growth in comparison 15 to a control culture. IgG concentration is measured by the standardized ELISA and expressed in ig/mL, Carlone GM, et at., J Clin Microbiol 1992; 30: 154-9' and Gheesling LL, et al., J Clin Mi crobiol, 1994; 32: 1475-82. Antibodies against diphtheria, tetanus, pertussis, and polio virus types 1, 2, and 3 in serum from 18 weeks of age, are measured using standard methods. Reactogenicity is 21 evaluated for each study group following each dose administered based on the proportion of infants who had at least one local reaction within 30 minutes of administration, or one local or systemic reac tion within 24 or 72 hours following administration. 5 Immune response is expressed as antibody concentrations for ELISA and geometric mean titers (GMT) of the inhibitory dilution for SBA. Antibody levels have been considered protective based on 2 g/mL according to ELISA and 1:4 for SBA using human complement, Goldschneider I, et al., J. Exp. Med., 1969, 129: 1327-48 and Lepow ML, et al., Pediatrics 1977; 60: 673-680. In addition, the results present the percent of infants with ELISA antibody concentration >2 ltg/mL and an SBA 10 titer of 1:128, Jodar L, el at., Biologicals 2002; 30: 323-9. Confidence intervals are calculated for GMT and antibody concentrations. Groups are compared by ANOVA analysis of variance according to the distribution of log titers for SBA against serogroup A and C at visit 6. The Student-Newman-Keuls test is used for multiple com 15 parisons. The anamnestic response is evaluated by comparison of percentages and 95% confidence intervals and the ratio of GMTs of infants with serologic protection compared with the baseline of SBA and ELISA for group 6. No severe adverse event is attributed to vaccine given by the study. Table 5 shows the SBA titers for serogroups A and C at 18 weeks, 10 months, 24 months and one 20 week after 24 month time period are provided in Table 8, below, for the six groups. The proportion of subjects having SBA titers 1: 12S are provided for each of the Groups. 22 Table 5 Serum bactericidal activity serogroup A and C polysaccha rides V isit 4 (18 Visit 6 (10 Visit 8 (24 Visit 9 (24 months weeks) months) months) + 1 week) GMT % GMT % GMT %> GMT(n) %? (n) 1/128 (n) 1/128 (n) 1/128 1/128 Serogroup A 87.4 56.1 309 88.6 48.3 38.1 3351 100 Group 1 (55) [45.7 - (78) [80.1 - (32) [27.7 - (76) [95.3 [62.2 - 66.1] [229 - 94.4] [30.6 - 49.3] [25S5 - 100] 117] 417] 76.41 43451 84.6 55.7 7.65 6.5 35.3 38.8 1421 95.9 Group 2 (54) [45.2 - (6) [2.4 - (33) [28.4 - (71) [88.6 [61.4 - 65.8] [5.88 - 13.7] [21.8 - 50.0] [978 - 99.2] 117] 9.94] 57.0] 2066] 152 68.1 415 S5.4 81.1 47.9 2761 100 Group 3 (64) [57.7 - (76) [76.3 - (35) [36.1 - (65) [94.5 [107 - 77.3] [302 - 92.0] [49.5 - 60.0] [2182 - 100] 215] 570] 133] 3492] 9S.3 60.8 S.37 4.3 55.5 44.6 2376 100 Group (59) [50.4 - (4) [1.2 - (37) [33.7 - (70) [94.9 [69.3 - 70.6] [6.66 - 10.8] [33.2 - 55.9] [1809 - 100] 139] 10.5] 92.9] 31211 5.19 (3) 3.0 129 61.9 69.3 48.3 2549 100 Group 5 [4.38 - [0.6- (60) [51.4 - (42) [37.4 - (76) [95.3 6.16] 8.4] [84.2 - 71.5] [41.8 - 59.21 [1913 - 100] 197] 115] 3397] Group 6 4.65 (2) 2.1 7.22 4.5 32.8 36.6 1250 97.3 [4.08 - [0.3 - (4) [1.3 - (30) [26.2 - (71) [90.5 5.29] 7.3] [5.63 - 11.2] [20.1 - 48.0] [883 - 99.7] 9.27] 53.7] 1770] Serogroup C I _ 289 83.7 215 72.7 8.41 9.5 711 (72) 94.7 Group 1 (82) [74.8 - (64) [62.2 - (8) [4.2 - [482 - [87.1 [205 - 90.4] [138 - 81.7) [6.01 - 17.9] 1049] 98.5] 406] 337] 11.S] 304 85.6 6.98 8.8 12.7 22.4 617 (61) 82.4 Group 2 (83) [77.0 - (8) [3.9 - (19) [14.0 - [383 - [71.8 [222 - 91.9] [5.43 - 16.6] [8.22 - 32.7] 996] 90.3] 415] 8.971 19.7) 1 1 i 111 63.8 553 85.4 16.6 24.7 1655 95.4 Group 3 (60) [53.3 - (76) [76.3 - (18) [15.3 - (62) [87.1 [74.4 - 73.5] [373 - 92.0] [9.68 - 36.1] [1064 - 99.0] 166] 821] 28.5] 2574] 79.9 56.7 6.53 7.6 16.S 24.1 1855 92.9 Group 4 (55) [46.3 - (7) [3.1 - (20) (15.4 - (65) [84.1 [53.7 - 66.7] [5.32 - 15.1] [10.3 - 34.7) [1146 - 97.6] 1 19] 8.13] 27.4] 3003] 6.83 (7) 6.9 19.1 25.8 13.4 21.8 2244 98.7 Group 5 [5.41 - [2.8 - (25) ,[17.4 - (19) [13.7 - (75) [92.9 2-3 8.621 13.8] [13.4 - 35.7] [8.53 - 32.0] [1579 - 100] 27.31 _ 21.1] 3188] 5.29(2) 2.1 9.97 11.4 5.33 3.7 68.4(38) 52.1 Group [4.44- [0.3 - (10) [5.6 - (3) [0.8 - (42.1 - [40.0 6.30] 7.3] [7.31 - 19.9] [4.41- 10.3] 111] 63.9] 1 1_ 13.6] 6.45] GMT geometric mean titer, [95% confidence interval] 24 Table 6 shows ELISA results for each Group. Table 6 ELISA antibody concentrations to group A and C polysaccharide Visit 4 (18 weeks) Visit 6 (10 months) Visit 8 (24 Visit 9 (24 mos + 1 months) wk) GMC %2 GMC % GMC %> GMC %> 2pg-mL 2ttg-mL 2pg-mL- 2pg.mLl Serogro upA 3.84 84.7 3.01 67.0 0.35 8.3 10.0 98.7 Group 1 [3.37 - [76.0 - [2.42 - [56.2 - [0.27 - [3.4 - [7.80 - [92.9 4.38] 91.2] 3.74] 76.7] 0.46] 16.4] 12.9] 100] Group 2 3.90 75.3 0.24 0 0.39 21.2 6.78 87.8 [3.34 - [65.5 - [0.20 - [0 - 3.9] [0.27 - [13.1 - [5.21 - [78.2 4.56] 83.5] 0.291 0.56] 31.41 8.83] 94.3] 4.82 86.2 3.68 71.9 0.66 27.4 12.0 92.3 Group 3 [4.05 - [77.5 - [2.92 - [61.4 - [0.43 - [17.6 - [9.30 - [83.0 5.73] 92.4] 4.64) 80.9) 1.01] 39.1] 15.61 97.5] 3.87 80.4 0.25 1.1 0.46 20.5 9.04 91.4 Group 4 [3.27 - [71.1 - [0.21 - [0- 5.9] [0.31 - [12.4 - [6.98 - [82.3 4.58] 87.8] 0.29 0.67] 3 11.7] 96.8] 0.42 9.9 2.47 60.8 0.52 20.7 13.2 93.4 Group [0.33 - [4.9 - [1.93 - [50.4 - [0.35 - (12.7 - [10.3 - [85.3 0.53] 17.5] 3.18] 70.6] 0.77] 30.7 _ 17.0] 97.8] 0.47 10.3 1.64 44.3 0.56 20.7 5.74 82.2 Group 6 [0.38 - [5.1 - [1.28 - 133.7 - [0.41 - [12.6 - [4.49 - [71.5 0.59] 18.1] 2.10] 55.3] 0.77] 31.1] 7.34] 90.2] Serogro upC 9.34 96.9 6.70 85.2 0.44 11.9 9.79 76.3 Group 1 [7.98 - [91.3 - [5.27 - [76.1 - [0.32- [5.9 - [6.74 - [65.2 10.9) 99.4] 8.50] 91.9) 0.60] 20.8] 14.20] 85.31 Group 2 92.8 0.79 23.9 0.64 27.1 9.73 82.4 [7.88 - [85.7 - [0.63 - [15.6 - [0.42 - [18.0 - [6.82 - [71.8 11.3] 97.0] 0.99] 33.9] 0.96] 37.8] 13.9] 90.31 Group 3 84.0 8.62 87.6 0.73 24.7 15.8 92.3 [4.60 - [75.0 - [6.70 - [79.0 - [0.48 - [15.3 - [11.6 - [83.0 6.78] 90.8] 1 i.] 93.7] 1.11] 36.1] 21.6] 97.5] Group 4 4.38 79.4 0.68 15.2 0.48 15.7 17.0 94.3 [3.60 - [70.0 - [0.55 - (8.6 - [0.34 - [8.6 - [12.5 - [86.0 5.33] 86.9] 0.84] 24.2] 0.67] 25.3] _23.2_ 98.4 Group 5 0.38 9.9 1.95 50.5 0.31 18.4 9.18 90.8 [0.30 - [4.9- [1.58 - [40.2 - [0.21 - [10.9- [7.03 - [81.9 0.49] 17.5] 2.41] 60.8] 0.44] 28.1] 12.0] 96.2] Group 6 0.33 4.1 7.84 90.9 0.48 13.4 2.61 54.8 [0.27 - [1.1 - [6.45 - [82.9 - [0.35 - [6.9 - [1.93;3.5 [42.7 0.41] 10. 9.52] 96.0] 0.64] 22.7] 3_ GMC geometric mean concentrations, [95% confidence interval) Response to EPI vaccinations There is no difference in antibody concentrations against the EPI vaccines (diphtheria, teta 5 nus, polio virus 1,2, and 3, pertussis) between the 6 groups. The results are provided below in Tables 7-18. The A/C Conjugate does not affect immunogenicity to other antigens included in the EPI pro gram. Table 7 10 Descriptive results of Anti-Diphtheria antibodies (Seroneutralisation - IU/nL) at V4 -Per protocol analysis Anti-Diphtheria (SN - Group#1 Group#2 Group#3 Group#4 Group#5 Group#6 rU/mL) (Injected) (Injected) (Injected) (Injected) (Injected) (Injected) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 63 (=63-0) 64 (=65-1) 64 (=64-0) 69 (=69-0) 81 (=81-0) 63 (=63-0) Log 10 Dist. {flU/mL} Mean -0.442 -0.627 -0.462 -0.425 -0.543 -0.567 Standard Deviation 0.488 0.547 0.700 0.589 0.493 0.503 Distribution {lU/mL) GMT 0.361 0.236 0.345 0.376 0.286 0.271 [95% CI] (0.272;0.479] [0.1 73;0.324] [0.231;0.51 [0.271;0.52 [0.223;0.36 [0.203;0.36 6] 1] 8] 3] Minimum;Maxirnuim 0.010;2.56 0.020;5.12 0.005;10.2 0.020;5.12 0.020;5.12 0.020;2.56 Median=Q2 0.320 0.160 0.320 0.320 0.320 0.320 Ql;Q3 {Quantiles) 0.160;0.640 0.080;0.640 0.160; 1.28 0.160; 1.28 0.160;0.640 0.160;0.640 >= 0.01 IU/mL % (n) 100(63) 100(64) 98.4(63) 100(69) 100 (8 1) 100(63) [95% CI] [94.3;100] [94.4;100] [91.6;100] [94.S;100) [95.5;100) {94.3;100] >= 0.1 RJI/mL % (n) 85.7 (54) 70.3 (45) 76.6 (49) 81.2 (56) 79.0 (64) 77.8 (49) (95% CI] [74.6;93.3] [57.6;8 1.11 [64.3;86.2] [69.9;89.6] [68.5;87.3] [65.5;87.3) >= 1 IU/mL __ % (n) 22.2 (14) 12.5 (8) 29.7(19) 29.0(20) 11.1 (9) 15.9(10) [95% CI) [1 2.7;34.5) [5.6;23.2] [ 18.9;42.4) [18.7;41.2] [5.2;20.0) [7.9;27.3] 26 Table 8 Descriptive results of Anti-Diplitheria antibodies (Seroneutralisation - IU/mL) at V4 Intent-to-treat analysis Anti-Diphtheria (SN - Group#1 Group#2 Group#3 Group#4 Group#5 Group#6 II/inL) (Randoinise(Randoinise (Randonise (Randomised (Randonise (Randonised) d) d) d) ) d) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 98 (=104-6)96 (=103-7) 94 (=103-9) 97 (=103-6) 101 (=103- 97 (=102-5) 2) Logl0 Dist. {U/inL) Mean -0.501 -0.639 -0.476 -0.427 -0.522 -0.594 Standard Deviation 0.510 0.535 0.642 0.589 0.487 0.506 Distribution (RJ/mL} GMT 0.316 0.230 0.334 0.374 0.301 0.255 [95% CI) [0.249;0.39 [0.179;0.29 [0.247;0.453 [0.285;0.492] [0.241;0.375 [0.201;0.322] 9] 5] ] ] Minimun;Maxinum 0.010;2.56 0.020;5.12 0.005;10.2 0.020;10.2 0.020;5.12 0.020;2.56 Median=Q2 0.320 0.160 0.320 0.320 0.320 0.320 Qi;Q3 {Quantiles} 0.1 60;0.640 0.080;0.640 0.160;0.640 0.160; 1.28 0.160;0.640 0.160;0.640 >= 0.01 IU/inL % (n) 100(98) 100(96) 98.9(93) 100(97) 100(101) 100(97) [95% Cl] [96.3;1001 [96.2;100] [94.2; 1001 [96.3; 100] [96.4; 1001 (96.3; 1001 >= 0.1 IU/nL % (n) 81.6 (SO) 69.8 (67) 77.7 (73) S2.5 (80) 80.2 (81) 76.3 (74) [95% CI] (72.5;88.7] [59.6;78.7] [67.9;85.61 [73.4;89.4] [71.1;87.5] [66.6;84.3] >= 1IU/nL % (n) 20.4 (20) 11.5(11) 24.5 (23) 27.8 (27) 12.9 (13) 15.5 (15) (95% CII [12.9;29.71 (5.9;19.6] [16.2;34.41 [19.2;37.91 (7.0;21.0) [8.9;24.21 5 Table 9 Descriptive results of Anti-Tetanus antibodies (ELISA - IU/niL) at V4 - Per protocol analysis Anti-Tetanus (ELISA - Group#1 Group#2 Group#3 Group#4 Group#5 Group#6 (U/mL) (Injected) (Injected) (Injected) (Injected) (Injected) (Injected) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 62 (=63-1) 63 (=65-2) 64 (=64-0) 68 (=69-1) 79 (=81-2) 63 (=63-0) LoglO Dist. {IU/nL} Mean 0.692 0.583 0.557 0.581 0.487 0.386 Standard Deviation 0.268 0.347 0.328 0.364 0.356 0.348 Distribution {IU/mL} GMT 4.92 3.82 3.61 3.81 3.07 2.43 [95% CI] (4.21;5.76] [3.13;4.68 [2.99;4.36] [3.11;4.66] [2.56;3.69 [1.99;2.98] ] ] Minimuin;Maximum 1.00;15.3 0.150;17.9 0.427;17.9 0.075;13.7 0.431;20.7 0.243;13.8 Median=Q2 4.97 4.31 4.13 4.46 3.28 2.74 QI;Q3 {Quantiles) 3.61;7.56 2.14;6.15 2.21;6.43 2.48;6.60 1.84;5.36 1.60;4.18 >= 0.0 1 IU/mL % (n) 100 (62) 100 (63) 100 (64) 100 (68) 100 (79) 100 (63) [95% CI] [94.2;100) [94.3;100] [94.4; 1001 [94.7;1001 [95.4;100] [94.3;1001 >= 0.1 J/nL % (n) 100 (62) 100 (63) 100 (64) 98.5 (67) 100 (79) 100 (63) [95% C1] [94.2;100) [94.3;100] [94.4;100] [92.1;100] [95.4;100] [94.3;100] >= 1 rU/mL % (n) 100 (62) 95.2 (60) 93.8 (60) 97.1 (66) 89.9 (71) 88.9 (56) [95% CI] [94.2;100) [86.7;99.0 [84.8;98.3) [89.8;99.6] [81.0;95.5 [78.4;95.4) ] ] 28 Table 10 Descriptive results of Anti-Tetanus antibodies (ELISA - lU/nL) at V4 - Intent-to-treat analysis Anti-Tetanus (ELISA - Group#I Group#2 Group#3 Group#4 GroupN5 Group#6 TU/mL) (Randomised (Randomised (Randonise (Randomise (Randomise (Randomised ) d) d) d) ) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All- 97 (=104-7) 95 (=103-8) 92 (=103- 96 (=103-7) 99 (=103-4) 97 (=102-5) Missing) 11) LoglO Dist. {IU/mL} Mean 0.656 0.559 0.510 0.565 0.466 0.418 Standard Deviation 0.306 0.358 0.406 0.343 0.343 0.344 Distribution {IU/mL) GMT 4.52 3.62 3.24 3.67 2.93 2.62 (95% C1] [3.93;5.21] [3.06;4.291 [2.67;3.93] [3.13;4.31] (2.50;3.43] (2.23;3.071 Minimum;Maximu 0.596;15.3 0.075;17.9 0.075;17.9 0.075;13.7 0.431;20.7 0.243;18.1 m Median=Q2 4.73 4.10 4.00 3.90 3.02 2.82 QI;Q3 (Quantiles} 2.89;7.47 2.58;5.91 2.08;6.07 2.39;6.47 1.81;5.03 1.66;4.33 >= 0.01 R/mL % (n) 100 (97) 100 (95), 100 (92) 100 (96) 100 (99) 100 (97) [95% CI] [96.3:100) [96.2;100] [96.1;100] {96.2;100] [96.3;100) [96.3;100] >= 0.1 IU/mL % (n) 100 (97) 98.9 (94) 97.8 (90) 99.0 (95) 100 (99) 100 (97) [95% CI] [96.3;100] [94.3;100] [92.4;99.7] [94.3;100] [96.3;100] [96.3;100] >= I U/nL % (n) 95.9 (93) 94.7 (90) 90.2 (83) 95.8 (92) 90.9 (90) 90.7 (88) [95% CI] [89.8;98.9] [88.1;98.3] [82.2;95.4] [89.7;98.9] [83.4;95.8] [83.1;95.7] Table 11 Descriptive results of Anti-Poliovirus type 1 antibodies (1/dil.) at V4 - Per protocol analysis \nti-Polio I (1/dil.) Group/l Group#2 Group#3 Group#4 GroupN5 GroupN6 (Injected) (Injected) (Injected) (Injected) (Injected) (injected) S SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 61 (=63-2) 62 (=65-3) 63 (=64-1) 68 (=69-1) 79 (=81-2) 63 (=63-0) LoglODist. {I/dil.) Mean 1.76 1.87 1.89 2.05 1.93 1.99 Standard Deviation 0.758 0.864 0.891 0.838 0.905 0.812 Distribution (1/dil.} GMT 57.8 74.0 78.0 112 84.8 96.7 [95% CI) [37.0;90.4] [44.7;123) [46.5;131] [69.9;178] [53.1;135] [60.4,1551 Minimum;Maximum 2.00;2048 2.00;23170 2.00;5793 2.00;8192 2.00;5793 2.00;4096 Median=Q2 64.0 90.5 90.5 90.5 128 128 QI;Q3 {Quantiles} 22.6;181 22.6;181 22.6;362 45.3;512 32.0;256 22.6;256 >= 4 /dil. % (n) 91.8 (56) 91.9 (57) 90.5 (57) 95.6 (65) 87.3(69) 96.8(61) [95% C1] [81.9;97.3] [82.2;97.3] [80.4;96.4]1[87.6;99.11 f7].0;93.81 [89.0;99.61 30 Table 12 Descriptive results of Anti-Poliovirus type I antibodies (1/dil.) at V4 - Intent-to-treat analysis Anti-Polio I (1/dil.) Group#ll Group#2 Group#3 Group#4 Group#5 Group#6 (Randomise (Randomised (Randomise (Randomise (Randomised (Randonised d) ) d) d) ) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All- 95 (=104-9) 94 (=103-9) 91 (=103- 96 (=103-7) 99 (=103-4) 96 (=102-6) Missing) 12) Log10 Dist. {i/dil.}_____ _______ ______ Mean 1.78 1.85 1.88 1.96 1.89 1.96 Standard 0.769 0.890 0.867 0.891 0.906 0.830 Deviation Distribution {1/dil.} _ GMT 59.9 71.2 75.4 91.5 76.8 90.5 [95% CI] [41.8;86.0] [46.8;108] [49.8;114] [60.4;139] [50.6;116] [61.5;133] Mininun;Max 2.00;5793 2.00;23170 2.00;5793 2.00;8192 2.00;5793 2.00;S192 imurn Median=Q2 64.0 90.5 64.0 90.5 90.5 90.5 QI;Q3 16.0;181 22.6;256 22.6;256 26.9;512 22.6;256 22.6;256 {Quantiles}_ >=4 1/dil. % (n) 92.6 (88) 89.4 (84) 91.2 (83) 92.7 (89) 87.9 (87) 96.9 (93) [95% C1I [85.4;97.0] [81.3;94.81 [83.4;96.11 [85.6;97.0] [79.8;93.61 [91.1;99.4] Table 13 Descriptive results of Anti-Poliovirus type 2 antibodies (1/dil.) at V4 - Per protocol analysis Anti-Polio 2 (1/dil.) Group#1 Group#2 Group#3 Group#4 Group#5 Group#6 (Injected) (Injected) (Injected) (Injected) (Injected) (Injected) S SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 62 (=63-1) 63 (=65-2) 62 (=64-2) 66 (=69-3) 79 (=81-2) 63 (=63-0) Log10 Dist. {I/dil.} Mean 2.46 2.66 2.61 2.77 2.59 2.59 Standard Deviation 0.664 0.756 0.712 0.656 0.748 0.600 Distribution (1/dil.} GMT 286 456 407 584 388 387 [95% CI] [194;422] [294;707] [269;617] [403;846] [264;571] [273;548] Mininum;Maxinum 2.00;8192 2.00;8192 2.00;8192 11.3;32768 4.00;13107 5.70;8192 2 N4edian=Q2 256 512 609 512 362 362 Q1;Q3 {Quantiles} 128;724 181;1448 181;1024 256;1448 181;1024 181:1024 >= 4 1/dil. % (n) 98.4 (61) 98.4 (62) 98.4 (61) 100 (66) 100 (79) 100 (63) [95% C] [91.3;100] [91.5;100] (91.3;100] [94.6,100] [95.4;100J [94.3;100] 32 Table 14 Descriptive results of Anti-Poliovirus type 2 antibodies (1/dil.) at V4 - Intent-to-treat analysis Anti-Polio 2 (1/dil.) Group#1 Group#2 Group#3 Group#4 Group#5 Group#6 (Randornise (Randornise (Randonised) (Randomise (Randomise (Randomised) d) d) d) d) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All- 97 (=104-7) 95 (=103-8) 90 (=103-13) 94 (=103-9)98 (=103-5) 97 (=102-5) Missing) Log1O Dist. {1/dil.__ Mean 2.50 2.65 2.61 2.73 2.52 2.59 Standard 0.621 0.823 0.714 0.685 0.824 0.672 Deviation Distribution {1/dil_. _ GMT 314 446 411 535 333 389 195% CI) 1235;419] [303;656) 1.291;580) [387;739] [227;487) [285;531) Minimum;Maxinu 2.00;8192 2.00;92682 2.00;92682 2.00;32768 2.00;13107 2.00;8192 m 2 Median=Q2 362 362 512 431 362 362 QI;Q3 128;724 181;1448 128;1024 181;1448 )28;1024 181;1024 (Quantiles_ >=41/dil. % (n) 99.0 (96) 97.9 (93) 98.9 (89) 98.9 (93) 96.9 (95) 99.0 (96) [95% Cl) {94.4;)00) 192.6;99.7) (94.0;100] [94.2;100) [91.3;99.4) [94.4;100) ,:K3 Table 15 Descriptive results of Anti-Poliovirus type 3 antibodies (1/dil.) at V4 - Per protocol analysis Anti-Polio 3 (1/dil.) Groupl1 Group#2 Group#3 Group#4 Group#5 Group#6 (Injected) (Injected) (Injected) (Injected) (Injected) (Injected) BS SCHEDULE > Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 58 (=63-5) 61 (=65-4) 61 (=64-3) 64 (=69-5) 75 (=81-6) 61 (=63-2) Log10 Dist. {l/dil.} Mean 2.11 2.13 1.98 2.07 2.31 2.14 Standard Deviation 0.776 0.962 0.927 0.828 0.753 0.824 Distribution (1/dil.} A I GMT 130 135 95.3 118 205 137 [95% CI) [81.0:207] [76.4;238) [55.2;165) [73.3;190] [138;306) [84.3;223] Minimum;Maximu 2.00;2048 2.00;23170 2.00;4096 2.00;5793 2.00;4096 2.00;4096 Median=Q2 181 181 128 152 256 181 Q1;Q3 (Quantiles} 64.0;362 32.0;512 22.6;362 64.0;362 90.5;724 64.0;512 >= 4 1/dil. % (n) 93.1 (54) 90.2 (55) 86.9 (53) 90.6 (58) 96.0 (72) 91.8 (56) [95% C1] [83.3;9S.1] [79.8;96.3] [75.8;94.2] [80.7;96.5] [88.8;99.2 [81.9;97.31 34 Table 16 Descriptive results of Anti-Poliovirus type 3 antibodies (1/dil.) at V4 - Intent-to-treat analysis Anti-Polio 3 Group#11 Group#2 Group#3 Group#4 Group#5 Group#6 (Randomis (Randomised) (Randomise (Randomise (Random ise (Randomised) ed) d) d) d) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All- 93 (=104- 92 (=103-11) 90 (=103- 92 (=103- 95 (=103-8) 94 (=102-8) Missing) 11) 13) 11) Log10 Dist. {I/dil.), Mean 2.10 2.10 2.02 2.11 2.26 2.10 Standard 0.795 0.928 0.897 0.871 0.759 0.812 Deviation Distribution {1/dil.} _ _ _ _ _ _ _ _ _ _ _ _ _ GMT 125 127 106 128 182 126 [95% Cl) (85.9;183] [81.6:198] [68.5;163] [84.5;194] [128;260] [86.0;185) Minimum; 2.00;5793 2.00;23170 2.00;4096 2.00;5793 2.00;4096 2.00;4096 Maximum Median=Q2 181 181 128 181 181 181 QI;Q3 45.3;362 45.3;431 45.3;362 76.1;362 90.5;512 64.0;362 (Quantiles} >=4 _/dil. % (n) 93.5 (87) 89.1 (82) 88.9 (80) 89.1 (82) 94.7 (90) 89.4 (84) {95% C1) {86.5;97.6) [80.9;94.7) [80.5;94.5) 180.9;94.7) [88.1;98.3) [81.3;94.8) Table 17 Descriptive results of Anti-Agglutinin against Pertussis (1/dil.) at V4 - Per protocol analysis Anti-Agglut. Pertussis (1/dil.) Group#1 Group#2 Group#3 Group#4 Group#5 Group#6 (Injected) (Injected) (Injected) (Injected) (Injected) (Injected) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 62 (=63-1) 63 (=65-2) 61 (=64-3) 67 (=69-2) 77 (=81-4) 63 (=63-0) LoglO Dist. {I/dil.) Mean 2.43 2.42 2.40 2.44 2.38 2.42 Standard Deviation 0.423 0.494 0.566 0.495 0.551 0.382 Distribution I 1/dil.) I_____ _____ GMT 271 265 250 275 240 262 [95% Cl] [211;347) [199;352] 1179;349) 1208;363) [180;321) [210;327] Minimum;Maximun 16.0;2048 4.00;4096 4.00;2048 16.0;2048 2.00;2048 16.0;1024 Median=Q2 256 256 256 256 256 256 QI;Q3 (Quantiles) 128;512 128;512 128;512 128;512 128:512 128;512 >= 40 1/dil. % (n) 93.5 (58) 93.7 (59) 91.8 (56) 91.0 (61) 90.9 (70) 98.4 (62) [95% Cl] [84.3;98.2] [84.5;98.2] [81.9;97.31 [81.5;96.6] [82.2;96.3 [91.5;100] 36 Table 18' Descriptive results of Anti-Agglutinin against Pertussis (1/dil.) at V4 - Intent-to-treat analysis Anti-Agglut. Pertussis (lidil.) Group#l Group#2 Group#3 Group#4 Group#5 Group#6 (Randomise (Randoimise (Randomis (Randomise (Randomised) (Randomise d) d) ed) d) d) BS SCHEDULE Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 Visit V4 N Data (=All-Missing) 96 (=104-8) 94 (=103-9) 90 (=103- 94 (=103-9) 97 (=103-6) 97 (=102-5) 13) Log]ODist. (1/dil. Mean 2.44 2.45 2.41 2.42 2.36 2.41 Standard Deviation 0.468 0.473 0.524 0.508 0.550 0.431 Distribution { lidil.} _ GMT 277 282 256 266 232 254 [95% CI] 1223;345) [225;352] (199;330] [209;338] [179;299] [208;310] Mininiurn;Maximuni 16.0;4096 4.00;4096 4.00;2048 2.00;2048 2.00;2048 16.0;2048 Median=Q2 256 256 256 256 256 256 QI;Q3 (Quantiles) 128;512 128;512 128;512 128;512 128;512 128;512 >= 40 1/dil. %(n) 92.7 (89) 94.7 (89) 92.2 (83) 91.5 (86) 90.7 (88) 94.8 (92) [95% Cl) [85.6;97.01 [88.0;98.3] [84.6;96.8 [83.9;96.3) [83.1;95.71 t8 .4;98.31 For serogroup A, mean SBA titers at 10 months of age did not differ between children who received 5 four A/C Conjugate doses (6, 10, 14 weeks and 9 months) or two doses (14 weeks and 9 months), but are significantly higher than titers of each of the other schedules. For serogroup C, A/C Conjugate at 14 weeks and 9 months induced higher mean SBA titers than did the other regimens. Administration of A/C PS at 24 months led to significantly higher SBA titers in A/C Conjugate recipients, including the two groups receiving single dose conjugate schedules. While responses are lower for serogroup C 10 than A, there is no evidence of hyporesponsiveness. Meningococcal A/C conjugate vaccine is safe and immunogenic in young infants, particularly when two doses are administered at 14 weeks and 9 months of age. A single dose of A/C Conjugate in the first year of life appears to induce memory. 15 This study demonstrates that iiiunogenicity against serogroups A and C is obtained by a number of different administration methods. For example, immunogenicity against serogroups A and C is ob tained when children are vaccinated with an A/C conjugate once at 14 weeks of age and a second dose at 9 months of age. Two primary doses of an A/C conjugate given at 6 and 10 weeks of age did not 20 seem to provide any additional benefit. Injection of a single dose, either at 14 weeks or 9 months of age, appeared to provide sufficient long-term protection, based on response to the polysaccharide vac cination at 24 months of age.

A two-dose schedule, whereby the A/C conjugate vaccine is administered at 14 weeks, the time of the DTP3, and again at 9 months, when measles vaccine is given, resulted in immunogenicity against A and C serogroups. 5 This study demonstrates that A/C conjugate vaccine provides lasting immunologic memory for both serogroup A and C. Borrow R el al., J Infect Dis 2002; 186: 1353-7 have shown comparable results for serogroup C conjugate alone, for infants vaccinated at 2, 3, and 4 months in comparison to those 13-16 months or 4 years of age. Although substantial experience is accumulating in the U.K. for a three dose series of serogroup C meningococcal conjugate vaccine in infants, this study suggests that 10 administration of multiple doses in the first year of life may not be necessary, at least for some conju gate formulations. This study also demonstrates that administering A/C Conjugate concomitantly with routine infant immunizations such as DTP and OPV, does not interfere with immune response to the other antigens. 15 38

Claims (17)

1. A method of inducing an immunological response in a patient to capsular polysaccha rides A and C of N. meningitidis comprising administering an immunologically effective amount of an 5 aluminum-free immunological composition to the patient, wherein the composition comprises two protein-polysaccharide conjugates, the first conjugate comprising a capsular polysaccharide of sero group A of N. meningitidis conjugated to one or more a carrier protein(s) and a second conjugate comprising a capsular polysaccharide of serogroup C of N. meningitidis conjugated to one or more a carrier protein(s). 10

2. The method of claim 1, wherein the carrier protein is a diphtheria toxoid.

3. The method of claim 2, wherein the carier protein and polysaccharide are covalently attached with a linker.

4. The method of claim 3, wherein the linker is adipic dihydrazide.

5. The method of claim 1, wherein the capsular polysaccharides A and C have an aver 1 5 age size of between 5 and 100 kDa.

6. The method of claim 1, wherein the capsular polysaccharides A and C have an aver age size of between 10 and 75 kDa.

7. The method of claim 1, wherein the capsular polysaccharides A and C have an aver age size of between 10 and 50 kDa. 20

8. The method of claim 1, wherein the capsular polysaccharides A and C have an aver age size of between 10 and 30 kDa.

9. The method of claim 1, wherein the capsular polysaccharides A and C have an aver age size of between 10 and 25 kDa.

10. The method of claim 1, wherein the composition comprises an adjuvant. 25

11. The method of claim 1, wherein the immunological composition is administered to the patient in a single dose.

12. The method of claim 11, wherein the patient is less than 12 months of age at the time the inimunological composition is administered.

13. The method of claim 1, wherein the immunological composition is administered on the same day or within six months of administration of a vaccine for diphtheria, tetanus, poliovirus, or pertussis.

14. The method of claim 13, wherein the immunological composition is administered on 5 the same day or within three months of administration of a vaccine for diphtheria, tetanus, poliovirus, or pertussis.

15. The method of claim 14, wherein the immunological composition is administered on the same day or within one month of administration of a vaccine for diphtheria, tetanus, poliovirus, or pertussis. 10

16. The method of claim 15, wherein the immunological composition is administered on the same day of administration of a vaccine for diphtheria, tetanus, poliovirus, or pertussis.

17. The method of claim 14, wherein the vaccine is a poliovirus type 1, 2 or 3. Dated 29 September, 2011 Sanofi Pasteur, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON 40