AU2007203489A2 - Multivalent meningococcal derivatized polysaccharide-protein conjugates and vaccine - Google Patents

Description

c MULTIVALENT MENINGOCOCCAL DERIVATIZED POLYSACCHARIDE-PROTEIN CONJUGATES AND VACCINE Cross-Reference to Related Applications C 5 The entire contents of the specification accompanying International (PCT) Patent Application No. PCT/US2005/046886 are incorporated herein by reference.

00 0 Field of the Invention ec The present invention relates to the field of medicine generally, and more specifically

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C1 10 to microbiology, immunology, vaccines and the prevention of infection by a bacterial O pathogen by immunization.

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Background of the Invention Neisseria meningitidis 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 1 to 5 per 100,000 in the developed world, and from 10 to 25 per 100,000 in developing countries (Reido, F. et al., J. Ped. Infect. Dis., 14, pp. 643-657 [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 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 immunological specificity of the capsular polysaccharide (Frasch, C. et al., Rev. Infect. Dis., 7, pp. 504-510 [1985]). Of these thirteen serogroups, five cause the majority of meningococcal 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.

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 subepithelial tissue of the nasopharynx. Carriage of meningococci can last for months. Spreading of meningococci occurs by direct contact or via air droplets. Meningococci Sbecome invasive by passing through the mucosal epithelium via phagocytic vacuoles as a Sresult of endocytosis. Host defense of invasive meningococci is dependent upon complementmediated bacteriolysis. The serum antibodies that are responsible for complement-mediated Sbacteriolysis are directed in large part against the outer capsular polysaccharide.

Vaccines based on meningococcal polysaccharide have been described which elicit an Simmune response against the capsular polysaccharide. These antibodies are capable of 00 complement-mediated bacteriolysis of the serogroup specific meningococci. The 0 meningococcal polysaccharide vaccines are shown to be efficacious in children and adults P (Peltola, et al., New Engl. J. Med., 297, pp. 686-691 [1997]; and Artenstein, M. et al., 0 10 New Engl. J. Med., 282, pp. 417-420 [1970]), but the efficacy is limited in infants and young children (Reingold, A. et al., Lancet, 2, pp. 114-118 [1985]). Subsequent doses of the polysaccharide in younger populations elicited a weak or no booster response (Goldschneider, et al., J. Infect. Diseases, 128, pp. 769-776 [1973] and Gold, et al., J. Infect. Diseases, 136, S31-S35 [1977]). The duration of protection elicited by the meningococcal polysaccharide vaccines is not long lasting, and has been estimated to be between 3 to 5 years in adults and children above four years of age (Brandt, B.L. and Artenstein, J. Infect.

Diseases, 131, pp. S69-S72 [1975], Kyhty, et al., J. Infect. Diseases, 142, pp. 861-868 [1980], and Cessey, et al., J. Infect. Diseases, 167, pp. 1212-1216 [1993]). For children from one to four years old the duration of protection is less than three years (Reingold, et al., Lancet, 2,pp. 114-118 [1985]).

Polysaccharides are incapable of binding to the major histocompatibility complex molecules, a prerequisite for antigen presentation to and stimulation of T-helper lymphocytes, they are T-cell independent antigens. Polysaccharides are able to stimulate B lymphocytes for antibody production without the help of T-helper lymphocytes. As a result of the Tindependent stimulation of the B lymphocytes, there is a lack of memory induction following immunization by these antigens. The polysaccharide 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.

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 C,1 activated by helper T cells specific for peptides that are a part of the conjugated carrier Sprotein. The T-helper response to the carrier protein serves to augment the antibody production to the polysaccharide.

The serogroup B polysaccharide has been shown to be poorly to non-immunogenic in the human population (Wyle, el al., J. Infect. Diseases, 126, pp. 514-522 [1972]).

SChemical attachment of this serogroup polysaccharide to proteins has not significantly altered 00 Sthe immune response in laboratory animals (Jennings, H.J. and Lugowski, Immunol., 0 127, pp. 1011-1018 [1981]). The reason for the lack of immune response to this serogroup polysaccharide is thought to arise from structural similarities between the serogroup B O 10 polysaccharide and polysialylated host glycoproteins, such as the neural cell adhesion molecules.

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.

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 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 ofN. meningilidis and provides no protection against infection by other serogroups.

Thus, a need exists for a meningococcal conjugate vaccine capable of conferring broad, longlived 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 which immunogenic polysaccharides from the major pathogenic serogroups of N. meningitidis have been converted to T-dependent antigens through conjugations to carrier proteins.

FDA licensure of vaccines for meningococcal polysaccharides has been based on bactericidal assays with baby rabbit complement (SBA-BR) performed on blood samples of those immunized with the licensed vaccine. A number of government and expert panels have published current requirements and recommendations for assessing meningococcal polysaccharide vaccines on such assays.

SLicensure is granted because human immunity to meningococcal disease has been shown to correlate well with the level of complement-mediated bactericidal antibody detected by the Serum Bactericidal Assay (SBA) (Goldschneider, et al., J. Exp. Med., 129:1307- 1326 [1969] and Goldschneider, et al., J. Exp. Med., 129:1327-1348 [1969]). A surrogate level of a 1:4 SBA titer against serogroup C has been established using a human complement Sin the assay (SBA-H). However, licensing requirements for meningococcal polysaccharide 00 vaccines are based on the induction of serum bactericidal responses using baby rabbit 0 complement (SBA-BR) as the source of complement in the assay (World Health Organization.

t 1976. Requirements for meningococcal polysaccharide vaccine. World Health Organization technical report series, no. 594. World Health Organization, Geneva, Switzerland (WHO 1976). According to this recommendation, the antibody titers of the sera from at least 90% of subjects vaccinated with meningococcal polysaccharide vaccine should show a 4-fold or greater rise 2-4 weeks after immunization when tested against the following target strains or equivalent strains: Al for serogroup A, Cll for serogroup C, S-1975 for serogroup Y, and S- 4383 for serogroup W-135 (WHO 1976, WHO 1981, Bureau of Biologics, Food and Drug Administration July 17, 1985). The Bureau of Biologics adopted the WHO recommendation and the meningococcal polysaccharide vaccines, groups A and C combined and groups A, C, Y, and W-135 combined, are licensed in the United States based upon this requirement. In order to facilitate interlaboratory comparisons of the bactericidal activity induced by meningococcal vaccines, a standardized SBA using baby rabbit complement (SBA-BR) is established through a multilaboratory study (Maslanka et al., Clin. Diagn. Lab.

Immunol., 4:156-167 (1997).

As data from meningococcal conjugate C vaccines started to become available, concerns began to emerge that the use of rabbit complement in the assay may lead to falsely high SBA titers. Following a March 1999 meeting to clarify and resolve issues relating to the laboratory assay for the analysis of human serum for meningococcal serogroups A and C specific antibodies, the WHO Expert Committee on Biological Standardization recommended that the SBA with baby rabbit complement be used for measuring antibody responses to serogroup C (The World Health Organization.1999. Standardization and validation of serological assays for the evaluation of immune responses to Neisseria meningitidis serogroup A/C vaccines. Geneva, WHO/V&B/99.19 (WHO 1999)). In an effort to avoid overestimating 4 protection using baby rabbit complement, the WHO recommended that a study be undertaken 1 to correlate the threshold titers measured by the SBA assay using baby rabbit complement relative to SBA titers measured using human complement. A follow-up meeting is held and results presented to support a general conclusion that a SBA titer of 1:8 using baby rabbit complement correlates with an absence of protection against serogroup C and that an SBA titer of >=1:128 using baby rabbit complement correlates well to the protective SBA titer of 00 1:4 using human complement. No information is provided for corresponding correlate SBA- O BR titers for other meningococcal serogroups, such as A, Y or W-135 or for polysaccharide conjugates.

SBA titers between 1:8 and 1:64 using baby rabbit complement do not necessarily correlate well with the protective SBA titer of 1:4 using human complement (Jodar, et al., Biologicals, 30:323-329 [2002]). The WHO Expert Committee recommended that post vaccination SBA-BR titers of 1:8, 1:16, 1:32 and 1:64 be reassessed using human complement. Other measures to resolve the uncertainties of the SBA-BR titers of 1:8, 1:16, 1:32, and 1:64 included the assessment of four-fold rise in antibody SBA titers between preand post-vaccination. Demonstration of memory as a correlate of protection is also offered, however the Expert Committee recognized that the available data for these surrogates are either inadequate or limited.

An SBA-BR titer higher than 1:8 is a better indicia of human immunity to meningococcal disease, as is a four-fold rise or higher, of SBA-BR titer from preimmunization to post-immunization period of about 15 to about 45 days after immunization.

In one embodiment, the present invention provides a method of immunizing a human patient with a multivalent meningococcal polysaccharide conjugate composition, wherein the human patient has a serum SBA-BR titer of 1: 16 or higher, preferably, of 1:32 or higher, and more preferably, 1:64 or higher, and even more preferably, 1:128 or higher. In still further embodiments, the present invention provides a method of immunizing a human patient with a meningococcal polysaccharide conjugate composition, wherein the human patient has fourfold rise, or higher, in antibody SBA titers between pre- and post-vaccination.

In still another embodiment, the present invention provides a method of providing immunity to a human patient against multiple serogroups ofN. meningococcal by immunizing the human patient with a multivalent meningococcal polysaccharide conjugate composition, CK, wherein the composition comprises two or more polysaccharides selected from N.

w meningococcal serogroups A and W-135; Y and W-135; C and Y; C and W-135; A, C and Y; A,C and W-135; C,Y and W-135; A,Y and W-135; and A,C,Y and W-135.

In still further embodiments, the present invention provides a method of providing immunity to a human patient against multiple serogroups of N. meningococcal and by Simmunizing the human patient with a multivalent meningococcal (purified) polysaccharide 00 conjugate composition, wherein the polysaccharide is derivatized to less than 100,000 Daltons.

O In one embodiment of the invention, the purified polysaccharide is depolymerized to an ("1 Saverage polysaccharide size of about 5,000 to about 75,000 Daltons; preferably, to an average polysaccharide size of about 7,000 to about 50,000 Daltons; more preferably, to an average polysaccharide size of about 8,000 to about 35,000 Daltons; even more preferably, to an average polysaccharide size of about 12,000 to about 25,000 Daltons. In one embodiment of the invention, the average polysaccharide size in the composition is about 15,000 to about 22,000 Daltons.

SUMMARY OF THE INVENTION The present invention provides a method of providing human immunity to meningococcal disease caused by pathogenic Neisseria meningitidis by administration of immunological compositions of meningococcal polysaccharide-protein conjugates.

In one embodiment of the invention, the immunological composition comprises two or more protein-polysaccharide conjugates, wherein each of the conjugates comprises a capsular polysaccharide from N. meningitidis conjugated to a carrier protein. In a preferred embodiment, the immunological composition comprises two or more distinct proteinpolysaccharide conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a different serogroup of N. meningilidis conjugated to a carrier protein.

The present invention provides a method of providing human immunity to meningococcal disease caused by pathogenic Neisseria meningitidis comprising administration of an immunological composition comprising two or more distinct proteinpolysaccharide conjugates, wherein each of the conjugates comprises a capsular polysaccharide from a different serogroup ofN. meningitidis conjugated to a carrier protein.

l The present invention provides a method of providing human immunity to meningococcal disease caused by pathogenic Neisseria meningilidis comprising administration of meningococcal polysaccharide-protein conjugates. The present invention c, provides multivalent meningococcal vaccines comprised of immunologically effective amounts of from two to four distinct protein-polysaccharide conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and 00 wherein each capsular polysaccharide is selected from the group consisting of capsular O polysaccharide from serogroups A, C, W-135 and Y. The present invention further provides a method of inducing an immunological response to capsular polysaccharide of N. meningitidis O 10 comprising administering an immunologically effective amount of the immunological composition of the invention to a human. In one embodiment, the multivalent meningococcal vaccine comprises immunologically effective amounts of two distinct protein-polysaccharide conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and wherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W-135 and Y, more preferably, comprises capsular polysaccharides A and W-135, A and Y, C and W-135, C and Y, and W-135 and Y. In one embodiment, the multivalent meningococcal vaccine comprises immunologically effective amounts of three distinct protein-polysaccharide conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and wherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W-135 and Y, more preferably, comprises capsular polysaccharides A, C and W-135, A, C and Y, C, Y and W-135, C, W-135 and Y, and A, W-135 and Y. In another embodiment, the multivalent meningococcal vaccine comprises immunologically effective amounts of four distinct protein-polysaccharide conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and wherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W-135 and Y.

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.

C1 The present invention provides a multivalent meningococcal vaccine comprised of Simmunologically effective amounts of from two to four distinct protein-polysaccharide conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and wherein each capsular polysaccharide is selected from the group consisting of capsular polysaccharide from serogroups A, C, W-135 and Y.

SThe present invention provides a method of protecting a human or animal susceptible 00 to infection from N. meningitidis comprising administering an immunologically effective dose 0 of the vaccine of the invention to the human or animal.

SIn still further embodiments, the present invention provides methods of boosting the response elicited following a first, second, third, etc., dose of a meningococcal vaccine or immunogenic composition. In some of these embodiments, the first (or more) dose of the meningococcal vaccine or immunogenic composition comprises capsular polysaccharides from one or more serogroups of N. meningitidis (serogroups A, B, C, Y, W-135, etc.). In some other of embodiments, the first (or more) dose of the meningococcal vaccine or immunogenic composition comprises capsular polysaccharides from one or more serogroups of N. meningitidis serogroups A, B, C, Y, W-135, etc.) conjugated to one or more carriers carrier proteins). In preferred embodiments, the carrier proteins are immunogenic and/or provide additional therapeutic or other benefits. In some preferred embodiments, a primary response in a subject a human) elicited after one or more doses of a meningococcal vaccine or immunogenic composition is boosted by one or more subsequent doses of a vaccine or immunogenic composition comprising capsular polysaccharides from 1, 2, 3, 4, or more, different serogroups of N. meningitidis. The present invention is not limited however to administration of one or more priming doses of a vaccine or immunogenic composition comprising non-conjugated capsular polysaccharides, nor is the present invention intended to be limited to the administration of one or more boosting doses comprising conjugated capsular polysaccharide-carrier protein immunogenic compositions or vaccines. Likewise, the present invention is not intended to be limited by the temporal spacing of the priming or boosting doses.

In some embodiments, the present example provides methods of administering the disclosed tetravalent C, Y, and W-135) meningococcal diphtheria toxoid conjugate composition (formulated as for example a human vaccine) in subjects who had previously Sreceived one or more does of a monovalent meningococcal conjugate vaccine composition 4) serotype A, B, C, Y, and/or W-135). The present methods are not intended to be limited, by the subject's age at the time they received either the monovalent meningococcal conjugate vaccine or one or more subsequent doses of the disclosed tetravalent immunological composition. For example, in certain embodiments, suitable subjects for the present administration methods and regimes include, but are not limited to, infants, toddlers, children, 00 71 adolescents, teenagers, and adults. In some preferred embodiments, suitable subjects include O from about 1 month to 55 year olds, or from about 12 month to 20 year olds. In particularly preferred embodiments, suitable subjects include 24 month to 5 year olds.

0 10 The present invention provides, in some preferred embodiments, methods for (Nl administering an immunogenic composition to a subject comprising, providing: a subject, wherein said subject has been vaccinated with a dose of a monovalent meningococcal conjugate vaccine; a tetravalent meningococcal polysaccharide protein conjugate composition; and administering said tetravalent meningococcal polysaccharide protein conjugate composition to said subject at least twelve months after said was vaccination with said monovalent meningococcal conjugate vaccine.

In additional embodiments, the present invention provides methods for administering an immunogenic composition to a subject comprising, providing: a subject, wherein said subject has been vaccinated with a dose of a monovalent meningococcal conjugate vaccine; a tetravalent meningococcal polysaccharide protein conjugate vaccine; and administering said tetravalent meningococcal polysaccharide protein conjugate vaccine to said subject at least twelve months after said was vaccination with said monovalent meningococcal conjugate vaccine.

Still additional embodiments provide methods for administering an immunogenic composition to a subject comprising, providing: a subject, wherein said subject has been vaccinated with a protective antigen against N. meningilidis serogroup C; a tetravalent meningococcal polysaccharide protein conjugate vaccine; and administering said tetravalent meningococcal polysaccharide protein conjugate vaccine to said subject at least twelve months after said was vaccination with said monovalent meningococcal conjugate vaccine.

Other embodiments of the present invention provide methods for administering an immunogenic composition to a subject comprising, providing: a subject, wherein said subject c has been vaccinated with a monovalent N. meningitidis serogroup C meningococcal vaccine; a Stetravalent meningococcal polysaccharide protein conjugate composition; and administering said tetravalent meningococcal polysaccharide protein conjugate vaccine to said subject at least twelve months after said was vaccination with said monovalent meningococcal conjugate vaccine.

Yet another embodiment provides a method of preventing meningococcal disease in a 00 subject comprising administering to said subject a therapeutically effective dose of a O tetravalent meningococcal conjugate vaccine about twelve months after said subject has been vaccinated with a monovalent meningococcal conjugate vaccine.

0 10 In addition to the compositions and methods already described, the present invention further provides a method of preventing meningococcal disease in a subject comprising administering to said subject a therapeutically effective dose of a tetravalent meningococcal conjugate vaccine from about 1 to 120 months after said subject has been vaccinated with monovalent N. meningitidis serogroup C meningococcal vaccine.

Still further embodiments of the present invention provide methods of stimulating and/or inducing an immunologic response in a subject comprising administering to said subject a therapeutically effective dose of a tetravalent meningococcal conjugate vaccine at least twelve months after said subject has been vaccinated with at least one dose of a monovalent meningococcal conjugate vaccine one or more of serogroups A, C, B, Y, and/or W-135). In some embodiments, a suitable subject preferably a mammal, and more preferably a human) has received concomitant administrations of one or more monovalent polysaccharide-protein conjugated vaccines or polysaccharide non-conjugated vaccines comprises a protective antigen against N. meningitidis bacteria or a disease caused by N. meningitidis.

In certain embodiments, the compositions of the present invention comprise tetravalent meningococcal conjugate vaccines formulated as sterile 0.5ml liquid doses in an acceptable administration device such as a syringes single use), nasal injections, aerosolizers, nebulizers, transdermal devices, and the like.

Various embodiments of the present invention provide compositions of from about 0.5ml of a multivalent tetravalent) meningococcal conjugate vaccine comprising from about 0.01 Vig to about 10 pg of purified capsular polysaccharides from one or more serogroups N N meningitidis purified capsular polysaccharides from one or more of the serogroups A, S C, Y, or W-135) Additional embodiments of the present invention provide combination vaccine C compositions comprising one, two, three, or four distinct meningococcal proteinpolysaccharide conjugates and one or more antigens or immunogenic components from other vaccine compositions licensed vaccines). In still other embodiments, the present 00 invention provides immunogenic compositions comprising one, two, three, or four distinct O protein-polysaccharide conjugates and one or more antigens or antigenic components from vaccine licensed vaccines) compositions. The present invention contemplates, but is not O 10 limited to, combinations of from one to four distinct N. meningitidis protein-polysaccharide conjugates and immunogenic components antigens) from vaccines directed to preventing or ameliorating the effects of diseases not caused by N. meningitidis. For example, in some embodiments, the present invention provides immunogenic compositions comprising one, two, three, or four distinct protein-polysaccharide conjugates and one or more components from a licensed vaccine typically administered to travelers including, but not limited to, vaccines against typhoid Typhim yellow fever YF-VAX®), polio vaccine, and the like.

For example, in still other embodiments, the present invention provides combinations of from one to four distinct N. meningitidis protein-polysaccharide conjugates and immunogenic compositions from vaccines directed to preventing or ameliorating the effects of hepatitis A, B, C, D, or small pox and vaccinia, AIDS, tuberculosis, diphtheria, haemophilus influenzae, measles, mumps, pertussis, pneumococcal diseases, polio, rubella, tetanus, varicella, adenovirus diseases, anthrax, cholera, encephalitis Japanese, tick-borne, etc.), plague, rabies, typhoid, herpes, malaria, parasitic diseases, poxvirus diseases, respiratory syncytial virus diseases, rotavirus, alpha virus infections Venezuelan, Western, or Eastern equine encephalitis, Chikungunya virus disease, Ross River virus disease, etc.), Bunyavirus diseases Rift Valley fever, Hantavirus disease), Arenavirus diseases Junin virus disease), rickettsial diseases Q fever), tularemia diseases, brucellosis diseases, pseudomonas diseases, botulism, West Nile disease, and staphylococcus infections, among other viral, bacteria, and parasitic based diseases. In preferred embodiments, the non- N. meningitidis immunogenic compositions administered in conjunction concomitantly) c with the distinct N. meningitidis protein-polysaccharide conjugates of the present invention are Sderived from licensed vaccines.

The present invention is not limited to any particular concomitant combination of vaccines or immunogenic compositions, nor to methods of using any such concomitant combinations. Indeed, the present invention also provides combinations of from one to four distinct N. meningitidis protein-polysaccharide conjugates and immunogenic components from 00 unlicensed experimental) non-N. meningitidis vaccines or immunogenic compositions.

O Administration of combinations of one to four distinct N. meningitidis proteinpolysaccharide conjugates and immunogenic components from non-N. meningitidis vaccines O 10 or immunogenic compositions can be concomitant. In some embodiments, the concomitant administration of from one to four distinct N. meningitidis protein-polysaccharide conjugates and immunogenic components non-N. meningitidis vaccines or immunogenic compositions is accomplished at two or more sites using similar or dissimilar administration methods. In still some other of these embodiments, concomitant administration of from one to four distinct N.

meningitidis protein-polysaccharide conjugates and immunogenic components from non-N.

meningitidis vaccines or immunogenic compositions is accomplished by physically combining two or more compositions into a single aggregate composition which is then administered in one or more sites using or more administration routes.

Administration of a combination of from one to four distinct N. meningitidis proteinpolysaccharide conjugates and immunogenic components from non-N. meningitidis vaccines or immunogenic compositions can be carried out at two different times temporally separated by seconds, minutes, hours, days, etc. The present invention is not intended to be limited by the routes and/or timing of administration events.

In still other embodiments, the present invention provides methods of administering to a subject a human) one or more immunological compositions a human vaccine), such that the first immunological composition primes subject for a booster effect following administration of a second (or more) immunological composition. In certain of these embodiments, the first, second, third, or more, immunological compositions comprises a protein. Suitable protein carriers include, but are not limited to, diphtheria toxin, diphtheria toxoid, CRMI 97 tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal 12 Ssurface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpit 4 hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), and the like. In a preferred embodiment, the carrier protein comprises a diphtheria toxin, diphtheria toxoid, CRM 1 97 tetanus toxoid, exotoxin A, or outer membrane complex c (OMPC). In a particularly preferred embodiment, the carrier protein comprises a diphtheria toxin or diphtheria toxoid.

00 Additional embodiments provide for the use of at least one of the compositions S contemplated by the present invention in the treatment, prevention, or attenuation of a disease state in a mammal. In certain of these embodiments, the disease state is caused by a bacterial infection, for example, an infection caused by one or more serogroups of Neisseria meningitidis.

Still further embodiments provide for the use of at least one of the compositions contemplated by the present invention for the manufacture of a medicament for a the therapeutic application such as the treatment, prevention, or attenuation of a disease state in a mammal caused by bacterial infection of the mammal by one or more serogroups of Neisseria meningitidis.

In still other embodiments, the compositions of the present invention are provided in a kit a commercial package) with instructions for administration and/or storage of the contents of the kit ampoules, syringes, vials, patches, suppositories, salves, etc.

comprising meningococcal vaccine or meningococcal vaccine components). As used herein, the term "instructions for administering said immunologic composition (or a human vaccine) to a subject" includes instructions for using the compositions contained in a kit for prophylaxis of meningococcal infection of a disease caused by N. meningitidis. In some embodiments, the instructions further comprise a statement of the recommended or usual dosages of the compositions contained within the kit pursuant to 21 C.F.R. §201 et seq., and/or those of other applicable U.S. or international regulatory agencies. Additional information concerning labeling and instruction requirements applicable to the methods and compositions of the present are available at the Internet web page of the U.S.F.D.A.

As used herein, the term "third party" refers to any entity engaged in selling, warehousing, distributing, or offering for sale the compositions or methods of the present invention.

cl All patents, patent applications, and other publications recited herein are hereby Sincorporated by reference in their entirety except where they are in conflict with the disclosed invention.

(N

DESCRIPTION OF THE FIGURES Figures 1-8 show certain embodiments of the present invention.

00 0 DETAILED DESCRIPTION OF THE INVENTION C The present invention comprises an immunological composition of two or more

O

CN 10 distinct protein-polysaccharide conjugates, wherein each of the conjugates comprises a Scapsular polysaccharide conjugated to a carrier protein. Thus, the present invention includes CN compositions that comprise two or more different derivatized capsular polysaccharides conjugated to one or more carrier protein(s).

Capsular polysaccharides can be prepared by standard techniques known to those of skill in the art. In the present invention capsular polysaccharides prepared from serogroups A, C, W-135 and Y of N. meningitidis are preferred.

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.

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 mild oxidative conditions.

Native meningococcal polysaccharide is about 500,000 to 1,500,000 daltons. The present invention is directed to meningococcal polysaccharides of a smaller size. When purifying native polysaccharides, a certain percentage of the polysaccharides will be of a smaller size. However, to obtain a better yield, it is generally preferred to depolymerize, or derivatize the native meningococcal polysaccharide to a preferred size range, preferably less than 100,000 daltons. In one embodiment of the invention, the purified polysaccharide is depolymerized to an average polysaccharide size of about 5,000 to about 75,000 daltons; preferably, to an average polysaccharide size of about 7,000 to about 50,000 daltons; more cl preferably, to an average polysaccharide size of about 8,000 to about 35,000 daltons; even Smore preferably, to an average polysaccharide size of about 12,000 to about 25,000 daltons.

In one embodiment of the invention, the average polysaccharide size in the composition is Sabout 15,000 to about 22,000 daltons.

The depolymerization or partial depolymerization of the polysaccharides may then be followed by an activation step. By "activation" is meant chemical treatment of the 00 polysaccharide to provide chemical groups capable of reacting with the carrier protein. A O preferred activation method involves treatment with adipic acid dihyrazide in physiological saline at pH for approximately two hours at 15 to 30 deg. C. One process for activation is described in U.S. Pat. No. 5,965,714.

Once activated, the capsular polysaccharides may then be conjugated to one or more carrier proteins. 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 polysaccharides from serogroups A, C, W-135 and Y of N.

meningitidis are each separately conjugated to the same carrier protein species.

Carrier proteins may include bacterial toxins such as diphtheria toxin, inactivated bacterial toxins such as diphtheria toxoid, CRM 1 97 tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesin 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 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 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. An alternative carrier protein is Protein D which is an outer membrane surface exposed protein of H. influenza.

In one embodiment of the invention, the average ratio of each derivatized polysaccharide to carrier protein is about 1:1 to about 1:20 In a preferred embodiment of the invention, the average ratio of total derivatized polysaccharide to carrier protein is about S1:2 to about 1:10 and an even more preferred average ratio of each derivatized Spolysaccharide to carrier protein is about 1:2 to about 1:6 In a more preferred embodiment of the invention, the average ratio of total derivatized polysaccharide to carrier protein is about more preferably, even more preferably, 1:(4±0.25) After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates may be purified (enriched with respect to the amount of 00 polysaccharide-protein conjugate) by a variety of techniques. One goal of the purification step O is to remove the unbound polysaccharide from the polysaccharide-protein conjugate. One method for purification, involving ultrafiltration in the presence of ammonium sulfate, is O 10 described in U.S. Pat. No. 6,146,902. Alternatively, conjugates can be purified away from 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, P. W. Anderson, et. al. (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 derivatized 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 composition comprising derivatized capsular polysaccharides from serogroups A and C of N.

meningitidis separately conjugated to diptheria toxin or toxoid. More preferably the present invention is a tetravalent immunological composition comprising capsular polysaccharides from serogroups A, C, W-135 and Y of N. meningitidis separately conjugated to diptheria toxin or toxoid.

The present invention is directed, in part, to a composition of multicomponent, derivatized polysaccharide conjugates where each derivatized polysaccharide is present in about 0.5 to about 15 ig per dose. Thus, the composition may comprise a total derivatized polysaccharide pig of 1 ag to 60 jg. In a preferred embodiment, the relative amount of each derivatized polysaccharide in the composition is about equal within 50%; more preferably, within 30%; even more preferably, within 16 cN Preparation and use of carrier proteins, and a variety of potential conjugation procedures, are b 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 C information readily available in the general literature. Guidance can also be obtained from any one or all of the following U.S. patents, the teachings of which are hereby incorporated in their entirety by reference: U.S. Pat. Nos. 4,356,170; 4,619,828; 5,153,312; 5,422,427 and 00 5,445,817.

O Alternatively, the immunological compositions may be made by either culturing two or more N. meningilidis serogroups together and copurifying, depolymerizing, activating and conjugating the polysaccharides, or by culturing purifying the N. meningitidis serogroups separately and combining two or more purified polysaccharides before or after any of the steps of depolymerizing, activating and conjugating the polysaccharides.

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 also contain one or more adjuvants. Adjuvants include, by way of example and not limitation, aluminum adjuvants aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate or combinations thereof), Freund's Adjuvant (Complete or Incomplete), BAY, DC-chol, pcpp, monophoshoryl lipid A, CpG, QS-21, cholera toxin and formyl methionyl peptide. See, Vaccine Design, the Subunit and Adjuvant Approach, 1995 (M.

F. Powell and M. J. Newman, eds., Plenum Press, The adjuvant is preferably an aluminum adjuvant, such as aluminum hydroxide or aluminum phosphate.

Alternative adjuvants include oil-in-water emulsion formulations for example MF59 as described in PCT Publ. No. WO 90/14837), SAF, containing 10% Squalane, 0.4% Tween 5% pluronic-blocked polymer LI21, and thr-MDP, RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM); saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, 17 1 Mass.) may be used or particles generated there from such as ISCOMs (immunostimulating P complexes); cytokines, such as interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF).

In one embodiment of the invention, the protein-polysaccharide conjugates have an average 0glycosylation ratio (polysaccharide to protein ratio) of about 0.05 to about 2; more preferably, 00 an average ratio of about 0.08 to about 1.25; and even more preferably, an average ratio of 0 about 0.1 to about 0.9. In one preferred embodiment, the protein-polysaccharide conjugates have an average glycosylation ratio polysaccharide to protein ratio of about 0.2 to about 0.8; 0 10 more preferably, an average ratio of about 0.2 to about 0.6, and even more preferred embodiment, an average ratio of about 0.3 to about As demonstrated below, the vaccines and immunological compositions according to the invention elicit a T-dependent-like immune response in various animal models, whereas 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-like immune responses to N. meningitidis 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 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 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 between about 0.02 pg to about 5 pg per kg body weight. In a preferred composition and method of the present invention the dosage is between about 0.1 iag to 3 l.g 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 depend on the bacterial load at the time of diagnosis. Multiple injections administered over a period of days could be considered for therapeutic usage.

CK, The multivalent conjugates of the present invention can be administered as a single Sdose or in a series 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 CI currently recommended for other vaccines to prevent childhood diseases.

The booster dose will generate antibodies from primed B-cells, an anamnestic response. That is, the multivalent conjugate vaccine elicits a high primary following a 00 single administration of vaccine) functional antibody response in younger populations when Scompared to the licensed polysaccharide vaccine, and is capable of eliciting an anamnestic response following a booster administration), demonstrating that the protective immune response elicited by the multivalent conjugate vaccine of the present invention is long-lived.

Compositions of the invention can include liquid preparations for orifice, oral, nasal, anal, vaginal, peroral, intragastric, mucosal perlinqual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration injectable administration), such as sterile suspensions or emulsions. Intravenous and parenteral administration are preferred. Such compositions may be in admixture with a suitable carrier, diluent, or 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, pH- 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", 1985, or later additions, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

In one embodiment of the invention, a preferred route of administration is intramuscular or subcutaneous, with intramuscular route preferred. Administration may be by injection or by an alternative delivery device.

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 p-I. If digestive tract absorption is preferred, compositions of the invention can be in the "solid" form of pills, tablets, capsules, caplets and the like, including 19 cK1 "solid" preparations which are time-released or which have a liquid filling, gelatin Scovered 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 Sdispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size.

00 Liquid preparations are normally easier to prepare than gels, other viscous O compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, 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 compositions, 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.

In a preferred embodiment of the invention, the vaccine composition is formulated as a sterile liquid, pyrogen-free, phosphate-buffered physiological saline, with or without a preservative. In one preferred embodiment, the formula per dose, comprises about 0.3 to about mg sodium phosphate and about 3.5 to about 6.0 mg sodium chloride and up to 1.5 mL water. In one preferred embodiment, the formula per dose, comprises about 0.6 0.2 mg sodium phosphate and 4.4 0.2 mg sodium chloride and up to about 0.5 0.2 mL water.

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, liquid dosage for whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, methylcellulose), colors and/or flavors may also be present. The compositions can be isotonic, it can have the same osmotic pressure as blood and lacrimal fluid.

rcl The desired isotonicity of the compositions of this invention may be accomplished Susing sodium tartrate, propylene glycol or other inorganic or organic solutes. In one embodiment, the preferred isotonicity of the composition is obtained from sodium phosphate Ci or sodium chloride, or mixtures thereof. Sodium chloride is preferred particularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected level using a 00 Spharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is O readily and economically 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 concentration 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.

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 agent selected.

Those skilled in the art will recognize that the components of the compositions must be selected to be chemically inert with respect to the N. meningitidis polysaccharide-protein carrier conjugates.

The invention will be further described by reference to the following illustrative, nonlimiting examples setting forth in detail several preferred embodiments of the inventive concept. Other examples of this invention will be apparent to those skilled in the art without departing from the spirit of the invention.

The following abbreviations and Trademarks are: ACIP, Advisory Committee on Immunization Practices; AE, Adverse Event; Cetavalon

T

cetyltrimethylammonium bromide, CTAB; CFR, Code of Federal Regulations; CRF, Case Report Form; DTP, Diphtheria Tetanus Pertussis; ELISA, Enzyme Linked Immunosorbent Assay; FDA, Food and Drug Administration; GCP, Good Clinical Practice; GMC, Geometric Mean Concentration; GMT, 21 c Geometric Mean Titer; IgG, Immunoglobulin G; IgGI, Immunoglobulin G subclass 1; IgG2, Immunoglobulin G subclass 2; IgM, Immunoglobulin M; ICH, International Conference on Harmonization; IND, Investigational New Drug; IRB, Institutional Review Board; MenA/C- 'I Dt Bivalent (A and C) Meningococcal Polysaccharide Diphtheria Conjugate Vaccine; MenPS, Meningococcal group specific polysaccharide; mL milliliter; Menomune'

M

licensed Meningococcal A,C Y and W-135 polysaccharide vaccine; OD, Optical Density; PBS, 00 SPhosphate Buffered Saline; SAE, Serious Adverse Event; SBA, Serum bactericidal activity; O SBA-BR, Serum bactericidal activity assay performed using baby rabbit complement; SBA- HC, Serum bactericidal activity assay performed using human complement; SIDS, Sudden Infant Death Syndrome; TetraMenD, Tetravalent C, Y, and W-135) Meningococcal Polysaccharide Diphtheria Conjugate Vaccine; Td, Tetanus and Diphtheria vaccine; UAE, Unexpected Adverse Experience; URI, Upper Respiratory Infection; pg, Micrograms.

EXAMPLES

Example 1 Preparation of Neisseria meningitidis Serogroups A, C, W-135, and Y Purified Capsular Polysaccharides Powders Crude Paste Preparation Separately, Neisseria meningitidis serogroup A, C, W-135, and Y wet frozen seed cultures are thawed and recovered 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 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 containing 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. After the incubation period, the contents of the fermentor vessel are transferred to a 500 L tank, Cetavlon"M is added, and the material mixed for I hours. The Cetavlon treated growth is centrifuged at approximately 15,000 to 17,000 x g at a flow rate of approximately 30 to 22 (liters per hours. The crude polysaccharide is precipitated from the supernatant with a second Cetavlon' TM precipitation. CetavlonTM is added to the supernatant and the material mixed for at least 1 hour at room temperature. The material is stored at 1 to 5 deg. C. for 8 to 12 hours. The precipitated polysaccharide is collected centrifugation at approximately 45,000 to 50,000 x g at a flow rate of 300 to 400 ml per minute. The collected paste is stored at -60 deg. C. or lower until further processed.

00 O Purified Polysaccharide Powder Preparation 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 extract. A second volume of 0.9 M calcium chloride is added to the paste, and blended to yield a homogeneous 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 ultrafiltration using 10-30 kDa MWCO spiral would ultrafiltration units.

Magnesium chloride is added to the concentrated, and the pH adjusted to 7.2 to using sodium hydroxide. DNase and RNase are added to the concentrate, and incubated at to 28 deg. C. with mixing for 4 hours. Ethanol is added to a concentration of 30 to Precipitated nucleic acid and protein are removed by centrifugation at 10,000 x g for 2 hours.

The supernatant is recovered and the polysaccharide precipitated by adding ethanol to and allowing it to stand overnight at 1 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 washed with alcohol. The polysaccharide is washed with 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 the polysaccharide-enzyme mixture, and placed on a platform shaker at I to 5 deg. C. for approximately 30 minutes. The mixture is centrifuged at 23 cN 10,000 x g for 15 to 20 minutes. The upper aqueous layer is recovered and saved. An equal Svolume of sodium acetate-phenol solution is added to the aqueous layer, and extracted as above. A total of four extractions are performed to remove protein and endotoxin from the C polysaccharide solution. The combined aqueous extracts are diluted up to ten fold with water for injection, and diafiltered against 10 volumes of water for injection. Calcium chloride is added to the diafiltered polysaccharide. The polysaccharide is precipitated overnight at I to 00 deg. C. by adding ethanol to 80%. The alcohol supernatant is withdrawn, and the O polysaccharide collected by centrifugation at 10,000 x g for 15 minutes. The purified polysaccharide is washed two times with ethanol, and once with acetone. The washed powder is dried under vacuum in a desiccator. The dried powder is stored at -30 deg. C. or lower until processed onto conjugate.

Example 2 Depolymerization of Neisseria meningitidis serogroups A,C, W135, and Y Purified Capsular Polysaccharide Powder Materials used in the preparation include purified capsular polysaccharide powders from Neisseria meningitidis serogroups A, C, W-135, and Y (prepared in accordance with Example sterile 50 mM 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).

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 mM sodium acetate buffer, pH 6.0 is added to the polysaccharide to yield a concentration of g polysaccharide per liter. The polysaccharide solution is allowed to mix at 1 to 5 deg. C.

for 12 to 24 hours to effect solution. 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 polysaccharide solution is heated to 55 deg.

An aliquot of 30% hydrogen peroxide is added to the reaction 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 polysaccharide over time. Every 15 to 20 minutes, aliquots are removed from the reaction mixture and injected onto a HPSEC column to measure the molecular size of the 24 cN polysaccharide. When the molecular size of the polysaccharide reached the targeted molecular Ssize, the heating unit is turned off and the polysaccharide solution rapidly cooled to 5 deg. C.

by circulation through an ice water bath. The depolymerized 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 concentrated depolymerized polysaccharide solution is diafiltered against 10 volumes of sterile physiological saline (0.85% 00 Ssodium chloride). The depolymerized polysaccharide is stored at I to 5 deg. C. until the next process step.

The molecular size of the depolymerized polysaccharide is determined by passage through a gel filtration chromatography column sold under the tradename "Ultahydrogel.TM.250" that is calibrated using Dextran molecular size standards and by multi-angle laser light scattering. The quantity of polysaccharide is 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 Svennerholm, L. (1955) Biochimica Biophysica Acta 24, pp 6 0 4 6 1 1. The O-acetyl content is determined by the method of Hesterin, S. (1949) Journal of Biological Chemistry 180, p249. Reducing activity is determined by the method of Park, J. T. and Johnson, M. J. (1949 Journal of Biological Chemistry 181, pp 14 9 15 1 The structural integrity of the depolymerized polysaccharide is determined by protein .sup.lH and .sup.13C NMR.

The purity of the depolymerized polysaccharide is determined by measuring the LAL (endotoxin) content and the residual hydrogen peroxide content.

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, C, W-135, and Y from Neisseria meningitidis (prepared in accordance with Example 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 I M sodium chloride, and sterile physiological saline (0.85% sodium chloride).

rCl Each serogroup polysaccharide is derivatized in a separate reaction. A stainless steel °P tank is charged with the purified depolymerized polysaccharide, and diluted with sterile 0.85% physiological saline to achieve a final reaction concentration of 6 g polysaccharide per liter.

To this solution is added a concentrated 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 concentrated aliquot dissolved in sterile 0.85% 00 physiological saline, to achieve a reaction concentration of Ig per liter. The pH is adjusted to and this pH is maintained for 2 hours using sterile IN hydrochloric acid and sterile IN sodium hydroxide at room temperature (15 to 30 deg. After two hours, a concentrated O 10 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. for 44 hours.+-.4 hours while maintaining the pH at Following this reaction period, the pH is adjusted to and the derivatized polysaccharide is concentrated to 12 g polysaccharide per liter by connecting the reaction tank to a ultrafiltration unit equipped with a 3000 MWCO regenerated cellulose cartridges. The concentrated derivatized polysaccharide is diafiltered against 30 volumes of I M sodium chloride, followed by 10 volumes of 0.15 M sodium chloride. The tank is disconnected from the ultrafiltration unit and stored at 1 to 5 deg. C. for 7 days. The tank is reconnected to an ultrafiltration unit equipped with 3000 MWCO regenerated cellulose cartridges, and diafiltered against 30 volumes of I M sodium chloride, followed by 10 volumes of 0.15 M sodium chloride.

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 depolymerized 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, pp 2 82 2 8 8 The structural integrity of the derivatized polysaccharide is determined by proton 'H and 3

C

NMR. The purity of the derivatized polysaccharide is determined by measuring the level of unbound hydrazide, the LAL (endotoxin) content, and the residual cyanoborohydride content.

S Example 4 Preparation of Carrier Protein Preparation of Crude Diphtheria Toxoid SProtein Lyophilized seed cultures are reconstituted and incubated for 16 to 18 hours. An aliquot from the culture 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 00 culture flask is incubated at 34.5 to 36.5 deg. C. on a rotary shaker for 14 to 22 hours. The O cultures from the 4-liter flask are used to inoculate a fermenter containing growth media. The ('i fermenter is incubated at 34.5 to 36.5 deg. C. for 70 to 144 hours. The contents of the 10 fermenter are filtered through depth filters into a collection vessel. An aliquot of formaldehyde ('i solution, 37% is added to the harvest to achieve a concentration of 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 solution, 37%, is added to each 20 liter bottle to achieve a concentration of 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 7 days on a shaker. An aliquot of formaldehyde solution, 37%, is added to each 20 liter bottle to achieve a concentration of The p-I 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 1 to 5 deg. 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 liter bottles are combined into a purification tank. T'he 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 1 hours, and is then filtered through a depth filter cartridge into a second purification tank. Solid ammonium sulfate is added to the filtrate to achieve 70% of saturation. The pl-I 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 washed 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 p-I is adjusted to 6.8 to 7.2, and ammonium sulfate is added to 40% of saturation. The pH of the solution is 27 cl adjusted to 7.0 to 7.2, and the solution is allowed to stand for 16 hours. The precipitate is b removed by filtration and discarded. Ammonium sulfate is added to the filtrate to 60% of saturation, 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 remove undissolved protein, and diafiltered against 0.85% physiological saline.

00 O Concentration and Sterile Filtration of the Purified Diphtheria Toxoid Protein The protein solution is concentrated to 15 g per liter and diafiltered against 10 volumes O 10 of 0.85% physiological saline suing a 10,000 MWCO regenerated cellulose filter cartridge.

The concentrated protein solution is sterilized by filtration through a 0.2 micron membrane.

The protein solution is stored at I to 5 deg. C. until processed onto conjugate.

The protein concentration is determined by the method of Lowry, O. H. et. al (1951) Journal of Biological Chemistry 193, p265-275. The purity of the protein is measured by sterility, LAL (endotoxin) content, and residual formaldehyde content.

Example 5 Preparation of Monovalent Conjugates of Neisseria meningitidis Serogroups A, C, W-135, and Y Polysaccharide to Diphtheria Toxoid Protein Materials used in this preparation include adipic acid derivatized polysaccharide from Neisseria meningitidis serogroups A, C, W-135, and Y (prepared in accordance with Example sterile diphtheria toxoid protein (prepared in accordance with Example EDAC, ammonium sulfate, sterile IN hydrochloric acid, sterile IN sodium hydroxide, and sterile physiological saline 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 derivatized polysaccharide at a reaction concentration of 700 to 1000 .mu.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. Physiological saline 0.85%, is used to dilute the starting materials to the target reaction concentrations and the pH is adjusted to I. An aliquot of EDAC is added to the polysaccharide protein mixture to achieve a reaction concentration of 2.28 to 2.4 g per liter. The pH of the reaction is kept at for 2 hours 28 c at 15 to 30 deg. C. After two hours, the pH is adjusted to using sterile IN sodium Shydroxide, and the reaction is stored at 1 to 5 deg. C. for 16 to 20 hours.

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. Solid ammonium sulfate is added to 60% of saturation (for serogroups A, W-135 and Y) and 50% of saturation (for serogroup The conjugate reaction mixture is diafiltered 00 I against 20 volumes of 60% of saturated ammonium sulfate solution (for serogroups A, W-135 O and Y) and 50% of saturated ammonium sulfate solution (for serogroup followed by 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 capsule containing a 0.22 micron filter.

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 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 is measured by multi-angle laser light scattering. The antigenic character of the conjugate is measured by binding to antipolysaccharide serogroup specific antibody using double-sandwich ELISA method. The purity of the conjugates is determined by measuring the amount of unbound (unconjugated) polysaccharide by elution 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 a Multivalent Meningococcal A, C, W-135, and Y Polysaccharide Diphtheria Toxoid Conjugate Vaccine Materials used in this preparation include, serogroups A, C, W-135, and Y polysaccharide-diphtheria toxoid conjugates that are prepared in accordance with Example sterile 100 mM sodium phosphate buffered physiological saline (0.85% sodium chloride).

cl An aliquot of sterile 100-500 mM sodium phosphate buffered physiological saline is added to physiological saline in a stainless steel bulking tank to yield a final vaccine concentration of 10 mM sodium phosphate. An aliquot of each of from two to four of the sterile monovalent meningococcal polysaccharide-diphtheria toxoid conjugates is added to the bulking tank containing 10 mM sterile sodium phosphate physiological saline to yield a final concentration of 8 pg of each serogroup polysaccharide per milliliter of buffer. The formulated 00 Stetravalent conjugate is mixed and filtered through a 0.2 .mu.m filter into a second bulking tank.

The quantity of each serogroup polysaccharide present in the multivalent formulation is determined by component saccharide analysis using high pH anion-exchange chromatography with pulsed amperometric detection. The quantity of protein is measured by the method of Lowry. Th pH of the vaccine is measured using a combination electrode connected to a pH meter. The antigenic character of the multivalent conjugate vaccine is measured by binding to anti-polysaccharide serogroup specific antibody using a doublesandwich ELISA method. Immunogenicity of the multivalent conjugate vaccine is measured the ability of each conjugate present in the vaccine to elicit both a primary and booster antipolysaccharide IgG immune response in an animal model. The purity of the multivalent conjugate vaccine is determined by measuring the amount of unbound (unconjugated) polysaccharide using high pH anion-exchange chromatography with pulsed 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 Preparation of conjugate adsorbed to aluminum hydroxide. Materials used in this preparation include serogroups A, C, W-135, and Y polysaccharide-diphtheria toxoid conjugates that are prepared in accordance with Example 5, sterile physiological saline (0.85% sodium chloride), and sterile aluminum hydroxide in physiological saline (0.85% sodium chloride).

An aliquot of each of the sterile monovalent meningococcal polysaccharide diphtheria toxoid conjugates is added to the bulking tank containing physiological saline to yield a final concentration of 8 pg of each serogroup polysaccharide per milliliter of buffer. An aliquot of c1 sterile aluminum hydroxide in physiological saline (0.85% sodium chloride) is added to the Smultivalent conjugate vaccine to achieve a final concentration of 0.44 mg aluminum ion per milliliter vaccine.

Example 8 Preparation of Aluminum Phosphate-adjuvanted Conjugate 0Materials used in this preparation include serogroups A, C, W-135, and Y 00 polysaccharide-diphtheria toxoid conjugates that are prepared according to Example 5, sterile 0 physiological saline (0.85% sodium chloride), and sterile aluminum phosphate in physiological saline (0.85% sodium chloride).

An aliquot of each of the sterile monovalent meningococcal polysaccharide-diphtheria toxoid conjugates is added to the bulking tank containing physiological saline to yield a final concentration of 8 pg of each serogroup polysaccharide per milliliter of buffer. An aliquot of sterile aluminum phosphate in physiological saline (0.85% sodium chloride) is added to the multivalent conjugate vaccine to achieve a final concentration of 0.44 mg aluminum ion per milliliter vaccine.

Example 9 General Description of Materials and Methods Used in Human Clinical Studies Immunogenicity of a Tetravalent Derivatized Conjugate Vaccine The conjugate vaccine is studied for its ability to elicit an immune response in humans under a number of different clinical protocols. The following studies summarize the results.

The materials and methods used in each of the following studies, unless indicated otherwise, are: TetraMenD TetraMenD vaccine comprises four meningococcal capsular polysaccharides of serogroups A, C, Y, and W-135, 4 p.g of each polysaccharide, covalently attached to a total of 48 p.g diphtheria toxoid protein. The vaccine is formulated in sterile, pyrogen-free, phosphatebuffered physiological saline, with no preservative. The formula comprises 0.6 mg sodium phosphate, 4.4 mg sodium chloride and up to 0.5 mL water.

CN Menomune® ;Z Menomune® is licensed in the United States and elsewhere for use among persons aged 2 years and older. Menomune is a freeze-dried preparation, each dose of vaccine containing c- 50 p.g of each A, C, Y and W-135 polysaccharide as antigens, reconstituted with a diluent of isotonic sodium chloride solution preserved with thimerosal and given subcutaneously as a mL dose. Each 0.5 mL dose of vaccine contains 2.5 mg to 5 mg of lactose as a stabilizer.

00 SMenomune® A/C/Y/W-135, Meningococcal Polysaccharide Vaccine, Groups A, C, Y, and Cc O W-135 Combined, for subcutaneous use, is a freeze-dried preparation of the group-specific polysaccharide antigens from Neisseria meningitidis, Group A, Group C, Group Y, and Group O 10 W-135. The diluent is sterile, pyrogen-free, distilled water. After reconstitution of the lyophilized product with diluent as indicated on the label, each 0.5 mL dose is formulated to contain 50 pg of "isolated product" from each of the serogroups A, C, Y, and W-135 in isotonic sodium chloride solution.

Tetanus and Diphtheria Toxoids Adsorbed for Adult Use® (referred to subsequently as Td) Is a sterile suspension of alum precipitated toxoid in an isotonic sodium chloride solution containing sodium phosphate buffer to control pH. The vaccine is for intramuscular injection. Each 0.5mL dose is formulated to contain 5Lf of tetanus toxoid, 2Lf of diphtheria toxoid and not more than 0.28 mg of aluminum by assay. Tetanus and diphtheria toxoids induce at least 2 units and 0.5 units of antitoxin per mL respectively in the guinea pig potency test. At visit 1, Td is administered to all participants as a single 0.5mL dose by intramuscular injection using a one inch 25 gauge needle into the deltoid of the left arm. Each 0.5mL dose contains 5Lf of tetanus toxoid and 2Lf of diphtheria toxoid.

Sera Samples Blood specimens are drawn on the Days indicated after the baseline. For example, if the protocol indicates three time points, DO, D28 and 6 Month, then blood specimens are drawn on Day 0 prior to vaccination (baseline), at Day 28 post-vaccination (to assess primary immune response, and at 6 month post-vaccination (to assess the longevity of the immune Tl response). Approximately 5 mL of whole blood is collected from each subject at each time Spoint. The whole blood is centrifuged within four hours of collection. The serum is removed and stored at -20 deg. C. A 'Day 28' blood sample is taken at least 28 days but not yet 57 days after the Day 0 injection. A "6 month" blood sample is taken at 6 months plus or minus 28 days after the Day 0 injection. Thus, a Day 28 sera represents a sera is drawn between day 28 to day 56 after DO; and a 6 month sera represents a sera is drawn between day 149 to day 217 after DO.

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Assay Techniques The present studies utilize a number of standard immunological assays. The following descriptions summarize the methodologies used herein. However, other similar assays, including variations of those presented herein, are well known by those in the art and may be utilized.

Anti-Meningococcal Antibody Determination by a Serum Bactericidal Assay using Baby Rabbit Complement (SBA-BR) Functional antibody activity for anti-meningococcal antibody to serogroups A, C, Y, and W-135 is measured using a serum bactericidal assay. Two-fold dilutions of test sera are prepared in sterile 96-well microtiter plates. Serogroup specific meningococcal bacteria along with baby rabbit complement are added to the serum dilutions and allowed to incubate. After this incubation period, an agar overlay medium is added to the serum/complement/bacteria mixture, allowed to harden, and then incubated overnight at 37 0 C with 5% CO 2 Bacterial colonies present in the wells are counted. The endpoint titer is determined by the reciprocal serum dilution yielding 50% killing as compared to the mean of the complement control wells. The limit of detection for this assay, using rabbit complement, is a titer of 8.

IgG Anti-Meningococcal Antibody Determination IgG antibody activity for anti-meningococcal antibody to serogroups A, C, Y, and W- 135 is measured using an indirect ELISA. This procedure involves reacting antibody in sera with excess meningococcal group specific polysaccharide (MenPs) antigen adsorbed to plastic microtiter wells by methylated human serum albumin. The amount of bound antibody is oi determined by a reaction with peroxidase-labeled mouse anti-human IgG specific monoclonal antibody. A subsequent reaction using peroxidase substrate generates a chromogenic product that is measured spectrophotometrically. The resulting optical density (OD) correlates with the amount of IgG antibody in the serum that is bound to the meningococcal polysaccharide on the microtiter plate. The amount of IgG antibody is then calculated by comparison to a 00 reference (Lot CDC 1992 or equivalent) with an assigned value using a 4-parameter logistic curve method.

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IgM Anti-Meningococcal Antibody Determination IgM antibody activity for anti-meningococcal antibody to serogroups A, C, Y, and W- 135 is measured using an indirect ELISA. This procedure involves reacting antibody in sera with excess MenPs antigen adsorbed to plastic microtiter wells by methylated human serum albumin. The amount of bound antibody is determined by a reaction with peroxidase-labeled mouse anti-human IgM specific monoclonal antibody. A subsequent reaction using peroxidase substrate generates a chromogenic product that is measured spectrophotometrically. The resulting OD correlates with the amount of IgM antibody in the serum that is bound to the meningococcal polysaccharide on the microtiter plate. The amount of IgM antibody is then calculated by comparison to a reference (Lot CDC 1992 or equivalent) with an assigned value using a 4-parameter logistic curve.

High Avidity Anti-Meningococcal IgG Antibody Determination High avidity IgG antibody activity for anti-meningococcal antibody to serogroups A, C, Y, and W-135 will be measured at Aventis Pasteur Inc. using a modified ELISA. This assay is currently under development at Aventis Pasteur Inc. and will be qualified prior to testing of clinical specimens. Briefly, 96 well microtiter plates are coated with MenPs antigen. After aspirating and ishing the coated plates, serial dilutions of clinical sera are prepared directly in the plates, using phosphate buffered saline (PBS) serum diluting buffer containing 75 mM ammonium thiocyanate, and allowed to incubate overnight. The amount of bound antibody is determined by a reaction with peroxidase-labeled mouse anti-human IgG specific monoclonal antibody. A subsequent reaction using peroxidase substrate generates a 34 l chromogenic product that is measured spectrophotometrically. The resulting OD correlates tb with the amount of high avidity IgG antibody in the serum that is bound to the meningococcal polysaccharide on the microtiter plate. The amount of high avidity IgG antibody is then calculated by comparison to a reference (Lot CDC 1992 or equivalent) using a 4-parameter logistic curve.

00 SIgGI and IgG2 Subclass Meningococcal Antibody Determination O IgGI and lgG2 subclass antibody distribution for anti-meningococcal antibody to serogroups A, C, Y and W-135 is measured using an ELISA. Antibody present in serial O 10 dilutions of sera is reacted with MenPs antigen adsorbed to the wells of microtiter plates. The amount of bound antibody will be determined using anti-human IgGI Fc or IgG2 Fc specific reagents. A subsequent reaction with enzyme substrate generates a chromogenic product that is measured spectrophotometrically. The resulting OD correlates with the amount of IgGI or IgG2 antibody in the serum that is bound to the meningococcal polysaccharide on the microtiter plate. The amount of antibody will be reported as the IgGl:IgG2 ratio in the serum specimen or as the concentration of IgGI or lgG2 in the specimen if a suitable reference is available.

Anti-Diphtheria Antibody Determination By Metabolic Inhibition of VERO Cells Anti-diphtheria antibody responses are measured by the ability of the test sera to protect VERO cells from a diphtheria toxin challenge. Using sterile 96-well microtiter plates, two-fold dilutions of test sera, beginning with a 1:4 dilution, are challenged with diphtheria toxin and allowed to incubate. VERO cells are then added, the wells sealed with sterile mineral oil and incubated for six to eight days. Antibody levels are then determined by observing a color change of the pH indicator in the media resulting from the byproducts of cell metabolism. Results are reported as International Units/mL by comparison to a calibrated WHO reference serum and determined by the highest serum dilution that allows cell metabolism in the presence of the challenge dose of diphtheria toxin. The lower limit of detection is determined by the minimum detectable antitoxin level of the reference serum, and the starting dilution of the test sera, and is typically 0.005 IU/mL.

c1 Anti-Tetanus Antibody Determination By Elisa SAnti-Tetanus antibody levels are determined by an indirect Enzyme Linked Immunosorbent Assay (ELISA). The method involves reacting antibody in test sera with tetanus toxoid adsorbed to plastic microtiter wells. The amount of bound antibody is determined by a reaction with Goat Anti-Human IgG-specific antibody conjugated to alkaline phosphatase. A subsequent reaction with alkaline phosphatase substrate generates a 00 chromogenic product that is measured spectrophotometrically. The OD (optical density) O correlates with the amount of antibody in the serum dilution that binds to the antigen coated microtiter plate. The antibody concentration is calculated by comparison to an international O 10 human reference (WHO Lot TE-3) with assigned unitage by a Parallel Line Analysis method.

Results are reported as International Units per milliliter (IU/mL). The minimum level of quantitation for the anti-tetanus IgG ELISA is 0.01 IU/mL, with samples resulting in values lower than this level reported as 0.01 IU/mL.

Adverse Event (AE) is defined as "any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and that does not necessarily have a causal relationship with this treatment. An adverse event can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product." (ICH guidelines, GCP (E6) Serious Adverse Experience (SAE) is "any adverse drug experience occurring at any dose that results in any of the following outcomes: death, a life-threatening adverse drug experience, inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant disability/incapacity, or a congenital anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgement, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse." (21 CFR Ch. 1, §312.32(a)).

C Unexpected Adverse Experience (UAE) is "any adverse drug experience, the specificity or severity of which is not consistent with the current investigator's brochure; or, if ;an investigator brochure is not required or available, the specificity or severity of which is not consistent with the risk information described in the general investigational plan or elsewhere in the current application, as amended." (21 CFR Ch. 1, §312.32(a)).

The studies conducted in accordance with standard clinical practice, and the criteria for 00 enrollment or exclusion of patients in the studies are: SInclusion criteria for patients: I. Participant is healthy, as determined by medical history and physical examination.

2. Participant is at least 11 years of age but not yet 19 years of age at the time of vaccination.

3. Parent/guardian or participant has signed Institutional Review Board (IRB) approved informed consent form where applicable.

4. Participant has signed Institutional Review Board (IRB) approved assent form where applicable Exclusion criteria for patients: I. Serious chronic disease cardiac, renal, neurologic, metabolic, rheumatologic, etc.).

2. Known or suspected impairment of immunologic function.

3. Acute medical illness with or without fever within the last 72 hours or an oral temperature 38 0 C (100.4 0 F) at the time of inclusion.

4. History of documented invasive meningococcal disease or previous meningococcal vaccination.

Administration of immune globulin, other blood products within the last 3 months, or oral or injected corticosteroids or other immunomodulatory therapy within 6 weeks of the study vaccine. Individuals on a tapering dose schedule of oral steroids lasting 7 days may be enrolled in the trial as long as they have not received more that one course within a two week period prior to enrollment.

6. Antibiotic therapy within the 72 hours prior to vaccination 37 cl 7. Received any vaccine in the 28-day period prior to enrollment, or scheduled to receive Sany vaccination in the 28-day period after enrollment, except where the study notes additional vaccinations.

8. Suspected or known hypersensitivity to any of the vaccine components.

9. Unavailable for the entire study period or unable to attend the scheduled visits or to 0comply with the study procedures.

10. Enrolled in another clinical trial.

11. Any condition which, in the opinion of the investigator, would pose a health risk to the Sparticipant or interfere with the evaluation of the vaccine.

C1 10 12. In females, a positive or equivocal urine pregnancy test at the time of vaccination.

Example 10 Study A -Dosage Study Study A is an unblinded, open-label, dose-escalation trial of three dosage levels of TetraMenD vaccine, administered to participants in three age groups. Ninety healthy adults (18 to 55 years of age) are enrolled in Stage I and received a single injection of TetraMenD vaccine. Thirty healthy children (12 to 22 months of age) are enrolled in Stage II and received 2 injections of a single dosage level of TetraMenD vaccine. Ninety healthy infants (6 to 12 weeks of age) are enrolled in Stage III and received 3 injections of a single dosage level of TetraMenD vaccine.

Stage IDosage Study in Adults 18 to 55 Years This clinical trial is an unblinded, open-label, dose-escalation trial of three dosage levels of TetraMenD vaccine, which is administered to participants in three age groups. In Stage 1, ninety healthy adults (18 to 55 years of age) receive a single injection of TetraMenD vaccine.

For adult participants, serum specimens for serologic analysis are obtained at baseline (day 0) prior to TetraMenD administration, and at day 28 after TetraMenD administration. All available specimens are analyzed for SBA against meningococcal polysaccharide serogroups A, C, Y, and W-135, and by ELISA for IgG antibody against these same serogroups. The SBA and IgG ELISA findings for all serogroups are summarized below.

CN One key immunogenicity endpoint is the proportion of participants with a 24-fold rise t from baseline. To determine what effect the baseline SBA titer had on the proportion with a >4-fold rise in SBA, a subgroup analysis is performed for adults whose baseline titer for each Cl particular antigen is less than 1:64, and adults whose baseline titer for that particular antigen is at least 1:64.

SThe safety profile of TetraMenD is comparable to that of Menomune®. The results of this Study are summarized in the following Tables.

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0~ Table A-I: Stage I (Adults)- Distribution of SBA Titers at Baseline (Day 0) by Tetr~ enD Dosge L veln* of Participants with Titer Result§ I <8 to 512 Serogroup dosage level <8 32 512 SBA 1fg 26 114 9 (15.4) (34.6) 4p28 2 1 3 1 2 3 1~g 2 (10.7) (10.7) 27 I 1 2 2 2 6 (22.2) ,SBA

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I t 6 15 1 1 6 I t 6(57.7) (23.1)(38 4A28 18 1 1 3 2 4i28(64.3) (10.7) (7.1) 1 g 27 17 1 2 2 3 1 g 27(63.0)1___ (11. 1) SBA I Ag 26 15 1 4 2 3 1 (15.4) (11.5) (3.8) 4g28 15 4 3 3 1 1 4g28(53.6) 1-(14.3) 10.7) (10.7) (3.6) Pg 27 10 1 2 2 6 2 2 1(22.2) 1(7.4) SBA (W-135) 1 Ag 26 18 1 1 I 1 1 1 (69.2) (3.8) 4 Ag 28 13 8 2 1 1 I I (28.6) (3.6) jig 19 (70.4) (3.7) 2 (7.4) (3.7) (3.7) (3.7) (3.7)

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Table A-I: Stage I (Adults)- Distribution of SBA Titers at Baseline (Day 0) by TetraMenD Dosage Level 11* ofPriiat with Titer Result§ Serogroup dosage level N

DO

1024 2048 4096 8192 16384 32768 65536 I Ag 26 (23.1) (3.8) 4 Ag 28 5 10 1 (17.9) (3 5.7) 5 6 2 ftg 27 (22.2) 1 jig 26 4 Ag 28 3 (10.7) 10Rg27 I 4 Ag 28 jig 27 SBA 1 jig 26 jig (3.7) Table A-2: Serogroup dosage leve Stage I (Adults) Distribution of SBA Titers at Day 28Pot Injection, by TetraMenD Dosage Level (Per-Protocol Population) In* of Participants with Titer ResuWt 8 o 512

I

N 8 1 8 116 132 164 1128 1256] 512 SBA In26 1 1 2 3.8) (7.7) 4 pig 278 SBA 3 4 1 ttg 26 1 .5 (15.4) (19.2) 4192 2 -11 4jig28 (3.6) 10fg2 2 1 4 ig27 (14.8) SBA 4 1 4 5 4 1 2 1 jig 26 (1 5.4) (15.4) (19.2) (15.4) (7.7) 4 pg 28 3 14 1 1 3 (14.3) (10.7) 1 g27 3 -2 5 1 4 Ijg27 (11.1) (18 (14.8) SBA (W-135) I t 6 3 -2 1 1 4 I i 6 (11.5) (15.4) 4 tg 28 4 1 2 3 (14.3)1_ (10.7) pig 2 (7.4) 2 (7.4) 2 (7.4) Table A-2: Stage I (Adults) Distribution of SBA Titers at Day 28Pot Injection, by TetraMenD Dosage Level (Per-Protocol Population) I of Participants with Titer Result§ 1024-65536 Serogroup dosa2e level N D28 1024 2048 4096 8192 16384 32768 65536 SBA 1__ 7 6 7 2 I jig 26 (26.9) (23.1) (267.9 (7.7) 1 5 88 26 11 1 4g28 (17.9) (28.6) (39.3) (3.6) 7 1 1 4 5 13 2 1 g 27 (14.8) (18.5) (48.1) (3.7) SBA 6 2 1 I jig 26 (1 9.2) (23.1) 4P28 6 7 4 4 2 4i28(21.4)1(25.0) (14.3) (14.3) (7.1) 7 3 5 4 2 jig 27 (11.1) (18.5) (14.8) (18.5) SBA IP26 1 31 I ji 26 (11.5) (3.8) 49 28 4 6 1 2 2 4g28(14.3) (21.4) 7.1) (7.1) toP 7 1 4 4 2 1 jig 27 1(14.8) (14.8) SBA (W-135) 1P26 3 9 2 1 1 jig 26 (11.5) (34.6) 3 10 4 1 4jig 28(10 .7)1(35.7) (14.3) (36 jig 7 (25.9) (3.7) 5 (18.5) 8 (29.6) Table A-3: Stage I (Adults) Proportions Achieving SBA Thresholds at Baseline and at Day 28 Post-Injection, By TetraMenD Dosage Level (Per-Protocol Pnnul:tion 00 Sero- Achieving Threshold group NDO/N >1:8 1:16 >_1:32 ___1:64 dosage 8 Day 0 Day Day Day Day 0 Day level 8Day 0 D 28 28 28 SBA (A) I jg 26/26 96.2 100.0 92.3 100.0 92.3 100.0 88.5 100.0 4 tg 28/28 92.9 100.0 92.9 100.0 92.9 100.0 89.3 100.0 pg 27/27 100.0 100.0 96.3 100.0 96.3 100.0 92.6 100.0 SBA (C) I tg 26/26 42.3 88.5 42.3 88.5 42.3 88.5 38.5 88.5 4 pig 28/28 35.7 100.0 32.1 96.4 32.1 96.4 28.6 96.4 fg 27/27 37.0 100.0 37.0 100.0 33.3 96.3 33.3 96.3 SBA (Y) I n 26/26 42.3 84.6 38.5 80.8 23.1 80.8 15.4 65.4 4 pg 28/28 46.4 89.3 46.4 85.7 46.4 85.7 32.1 71.4 fg 27/27 63.0 88.9 59.3 88.9 51.9 88.9 44.4 81.5 SBA (W- 135) 1 tg 26/26 30.8 88.5 26.9 88.5 26.9 88.5 23.1 80.8 4 pg 28/28 53.6 85.7 25.0 85.7 17.9 85.7 14.3 85.7 tg 27/27 29.6 100.0 25.9 100.0 18.5 100.0 18.5 100.0 N: number of evaluable participants at each time point (day 0; day 28) Table A-4: Stage I (Adults) SBA and IgG ELISA Results at Baseline and at Day 28 Post-Injection, by TetraMenD Dosage Level (Per-Protocol Population) Serogroup ND0 GMT/GMC* (95% CI) Mean Fold T >4-Fold dosage level /ND 28 Day 0 Day 28 at Day 28 (95% CI) 1 SBA (A) 26/26 460.2 3054.9 6.6 1g 26/2 (223.0-949.7) (1872.9-4982.9) (2.9-15.4) 65.4 487.3 4g83 6720.2 13.8 4 pg 28/28 (231.2- (4666.5-9677.7) (6.0-31.7) 71.4 1027.2) pig 27/27 525.3 (286.6-962.9) 10865.1 (7651.5- 15428.2) 20.7 (11.7-36.6) 96.3 Table A-4: Stage I (Adults) SBA and IgG ELISA Results at Baseline and at Day 28 Post-Injection, by TetraMenD Dosage Level (Per-Protocol Population) 00 Table A-4: Stage I (Adults) SBA and IgG ELISA Results at Baseline and at Day 28 Post-Injection, by TetraMenD Dosage Level (Per-Protocol Population) Day 0: Baseline blood sample drawn prior to vaccination.

Day 28: Blood sample drawn 28 days following vaccination.

4-fold rise: the percent of adults who had a 4-fold rise in GMT at day 28 in comparison to day 0.

N: number ofevaluable participants *GMTs are computed for the SBA data; GMCs are computed for the IgG ELISA data.

Table A-5 presents the GMT by dose, patient age and serogroup.

Table A-5 GMT by Dose, patient age and Serogroup Age Dose No. of Blood A GMT C GMT W GMT Y GMT TetraMenD Subjects Day 19 1 pg 2 0 1024.00 512.00 45.25 8.00 1 ftg 2 28 2896.31 2048.00 2048.00 362.04 4 pg 2 0 2048.00 64.00 16.00 181.02 4 pug 2 28 8192.00 8192.00 2048.00 2048.00 tg 10 0 1097.50 34.30 21.11 128.00 tg 10 28 18820.27 3821.70 2352.53 1176.27 1 pg 3 0 322.54 10.08 10.08 12.70 1 pg 3 28 3251.00 203.19 2048.00 203.19 4 ug 2 0 512.00 4.00 4.00 4.00 4 ug 2 28 5792.62 128.00 22.63 22.63 Rg I 0 128.00 4.00 4.00 4.00 ug 1 28 16384.00 4096.00 128.00 2048.00 21 1 pg 2 0 1024.00 22.63 32.00 256.00 I pg 1 28 8192.00 8192.00 2048.00 1024.00 4 g 3 0 812.75 12.70 4.00 256.00 4 g 3 28 10321.27 1024.00 5160.64 8192.00 22 1 Ag 2 0 1024.00 4.00 11.31 16.00 1 pg 2 28 4096.00 362.04 2048.00 512.00 4 g 2 0 128.00 45.25 5.66 16.00 4 g 2 28 8192.00 2896.31 1024.00 2048.00 jig 2 0 2048.00 90.51 4.00 22.63 jig 2 28 16384.00 8192.00 512.00 512.00 23 1 jg -1 0 512.00 2048.00 2048.00 16.00 1 ttg 1 28 512.00 2048.00 2048.00 64.00 4 pg 2 0 1448.15 4.00 4.00 45.25 4 g 2 28 4096.00 1448.15 1024.00 256.00 jig 1 0 1024.00 128.00 4.00 64.00 jLg 1 28 16384.00 512.00 8192.00 2048.00 24 4 Ag 2 0 181.02 11.31 90.51 4.00 4 g 2 28 11585.24 2896.31 1448.15 512.00 jig 1 0 1024.00 4.00 4.00 4.00 jAg 1 28 16384.00 2048.00 4096.00 4.00 1 tg 1 0 128.00 4.00 4.00 4.00 1 pg 1 28 4096.00 4096.00 2048.00 32.00 jAg 2 0 1024.00 32.00 4.00 22.63 jig 2 28 8192.00 2896.31 1448.15 2048.00 26 1 pg 2 0 2048.00 4.00 64.00 4.00 1 jAg 2 28 4096.00 512.00 2896.31 1448.15 Lg 2 0 362.04 45.25 22.63 4.00 pg 2 28 5792.62 2048.00 1448.15 32.00 27 1 pg 1 0 512.00 128.00 1024.00 4.00 I pg 1 28 4096.00 1024.00 4096.00 4.00 4 tg 2 0 90.51 22.63 64.00 16.00 4 pjg 2 28 16384.00 4096.00 1448.15 90.51 Lg 1 0 512.00 4.00 128.00 64.00 10 g 1 28 8192.00 16384.00 8192.00 16384.00 28 1 g 0 4.00 4.00 4.00 4.00 1 g 28 16384.00 1024.00 2048.00 256.00 4 tg 2 0 724.08 64.00 8.00 11.31 4 pg 2 28 5792.62 1448.15 2048.00 2048.00 g 0 64.00 4.00 4.00 4.00 1g 1 28 4096.00 16.00 1024.00 4.00 4 g 1 0 32.00 4.00 16.00 32.00 4 pg 1 28 16384.00 16384.00 8192.00 4096.00 31 1 g 1 0 32.00 4.00 4.00 4.00 I pig i 28 8192.00 512.00 4.00 4.00 32 4 Lg 2 0 724.08 64.00 8.00 11.31 4 g 2 28 4096.00 1024.00 1148.15 128.00 33 1 Ag 2 0 2896.31 4.00 4.00 22.63 1 n 2 28 2048.00 2048.00 724.08 512.00 4 g 1 0 1024.00 4.00 8.00 4.00 4 pg 1 28 32768.00 1024.00 4096.00 8192.00 34 4 1Lg 1 0 64.00 128.00 4.00 32.00 4 ftg 1 28 16384.00 8192.00 2048.00 512.00 1 lg 2 0 724.08 16.00 5.66 32.00 1 fag 2 28 2896.31 362.04 724.08 181.02 4 fag 2 0 90.51 5.66 11.31 4.00 4 ftg 2 28 5792.62 5792.62 32.00 181.02 36 10 ftg 2 0 256.00 22.63 4.00 4.00 fag 2 28 11585.24 512.00 5792.62 256.00 37 1 ag 1 0 4096.00 4.00 4.00 32.00 1 pg 1 28 8192.00 2048.00 2048.00 64.00 pg 1 0 2048.00 4.00 8.00 4.00 lag 1 28 16384.00 2048.00 4096.00 64.00 39 1 2 0 512.00 4.00 45.25 4.00 1 g 2 28 5792.62 724.08 512.00 11.31 4 Ag 1 0 8192.00 4.00 8.00 4.00 4 g I 28 16384.00 2048.00 256.00 1024.00 1 pg 1 0 2048.00 4.00 4.00 4.00 I Ag 1 28 8192.00 4.00 128.00 64.00 41 1 g 2 0 724.08 128.00 4.00 11.31 I ttg 2 28 2896.31 362.04 128.00 128.00 42 4 ttg 2 0 362.04 45.25 22.63 128.00 4 ag 2 28 2048.00 4096.00 2896.31 724.08 tag 1 0 128.00 4.00 4.00 8.00 Ag 1 28 8192.00 2048.00 512.00 512.00 44 1 ftg 1 0 8.00 32.00 4.00 4.00 I pg 1 28 2048.00 512.00 4.00 8.00 4 tag 1 0 512.00 4.00 16.00 4.00 4 pig 1 28 2048.00 512.00 2048.00 8.00 4 ag 2 0 2048.00 4.00 5.66 4.00 4 tg 2 28 2896.31 128.00 4.00 16.00 fag 2 0 724.08 45.25 128.00 11.31 pg 2 28 2896.31 724.08 2896.31 181.02 46 1 1tg 1 0 2048.00 128.00 4.00 4.00 1 ttg 1 28 2048.00 256.00 4.00 4.00 47 10 ttg 1 0 8.00 4.00 4.00 16.00 ftg 1 28 1024.00 128.00 512.00 4096.00 48 1 g 1 0 128.00 512.00 4.00 16.00 I pg 1 28 128.00 2048.00 512.00 128.00 49 10 g 1 0 32.00 512.00 4.00 4.00 S_ 10 pg 1 28 8192.00 16384.00 4096.00 64.00 b) 52 10 pg 1 0 512.00 4.00 4.00 16.00 10 ug 1 28 32768.00 256.00 1024.00 32.00 54 1 pg 1 0 512.00 4.00 4.00 16.00 1 1 Ig I 28 256.00 4.00 32.00 64.00 Stage II Dosage Study in Toddlers Aged 12 Months to 22 Months 00 This clinical trial is an unblinded, open-label, dose-escalation trial of three dosage c levels of TetraMenD vaccine, which is administered to participants in three age groups. In N 5 Stage II, thirty healthy children (12 to 22 months of age) receive two injections of a single 0 dosage level of TetraMenD vaccine.

C1 For toddler participants, serum specimens for serologic analysis are obtained at three timepoints: at baseline (day 0) prior to TetraMenD injection at day 60 after enrollment days after injection #1 and immediately prior to TetraMenD injection and at day 90 after enrollment (30 days after injection All available specimens are analyzed for SBA against meningococcal polysaccharide serogroups A, C, Y, and W-135, and by ELISA for IgG antibody against these same serogroups. The SBA and IgG ELISA findings for all serogroups are summarized below. The results are summarized in the following Tables.

Table A-6: Stage 11 (Toddlers) Distribution of SBA Titers at Baseline (Day 0) by TetraMenD Dosage Level (Per-Protocol 11* of Participants with Titer Result* to 256 Serogroup ND~t dosage level 8 16 32 64 128 256 SBA I__ OR 4 2 1 1 Ijitg 9 (22.2) (11.1) (11.1) _t ig 10 (80.0) SBA 1 P9 9 ijig (100.0)__ 4 pg 8- (12.5)1 fig 10 7 2 1(70.0) (10.0) 20.0)

SBA(Y)__

1 Ag 9 4 1 1 2 1 (11. 1) (11. 1) (22.2) (11.1) 4jig 8 (62.5) (25.0) 10Pg 10 8 11 ig1 (80.0) (10.0)1 (10.0)1_ SBA (W-135) Ijig(77.8) 4 pg 8 1 (80.0) (110 (10.0 jig (80.0) (10.0) (10.0) Table A-6: Stage 11 (Toddlers) Distribution of SBA Titers at Baseline (Day 0) by TetraMenD Dosage Level (Per-Protocol ;ZPopulation)_ ii* of Participants with Titer Result§ to 65536 Serog rou 00level SBA rI fig 9 4 pig 8 Rg 10 1 SBA 4 g 8 0 g 10 SBA I1tg 9~ fg 51 Table A-7: Stage 11 (Toddlers) Distribution of SBA Titers 60 Days After the 1st Injection (Day 60), by TetraMenD Dosage Level (Per-Protocol Population) n* of Participants with Titer ResuWt to 256 Serogrou p ND 6 Ot 8 dosage level __<816 32 64 128 256 SBA I Ag 9 4 pg 8 (12.5) jig 10 -(00 SBA 1 9g 3 1 1 2 1 (33.3) (11. 1) (11.1) (22.2 (11.1) 4jig8 (12.5) (25.0)1(25.0) (12.5) 1 g10 2 1 1 2 1 ig(20.0)1 (10.0) 20.0) (10.0) SBA 1 9g 2 1 2 1 2 1 (11. 1) (22.2) (22.2) (11.1) 4 Ig 8 1 (12.5) (12.5) (37.5)1 10jg(40.0) (20.0)1(10.0) (30.0) SBA (W-135) I (62.58 (12.5) (12.5) jig (20.0) (10.0) 3 (30.0) (20.0)

I

Table A-7: Stage II (Toddlers) Distribution of SBA Titers 60 Days After the 1st Injection (Day 60), by TetraMenD Dosage Level (Per-Protocol Population) n* of Participants with Titer Result 512 to 65536 Table A-8: Stage 11 (Toddlers) Distribution of SBA Titers Days After the 2nd Injection (Day 90), by TetraMenD Dosage Level Population) jn* of Participants with Titer Result§ to 256 Serogroup ND9o t <8 8 1 32 6 18 dosage level k8 8 1 32 6 18 SBA jig 10 SBA (C) 4 Ag 8 (12.5) (25.0) (12.5) 0 (30.0) (10.0) (10.0)1 (20.0) SBA I lig 9- 32 (22.2) (11.1) 4jg (12.5)1 (25.0) (12.5) 1 g 10 1 1 3 2 2 ig 10 (10.0) (10.0) (30.0) SBA (W-135)_ I Ag 9 1 1 11 2 (1.1 (1.1)(111) (11.1) (22.2) 4 pg 8 1_ 1_ (12.5) (12.5) jig

I

(10.0) (00 3 (30.0)

I

(10.0) Table A-8: Stage 11 (Toddlers) Distribution of SBA Titers 30 Days After the 2nd Injection (Day 90), by TetraMenD Dosage Level Population) n* of Participants with Titer Result§ to 65536 Serogroup NI) 9 0 t dosage level 512 1024 2048 4096 8192 16384 32768 65536 -SBA Ijig 4jg 8 (12.5) (25.0) jig 10 2 4 4 (40.0) (40.0) -SBA 1 jig 8 4jig 8(25.0) (25.0) l0 Pg 10 2 2 (10.0) 1_ -SBA 1 jig 9 4 jig 8 2 1 1 (25.0) (12.5) (12.5) jig 10 (1 SBA (W-135) 1ji 9 2 1 (22.2)1 (11.1) 1 1__ 4 jig 81 (50.0)1(12.5) (12.5) jig (10.0) (00

I

(10.0) 2 (20.0) C1 Table A-9: Stage II (Toddlers) -Proportions Achieving SBA Thresholds at OJI Baseline, 60 Days Post-Injection and 30 Days Post-Injection by TetraMenD Dosage Level (Per-Protocol Population) Achieving Threshold Achieving Threshold SSerogroup No/ND6o/ >1:8 1:16 dosage ND90 Post- Post- B l Post- Postlevel Baseline Baseline (DO) Inj #1 Inj #2 (DO) Inj #1 Inj #2 00 (D60) (D90) (D60) e¢3 SBA Serogroup A 0 1 g 9/9/9 55.6 100.0 100.0 33.3 100.0 100.0 4 g 8/8/8 12.5 87.5 100.0 12.5 87.5 100.0 jg 10/10/10 20.0 100.0 100.0 20.0 100.0 100.0 BI Sg 9/9/9 0.0 66.7 77.8 0.0 55.6 77.8 4 Ftg 8/8/8 12.5 100.0 100.0 12.5 87.5 100.0 Ig 10/10/10 30.0 80.0 70.0 30.0 80.0 60.0 SBA Serogroup 1 Lg 9/9/9 55.6 77.8 100.0 44.4 77.8 100.0 4 g 8/8/8 37.5 87.5 100.0 12.5 87.5 100.0 pg 10/10/10 20.0 60.0 90.0 10.0 60.0 90.0 SBA Serogroup 1 Ag 9/9/9 22.2 44.4 88.9 22.2 44.4 77.8 4 g 8/8/8 12.5 37.5 100.0 12.5 37.5 100.0 Ag 10/10/10 20.0 80.0 100.0 20.0 80.0 100.0 STable A-9: Stage 11 (Toddlers) -Proportions Achieving SBA Thresholds at J Baseline, 60 Days Post-Injection and 30 Days Post-Injection by TetraMenD Dosage Level (Per-Protocol Population) Achieving Threshold Achieving Threshold Serogroup NI)o/ND60/ &dosage r- dosage ND9o0 Post- Post- Post- Postlevel Baseline Baseline Inj #1 Inj #2 Inj#1 Inj #2 0_ (D60) (D90) (DO) (D60) Serogroup I lug 9/9/9 33.3 100.0 100.0 33.3 100.0 100.0 4 g 8/8/8 12.5 87.5 100.0 12.5 87.5 100.0 S10 Pg 10/10/10 20.0 100.0 100.0 10.0 100.0 100.0 SBA Serogroup C I lg 9/9/9 0.0 55.6 66.7 0.0 44.4 66.7 4 Lg 8/8/8 12.5 87.5 100.0 12.5 87.5 100.0 flg 10/10/10 20.0 80.0 60.0 20.0 70.0 50.0 T SBA Serogroup Y Slug 9/9/9 33.3 66.7 100.0 11.1 44.4 66.7 4 ug 8/8/8 0.0 75.0 100.0 0.0 75.0 87.5 Pg 10/10/10 10.0 40.0 80.0 10.0 30.0 50.0 Serogroup 1 g 9/9/9 22.2 44.4 66.7 22.2 44.4 66.7 4 ug 8/8/8 12.5 37.5 100.0 12.5 37.5 100.0 ug 10/10/10 20.0 80.0 100.0 10.0 70.0 90.0 00 Table A-IO: Stage 11 (Toddlers) SBA and IgG ELISA Results Among Toddlers by TetraMenD Dosage Level At Baseline, 60 Days Post-Injection and Post-Injection #2 (Per-Protocol Population Sero- Mean Fold T Foldt group GMT/GMC* (95% CI) (from baseline) (from baseline) &oag .asein Post- Post-Inj Post- IPost- Post- Postlevel (DO) Inj #1 #2 Inj #1 Inj #2 Inj #1 Inj #2 (D90) (D60) j(D90) (D60) SBA 17.3 573 2389.1 SLpg 9/9/9 (3.9 (214.5 (1674.8- 34.6 138.2 100.0 100.0 77.0) 1663.1)1 3407.9) 8.7 1380 3158.4 4 pg 8/8/8 (1857.4- 152.2 362.0 75.0 87.5 55.1) 9805)5370.7) 9.2 1482 2048.0 I0 Pg 10/10/10 (1155.2- 157.6 222.9 90.0 90.0 38.7) 23.)3630.7) SBA 29.6 69.1 1 Ag 9/9/9 (14.8- 7.4 17.3 55.6 77.8 4.0) 148.0) 322.6) 6.7 117.4 304.4 4 pg 8/8/8 (37.7- (128.5 17.4 45.3 75.0 100.0 23.0) 365.3) 721.1) 97.0 68.6 l0 Pg 10/10/10 (22.9- (11.0- 12.1 8.6 80.0 50.0 18.6) 1411.4) 1 428.6) 1 SBA 10.9 34.6 174.2 Sipg 9/9/9 (11.0- (52.7- 3.2 16.0 44.4 77.8 25.4) 108.5) 575.2)_ 5.7 98.7 304.4 4 pg 8/8/8 (20.4- (100.7- 17.4 53.8 87.5 100.0 8.8) 478.0) 920.1) pg 10/10/10 5.7 (3.0- 10.6) 14.9 (6.1 36.3) 48.5 (18.9- 124.3) 50.0 80.0 Table A-10: Stage II (Toddlers) SBA and IgG ELISA Results Among Toddlers by TetraMenD Dosage Level At Baseline, 60 Days Post-Injection and Post-Injection #2 (Per-Protocol Population Sero- Mean Fold T 4 Foldt group GMT/GMC* (95% CI) (from baseline) (from NDo/ND6o/ND9O baseline) dosage Baseline Post- Post-Inj Post- Post- Post- Postlevel (DO) Inj #I #2 Inj #1 Inj #2 Inj #1 Inj #2 (D90) (D60) (D90) (D60) SBA (W-135) 9.3 20.2 101.6 1 Pg 9/9/9 (18.1 2.2 10.9 33.3 66.7 36.1) 108.7) 571.0)_ 6.2 22.6 430.5 4 pg 8/8/8 (172.2- 3.7 69.8 37.5 100.0 17.2) 184.9) 1076.3) 90.5 274.4 jig 10/10/10 (20.2- (97.9- 13.0 39.4 70.0 100.0 16.6) 406.1) 769.2) IgG ELI A 0.3 0.8 1.9 1 jig 9/9/9 (0.1 (0.3 (0.6 2.6 6.3 22.2 44.4 0.7) 1.9) 0.2 2.1 4.4 4 pg 8/8/8 (0.1 (2.1 12.4 26.1 87.5 100.0 0.4) 4.8) 9.1) 0.2 4.4 6.2 Jig 10/10/10 (0.1 23.4 33.1 100.0 100.0 0.3) 1 6.5) 9.1) 1 IgG ELIS 0.1 0.3 ijig 9/9/9 (0.1 (0.1 2.6 3.8 33.3 44.4 0.2) 0.9) 1 0.2 1.0 4 ftg 8/8/8 (0.0 (0.3 (0.6 5.6 8.3 75.0 87.5 0.7) 3.1) 3.6) jig 10/10/10 0.2 (0.1 0.3) 0.7 (0.3- 1.5) 1.2 (0.7- 60.0 70.0 Table A-10: Stage 11 (Toddlers) SBA and IgG ELISA Results Among Toddlers by TetraMenD Dosage Level At Baseline, 60 Days Post-injection and Post-Injection #2 (Per-Protocol Popula) Sero- Mean Fold T 4 Foldt group GMT/GMC* (95% CI) (from baseline) (from baseline) &oag BaselineD~o Post- Post-Inj Post- Post- Post- Postlevel (DO) lnj #1 #2 Inj #1 Inj H2 Inj #1 Inj #2 (D90) (D60) (D90) (D60) IgG ELISA 0.4 0.7 1.4 1 jig 9/9/9 1.9 3.8 11.1 33.3 1.0) 0.3 1.2 4.

4 pg 8/8/8 4.4 16.4 37.5 100.0 12.8) 0.2 0.8 1.8 jig 10/10/10 -4.3 10.2 70.0 90.0 (0.7-4.5) 1.3) IgG ELISA (W-135) 0.2 0.3 0.8 1 Pg (0.4 1.9 5.2 22.2 55.6 1.6) 01 0.6 4tg 888(0.1-0.2) (0.8 5.3 12.7 62.5 100.0 1.9) 3.1) 0.1 0.5 1.3 l0/lO/l0 4.5 11.8 60.0 80.0 1 1 1(0.8-2.2)1 1 Day 0: Baseline blood sample drawn prior to injection 1.

Day 60: Blood sample drawn 60 days following injection #1 and Day 90: Blood sample drawn 30 days following injection 112.

prior to injection #2.

!4-fold rise: Post-lnj the percentage of toddlers which had a :4-fold rise in GMT at day 60 in comparison to day 0; Post-Inj the percentage of toddlers which had a 4-fold rise in GMT at day 90 in comparison to day 0.

N: number of evaluable participants *GMTs are computed for the SBA data; GMCs are computed for the IgG ELISA data.

Table A-I I summarizes the GMT by Dose, Patient Age and Serogroup Table A-I1 Summary of GMT by Patient Age and Serogroup Age Dose No. of Blood A GMT C GMT W GMT Y GMT TetraMenD Subjects Day 12 4 tg 4 0 13.45 4.00 4.00 4.00 4 Ag 4 60 1448.15 107.63 32.00 53.82 4 ttg 4 90 4096.00 215.27 724.08 304.44 lg 6 0 16.00 12.70 10.08 4.00 lPg 6 60 1448.15 64.00 101.59 12.70 tg 6 90 2298.80 80.63 256.00 40.32 13 4 g 1 0 4.00 4.00 4.00 16.00 4 pg 1 60 4.00 64.00 4.00 128.00 4 ttg 1 90 2048.00 64.00 512.00 128.00 Ag 1 0 4.00 4.00 4.00 64.00 Ag 1 60 4096.00 256.00 512.00 4.00 Rg 1 90 4096.00 1024.00 1024.00 16.00 14 10 Ag 2 0 4.00 4.00 4.00 5.66 Ag 2 60 724.08 181.02 128.00 32.00 fag 2 90 1024.00 5.66 512.00 128.00 4 pg 2 0 90.51 4.00 4.00 5.66 4 pg 2 60 2896.31 45.25 32.00 256.00 4 tg 2 90 2896.31 362.04 181.02 362.04 16 4 Ag 2 0 4.00 4.00 4.00 4.00 4 ag 2 60 2896.31 90.51 4.00 45.25 4 pg 2 90 4096.00 256.00 256.00 128.00 18 4 g 0 4.00 256.00 128.00 8.00 4 g 60 2048.00 512.00 2048.00 1024.00 4 Ig 1 90 2048.00 1024.00 2048.00 2048.00 Pg 1 0 4.00 4.00 4.00 4.00 lPg 1 60 2048.00 128.00 4.00 32.00 Lg 1 90 2048.00 256.00 32.00 64.00 Stage III Dosage Study in Infants This clinical trial is an unblinded, open-label, dose-escalation trial of three dosage levels of TetraMenD vaccine, which is administered to participants in three age groups. In Stage Ill, ninety healthy infants (6 to 12 weeks of age) receive three injections of a single dosage level of TetraMenD vaccine.

Infant participants received TetraMenD injections at age 2 months (injection at age 4 months (injection and at age 6 months (injection Serum specimens for rCl serologic analysis are obtained at two time points: at age 6 months (2 months following injection and at age 7 months (one month after injection All available specimens are analyzed for SBA against meningococcal polysaccharide serogroups A, C, Y, and W-135, and C, by ELISA for IgG antibody against these same serogroups. The SBA and IgG ELISA findings for all serogroups are summarized below. The results are summarized in the following Tables.

00 1O Table A-13: Stage III (Infants) Distribution of SBA Titers at Age 7 months (one month post 3rd dose), by TetraMenD Dosage Level (Per-Protocol Population) n* of Participants with Titer Result§ Serogrou dosage N~<8 8 16 32 64 128 256 level SBA 2 3 2 2 4 1 1 1 g 2 (87 (17.4) (4.3) 4 jig 24 2 2 2 6 3 3 (25.0) (12.5) (12.5) 10P 1 3 2 3 3 3 1 g 21(14.3) 1(9.5) (14.3)1(14.3) (14.3) (23.8), SBA IA 23 5 1 3 2 4 5 2 1 i 3(21.7) (13.0) (17.4) (21.7) (8.7) 4 t 4 11 1 1 2 4 1 3 4g 24(45.8) 1(4.2) 1(16.7) (12.5)1 fig 21 6 1 3 3 5 2 (14.3) (14.3) (23.8) SBIA I i 3 7 2 3 4 3 2 1 I i 3(30.4) 1(13.0) (17.4) (13.0) (4.3) 4 ftg 24 8 3 5 4 3 1 (12.5) (20.8)1(16.7) (12.5) (4.2) 1 4 1 5 4 4 1 1 1jg 21 (19.0) (23.8) (19.0) (19.0) (4.8) SBA (W- 135) 1__1 1 t 3 5 1 3 3 5 3 2 iji 23(21.7) (43 (13.0) (13.0) (21.7) 13.0) E(8.7 4A 24 9 4 2 1 1 6 1 4 i 4(37.5)1(16.7), 1(25.0), (4.2) jig 5 (23.8) 3 (14.3) 3 (14.3) 4 (19.0) 3 (14.3) 2 Table A-13: Stage III (Infants) Distribution of SBA Titers at Age 7 months (one month post 3rd dose), by TetraMenD Dosage Level (Per-Protocol Population) n* of Participants with Titer ResuWt Serogrou dosage N~m' 512 1024 2048 4096 8192 16384 32768 65536 level SBA Rg 23 3 2 I jig 23(13.0) (8.7) 4 ttg 24 3 3 4jig 24(12.5) (12.5) l0 Ag 21 2 1_ SBA I Ag 23 (4.3) 4 jig 24 jg 21(4.8) SBA 1 ji 23(4.3) jig 21 (48 SBA (W- 135) 1 jig 23 jig (4.8) Ni Table A-14: Stage III (Infants) Proportions Achieving SBA Thresholds at tb! Age 6 months (pre 3 rd dose) and at Age 7 months (post 3 rd dose) by TetraMenD Dosage Level (Per-Protocol Population) Serogroup Achieving Threshold_____

N

6 m/N 7 m 1:8 1:16 >1:32 1:64 dosage 6 7 6 7 6 7 mos s mos mo 6 mos 7 mos level mos mos mos mos mos mos6o SSBA lAg 22/23 54.5 78.3 36.4 65.2 27.3 56.5 13.6 47.8 S4 Ag 23/24 69.6 91.7 69.6 91.7 56.5 83.3 30.4 75.0 r, 10 Pg 21/21 85.7 85.7 66.7 85.7 52.4 76.2 19.0 61.9 r- SBA 1 Ag 22/23 54.5 78.3 50.0 73.9 45.5 60.9 40.9 52.2 4 g 23/24 60.9 54.2 52.2 50.0 47.8 45.8 43.5 37.5 Ag 21/21 85.7 71.4 81.0 66.7 71.4 66.7 61.9 52.4 SBA I Ag 22/23 40.9 69.6 27.3 60.9 18.2 47.8 9.1 30.4 4 tg 23/24 34.8 66.7 26.1 54.2 21.7 54.2 21.7 33.3 jg 21/21 47.6 81.0 42.9 76.2 23.8 52.4 4.8 33.3 SBA 1 Ag 22/23 27.3 78.3 18.2 73.9 4.5 60.9 4.5 47.8 4 g 23/24 30.4 62.5 21.7 45.8 17.4 37.5 8.7 33.3 pg 21/21 42.9 76.2 38.1 76.2 23.8 61.9 19.0 47.6 N: number ofevaluable participants at each time point (6 months of age; 7 months of age) Table A-15: Stage III (Infants) SBA and IgG ELISA Results Among Infants At Age 6 months (pre 3 rd dose) and at Age 7 months (post 3 rd dose), by TetraMenD Dosage Level (Per-Protocol Population) Serogroup GMT/GMC* (95% CI) dosage N 6 m/N m Slevel Age 6 mos Age 7 mos SBA (A) Ig 22/23 11.3 40.7 (6.2-20.6) (17.6-94.0) 4Ig 23/24 25.1 101.6 4_g /4 (12.9-49.0) (51.9-199.0) 18.9 68.4 gg 21/21 18.9 68.4 (12.2-29.1) (32.2-145.1) Table A-15: Stage III (Infants) SBA and IgG ELISA Results Among Infants At Age 6 ;Z ~months (pre 3 ddose) and at Age 7 months (post 3 ddose), by TetraMenD Dosage Level (Per-Protocol Population) Serogroup GMT/GMC* (95% CI) dosage N 6 m/N 7 m g ms Ae o 00 ~~level Ag___s Ae o SBA 1 19.3 37.2 c-I 1 jg 22/23 (9.3-40.1) (18.6-74.5) f 24.4 19.6 4 jg 3/4 11.0-54.1) (9.4-40.6) Table A-15: Stage III (infants) SBA and IgG ELISA Results Among Infants At Age 6 months (pre Yd dose) and at Age 7 months (post 3 rd dose), by TetraMenD Dosage Level (Per-Protocol Population) IgG ELISA (W-135) 1 Itg 20/20 0.5 1.1 (0.8-1.6) 4 jg 20/21 0.5 0.9 (0.6-1.3) ig 18/19 0.9 (0.8-2.5) N: number of evaluabic participants *GMTs are computed for the SBA data; GMCs are computed for the IgO EIISA data.

Table A- 16 presents a summary of GMT by patient age and serogroup Table A-16 Summary of GMT by Patient Age and Serogroup Age (weeks) Dose No. of Blood A C W Y TetraMenD Subject Day GMT GMT GMT GMT

S

7 4, ig 1 6-12 512.00 128.00 4.00 4.00 weeks 6 month 512.00 128.00 4.00 32.00 8 1 pg 1 6-12 4.00 4.00 4.00 4.00 weeks I g16 month 132.00 16.00 16.00 4.00 4 g6 6-12 11.31 7.13 5.66 4.49 weeks 6 6 month 71.84 7.13 22.63 6.35 ptg 1 6-12 9.51 19.03 5.66 9.51 weeks pig 1 6 month 32.00 38.05 19.03 32.00 9 ijitg 13 6-12 11.02 18.78 5.51 7.58 I pg 14 6 month 155.17 55.17 40.99 22.63 4 pg 13 6-12 30.34 27.27 7.19 14.38 weeks 4 i 13 6 month 60.66 19.80 14.38 27.27 jig 1 6-12 21.53 35.33 10.25 7.61 weeks fig 1 6 month 70.66 26.25 26.25 22.63 1 Ag 10 6-12 19.70 29.86 12.13 7.46 weeks I A 10 6 month 39.40 19.70 45.25 14.93 4 pg 1 6-12 12.13 32.00 4.00 4.00 weeks 4 pg~ 1 6 month 114.04 22.63 28.51 25.40 jig 1 6-12 19.50 43.07 8.00 14.49 weeks jig 1 6 month 52.50 35.33 39.01 32.00 11 1 jg1 6-12 4.00 4.00 4.00 16.00 weeks Iji~g 1 6 month 8.00 4.00 4.00 32.00 4 jig 1 6-12 512.00 512.00 64.00 64.00 weeks 4fg1 6 month 1024.00 256.00 128.00 128.00 12 4jig 1 6-12 32.00 4.00 4.00 64.00 weeks 4aJ!-~ 1 6 month 11024.00 64.00 256.00 256.00 13 10 jig 1 6-12 512.00 45.25 64.00 22.63 weeks fig 1 6 month 724.08 190.51 256.00 181.02 C Pediatric Vaccines Administered Concomitantly with TetraMenD in Infants nInfants currently receive routine pediatric vaccinations per current ACIP recommendation and local practice. In this study, infants receive TetraMenD with pediatric q vaccinations. DTacP (Tripedia®) and Hib (ActHIB®) are administered at ages 2, 4, and 6 months. Either IPV or OPV may be given; IPV is administered with the first and second 0\ injections of TetraMenD (at ages 2 and 4 months). Hepatitis B vaccine is given per local 00 practice; hepatitis B vaccine is administered at age 2 months to some participants, but not 0 administered to any participant at ages 4 months or 6 months. During the conduct of the infant 7 stage of this trial, RotaShield® became licensed and received an ACIP recommendation for routine use. A single participant received RotaShield® at ages 4 months and 6 months in the context of this trial.

Antibody responses to routinely administered pediatric vaccine antigens are assessed at age 6 months and 7 months. The results are summarized in separate Tables.

Infants participating in this trial received DTacP and PRP vaccines at 2, 4, and 6 months of age; the 7-month blood draw occurred one month after the third injection of these vaccines. For each of these vaccine antigens (diphtheria, tetanus, pertussis FHA, pertussis PT, and PRP), the observed antibody levels do not demonstrate a statistically significant difference among the 3 TetraMenD dosage groups (all p-values (See Table A-17).

In the context of this trial, IPV is administered at 2 months and at 4 months of age.

The 7-month blood draw occurs three months after the second injection of IPV. For polio type I and polio type 2, the observed GMTs, proportions with NA and proportions with NA >1:8 do not demonstrate a statistically significant difference among the 3 TetraMenD dosage groups (all p-values At least 95.0% of all 3 TetraMenD dosage groups demonstrate protection against polio types 1 and 2 by proportion with NA 11:8. For polio type 3, the GMTs in the 1 pg, 4 pg, and 10 pg groups are 562.7, 164.0, and 113.3, respectively. The difference among the groups in the polio type 3 GMTs is statistically significant (p=0.001, ANOVA). However, all three TetraMenD dosage groups demonstrate protection against polio type 3 by proportion with NA >1:8 (100.0% [22/22], 100.0% [21/21], and 94.1% [16/17], respectively). These proportions are not statistically different (p=0.283, Fisher's exact test).

Moreover, the observed GMTs for the three polio serotypes are well within published ranges Sfollowing two doses of IPV at 2 and 4 months of age, the IPV vaccination schedule utilized in t this trial.

The 7-month blood draw occurs at a minimum of 5 months after the most recent hepatitis B vaccination. The observed levels of hepatitis B surface antibody by GMT and proportion 210 mlU/mL do not demonstrate a statistically significant difference among the 3 TetraMenD dosage groups (both p-values 0.649). Notably, no infants in this trial received 00 Shepatitis B vaccine at the 6-month visit, which is the earliest recommended age for the third dose of this vaccine. This may explain why the proportions of 7-month-old infants with hepatitis B surface antibody titers 210 mlU/mL are consistent with published ranges for detectable antibody following the initial doses of the vaccine, but lower than would have been expected for protective antibody levels following the complete three-vaccination series. The results of this Study are summarized in the following Tables.

Table A-17: Stage III (Infants) Immunogenicity of Concomitant Vaccines Among Infants At Age 7 Months, by TetraMenD Dosage Level (Per-Protocol Population) Antigen

N

7 m TetraMenD Dosage Level Immunologic 1 ag/4 p-value* Criteria p g/10 1 lg 4 pg 10 fig Diphtheria (IU/mL) 0.16 0.09 0.08 GMT (95% CI) 2 (0.10- (0.06- (0.05 0.150 0 .25) 0.14) 0.15) %>0.01 100.0 100.0 95.2 0.313 %>0.10 56.5 43.5 47.6 0.750 Tetanus (IU/mL) 1.52 1.26 1.23 GMT (95% CI) (1.08- (0.88- (0.88- 0.618 2.15) 1.78) 1.74) 0.01 0.10 Not 100.0 100.0 100.0

N

calculable Not 100.0 100.0 100.0 calculable Table A-17: Stage III (infants) Immunogenicity of Concomitant Vaccines Among Infants At Age 7 Months, by TetraMenD Dosage Level (Per-Protocol Population) Antigen

N

7 m Tetra enD Dosage Level Immunologic 1/I i jig 4 {p-value* _iaPg I _g _g Pertussis FHA ELISA 202/ 16 69.6 63.4 GMC (95% CI) 9 I614 (55.6- (43.5- 0.45 108.4) 87.2) 92.3) Pertussis PT ELISA j 2/2/1 66.4 56.5 80.0 GMC (95% CI) 202/ (43.6- (37.2- (56.7- 0.441 101.2) 85.8) 112.7) 1_ Pertussis PT CHO (titer) 222.9 256.0 332.0 GMT (95% CI) 202/ 175.2- (200.9- 0.476 379.6) -374.2) 548.7) Polio type I (titer) 169.9 122.1 93.7 GMT (95% CI) (95.3 (72.6 (46.5 0.3 303.0) 205.4) 188.7) NA 1I:4 0 100.0 100.0 100.0 calulbl NA 1>:8 1 100.0 95.5 1 95.0 1 0.760 Polio type 2 (titer) 183.9 220.7 211.2 GMT (95% CI) (96.2 (135.9 (107.3 0.893 22/21/1 351.4) 358.5) 415.6) Not____ NA 1>:4 8 100.0 100.0 100.0 Notulbl NA !1:8 100.0 100.0 100.0 Not calculable Polio type 3 (titer) GMT (95%'Y CI) NA 1I:4 NA 1:8 562.7 (363.3 871.8) 164.0 (97.8 274.8) 113.3 (44.6 287.7) 0.001 1 22/21/1 7 100.0 100.0 94.1 0.283 100.0 100.0 94.1 0.283

O

0 Table A-17: Stage 111 (Infants) Immunogenicity of Concomitant Vaccines Among Infants At Age 7 Months, by TetraMenD Dosage Level (Per-Protocol Population) Antigen N 7 m TetraMenD Dosage Level Antigen Immunologic 1 ,g/ 4 p-value* Criteria Pg/10 1 Pg 4 fg 10 p g PRP (~g/mL) 4.87 4.41 3.39 GMT (95% CI) (3.04- (2.52- (1.64- 0.648 23/22/1 7.78) 7.69) 6.99) 9 Not 20.15 100.0 100.0 100.0 N calculable >1.0 pg/mL 95.7 81.8 78.9 0.209 Hep B Surface Ab (mIU/mL 46.9 36.9 48.3 GMT (95% CI) 21/23/1 (12.9- (12.4- (28.1 0.916 9 170.1) 110.2) 83.2) >10 81.0 78.3 89.5 0.649 GMT comparisons use the exact test.

t p-value 0.05 F-test. Comparisons of percentages use the Fisher's Example 11 Study B One and Six Month Study in Children Aged 2 to This is a randomized, active-controlled sutyd of healthy children between the ages of 2 and 10, comparing a single dose of TetraMenD with a single dose of Menomune. Blood specimens are drawn on DO, before vaccination, D28 and at 6-months post DO. The overall safety of TetraMenD compared with Menomune is comparable. The results of this study are summarized in the following Tables.

Distribution of SBA-BR Antibody Titers Table B-I shows the frequency distribution of baseline, Day 28 and Month 6 SBA-BR antibody titers for each serogroup.

Table B-l: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers <8 to 512 Test Test Test Test Group <8 8 16 32 64 128 256 512 ype Date n n n n n TetraMenD 280 20 15 24 44 63 64 56 (638) (8.8) (43.9) (10.0) Day 0 Menomune 281 26 12 52 43 64 49 56 (655) (42.9) TetraMenD 3 0 2 4 11 30 35 69 (637) (10.8) SBA y 2 Menomune 11 6 3 8 18 45 66 (654) (13.8) (10.1) TetraMenD 17 6 1 9 16 31 52 64 (607) (10.5) Month (8.6) 6 Menomune 99 13 15 23 29 52 70 99 (623) (15.9) (15.9) (11.2) Table B-1: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers -8 to 512 Test Tlest Group 8 8 16 32 64 128 256 512 Type Date n In n n n n n n TetraMenD 339 30 18 30 31 51 61 37 (638) 5.8) Day 0 (53.1) 8.0) (9.6) Menomune 368 34 12 21 43 54 51 (655) 3.8) (56.2) 3.2) 8.2) Tetra~lenD 24 10 13 27 44 85 102 97 (636) (15.3) SBA Da 8( 3.8) (13.4) (16.0) Da 8 Menomune 69 10 6 34 55 87 90 96 (653) (14.7) (10.6) 5.2) (13.3) (13.8) letra~lenD 85 19 17 35 67 90 88 66 (607) (10.9) Month (14.0) 5.8) (11.0) (14.8) (14.5) 6 Menomune 185 30 14 35 54 68 61 73 (623) (11.7) 5.6) (10.9) Table B-i: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers <8 to 512 Test Test Group 8 8 16 32 64 128 256 512 Type Date n n n n n n n(% TetraMenD 88 13 17 40 82 113 107 68 (637) (10.7) Day 0 (13.8) (12.9) (17.7) (16.8) Menomune 96 11 12 42 81 116 124 66 (654) (10.1) (14.7) (12.4) (17.7) (19.0) Tetra~lenD 11 3 5 8 19 69 100 121 (636) (19.0) SBA Da 8(1.7) (10.8) (15.7) D 28 Menomune 16 4 7 20 43 85 121 102 (654) (15.6) (1.I1) (13.0) (18.5) Tetra~lenD 25 3 3 17 23 46 72 110 (608) (18.1) Month 4.1) (11.8) 6 Menomune 62 17 7 24 38 73 98 114 (622) (18.3) (10.0) 1) (11.7) (15.8) 00 Table B-i: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers <8 to 512 Test 'rest G rou p 8 8 16 32 64 128 256 512 Type Date n n n n n n n n(% TetraMenD 401 28 24 21 51 45 43 (638) (2.4) Day 0 (62.9) Menomune 403 36 22 30 52 48 34 17 (654) (2.6) (61.6) 4.6) 8.0) (5.2) TetraMenD 22 2 1 9 24 39 73 108 SBA (636) (17.0) Da 8( 3.5) 1.4) 3.8) (11.5) Da 8 Menomune 43 3 4 8 33 61 88 130 135) (653) (19.9) 1.2) 5.1) (13.5) TetraMenD 46 9 3 10 31 69 96 107 (607) (17.6) M'onth 1.5) 1.6) 5.1) (11.4) (15.8) 6 Menomune 82 12 11 23 66 111 120 100 (624) (16.0) 1.9) 3.7) (10.6) (17.8) (19.2) Table B-i: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers 1024 to >65536 Test Trest Group }1024 2048 409618192 T16384 T32768 [656>65536 Type Date n n n n( n(%

SBA

(A)

Tet ra Men D (638) 40 (6.3) 26 0 (0.0) 0 (0.0) 0 (0.0) (4.1) (0.3) (0.5) (0.2) Day 0 1~ 1- 1- Menomune (655) 51 7.8) 19 0 (0.0) 0 (0.0) 0 (0.0) (2.9) (0.0) (0.2) (0.2) TetraMenD 120 151 79 71 39 20 1 1 (637) (0.2) Day 28 (18.8) (23.7) (12.4) (11.1) (0.2) Menomune 122 162 62 44 16 1 0 0 (654) (0.0) (24.8) (0.0) Mth retraMenD 129 139 48 52 27 16 0 0 6 (21.3) (22.9) Table B-i: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers 1024 to >65536 TIest Test Group 1024 2048 4096 8192 16384 32768 65536 >65536 Type Date n n n n n n n n(% Menomune 94193 17 9 8 2 0 0 (623) I(0.0) Table B-I: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers 1024 to >65536 Test Test Group 1024 2048 4096 8192 16384 32768 65536 >65536 Type Date n n n n n n n n(% TletraMenD 22 16 3 0 0 0 0 0 (638) (0.0) Day 0 (0.0) Menomune 20 22 3 0 1 1 0 0 (655) (0.0) 3.1) (0.0) TetraMenD 95 92 19 16 6 6 0 0 (636) (0.0) SB A Da 8(14.9) (14.5) 3.0) 2.5) 0.9) 0.9) (0.0) Da M8Nenomune 86 91 10 9 7 3 0 0 (653) (0.0) (13.2) (13.9) 1.5) 1.4) 1) (0.0) T1etraMenl) 62 56 17 3 1 1 0 0 (607) (0.0) Month 2.8) 0.5) (0.0) 6 Menomune 42 45 6 7 1 I 1 0 (623) (0.0) 1.0) C1.1) Table B-i: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers 1024 to >65536 Test Test Group 1024 2048 4096 192 16384 32768 65536 >65536 Type Date n n n n n n n(% TetraMenD 62 37 5 2 3 0 0 0 (637) (0.0) Day 0 0.3) 0.5) (0.0) Menomune 50 43 10 2 1 0 0 0 (654) (0.0) (0.0) TetraMenD 111 113 29 21 17 9 0 0 (636) (0.0) SBA Day (17.5) (17.8) 1.4) (0.0) Da 8 Menomune 127 86 23 13 5 2 0 0 (654) (0.0) (13.1) 3.5) 0.3) (0.0) TetraMenD 123 111 34 22 13 6 0 0 (608) (0.0) Month (18.3) 5.6) 1.0) (0.0) 6 Menomune 80 84 13 7 3 2 0 0 (622) (0.0) (12.9) (13.5) Table B-l: Distribution of SBA-BR Antibody Titers at Day 0, Day 28, and Month 6 After Vaccination (Per-Protocol Population) SBA-BR Titers 1024 to >65536 Test Test Group 1024 2048 4096 8192 16384 32768 65536 >65536 Type Date(%) TetraMenD 4 5 1 0 0 0 0 0 (638) (0.0) (Da (0.0) Day 0 Menomune 6 3 2 0 1 0 0 0 (654) (0.0) (0.0) TetraMenD 108 160 36 32 16 6 0 0 SBA (636) (0.0) (25.2) (0.0) a Menomune 117 129 24 9 3 1 0 0 135) (653) (0.0) (17.9) (19.8) (0.0) TetraMenD 101 92 16 16 8 3 0 0 (607) (0.0) Month (16.6) (15.2) (0.0) 6 Menomune 67 25 5 2 0 0 0 0 (624) (0.0) (10.7) (0.0) Table B-2 summarizes of Geometric Mean Titer (GMT) by Subject Age and Serogroup for TetraMenD Table B-2 Summary of GMT by Subject Age and Serogroup for TetraMenD Age (in Blood No. of Serogroup Serogroup Serogroup Serogroup Year) Day Subjects A CGMT W Y-135 GMT GMT GMT 2 Day 0 264 20.59 16.04 8.75 91.23 Day 28 260 940.26 151.41 332.43 393.88 6 Month 244 460.92 64.92 153.52 312.95 3 Day 0 235 33.84 14.60 12.64 148.78 Day 28 228 1610.74 292.64 795.64 662.97 6 Month 228 1033.38 105.69 408.85 631.50 4 Day 0 35 74.99 29.56 9.95 128.00 Day 28 34 3479.60 795.86 1731.22 906.10 6 Month 29 3812.58 302.62 634.88 908.65 Day 0 27 118.51 32.00 29.63 121.59 Day 28 27 3010.02 612.79 1323.71 1050.63 6 Month 27 3010.02 406.37 679.07 948.10 6 Day0 38 38.40 34.42 13.83 112.66 Day 28 37 4414.32 950.07 2086.73 898.15 6 Month 37 3729.75 386.57 1005.00 849.06 7 Day 0 30 111.43 78.79 16.00 111.43 Day 28 29 3999.26 1612.60 1691.56 1537.33 6 Month 28 2375.94 524.83 1049.67 1638.97 8 Day 0 18 118.51 54.86 17.28 188.13 Day 28 16 6049.08 1878.02 2543.32 1069.34 6 Month 17 2837.86 453.05 1111.00 1418.93 9 Day 0 29 116.33 91.60 21.31 87.32 Day 28 28 4870.99 2825.49 3119.59 1521.66 6 Month 28 3995.85 974.54 1187.97 1412.75 Day 0 21 98.30 27.13 18.26 45.25 Day 28 21 4233.45 3251.00 2337.06 1333.45 6 Month 20 3821.70 989.12 1782.89 1499.22 Table B-3 shows the numbers and proportions of participants with a >4-fold rise in SBA-BR titer from baseline to Day 28 for the serogroups A, C, Y, and W-135. For each serogroup, these percentages are higher in the TetraMenD group than in the Menomune® group. The differences in the proportions are: -0.0397, -0.0452, -0.1092 and -0.0562, for serogroups A, C, Y, and W-135, respectively.

Table B-3: Summary of Primary Hypothesis Testing for the Per-Protocol Population 4-fold rise erogrou in SBA titer Upper One-sided TetraMenD Menomune® Difference n/N P, n/N P, (Pm CL of the Difference Serogroup A 558/636 0.8774 547/653 0.8377 -0.0397 -0.0077 Serogroup C 466/635 0.7339 449/652 0.6887 -0.0452 -0.0037 Serogroup Y 359/634 0.5662 298/652 0.4571 -0.1092 -0.0636 Serogroup W-135 578/635 0.9102 556/651 0.8541 -0.0562 -0.0267 n: number of participants with a 4-fold rise from baseline titer.

N: total number of participants in the used population.

P, and Pm: proportions of participants with a >4-fold rise in SBA Menomune® groups, respectively.

post-vaccination titer from the TetraMenD and The proportion of participants with SBA antibody titers 32 at Day 28 after vaccination is summarized in Table B-3.

Table B-3: Percentage and Number of Participants with an SBA-BR Antibody Titer 32 at Day 28 Post-Vaccination (Per-Protocol Population) TetraMenD Menomune 95% CI for 95% CI for the percentage (nt/N) the percentage 99.22 (98.18, 100.00) 96.94 (95.32, 100.00) Serogroup A (632 637) (634 654) 92.61 (90.29, 100.00) 86.98 (84.16, 100.00) Serogroup C (589 636) (568 /653) 97.01 (95.37, 100.00) 95.87 (94.05, 100.00) Serogroup Y (617 636) (627/ 654) 96.07 (94.25, 100.00) 92.34 (90.03, 100.00) Serogroup W-135 (611/ 636) (603/ 653) n/N.

t n: number of participants with titer>32 at Day 28 post-vaccination.

SN: total number of participants with valid blood sample at Day 28 in this group.

The proportion of participants with SBA antibody titers 128 at Day 28 after vaccination is summarized in Table B-4.

Table B-4: Percentage and Number of Participants with an SBA-BR Antibody Titer 128 at Day 28 Post-Vaccination (Per-Protocol Population) TetraMenD Menomune 95% CI for 95% CI for the percentage the percentage 96.86 (95.19, 100.00) 92.97 (90.73, 100.00) Serogroup A (617 637) (608 654) 81.45 (78.20, 100.00) 73.35 (69.79, 100.00) Serogroup C (518 636) (479 /653) 92.77 (90.47, 100.00) 86.24 (83.36, 100.00) Serogroup Y (590 636) (564 654) 90.88 (88.37, 100.00) 86.06 (83.17, 100.00) Serogroup W-135 (578 636) (562/ 653) n/N.

Sn: number of participants with titer>128 at Day 28 post-vaccination.

N: total number of participants with valid blood sample at Day 28 in this group.

Proportion of Participants with at least a 4-fold rise in SBA-BR Antibody Titers Table B-5 shows the proportion of participants with a >4-fold rise in Day 28 and Month 6 SBA antibody titers from baseline. Twenty-eight to 56 days after receiving TetraMenD, the majority of participants experienced a 4-fold rise in the SBA-BR antibody titer for each of the serogroups contained in the vaccine.

Table B-5: Percentage and Number of Participants with a >4-Fold Rise in Day 28 and Month 6 SBA-BR Antibody Titers From Baseline (Per-Protocol Population) TetraMenD Menomune® Test Date Test Type (n

T

N) (95% Cl) (n (95% CI) (n'/Nl) Day 28 87.7 84.9, 83.8 (80.7, 86.5) Serogroup A (558/636) 90.2) (547/653) SBA Month 6 79.1 (75.6, 56.9 (52.9, 60.8) (480/607) 82.2) (354/622) Day 28 73.4 (69.8, 68.9 (65.2, 72.4) Serogroup C (466/635) 76.8) (449/652) SBA Month 6 55.7 (51.6, 43.7 (39.8, 47.7) (338/607) 59.7) (272/622) Day 28 56.6 (52.7, 45.7 (41.8, 49.6) Serogroup Y (359/634) 60.5) (298/652) SBA Month 6 57.3 (53.3, 39.2 (35.3, 43.2) (348/607) 61.3) (243/620) Day 28 91.0 (88.5, 85.4 (82.5, 88.0) Serogroup W- (578/635) 93.1) (556/651) 135 SBA Month 6 82.7 (79.5, 69.5 (65.7, 73.1) (502/607) 85.6) (432/622) n/N.

t n: number of participants with 4-fold rise from baseline titer.

total number of participants in the used population.

Proportion of Participants with Undetectable Titers at Day 0 Achieving a >4-Fold Rise in Day 28 SBA-BR Antibody Titers In both treatment groups and for all vaccine serogroups, most participants with an undetectable SBA-BR titer at baseline achieved a >4-fold rise in Day 28 SBA titers.

(Table B-6) The proportions of participants with an SBA titer <8 at Day 0 who had a >4-fold

I

a oo 0

(N

1-^ rise from baseline to Day 28 ranged from 86.21% to 98.57% in the TetraMenD group; and from 75.00 to 94.64 in the Menomune® group.

Table B-6: Number and Percentage of Participants with Undetectable Titers at Day 0 Achieving a >4-Fold Rise in Day 28 SBA-BR Antibody Titers.

TetraMenD Menomune Serogroup 95% CI' 95% CI t (n/N) A 98.57 (96.37, 100.00) 94.64 (91.32, 100.00) (275 279) (265 280) C 87.87 (83.91, 100.00) 80.05 (75.59, 100.00) (297/ 338) (293 366) Y 86.21 (77.15, 100.00) 75.00 (65.12, 100.00) 87) (72/ 96) W-135 96.00 (93.59, 100.00) 89.53 (86.11, 100.00) (384/400) (359/401) *n The number of participants serogroup with titers 8 at Day 0 and titers 32 at Day 28 within each N The number of participants with titers 8 at Day 0 within each serogroup t Exact 95% confidence interval for the percentage SBA-BR Antibody GMTs and mean fold rises Table B-7 shows the SBA GMTs at baseline and on Day 28 and Month 6 after vaccination and the fold rises in SBA GMTs.

Table B-7: SBA-BR Serology Results at Baseline, Day 28, and Month 6 After Vaccination (Per-Protocol Population) TetraMenD Menomune Test Type Parameter* Bleed t Geometric CI) N Geometric (95% C Mean Mean Serogroup A SBA Titer Day 0 Day 28 Mont h6 638 35.44 29.77, 42.20) 637 1700.27 (1512.07, 1911.89) 607 1053.65 (912.93, 1216.07) 655 32.72 27.71, 38.63) 654 892.20 (789.97, 1007.66) 623 214.97 179.84, 256.97) Table B-7: SBA-BR Serology Results at Baseline, Day 28, and Month 6 After Vaccination (Per-Protocol Population) TetraMenD Menomune® Test Type Parameter" Bleed Geometric (95% CI) Nt Geometric (95% CI) Mean Mean Fold rise 638 1.00 1.00, 655 1.00 1.00, Day 0 1.00) 1.00) Day 636 35.18 29.72, 653 20.21 17.43, 28 41.65) 23.44) Mont 607 23.19 19.20, 622 5.04 4.21, h 6 28.00) 6.03) Serogroup Titer 0 638 20.63 17.59, 655 18.69 15.95, C SBA y24.20) 21.90) Day 636 353.85 (307.95, 653 230.71 (197.72, 28 406.58) 269.20) Mont 607 136.92 (116.40, 623 65.51 54.64, h6 161.06) 78.55) Fold rise 638 1.00 1.00, 655 1.00 1.00, Day 0 1.00) 1.00) Day 635 11.86 10.19, 652 8.40 7.23, 28 13.81) 9.77) Mont 607 4.49 3.85, 622 2.41 2.05, h 6 5.25) 2.83) Serogroup Titer 0 637 118.61 (102.49, 654 117.84 (101.98, Y SBA y137.27) 136.18) Day 636 636.70 (563.06, 654 408.10 (362.19, 28 719.97) 459.84) Mont 608 591.77 (514.65, 622 239.18 (204.91, h6 680.43) 279.17) Fold rise 637 1.00 1.00, 654 1.00 1.00, Day 1.00) 1.00) Day 634 4.83 4.25, 652 3.14 2.79, 28 5.49) 3.52) Mont 607 4.63 4.00, 620 1.85 1.60, h 6 5.37) 2.14) Serogroup W-135 SBA Titer Day 0 Day 28 Mont h6 12.09 749.78 362.25 10.62, 13.76) (657.37, 855.18) (311.67, 421.03) 12.15 424.75 136.06 10.69, 13.80) (371.47, 485.67) (118.08, 156.78) Fold rise Day 0 Day 28 1.00 40.24 1.00, 1.00) 34.30, 47.21) 1.00 22.98 1.00, 1.00) 19.73, 26.76) Table B-7: SBA-BR Serology Results at Baseline, Day 28, and Month 6 After Vaccination (Per-Protocol Population) TetraMenD Menomune® Test Type Parameter" Bleed N Geometric (95% C) Nt Geometric (95% CI) Mean Mean Month 607 19.19 16.31, 622 7.42 6.33, 6 22.56) 8.69) Titer or fold rise, where fold rise titer at Day 28/titer at Day 0 tN: total number of participants used in the calculation.

ELISA IgG for serogroups A, C, W-135, and Y Table B-8 shows the IgG GMCs at baseline and on Day 28 and Month 6 after vaccination and the fold rises in IgG GMCs.

Table B-8: IgG Serology Results at Baseline, Day 28, and Month (Per-Protocol Population) 6 After Vaccination TetraMenD Menomune Parame Geom Geomet Test Type N Bleed etric (95% CI) N t ric (95% CI) Mean Mean Serogroup Titer 115 0.36 0.31, 113 0.33 0.28, A (IgG) Day0.43) 0.38) ELISA 115 7.65 6.27, 110 6.81 5.51, Day 28 9.33) 8.42) Month 112 1.70 1.37, 109 4.53 3.60, 6 2.11) 5.70) Fold 115 1.00 1.00, 113 1.00 1.00, rise Day 0 1.00) 1.00) 115 21.00 16.60, 108 21.09 16.78, Day 28 26.58) 26.50) Month 112 4.58 3.65, 107 14.40 11.22, 6 5.75) 18.49) Serogroup Titer D 0 115 0.23 0.20, 113 0.25 0.22, C (IgG) a0.25) 0.29) ELISA 115 1.24 1.03, 110 7.62 6.33, Day281.50) 9.19) Month 111 0.36 0.31, 109 3.49 2.82, 6 0.43) 4.32) Fold 115 1.00 1.00, 113 1.00 1.00, rise Dy 0 1.00) 1.00)

I

Table B-8: IgG Serology Results at Baseline, Day 28, and Month 6 After Vaccination (Per-Protocol Population) TetraMenD Menomune Parame Geom Geomet Test Type Bleed t ter N etric (95% CI) Nt ric (95% CI) Mean Mean 115 5.50 4.58, 109 30.18 24.26, Day 286.62) 37.55) Month 111 1.60 1.36, 107 14.42 11.46, 6 1.89) 18.15) Serogroup Titer Da0 115 0.38 0.34, 114 0.34 0.31, Y (IgG) a0.43) 0.38) ELISA 115 1.54 1.26, 110 4.15 3.30, Day 28 1.88) 5.22) Month 112 0.76 0.65, 109 2.90 2.25, 6 0.89) 3.73) Fold 115 1.00 1.00, 114 1.00 1.00, rise y 1.00) 1.00) 115 4.04 3.30, 109 12.43 9.85, Day 28 4.93) 15.68) Month 112 1.98 1.68, 108 8.72 6.79, 6 2.32) 11.21) Serogroup Titer y 115 0.25 0.22, 113 0.22 0.19, W-135 y 0.28) 0.25) (IgG) 115 0.90 0.72, 110 2.53 2.06, ELISA Day2 1.12) 3.11) Month 112 0.55 0.47, 109 1.88 1.53, 6 0.65) 2.31) Fold 0 115 1.00 1.00, 113 1.00 1.00, rise Day1.00) 1.00) 115 3.60 2.90, 108 11.67 9.34, ay28 4.47) 14.58) Month 112 2.18 1.84, 107 8.70 6.96, 6 _2.58) 10.87) Titer or fold rise, where fold rise titer at Day 28/titer at Day 0 SN: total number of participants used in the calculation.

Twenty-eight to 56 days after receiving the study vaccination, TetraMenD, the majority of participants experience a 4-fold rise in the SBA-BR antibody titer for each of the serogroups contained in the vaccine. Overall, 77% of TetraMenD recipients experience a 4-fold rise in antibody titer across all serogroups. Higher pre-vaccination antibody levels are observed for serogroup Y than for C or W-135. This may be related to the fact that natural 87 cN exposure to serogroup Y at this age may be more common than previously thought. Higher b circulating antibody levels reflect recent natural exposure and may reduce the proportion of vaccine recipients exhibiting 4-fold or higher antibody responses. This clearly appears to be the case for serogroup Y responses when compared to other serogroups. The 4-fold rise for serogroup Y is 56.6% compared with 73.4% for serogroup C, 87.7% for serogroup A, and 91.0% for serogroup W-135. High pre-vaccination antibody levels are also observed for 00 serogroup A. This may be the result of intermittent exposure over a prolonged period of time O to several naturally occurring cross-reacting antigens.

To further evaluate the impact of pre-existing titers and to investigate the rate of seroconversion (as defined by the proportion of vaccine recipients who achieve a 4-fold rise in antibody titer when the pre-vaccination titer for any serogroup is a separate analysis is performed on participants who had pre-vaccination antibody titers of <l:8 to any one of the 4 serogroups contained in the vaccine. A titer of 1:8 by the SBA assay using baby rabbit as the complement source is considered to represent an undetectable level of circulating antibody. When participants are evaluated using this criterion, it is observed that there is a 98.6% seroconversion rate for serogroup A, 87.9% for serogroup C, 96.0% for serogroup W- 135, and 86.2% for serogroup Y after vaccination with TetraMenD.

Based on observations in military recruits, Goldschneider proposed that a minimum titer of 2 1:4 using an SBA assay with a human complement source correlated with protection from invasive disease against Serogroup C. However, because of the need for standardization of the assay and the lack of a reliable source of human complement, baby rabbit complement is suggested as an alternative source. Meningococci appear to be more sensitive to the baby rabbit complement than human complement, resulting in higher measured antibody titers.

Several authors have suggested that titers 1:128 using the rabbit complement assay are predictive of protection while titers of 1:8 are predictive of susceptibility at least for serogroup C. Although this level may be appropriate when evaluating polysaccharide vaccines, it may not be applicable for conjugate vaccines. Borrow suggested that, in subjects receiving a monovalent C conjugate vaccine who demonstrated post vaccination SBA titers between 8 and 64, the demonstration of a memory response using a reduced dose (10 p.g) of a meningococcal polysaccharide vaccine given several months later showed that these individuals are also protected, having achieved an antibody level >1:128. The results for subjects who received the TetraMenD vaccine with SBA-BR titers >1:128 for each serogroup are presented the Tables. When these criteria are applied to each of the serogroups contained in Sthe vaccine, overall, 96.2% of participants who received TetraMenD achieved a post-vaccination SBA-BR titer of 1:32 and 90.5% achieved a titer 1:128. A subset of sera from this clinical 0study is also used to evaluate the correlation between the SBA assay using baby rabbit 00 complement and human complement and the results are provided in a subsequent Study.

O Total IgG responses are significantly higher for serogroups C, Y, and W-135 in the 1' Menomune" group than in the group receiving TetraMenD. However, the post-vaccination O 10 SBA GMT levels for serogroups A, C, Y, and W-135, are significantly higher in the TetraMenD group. Table B-9 provides a comparison of GMC versus GMT titers by serogroup.

Table B-9 Comparison of IgG GMC and SBA GMTs Titers by Serogroup Serogroup Day 28 Results: IgG GMC SBA GMT TetraMenD 7.65 1700.3 A Menomune 6.81 892.2 TetraMenD 1.24 353.9 CMenomune 7.62 230.7 TetraMenD 1.54 636.7 Menomune 4.15 408.1 TetraMenD 0.90 749.8 W-135 Menomune' 2.53 424.8 The observation that the lower levels of IgG produced by the conjugate generated a higher level of bactericidal activity than the polysaccharide vaccine strongly suggests that the quality and affinity of the antibody response to the conjugate vaccine is superior to that generated by unconjugated polysaccharide vaccine. High affinity antibody is associated with functional activity and memory response. This effect has also been observed in several published studies. These data demonstrate that TetraMenD is highly immunogenic in children aged 2 to 10 years, the observed GMTs in the TetraMenD group are superior to those observed in the Menomune group for each of the four serogroups, and the titers achieved are predictive of protection. Finally, it appears that TetraMenD generates higher affinity antibody responses 43 for each serogroup contained in the vaccine.

Safety is monitored at 4 specific time points during the trial: Immediate reactions t>s (within 30 minutes of vaccination), solicited local and systemic reactions within the first 7 days post-vaccination, all adverse events in the 28-day period after vaccination and continuing AEs (from Days 0-28) and serious adverse events from Day 0 to 6 months post-vaccination 00 Sare reported.

O For all participants, most local solicited reactions for both treatment groups are reported as mild and resolved within 3 days of vaccination. The frequency of local reactions O 10 is similar for each treatment group. In the group receiving TetraMenD 58.8% reported at least one local reaction while the group receiving Menomune® 58.3% reported the same. In addition, experience with the monovalent C CRM 197 conjugate vaccine given intramuscularly to adolescents shows that the rate of local reactions is very similar to that observed for TetraMenD in this study.

The majority of reported AEs are not serious, reversible, and unrelated to vaccination.

There are no reports in this study of new onset bronchial asthma, diabetes mellitus, or autoimmune disease.

Example 12 Study C One Month Study in Children Aged 11 to 18 Study C is a randomized, active-controlled study of healthy children aged 11 to 18 years as of DO of a single dose of TetraMenD versus a single dose of Menomune®. Blood serum is drawn on DO, prior to vaccination and D28 and analyzed, and a subset of sera from patients is further evaluated as described in the results.

For all participants, most local solicited reactions for both treatment groups are reported as mild and resolved within 2 days of vaccination. The frequency of local reactions is more common in the group receiving TetraMenD than in the group receiving Menomune® This result is probably due to the nature of the conjugate vaccine (diphtheria carrier protein) rather than the route of administration (intramuscular). The results of this Study are summarized in the following Tables.

Table C-I shows the frequency distribution of baseline and Day 28 SBA-BR antibody titers for each serogroup.

Table C-l: Distribution of SBA-BR Antibody Titers at Day 0 and Day 28 After Vaccination (Per-Protocol Population) SBA-BR Titers <8 to 512 Test Test Group (N) Type Date <8 8 16 32 64 128 256 512 n n n n TetraMenD 81 19 4 9 33 76 86 51 (425) (19.1) (17.9) (20.2) (12.0) Day 0 SMenomune® 93 12 10 13 38 72 72 56 SBA (423) (22.0) (17.0) (17.0) (13.2) TetraMenD 1 1 Day (423) 28 Menomune® 8 12 19 (423) TetraMenD 157 37 18 24 36 40 39 (425) (36.9) (8.2) Day 0 Menomune* 152 35 15 19 40 46 42 SBA (423) (35.9) (10.9) (5.9) TetraMenD I 1 1 2 17 33 58 Day (423) (13.7) 28 Menomune® I 1 1 4 26 47 56 (423) (11.1)(13.2) TetraMenD 61 6 1 22 64 94 101 (425) (14.4) (15.1) (22.1) (23.8) (11.8) Day 0 Menomune® 47 3 7 27 74 94 85 51 SBA (423) (11.1) (17.5) (22.2) (20.1) (12.1) TetraMenD 1 1 23 53 71 Day (423) (12.5) (16.8) 28 Menomune® 1 2 11 59 81 (423) (13.9) (19.1) TetraMenD 165 37 28 36 60 56 22 (425) (38.8) (14.1) (13.2) Day 0 Menomune® 139 52 25 34 67 43 46 11 SBA (423) (32.9) (12.3) (15.8) (10.2) (10.9) (2.6)

(W-

-135 TetraMenD 4 1 I 1 19 34 63 Day (423) (14.9) 28 Menomune® I 1 1 2 12 21 51 (423) (5.0)(12.1) Table C-i: Distribution of SBA-BR Antibody Titers at Day 0 and Day 28 After Vaccination (Per-Protocol Population) SBA-BR Titers 1024 to >65536 Test Test Group 1638 Type Date 1024 2048 4096 8192 4 32768 65536 65536 n n n n n TetraMenD 32 29 2 1 2 DyO (425) 0(7.5) y Menomune 38 14 2 2 1 (423) (0.2) (BA etraMenD 36 66 90 108 63 37 4 2 Day (423) (15.6) (21.3) (25.5) (14.9 28 Menomunee 46 100 108 76 43 11 (423) (10.9) (23.6) (25.5) (18.0) (10.2 (2.6) TetraMenD 20 13 4 2 Dy0 (425) ~au Menomune& 23 22 2 1 1 SBA (423) TetraMenD 66 82 66 45 24 21 4 2 Day (423) (15.6) (19.4) (15.6) (10.6) 28 Menomune' 70 64 55 41 35 19 3 (423) (16.5) (15.1) (13.0) (0.7) TetraMenD 13 6 2 2 2 1 Dy0(425) (0.2) DaO Menomunee 24 6 2 1 1 1 SBA (0.2) TetraMenD 77 80 52 41 16 7 1 Day (423) (18.2) (18.9) (12.3) (0.2) 28 Menomune®, 90 74 53 35 11 6 (423) (21.3) (17.5) (12.5) TetraMenD 4 2 Day 0 (425) SAMenomunee 4 1 1 (W A (423) 135) TetraMenD 90 88 64 36 16 6 1 13) Day (423) (21.3) (20.8) (15.1) (0.2) 28 Menomune" 103 114 67 42 6 2 (24.3) (27.0) (15.8) Table C-2 summarizes GMT levels by Subject Age and Serogroup for TetraMenD Table C-2 Summary of GMT by Subject Age and Serogroup for TetraMenD Age Blood No. of Serogroup Serogroup Serogroup Serogroup (in Day Subjects A GMT C GMT W GMT Y-135 Year) GMT 11 Day 0 45 101.59 37.33 21.77 91.21 Day 28 45 4705.07 1372.15 1482.00 1024.00 12 Day 0 54 104.24 42.99 14.63 72.77 Day 28 53 5049.37 2157.99 1245.94 1198.00 13 Day 0 65 145.47 32.34 20.23 128.00 Day 28 65 7363.39 1880.53 2206.73 1782.89 14 Day 0 67 85.50 31.67 24.71 107.36 Day 28 67 5124.87 2006.06 1753.62 1159.35 Day 0 70 129.27 42.22 27.31 126.74 Day 28 68 4870.99 2090.18 1193.17 1193.17 16 Day 0 69 103.66 21.41 22.74 129.29 Day 28 69 7189.09 2357.27 1455.45 2068.68 17 Day 0 69 85.64 38.73 13.90 72.93 Day 28 67 4269.06 1665.21 841.27 904.45 18 Day 0 1 4 8 16 64 Day 28 1 8192.00 256.00 512.00 8192.00 Table C-3 shows the numbers and percentages of participants with a >4-fold rise in SBA-BR titer from baseline to Day 28 for the serogroups A, C, Y, and W-135. For each serogroup, these percentages are higher in the TetraMenD group than in the Menomune® group.

Table C-3: Numbers and Percentages of participants with a 24-fold rise in SBA- BR titer from Baseline to Day 28 4-fold rise TetraMenD Menomune® Upper bound of Difference the one sided 95% in SBA titer for n/N P, n/N Pm i th serogroup Proportion Proportion Difference Difference A 392/423 0.9267 391/423 0.9243 NA NA C 388/423 0.9173 375/423 0.8865 -0.0307 0.0029 Y 346/423 0.8180 339/423 0.8014 -0.0165 0.0278 W-135 409/423 0.9669 403/423 0.9527 -0.0142 0.0080 Frequency of SBA-BR Antibody Titers 32 The proportion of participants with SBA antibody titers 32 at Day 28 after vaccination is summarized in Table C-4.

Table C-4: Percentage and Number of Participants with an SBA Antibody Titer 32 at Day 28 Post-Vacci nation (Per-Protocol Population) TetraMenD Menomuneo 95% CI for 95% CI for the percentage the percentage Serogroup A (420/420 (99.13, 100.00) 10.0 (99.13, 100.00) (423/423)_ Serogroup C 9.3 (98.30, 100.00) 9.3 (9.0100) (421/423) (421/423) 9.0 0.0 SrgopY 99.76 (98.69, 100.00) 99.76 (98.69, 100.00) Seogou Y (422/423) Serogroup W-135 9.2 (97.26, 100.00) 9.3 (98.30, 100.00) n: number of participants with titer>_32 at Day 28 post-vaccination.

N: totai number of participants with valid blood sample at Day 28 in this group.

Frequency of SBA-IIR Antibody Titers> 128 The proportion of participants with SBA antibody titers 128 at Day 28 after vaccination is summarized in Table Table C-5: Percentage and Number of Participants with an SBA Antibody Titer 128 at Day 28 Post-Vaccination (Per-Protocol Population) TetraMenD Menomune 95% CI for 95% CI for the percentage the percentage 99.76 100.00 Serogroup A (42 (98.69, 100.00) (42 (99.13, 100.00) 98(422/423) (423/423) Serogroup C (97.26, 100.00) (96.62, 100.00) (418/423) (416/423) 99.53 99.29 Serogroup Y (98.30, 100.00) (97.94, 100.00) (421/423) (420/423) 98.58 98.82 Serogroup W-135 (96.94, 100.00) (97.26, 100.00) (417/423) (418/423) n/N expressed as a percentage.

t n: number of participants with titer 128 at Day 28 post-vaccination.

total number of participants with valid blood sample at Day 28 in this group.

Percentage of Participants with 4-fold Rise in SBA-BR Antibody Titers Table C-6 shows the proportion of participants with a >4-fold rise in Day 28 SBA antibody titers from baseline.

Table C-6: Percentage and Number of Participants with a >4-Fold Rise in Day 28 SBA Antibody Titers From Baseline TetraMenD Menomune Test Type (tTe p(95% CI) (n t /N (95% CI) (nt/N 92.7 92.4 Serogroup A SBA 92 (89.8, 95.0) 942 (89.5, 94.8) (392/423) (391/423) 91.7 88.7 Serogroup C SBA 9 (88.7, 94.2) .7 (85.2, 91.5) (388/423) (375/423) 81.8 80.1 Serogroup Y SBA (77.8, 85.4) (76.0, 83.8) (346/423) (339/423) 96.7 95.3 Serogroup W-135 SBA 9 (94.5, 98.2) 3 3 (92.8, 97.1) (409/423) (403/423) n/N expressed as a percentage..

n: number of participants with 4-fold rise from baseline titer.

N: total number of participants in the used population.

Percentage of Participants with Undetectable Titers at Day 0 achieving a >4-Fold Rise in Day 28 SBA-BR Antibody Titers In both treatment groups and for all vaccine serogroups, most participants with an undetectable 8) SBA titer at baseline achieve a >4-fold rise in Day 28 SBA titers. The proportions of participants with an SBA titer <8 at Day 0 who had a >4-fold rise from baseline to Day 28 range from 98.17% to 100.0% (Table C-7).

Table C-7: Number and Percentage of Participants with Undetectable Titers at Day 0 Achieving a >4-Fold Rise in Day 28 SBA-BR Antibody Titers.

00 00 Cc, F1- 0

(N

0 TetraMenD Menomune® Serogroup Percent n/N* 95% CI Percent n/N 95% CIt A 100.00 81/81 (95.55, 100.00) 100.00 93/93 (96.11, 100.00) C 98.71 153/155 (95.42, 100.00) 99.34 151/152 (96.39, 100.00) Y 98.17 161/164 (94.75, 100.00) 99.28 138/139 (96.06, 100.00) W-135 98.36 60/61 (91.20, 100.00) 100.00 47/47 (92.45, 100.00) *n The number of participants with titers 8 at Day 0 and titers 32 at Day 28 within each serogroup N The number of participants with titers 8 at Day 0 within each serogroup Exact 95% confidence interval for the percentage SBA-BR Antibody GMTs and Mean Fold Rises Table C-8 shows the SBA GMTs at baseline and on Day 28 after vaccination and the fold rises in SBA GMTs.

Table C-8: SBA Serology Results at Baseline and at Day 28 After Vaccination (Per- Protocol Population) TetraMenD Menomune Test Type Parameter' Bleed t Geometric (95% C) N Geometric (95% C Mean Mean (87.60, (73.05, Day 0 425 106.28 423 88.67 (73.

128.95) 107.64) Titer Serogroup D (4920.12, 3245.6 (2909.97, Se Day 28 423 5483.21 423 A SBA 6110.74) 7 3260.11) (36.98, (26.62, Fold rise Day 28 423 44.92 (36.98 423 31.43 (26.62 54.57) 37.10) (27.54, (30.40, Day 0 425 33.71 41.28) 423 37.39 41.28) 45.98) Titer Serrooup Day 28 423 1924.36 (1662.08, 1638.8 (1405.55, C SBA 2228.03) 7 1910.93) (36.40, (28.31, Fold rise Day 28 423 43.83 .40 423 34.17 (2831 52.78) 41.24) (87.80, (96.03, Day 0 425 103.21 423 111.91 (96.

121.32) 130.41) TIiter Serogroup Day 28 423 13226 (1161.85, 1228.2 (1088.20, Y SBA 1504.82) 7 1386.37) (9.94, (8.76, Fold rise Day 28 423 11.62 (994 423 10.16 (876 13.60) 11.79) (17.70, (20.40, Day 0 425 20.70 423 23.90 (20.0 24.22) 28.02) Serogroup Titer 2 2 Scrogroup (1232.07 1544.9 (1383.63 W-135 Day 28 423 1407.22 (1232.07, 423 544.9 (1383.63, SBA 1607.27) 9 1725.16) (44.36, (44.32, Fold rise Day 28 423 51.98 60.90) 423 51.47 4 4 60.90) 59.76) Titer or fold rise, where fold rise titer at Day 28/titer at Day 0 SN: total number of participants used in the calculation.

ELISA IgG for serogroups A, C, W-135, and Y Table C-9 shows the IgG GMCs (in pg/mL) at baseline and on Day 28 after vaccination and the fold rises in IgG GMCs.

Table C-9: IgG Serology Results at Baseline and at Day 28 After Vaccination (Per- Protocol Population) TetraMenD Menomune Test Parameter Bleed Geometric Geometric (95% C Type Nt Mean (95% CI) Nt Mean (p (lIg/mL) g/mL) (0.61, (0.45, Day 0 82 0.84 0 61 79 0.62 0.

4 Sero- T 1.

16 0.

84 Titer group Day 28 82 1809 (13.56, 11.61(8.81, A (IgG) 24.12) 15.29) ELISA (16.35, (14.23, ELISA Fold rise Day 28 82 21.49 8.2) 79 18.87 25.0 28.24) 25.00) (0.23, (0.24, Sero Day 0 82 0.27 0.31) 79 0.30 0.37) Sero- 0.31) 0.37) Titer group Day 2(3.85, 79 8. (5.37, C (IgG) 7.97) 12.18) ELISA (14.74, (18.93, ELSA Fold rise Day 28 82 20.78 29.28) 79 26.97 8.41) (0.32, (0.30, Sero- Day 0 82 0.41 0.3 79 0.39 0.0 Sero- 0.53) 0.50) Titer group (2.74, (6.58, (group Day 28 82 4.41 74 79 9.17 (.58 Y (IgG) 7.08) 12.78) ELISA (7.32, (16.93, ELISA Fold rise Day 28 82 10.81 15.95) 79 23.55 32.75) (0.20, (0.19, Sero- Day 0 82 0.24 0.29 79 0.24 0.1)9 group Titer 0.29) 0.30) group Titer (2.02, (3.47 W-135 Day 28 82 2.95 79 4.93 7 (gG) 4.30) 7.00) (IS) (8.48, (14.62, ELISA Fold rise Day 28 82 12.26 7.7) 79 20.40 2.46 17.73) 28.46) Titer or fold rise, where fold rise titer at Day 28/titer at Day 0 tN: total number of participants used in the calculation.

ELISA IgM for serogroups A, C, Y, and W-135 Table C-10 shows the IgM GMCs at baseline and on Day 28 after vaccination and the fold rises in IgM GMCs. Table C-10: IgM Serology Results at Baseline and at Day 28 After Vaccination (Per-Protocol Population) STetraMenD Menomune Test Parameter Type Bleed Nt Geometric (95% C) Geometric (95% CI) I Mean 5% Mean Serogroup A (IgM)

ELISA

Day 0 1.66 (1.34, 2.06) (1.15, 1.75) Titer 4 4 I Day 28 l7.80 12.00 Day 28 17.80 (14.67, 21.59) 12.00 (9.67, 14.89) Table C-10 shows the IgM GMCs at baseline and on Day 28 after vaccination and the fold rises in IgM GMCs. Table C-10: IgM Serology Results at Baseline and at Day 28 After Vaccination (Per-Protocol Population) TetraMenD Menomune® Test Parameter Type Bleed t Geometric (95% CI) N Geometric (95% CI) Mean Mean Fold rise Day 28 79 11.22 (14.7) 79 8.47 10.16) Ser_ 14.73) 10.16) (0.14, (0.12, Day 0 82 0.19 0.24) 79 0.16 0.22) Sero- 0.24) 0.22) Titer group (1.20, (1.39, group Day 28 80 1.55 (1.20, 79 1.71 (1.39, C (IgM) 2.00) 2.10) ELISA(6.34 (8.04 ELSA Fold rise Day 28 80 8.42 6.3, 79 10.60 11.18) 13.97) (0.29, (0.32, Day 0 82 0.37 0.46) 79 0.40 0.50) Sero- Te0.46) 0.50) Titer group (2.81, (2.85, group Day 28 80 3.47 2 8 1 79 3.45 2 85 Y (IgM) 4.27) 4.17) ELISA(7.43 (6.79 ELISA Fold rise Day 28 80 9.47 79 8.65 (6.

12.05) 11.03) (0.15, (0.16, Ser- Day 0 82 0.17 0.1, 79 0.18 0.21) ogroup Titer 0.20) (1.41 W-135 Day 28 82 1.92 79 1.68 (1.4 (IgM) 1-2.29) 1.99) (9.06, (7.61, ELISA Fold rise Day 28 82 11.01 79 9.16 (7.6 13.39) 11.03) Titer or fold rise, where fold rise titer at Day 28/titer at Day 0 N: total number of participants used in the calculation.

Twenty-eight to 56 days after receiving the study vaccination, TetraMenD, the majority of participants experience a 4-fold rise in the SBA-BR antibody titer for each of the serogroups contained in the vaccine. Overall, 90.7% of TetraMenD recipients experience a 4-fold rise in antibody titer across all serogroups. Higher pre-vaccination antibody levels are observed for serogroup Y than for C or W-135. This may be related to the fact that serogroup Y is currently the most common serogroup associated with invasive meningococcal disease in this age group in the U.S. and that natural exposure to this serogroup may be more common.

Higher circulating antibody levels reflect recent natural exposure and may reduce the proportion of vaccine recipients exhibiting 4-fold or higher antibody responses. This appears to be the case for serogroup Y responses when compared to other serogroups. The 4-fold rise for serogroup Y is 81.8% compared with 91.7% for serogroup C and 96.7% for serogroup W- 135. High pre-vaccination antibody levels are also observed for serogroup A. This may be Sthe result of intermittent exposure over a prolonged time to several naturally occurring crossreacting antigens.

To further evaluate the impact of pre-existing titers and to investigate the rate of seroconversion (as defined by the proportion of vaccine recipients who achieve a 4-fold rise in 0antibody titer when the pre-vaccination titer for any serogroup is< a separate analysis is 00 Sperformed on participants who had pre-vaccination antibody titers of <1:8 to any one of the 4 O serogroups contained in the vaccine. A titer of 1:8 by the SBA assay using baby rabbit as Sthe complement source is considered to represent an undetectable level of circulating antibody. When participants are evaluated using this criterion, it is observed that there is a 100% seroconversion rate for serogroup A, 98.1% for serogroup C, 98.1% for serogroup W- 135, and 98.3% for serogroup Y after vaccination with TetraMenD.

As previously discussed in another Study, Goldschneider proposed that a minimum titer of 1:4 using an SBA assay with a human complement source correlated with protection from invasive disease against Serogroup C based on observations in military recruits.

However, because of the need for standardization of the assay and the lack of a reliable source of human complement, baby rabbit complement is suggested as an alternative source.

Meningococci appear to be more sensitive to the baby rabbit complement than human complement, resulting in higher measured antibody titers. Several authors have suggested that titers 2 1:128 using the rabbit complement assay are predictive of protection while titers of 1:8 are predictive of susceptibility at least for serogroup C. Although this level may be appropriate when evaluating polysaccharide vaccines, it may not be applicable for conjugate vaccines. Borrow suggested that, in subjects receiving a monovalent C conjugate vaccine who demonstrated post vaccination SBA titers between 8 and 64, the demonstration of a memory response using a reduced dose (10ltg) of a meningococcal polysaccharide vaccine given several months later showed that these individuals are also protected, having achieved an antibody level >1:128. The results for subjects who received the TetraMenD vaccine with SBA- BR titers 2 1:128 for each serogroup is presented in the Tables. When these criteria are applied to each of the serogroups contained in the vaccine, overall, 99.2% of participants who received TetraMenD achieved a post-vaccination SBA-BR titer of 1:128.

cK, IgG and IgM responses are evaluated in a subset of participants using a standard °P ELISA assay. Post-vaccination, the mean level of IgG antibody in the TetraMenD recipients is 2tg for each serogroup. IgM responses are very similar for each serogroup in both C, treatment arms. The IgG responses are generally higher for serogroups C, Y, and W-135 in the Menomune® group than in the group receiving TetraMenD. The post-vaccination SBA GMT levels for serogroups C, Y, and W-135, however, are very similar in each treatment 00 Sgroup, Table C-11.

Table C-11: Relative Contribution of IgG and IgM to Total Bactericidal Activity Day 28 IgG GMC* IgM GMC SBA GMT Serogroup Results: TetraMenD 18.09 17.80 5483.21 A Menomune® 11.61 12.00 3245.67 TetraMenD 5.54 1.55 1924.36 C Menomune® 8.08 1.71 1638.87 TetraMenD 4.41 3.47 1322.26 Y Menomune 9.17 3.45 1228.27 TetraMenD 2.95 1.92 1407.22 W-135 Menomune 4.93 1.68 1544.99 GMC units are pg/mL The observation that the lower levels of IgG produced by the conjugate generated a similar level of bactericidal activity as the polysaccharide vaccine strongly suggests that the quality and affinity of the antibody response to the conjugate vaccine is superior to that generated by the polysaccharide. It is the high affinity antibody that is associated with functional activity and memory response. This effect has also been observed in several published studies.

These data demonstrate that TetraMenD is highly immunogenic in the adolescent population. The GMTs are essentially equivalent for each of the four serogroups for both vaccines, and the titers achieved are predictive of protection, and it appears that TetraMenD generates higher affinity antibody responses for each serogroup contained in the vaccine.

Study D This study is a randomized, active-controlled study of healthy adults aged 18 to years as of DO of a single dose of TetraMenD versus a single dose of Menomune®. Blood serum is drawn on DO, prior to vaccination and D28 and analyzed.

Generally, the safety profile of TetraMenD is comparable to Menomune, specifically, the percentages reported for Solicited Local Reactions (Days Solicited Systemic Reactions (Days Unsolicited Adverse Events (Days 0-28) Unsolicited Significant Adverse Events and SAEs (Day 29-Month 6) Serious Adverse Events (Day 0-Month 6) are all within 2-3% of the percentages reported for Menomune. The results of the Study are provided in the following Tables.

Distribution of SBA-BR Antibody Titers Table D-l shows the frequency distribution of baseline and Day 28 SBA-BR antibody titers for each serogroup.

Table D-l: Distribution of SBA-BR Antibody Titers at Day 0 and Day 28 After Vaccination (Per-protocol Population) SBA-BR Titers <8 to 512 Test Test Test Test Group <8 8 16 32 64 128 256 512 Type Daenn n n n n n n n TetraMenD 156 36 15 37 96 122 176 217 (1279) (12.2) (13.8) (17.0) Day Menomune 144 35 11 41 77 105 134 201 SBA (1099) (13.1) (12.2) (18.3) TetraMenD 0 0 0 1 1 19 28 Day (1278) (3.9) 28 Menomune 1 0 0 0 0 10 23 51 (1099) (4.6) TetraMenD 343 124 73 65 91 115 142 120 (1279) (26.8) (11.1) (9.4) Day Menomune® 304 107 60 60 90 115 108 97 SBA (1099) (27.7) (10.5) (8.8) TetraMenD 2 1 3 4 6 45 60 110 Day (1278) (8.6) 28 Menomune® 3 5 4 1 4 32 51 67 (1099) (6.1) Table D-l: Distribution of SBA-BR Antibody Titers at Day 0 and Day 28 After Vaccination (Per-protocol Population) SBA-BR Titers <8 to 512 Test Test Tes t Test Group <8 8 16 32 64 128 256 512 Typne Dae n n n( n n TetraMenD 279 22 17 52 105 137 165 186 (1279) (21.8) (10.7) (12.9) (14.5) y Menomune 228 18 20 43 77 145 143 160 SBA (1099) (20.7) (13.2) (13.0) (14.6) TetraMenD 21 4 3 5 6 51 98 148 Day (1278) (11.6) 28 Menomune® 10 1 1 2 3 28 65 111 (1099) (10.1) TetraMenD 372 134 91 98 152 148 134 87 Day (1279) (29.1) (10.5) (11.9) (11.6) (10.5) (6.8) Day Menomune 328 114 62 92 144 145 115 63 SBA (1099) (29.8) (10.4) (13.1) (13.2) (10.5) (5.7) TetraMenD 9 6 2 7 13 67 116 203 135) Day (1278) (15.9) 28 Menomune® 3 3 3 1 7 38 67 133 (1099) (12.1) Table D-l: Distribution of SBA-BR Antibody Titers at Day 0 and Day 28 After Vaccination (Per-protocol Population) SBA-BR Titers 1024 to >65536 Test Test 1638 6553 Test Test Group 1024 2048 4096 8192 32768 65536 Type Date 4 6 n y p n TetraMenD 209 173 25 10 2 5 0 0 y0 (1279) (16.3) (13.5) (0.0) Menomune® 196 131 10 8 4 2 0 0 (1099) (17.8) (11.9) (0.0) SBA 179 SA TetraMenD 140 260 287 241 69 3 0 (1278) (10.9) (20.3) (22.4) (18.8) (0.0) Day 28 168 Menomune 115 194 266 209 168 60 1 1 (1099) (10.5) (17.7) (24.2) (19.0) (153 (0.1) TetraMenD 96 80 15 10 2 2 1 0 (1279) (0.0) Menomune® 70 65 8 12 3 0 0 0 (1099) (0.0) SBA 140 (C TetraMenD 138 213 225 178 119 21 13 (1278) (10.8) (16.7) (17.6) (13.9) 9 Day 2 Menomune® 133 162 190 199 120 100 15 13 (10.9 (1099) (12.1) (14.7) (17.3) (18.1) 0 (1.2) TetraMenD 180 111 11 8 4 2 0 0 (1279) (14.1) (0.0) Day 0 Menomune® 147 88 15 9 5 1 0 0 SBA (1099) (13.4) (0.0) (Y TetraMenD 211 216 221 145 94 51 2 2

(Y)

Day (1278) (16.5) (16.9) (17.3) (11.3) (0.2) Day 28 Menomune" 141 200 206 165 119 45 1 1 (10.8 (1099) (12.8) (18.2) (18.7) (15.0) 0 (0.1) SBA TetraMenD 43 17 1 0 1 1 0 0 (1279) (0.0) 135) D Menomune® 26 8 1 1 0 0 0 0 (1099) (0.0) Day TetraMenD 252 244 178 100 59 21 1 0 28 (1278) (19.7) (19.1) (13.9) (0.0) Table D-l: Distribution of SBA-BR Antibody Titers at Day 0 and Day 28 After Vaccination (Per-protocol Population) SBA-BR Titers 1024 to >65536 Test Test 1638 6553 Test Test Group 1024 2048 4096 8192 1638 32768 65536 6553 Type Date 4 6 n n n n n Menomune® 183 242 195 134 57 32 1 0 (1099) (16.7) (22.0) (17.7) (12.2) (0.0) Table D-2 provides a summary of the Geometric Mean Titer (GMT) by Subject Age and Serogroup for TetraMenD.

Table D-2 Summary of GMT by Subject Age and Serogroup for TetraMenD Age Blood No. of Serogroup Serogroup Serogroup Serogroup (in Day Subjects A C GMT W Y-135 Year) GMT GMT GMT 18 Day 0 127 238.47 45.63 42.04 111.67 Day 28 122 5170.42 2690.10 1613.23 2556.00 19 Day 0 132 224.51 40.96 37.66 244.18 Day 28 127 4421.24 2425.55 1786.78 2492.65 Day 0 107 193.76 69.62 41.73 108.16 Day 28 103 5080.24 3193.18 1766.17 1966.95 21 Day 0 106 239.80 57.27 32.42 122.27 Day 28 105 3911.03 2447.58 1521.66 1725.00 22 Day 0 84 371.11 47.95 34.18 175.14 Day 28 82 4649.72 3151.78 1729.45 3151.78 23 Day 0 81 219.45 54.40 50.80 180.25 Day 28 80 4664.48 3788.73 1620.81 1961.17 24 Day 0 66 223.33 71.84 42.49 124.03 Day 28 64 3922.34 3057.48 1479.87 1562.21 Day 0 62 289.50 48.94 29.26 148.02 Day 28 59 4771.87 3685.02 2121.47 1563.07 26 Day 0 29 131.10 37.83 17.19 73.87 Day 28 26 4936.36 4320.32 1170.01 2673.69 27 Day 0 20 315.17 45.25 28.84 445.72 Day 28 20 5042.77 5595.30 803.41 2702.35 28 Day 0 33 593.10 72.60 55.25 157.92 Day 28 33 6640.01 4948.33 1558.63 2091.47 29 Day 0 26 270.02 29.54 28.76 51.71 Day 28 26 4936.36 2534.86 1201.62 2278.46 Day 0 19 229.46 68.84 25.71 99.15 Day 28 19 5287.69 5687.92 1645.39 1529.61 31 Day 0 17 138.88 52.20 27.18 226.53 Day 28 17 2314.48 3340.58 1111.00 1966.18 32 Day 0 24 362.04 58.69 64.00 50.80 Day 28 24 4732.32 3545.24 1448.15 1933.05 33 Day 0 22 329.39 109.34 68.16 164.69 Day 28 22 2989.02 3499.00 1317.54 1749.50 34 Day 0 16 534.67 51.54 41.50 112.40 Day 28 15 3734.42 4096.00 741.00 1415.08 Day 0 17 369.50 156.95 19.62 48.11 Day 28 16 4096.00 6888.62 824.57 1649.14 36 Day 0 17 192.44 69.44 20.43 156.95 Day 28 17 3078.98 7864.70 1362.24 2410.80 37 Day 0 20 238.86 78.79 20.39 73.52 Day 28 19 1835.69 4406.03 637.28 1474.81 38 Day 0 24 203.19 90.51 15.54 78.34 Day 28 24 3158.45 4216.02 558.34 1824.56 39 Day 0 18 376.25 61.58 33.26 143.68 Day 28 18 4778.10 4096.00 1824.56 1970.63 Day 0 25 249.00 71.51 25.63 86.82 Day 28 25 3983.99 3769.09 916.51 1112.82 41 Day 0 26 242.71 60.68 23.24 131.46 Day 28 26 3681.69 2403.25 1201.62 1336.84 42 Day 0 24 128.00 71.84 25.40 32.94 Day 28 24 2435.50 2169.78 542.45 574.70 43 Day 0 23 144.40 51.83 33.99 72.70 Day 28 23 2453.92 1815.42 1120.89 1515.12 44 Day 0 27 198.04 101.59 47.03 99.02 Day 28 27 2647.42 3335.54 1194.53 998.05 Day 0 23 158.06 53.41 31.05 79.03 Day 28 23 2241.79 2686.11 453.85 1561.48 46 Day 0 28 204.87 55.17 21.53 68.93 Day 28 28 2205.89 1680.05 927.46 1217.75 47 Day 0 20 187.40 81.57 32.00 87.43 Day 28 20 2352.53 4544.80 652.58 1351.18 48 Day 0 32 94.52 38.05 25.22 139.58 Day 28 32 3158.45 2435.50 939.01 1299.51 49 Day 0 19 114.73 33.19 24.79 137.69 Day 28 19 2048.00 4096.00 951.95 1586.44 Day 0 16 145.76 94.52 34.90 98.70 Day 28 16 3922.34 2048.00 693.38 1024.00 51 Day 15 73.52 29.18 27.86 67.03 Day 28 15 1702.38 2702.35 280.79 370.50 52 Day 0 12 135.61 90.51 11.99 95.89 Day 28 12 2169.78 3251.00 542.45 542.45 53 Day 0 11 272.65 128.00 49.74 105.95 Day 28 11 1922.93 1922.93 423.81 350.81 54 Day 0 10 84.45 90.51 9.19 238.86 Day 28 10 2702.35 3104.19 222.86 1176.27 Day 0 6 71.84 22.63 57.02 50.80 Day 28 6 812.75 3649.12 512.00 724.08 Table D-3 shows the numbers and percentages of participants with a 24-fold rise in SBA-BR titer from baseline to Day 28 for the serogroups A, C, Y, and W-135. The numbers and percentages for the serogroups A, 1028/1278 C, 1131/1278 Y, 941/1278 and W-135, 1142/1278 in the TetraMenD group are comparable to those in the Menomune® group, with serogroups A, 929/1099 C, 985/1099 Y, 872/1099 and W-135, 1036/1099 Table D-3: Number and Percentage of participants with a 4-Fold Rise from Baseline in SBA-BR Titer by Serogroup TetraMenD Menomune® Upper one- 4-fold rise Difference Sided 97.5% in SBA-BR (PMenomune Confidence titer for n/Nt PTetraMenD n/N PMenomune® limit of the Serogroups PTetraMenD) Difference A 1028/1278 80.4 929/1099 84.5 4.1 7.1 C 1131/1278 88.5 985/1099 89.6 1.1 3.6 y 941/1278 73.6 872/1099 79.3 5.7 9.1 W-135 1142/1278 89.4 1036/1099 94.3 4.9 7.1 Testing the null hypothesis Ho: PMenomune PTetraMenD 20.10 versus Ha: PMenomune PTecraMenD 0. t n/N: n number of participants with a 24-fold rise from baseline titer N total number of participants in the per-protocol population.

SPrctraMenD: percentages of participants with a >4-fold rise from baseline in SBA-BR postvaccination titer from the TetraMenD group.

PMenomune®: percentages of participants with a 24-fold rise from baseline in SBA-BR postvaccination titer from the Menomune® group.

Frequency of SBA-BR Antibody Titers 32 The proportion of participants with SBA-BR antibody titers 32 at Day 28 after vaccination is summarized in Table D-4.

Table D-4: Percentage and Number of Participants with an SBA Antibody Titer 32 at Day 28 Post-Vaccination (Per-protocol Population) TetraMenD Menomune 95% CI for t 95% CI for (n/N) t the percentage the percentage 100.0 99.9 (99.49%, Serogroup A (99.77%, 100.00%) 9 (1278/1278) (1098/1099) 100.00%) SerogroupC 99.5 8.98989 (98 (1272/1278) (1087/1099) 99.43%) 97.8 98.9 (98.10%, Serogroup Y (96.85%, 98.54%) (1250/1278) (1087/1099) 99.43%) Serogroup 98.7 99.2 (98.45%, W-135 (1261/1278) (97 (1090/1099) 99.62%) t n: number of participants with a titer 32 at Day 28 participants with a valid blood sample at Day 28 in this group.

n/N post-vaccination N: total number of Frequency of SBA-BR Antibody Titers 128 The proportion of participants with SBA-BR antibody titers 128 at Day 28 after vaccination is summarized in Table Table D-5: Percentage and Number of Participants with an SBA Antibody Titer 128 at Day 28 Post-Vaccination (Per-protocol Population) TetraMenD Menomune 95% CI for 95% CI for (n/N)t the percentage the percentage 99.8 (99.44%, 99.9 (99.49%, SerogroupA (1276/1278) 99.98%) (1098/1099) 100.00%) 98.7 (97.97%, 98.5 (97.53%, erogroupC (1262/1278) 99.28%) (1082/1099) 99.10%) 96.9 (95.85%, 98.5 (97.53%, erogroupY (1239/1278) 97.82%) (1082/1099) 99.10%) Serogroup W- 97.1 (96.03%, 98.5 (97.53%, 135 (1241/1278) 97.95%) (1082/1099) 99.10%) n/N.

t n: number of participants with a titer 128 at Day 28 post-vaccination.

SN: total number of participants with a valid blood sample at Day 28 in this group.

Table D-6 Analysis of Treatment Effect on GMTs Adjusted by Baseline Covariate: Response of Titer Difference from Day 0 to Day 28 (Per-protocol Population)* Difference of Anti-Log of 95% CI for Estimate of Treatment Treatment Anti-Log of Serogroup Baseline Baseline Effect Effect* Treatment GMT GMT Effect (Menomune®- (Menomune®- effect TetraMenD) TetraMenD) (Menomune TetraMenD)

SBA

Serogroup A TetraMenD 223.6 -0.850 0.096 1.069 (0.973, 1.175) Menomune 203.9

SBA

Serogroup C TetraMenD 57.2 -0.772 0.130 1.094 (0.965, 1.240) Menomune 51.8

SBA

Serogroup Y TetraMenD 122.9 -0.743 0.469 1.384 (1.225, 1.563) Menomune 127.4

SBA

Serogroup W-135 TetraMenD 33.2 -0.766 0.576 1.491 (1.334, 1.666) Menomune 31.0 a r I Anti-Log or treatment TetraMenD) power.

ettect is calculated as 2 to the treatment etrect (Menomune-- Proportion of Participants with at least a 4-fold rise in SBA-BR Antibody Titers Table D-7 shows the proportion of participants with a 4-fold rise from baseline in Day 28 SBA antibody titers.

Table D-7: Number and Percentage of Participants with a 4-Fold Rise in Day 28 SBA Antibody Titers From Baseline TetraMenD Menomune Test Type (95% CI) (n t (95% CI) 80.4 (1028/1278) (78.16%, 84.5 (929/1099) (82.26%, SBA 82.58%) 86.62%) 88.5 (1131/1278) (86.62%, 89.6 (985/1099) (87.67%, SBA 90.20%) 91.37%) 73.6 (941/1278) (71.12%, 79.3 (872/1099) (76.83%, SBA 76.03%) 81.70%) 89.4 (1142/1278) (87.54%, 94.3 (1036/1099) (92.72%, SBA (W-135) 91.00%) 95.57%) n/N.

Sn: number of participants with 4-fold rise from baseline titer.

number of participants with blood draws within each scrogroup.

Proportion of Participants with Undetectable Titers 8) at Day 0 Achieving a 4-Fold Rise in Day 28 SBA-BR Antibody Titers Table D-8 shows the proportion of participants with undetectable titers 8) at Day 0 Achieving a 4-Fold Rise in Day 28 SBA-BR Antibody Titers. In both treatment groups and for all vaccine serogroups, most participants with an undetectable 8) SBA titer at baseline achieved a 4-fold rise in Day 28 SBA titers. The proportions of participants with an SBA titer 8 at Day 0 who had a 4-fold rise from baseline to Day 28 ranged from 90.7% to 100.0% in the TetraMenD group and from 96.9% to 99.3% in the Menomune® group.

Table D-8: Proportion of Participants with Undetectable Titers at Day 0 Achieving a >4-Fold Rise in Day 28 SBA-BR Antibody Titers TetraMenD Menomune Test Type (nt/N) (95% CI) (nt/N) (95% CI) 100.0 (156/156) (98.10%, 99.3 (143/144) (96.19%, SBA 100.00%) 99.98%) 99.4 (341/343) (97.91%, 97.7 (297/304) (95.31%, SBA 99.93%) 99.07%) 90.7 (253/279) (86.64%, 96.9 (221/228) (93.78%, SBA 93.82%) 98.76%) 96.5 (359/372) (94.10%, 99.1 (325/328) (97.35%, SBA (W-135) 98.13%) 99.81%) n/N.

t n: number of participants with titers 1:8 at Day 0 and titers 1:32 at Day 28 within each serogroup 1N: number of participants with titers 1:8 at Day 0 within each serogroup.

Table D-9 shows the SBA GMTs at baseline and on Day 28 after vaccination and the fold rises in SBA GMTs.

Table D-9: Summary of Geometric Mean of Antibody Titers (GMT) and Fold Rise of GMT by Serogroup (Per-protocol Population) 00 O 0 Test Param TetraMenD Menomune Test Param Type eter* N e GMT (95% CI) N GMT (95% CI)' Day 0 1279 223.6 (199.86' 1099 203.9 (180.53 Serogro Tr 250.08) 230.23) Serogro Titer (3646.33, 4108. (3827.43, upA Day 28 1278 3896.6 6 1099 4108. (3 SBA 4164.11) 9 4411.15) Fold (14.39, (16.39, FoRe Day 28 1278 16.0 1.4) 1099 18.4 2.6 Rise 17.84) 20.67) Day0 1279 57.2 (50.50, 1099 51.8 (45.47, 64.73) 59.11) Serogro Titer (2958.46, 3463. (3143.05 upC Day 28 1278 3235.2 5.6, 1099 3463. 3143.

SBA -3537.76) 4 3816.34)

SBA

Fold Day 28 1278 47. (41.74, (48.53, Rise Day 28 1278 47.1 53.05) 1099 55.1 62.67) Rise 53.05) 62.67) Day 0 1279 122.9 (10889 1099 127.4 (111.97, 138.72) 145.03) Serogro Titer upY Day 28 1278 1751.8 (1598.14, 1099 2446. (2235.36, SBA _1920.30) 7 2677.93) Fold (10.97, (14.68, Fod Day 28 1278 12.3 1. 1099 16.6 1 Rise 13.68) 18.83) (29.95 (27.90 Day 0 1279 33.2 (29.95 1099 31.0 (27.90, Serogrou Titer 36.73) 34.46) pW-135 Day 28 1278 1270.7 (1171.59, 1865. (1717.28, SBA 1378.22) 5 2026.48) Fold (28.35, (44.07, Fold Day 28 1278 31.4 8 3' 1099 48.9 44 0 Rise 34.70) 54.30) Titer or fold-rise, where fold rise titer at Day 28 Titer at Day 0 t N: number of participants with blood draws within each serogroup. Note: One Participant did not have a second blood sample done CI for the GMT is calculated based on an approximation to the normal distribution.

Twenty-eight to 56 days after receiving the study vaccination, TetraMenD, the majority of participants experience a 4-fold rise in the SBA-BR antibody titer for each of the l serogroups contained in the vaccine. The percentages of TetraMenD recipients obtaining a S4-fold rise in antibody titer are 80.4%, 88.5%, 73.6 and 89.4% for serogroups A, C, Y, and W-135, respectively. Higher pre-vaccination antibody levels are observed for serogroup Y than for C or W-135. This may be related to the fact that serogroup Y is currently the most common serogroup associated with invasive meningococcal disease in this age group in the U.S. and that natural exposure to this serogroup may be more common. Higher circulating 00 antibody levels reflect recent natural exposure and may reduce the proportion of vaccine O recipients exhibiting 4-fold or higher antibody responses. This appears to be the case for serogroup Y responses when compared to other serogroups. The 4-fold rise for serogroup Y is O 10 73.6% compared with 88.5% for serogroup C and 89.4% for serogroup W-135. High prevaccination antibody levels are also observed for serogroup A. This may be the result of intermittent exposure over a prolonged period of time to several naturally occurring crossreacting antigens.

To further evaluate the impact of pre-existing titers and to investigate the rate of seroconversion (as defined by the proportion of vaccine recipients who achieve a 4-fold rise in antibody titer when the pre-vaccination titer for any serogroup is< a separate analysis is performed on participants who had pre-vaccination antibody titers of 1:8 to any one of the 4 serogroups contained in the vaccine. A titer of 1:8 by the SBA assay using baby rabbit as the complement source is considered to represent an undetectable level of circulating antibody. When participants are evaluated using this criterion, it is observed that there is a 100% seroconversion rate for serogroup A, 99.4% for serogroup C, 96.5% for serogroup W- 135, and 90.7% for serogroup Y after vaccination with TetraMenD.

As previously discussed in another Study herein, based on observations in military recruits, Goldschneider proposed that a minimum titer of 1:4 using an SBA assay with a human complement source correlated with protection from invasive disease against Serogroup C. Baby rabbit complement is suggested as an alternative source, but meningococci appear to be more sensitive to the baby rabbit complement than human complement, resulting in higher measured antibody titers. Several authors have suggested that titers 2 1:128 using the baby rabbit complement assay are predictive of protection while titers of 1:8 are predictive of susceptibility at least for serogroup C. Although this level may be appropriate when N evaluating polysaccharide vaccines, it may not be applicable for conjugate vaccines. Borrow tb suggested that, in subjects receiving a monovalent C conjugate vaccine who demonstrated post vaccination SBA titers between 8 and 64, the demonstration of a memory response using a Sreduced dose (10pg) of a meningococcal polysaccharide vaccine given several months later showed that these individuals are also protected, having achieved an antibody level >1:128.

When this criterion is applied to all the serogroups contained in the vaccine, the percentages of 00 participants receiving TetraMenD who achieve a post-vaccination SBA-BR titer 1:128 are 0 99.8%, 98.7%, 96.9%, and 97.1% for serogroups A, C, Y, and W-135, respectively.

Example 13 Study E Td Booster Study in Children Aged 10 to 18 This study compares the tetanus and diphtheria toxoid (Td) booster response in the group receiving the experimental tetravalent Meningococcal Diphtheria Conjugate vaccine, TetraMenD, concomitantly with Td to the response in the group receiving Td with placebo, as measured by the proportion of participants who have an acceptable response in their respective tetanus and diphtheria titers. An acceptable response is defined as, 28 days following vaccination, at least a 4-fold rise from baseline in participants with a predefined low prevaccination titer and at least a 2-fold rise from baseline in participants with a predefined high pre-vaccination titer.

To compare the antibody response for serogroups A, C, Y, and W-135 in TetraMenD when administrated concomitantly with Td to the response when TetraMenD is administrated 28 days following Td vaccine, as measured by the proportion of participants with at least a 4fold rise in titer to each serogroup.

This is a randomized, modified double-blind, active-control multi-center trial, with a total of 1024 participants randomized to one of two treatment groups: A and B.

Day 0 Day 28 Day 56 VI V2 V3 Group A BS-I Td BS-2 Placebo BS-3 TetraMenD Group B BS-I Td Placebo BS-2 TetraMenD BS-3 ct, The age range of 11 to 17 years is chosen to capture those individuals who would normally P receive Td vaccine as part of the routine childhood immunization schedule. In addition, this age range has been identified as high risk for development of invasive meningococcal disease C, and would most likely be candidates for vaccination with the meningococcal conjugate vaccine once licensed. In order to properly evaluate safety, a modified double-blind design using a placebo control is utilized. For the first visit, the vaccination nurse is unblinded and 00 administered the vaccines in each arm according to protocol; TetraMenD (IM) or placebo in O the right arm and Td in the left. For the second visit, each treatment group received the vaccine in the left arm. The evaluation nurse is blinded monitored local and systemic reactions and adverse events.

The age range of II to 17 years is chosen to capture those individuals who would normally receive Td vaccine as part of the routine childhood immunization schedule. In addition, this age range has been identified as high risk for development of invasive meningococcal disease and would most likely be candidates for vaccination with the meningococcal conjugate vaccine once licensed.

Blood specimens (at least 5 mL whole blood) for serologic testing are drawn on Day 0 prior to vaccination (baseline) and at Day 28 post-vaccination 1. There is a third blood draw for participants 28 days after visit 2. At each of these time points, sera are assayed for meningococcal serogroups A, C, Y, and W-135, anti-diphtheria antibody and anti-tetanus antibody.

To evaluate antibody function in recipients of TetraMenD, all available specimens are assayed for SBA using baby rabbit complement (SBA-BR) against each vaccine serogroup.

One immunologic endpoint is the proportion of participants in each treatment group with a >4fold rise in SBA-BR titer. Anti-diphtheria antibody levels are measured by the ability of the test sera to protect Vero cells from a diphtheria toxin challenge. Anti-tetanus antibody levels are measured by an indirect Enzyme Linked Immunosorbent Assay (ELISA).

This study compares the antibody responses to TetraMenD for serogroups A, C, Y, and W-135 as measured by the GMTs in participants from an earlier study, Study C, who receive one dose of TetraMenD to the responses in participants who receive TetraMenD administered concomitantly with Td and 28 days following Td vaccination. Serum specimens for serologic analysis are obtained at baseline (Day 0) prior to vaccination and at Day 28 (window: +28 115 days) and 6 months after vaccination. Antibody titers to tetanus toxoid and diphtheria toxoid (Td) vaccine are measured pre- and 28 days post vaccination.

SBA-BR antibody titers for N. meningilidis serogroups A, C, Y, and W-135 are measured for all available serum specimens pre- and 28 days post vaccination. Overall, the safety profile of Group A and Group B are comparable. The results of this Study are summarized in the following Tables.

Table E-1 summarizes GMT levels by Subject Age and Serogroup.

1) Table E-1 Summary of GMT by Subject Age and Serogroup Age Dose No. of Blood A GMT C GMT W GMT Y GMT TetraMenD Subjects Day 1 Pg 1 0 2048.00 512.00 512.00 256.00 4 tg 1 28 8192.00 1024.00 4096.00 1024.00 Ptg 1 56 32768.00 4096.00 16384.00 8192.00 11 1 Pg 273 0 186.38 63.35 23.06 140.97 4 lig 267 28 2331.86 513.33 339.73 775.64 19lag 265 56 10668.96 2005.59 2285.81 2203.62 12 1 tg 236 0 185.87 56.24 27.23 138.56 4 ltg 229 28 1672.07 516.67 280.33 752.00 lg 226 56 10030.01 2522.93 2164.24 2231.65 13 1 jg 172 0 219.65 60.25 20.46 128.00 4 9 170 28 2562.84 664.66 372.52 898.74 .ig 168 56 10493.02 2700.12 2346.72 2289.34 14 1 pg 128 0 326.64 65.05 30.81 107.63 4 pg 126 28 1896.19 597.26 388.88 752.50 ltg 126 56 9044.70 2375.94 2248.77 1885.79 1 101 0 223.17 83.07 47.00 114.69 4 ltg 95 28 2352.53 716.20 504.58 776.05 1 0 gtg 94 56 9424.03 4575.06 2356.01 1988.48 16 1 lag 71 0 393.36 46.83 24.11 70.56 4 pg 70 28 2399.59 783.77 450.16 512.00 tg 69 56 8527.88 3154.48 1908.93 1833.75 17 1 itg 35 0 358.47 107.10 28.41 79.58 4 tg 35 28 2173.36 403.70 156.70 411.78 34 56 7398.11 3340.58 1418.93 1390.30 Table E-2 shows the numbers and proportions of participants with at least a 4-fold or 2-fold rise in tetanus and diphtheria antibody on Day 28.

Table E-2 Numbers and Proportions of Participants with at least a 4-fold or 2-fold rise in Tetanus and Diphtheria Antibody on Day 28 Td+TetraM 95% CI enD, Td+Placebo, Placebo TetraMenD for the n/N n/N Difference Antigen Response (Pb-Pa) Difference Tetanus 2-Fold (Pre-titer >5.3 0/24 2/23 IU/mL) (0.00) (8.70) 4-Fold (Pre-titer <5.3 399/439 417/448 IU/mL) (90.89) (93.08) Total Responders 399/463 419/471 2.78 (-1.45,7.01) (86.18) (88.96) Diphtheria 2-Fold(Pre-titer 44/47 42/49 >1.28 IU/mL) (93.62) (85.71) 4-Fold(Pre-titer 419/419 416/425 1.28 IU/mL) (100.00) (97.88) Total Responders 463/466 458/474 -2.73 (-4.51,-0.95) (99.36) (96.62) Tetanus and Diphtheria Antibody Titers and SBA Antibody Titers for Serogroups A, C, Y, and W-135 Table E-2 shows the numbers and proportions of participants with at least a 4-fold or 2-fold rise in tetanus and diphtheria antibody on Day 28. The differences in the proportions are: 2.78 and -2.73 for tetanus and diptheria, respectively.

Table E-2: Total Number and Proportion of Participants with at least 4-Fold or 2-Fold Rise Response in Tetanus and Diphtheria Antibody on Day 28 Following the Tetanus and Diphtheria Vaccination, Primary Hypothesis 1(Per-Protocol Population) Td+TetraMenD,Placebo Td+Placebo,TetraMenD Difference 95% CI for the Antigen Response n/N n/N (Pb-Pa) Difference Tetanus 2-Fold (Pre-titer >5.3 0/24 (0.00) 2/23 (8.70) IU/mL) 4-Fold (Pre-titer <5.3 399/439 (90.89) 417/448 (93.08) IU/mL) Total Responders 399/463 (86.18) 419/471 (88.96) 2.78 (-1.45,7.01) Table E-2: Total Number and Proportion of Participants with at least 4-Fold or 2-Fold Rise Response in Tetanus and Diphtheria Antibody on Day 28 Following the Tetanus and Diphtheria Vaccination, Primary Hypothesis I(Per-Protocol Population) Td+TetraMenD,Placebo Td+Placebo,TetraMenD Difference 95% Cl for the Antigen Response n/N n/N (Pb-Pa) Difference Diphtheria 2-Fold(Pre-titer >1.28 44/47 (93.62) 42/49 (85.71) IU/mL) 4-Fold(Pre-titer 1.28 419/419 (100.00) 416/425 (97.88) I U/m L) Total Responders 463/466 (99.36) 458/474 (96.62) -2.73 (-4.51,-0.95) Table E-3 shows the numbers and proportions of participants with at least a 4-fold rise in antibody titer to serogroups A, C, Y, and W-135 on Day 28.

Table E-3: Number and Proportion of Participants with a >4-Fold Rise in SBA-BR Titer on Day 28 Following the TetraMenD Vaccination, Primary Hypothesis 2 (Per- Protocol Population) Td+TetraMenD,Placebo Td+Placebo,TetraMenD Difference 95% CI for the Serogroup n/N n/N (Pb-Pa) Difference Serogroup A 419/466 (89.91) 433/478 (90.59) 0.67 (-3.11,4.46) Serogroup C 424/466 (90.99) 394/478 (82.43) -8.56 (-12.85,-4.27) Serogroup Y 399/466 (85.62) 311/478 (65.06) -20.56 (-25.89,-15.23) Serogroup W-135 448/466 (96.14) 419/478 (87.66) -8.48 (-11.91,-5.05) Table E-4 shows the number of participants with high diphtheria and tetanus titers at baseline and the number and proportion of participants with a 2-fold rise on Day 28.

Table E-4: Number of Participants with High Diphtheria and Tetanus Pre- Titers at Baseline and Number and Proportion of Participants with 2-Fold Rise on Day 28 Per-Protocol Population Td+TetraMenD,Placebo Td+Placebo,TetraMenD Baseline Titer 2 2-Fold Rise Baseline Titer 2 2-Fold Rise n/N n/N n/N n/N Tetanus 24/468 5.13 0/24 0.00 23/472 4.87 2/23 8.70 5.3 IU/ml Diphtheria 47/469 10.02 44/47 93.62 49/476 10.29 42/49 85.71 1.28 IU/ml Table E-5 shows the number of participants with low diphtheria and tetanus titers at baseline and the number and proportion of participants with a 4-fold rise on Day 28.

Table E-5: Number of Participants with Low Diphtheria and Tetanus Pre- Titers at Baseline and Number and Proportion of Participants with 4-Fold Rise on Day 28- Per Protocol Population Td+TetraMenD,Placebo Td+Placebo,TetraMenD Baseline Titer 4-Fold Rise Baseline Titer 4-Fold Rise n/N n/N n/N n/N Tetanus <5.3 IU/ml 444/468 94.87 399/439 90.89 449/472 95.13 417/448 93.08 Diphtheria 51.28 422/469 89.98 419/419 100.00 427/476 89.71 416/425 97.88 IU/ml

C-

O

O

Table E-6 shows the number and proportion of participants with a titer >1.0 IU/ml in tetanus and diphtheria antibody on Day 28 following tetanus and diphtheria vaccination given concomitantly with TetraMenD or Placebo.

Table E-6: Number and Proportion of Participants with Titer >1.0 IU/ml in Tetanus and Diphtheria Antibody on Day 28 Following Tetanus and Diphtheria Vaccination, (Per-Protocol Population) Td+TetraMenD,Placebo Td+Placebo,TetraMenD Difference 95% CI for the n/N n/N (Pb-Pa) Difference Tetanus 1.0 461/465 (99.14) 470/477 (98.53) -0.61 (-1.97,0.76) IU/ml Diphtheria 1.0 467/467 (100.00) 474/476 (99.58) -0.42 (-1.00,0.16) IU/ml Table E-7 shows the geometric mean antibody titers(GMTs) for tetanus and diphtheria on Day 28 following tetanus and diphtheria vaccination (given concomitantly with TetraMenD or Placebo).

Table E-7: Comparison of Geometric Mean Antibody Titers(GMTs) for Tetanus and Diphtheria on Day 28 Following Tetanus and Diphtheria Vaccination, (Per-Protocol Population)[1] Td+TetraMenD,Placebo Td+Placebo,TetraMenD GMT Ratio 95% CI for GMTa (95% CI) GMTb (95% CI) GMTb/GMTa GMT Ratio Tetanus 11.46 (10.79,12.18) 13.56 (12.73,14.44) 1.18 (1.08,1.29) Diphtheria 304.69 (221.69,418.78) 10.60 (9.23,12.18) 0.03 (0.02,0.05) Table E-8 shows the geometric mean antibody titers(GMTs) for SBA-BR for serogroups A, C, Y, and W-135 on Day 28 post TetraMenD vaccination. The GMT ratios are 0.92, 0.42, 0.39, and 0.32 for serogroups A, C, Y, and W-135, respectively.

Table E-8: Comparison of Geometric Mean Antibody Titers(GMTs) for SBA-BR on Day 28 Following the TetraMenD Vaccination, (Per-Protocol Population)[1] Td+TetraMenD,Placebo T'd+Placebo,TetraMenD GMT Ratio GMTb/GMT 95% CI for GMTa (95% CI) GMTb (95% CI) a GMT Ratio Serogroup A 11321.8 (10173.2,12600.0) 10391.4 (9523.1,11338.8) 0.92 (0.8,1.1) Serogroup C 5042.0 (4389.4,5791.7) 2136.0 (1810.8,2519.4) 0.42 (0.3,0.5) Serogroup Y 3387.3 (2978.2,3852.5) 1331.3 (1170.2,1514.6) 0.39 (0.3,0.5) Serogroup 4175.8 (3702.1,4710.1) 1339.1 (1161.8,1543.4) 0.32 (0.3,0.4) W-135 Table E-9 shows the geometric mean antibody titers(GMTs) for SBA-BR for serogroups A, C, Y, and W-135 on Day 28 post TetraMenD vaccination in the Td TetraMenD, Placebo group and the corresponding results from study MTA02. The GMT ratios are 0.48, 0.38, 0.34, and 0.39 for serogroups A, C, Y, and W-135, respectively.

Table E-9: Comparison of Geometric Mean Antibody Titers(GMTs) for SBA-BR on Day 28 Following the TetraMenD Vaccination in Group Td+TetraMenD,Placebo to the Corresponding Results from Study C, (Per-Protocol Population) Td+TetraMenD,Placebo Study C GMT GMT GMT Ratio Study GMTmta02/ 95% CI for GMTa (95% CI) C (95% CI) GMTa GMT Ratio Serogroup A 11321.8 (10173.2,12600.0) 5483.2 (4920.1,6110.7) 0.48 (0.4,0.6) Serogroup C 5042.0 (4389.4,5791.7) 1924.4 (1662.1,2228.0) 0.38 (0.3,0.5) Serogroup Y 3387.3 (2978.2,3852.5) 1322.3 (1161.9,1504.8) 0.39 (0.3,0.5) Serogroup 4175.8 (3702.1,4710.1) 1407.2 (1232.1,1607.3) 0.34 (0.3,0.4) W-135 Table E-10 shows the geometric mean antibody titers (GMTs) for SBA-BR for serogroups A, C, Y, and W-135 on Day 28 post TetraMenD vaccination in the Td Placebo, TetraMenD group and the corresponding results from study MTA02. The GMT ratios are 0.53, 0.90, 0.99, and 1.05 for serogroups A, C, Y, and W-135, respectively.

00

OO

CIA

Table E-10: Comparison of Geometric Mean Antibody Titers (GMTs) for SBA-BR on Day 28 Following the TetraMenD Vaccination in Group B to the Corresponding Results from Study C, Observational Hypothesis(Per-Protocol Population)[l] Td+Placebo,TetraMenD Study C GT GMT Ratio

GM'I

GMTmta02/ 95% CI for GMTb (95% CI) Study C (95% CI) GMTb GMT Ratio Serogroup A 10391.4 (9523.1,11338.8) 5483.2 (4920.1,6110.7) 0.53 (0.5,0.6) Serogroup C 2136.0 (1810.8,2519.4) 1924.4 (1662.1,2228.0) 0.90 (0.7,1.1) Serogroup Y 1331.3 (1170.2,1514.6) 1322.3 (1161.9,1504.8) 0.99 (0.8,1.2) Serogroup 1339.1 (1161.8,1543.4) 1407.2 (1232.1,1607.3) 1.05 (0.9,1.3) W-135 Table E-l I shows the distribution of SBA-BR antibody titers on Day 0 and Day 28 after the TetraMenD Vaccination by serogroup for the Per-Protocol Population (SBA-BR Titers <8 to 1024). While, Table E-12 shows the distribution of SBA-BR antibody titers on Day 0 and Day 28 after the TetraMenD Vaccination by serogroup for the Per-Protocol Population (SBA-BR Titers 2048 to 524288). Table E-13 provides additional data.

Table E-ll: Distribution of SBA-BR Antibody Titers on Day 0 and Day 28 After the TetraMenD Vaccination by Serogroup (Per-Protocol Population) (SBA-BR Titers <8 to 1024) SBA Titers <8 8 16 32 64 128 256 512 1024 Test Test Grou n n n n n n n nn n Test Test Grou Type Date p N Sero- Day0 Group 470 90 13 5 2 9 25 60 68 97 group A 19.1 2.8 1.1 0.4 1.9 5.3 12.8 14.5 20.6 A Group 478 53 6 2 0 5 36 74 73 103 B 11.1 1.3 0.4 0.0 1.0 7.5 15.5 15.3 21.5 Day Group 470 0 0 0 0 0 3 3 7 28 A 0.0 0.0 0.0 0.0 0.0 0.6 0.6 1.5 2.1 Group 478 0 0 0 0 0 0 1 4 6 B 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.8 1.3 Serogroup

C

Day 0 Group

A

Group 478 153 31 20 13 12 55 49 42 B 32.0 6.5 4.2 2.7 2.5 11.5 10.3 8.8 9.4 Table E-ll: Distribution of SBA-BR Antibody Titers on Day 0 and Day 28 After the TetraMenD Vaccination by Serogroup (Per-Protocol Population) (SBA-BR Titers <8 to 1024) SBA Titers <8 8 16 32 64 128 256 512 1024 Test Test Grou n n n n n n n n n Test Test Grou Type Date p N Day Group 470 1 0 0 2 1 5 11 26 39 28 A 0.2 0.0 0.0 0.4 0.2 1.1 2.3 5.5 8.3 Group 478 2 2 2 9 4 20 35 52 67 B 0.4 0.4 0.4 1.9 0.8 4.2 7.3 10.9 14.0 Sero- Day Group 470 101 5 6 22 30 53 81 67 52 group A 21.5 1.1 1.3 4.7 6.4 11.3 17.2 14.3 11.1 Y Group 478 93 1 2 8 20 84 69 71 69 B 19.5 0.2 0.4 1.7 4.2 17.6 14.4 14.9 14.4 Day Group 470 1 1 1 2 3 7 12 23 28 A 0.2 0.2 0.2 0.4 0.6 1.5 2.6 4.9 13.8 Group 478 4 3 1 2 5 20 38 69 B 0.8 0.6 0.2 0.4 1.0 4.2 7.9 14.4 17.8 Sero- Day0 Group 470 205 30 17 33 37 41 39 31 17 group A 43.6 6.4 3.6 7.0 7.9 8.7 8.3 6.6 3.6 W-135 Group 478 213 29 9 14 26 69 48 32 24 B 44.6 6.1 1.9 2.9 5.4 14.4 10.0 6.7 Day Group 470 1 0 0 0 1 4 7 24 62 28 A 0.2 0.0 0.0 0.0 0.2 0.9 1.5 5.1 13.2 Group 478 7 3 1 2 1 21 42 72 87 B 1.5 0.6 0.2 0.4 0.2 4.4 8.8 15.1 18.2 Group A: Vaccination 1 Td TetraMenD Vaccination 2 Placebo Group B: Vaccination I Td Placebo Vaccination 2 TetraMenD i 13 oO 0 tm- 0

(N

1-^ Table E-12: Distribution of SBA-BR Antibody Titers on Day 0 and Day 28 After the TetraMenD Vaccination by Serogroup (Per-Protocol Population) (SBA-BR Titers 2048 to 524288) SBA Titers Miss- 2048 4096 8192 16384 32768 65536 131072 524288 ing n n n n n n n n n n n Test Test Type Date Group N Sero- Day A 470 70 16 10 3 1 0 0 0 1 group 0 14.9 3.4 2.1 0.6 0.2 0.0 0.0 0.0 0.2 A B 478 75 29 13 4 5 0 0 0 0 15.7 6.1 2.7 0.8 1.0 0.0 0.0 0.0 0.0 Day A 470 29 60 109 102 117 17 10 0 3 28 6.2 12.8 23.2 21.7 24.9 3.6 2.1 0.0 0.6 B 478 38 75 112 147 83 7 5 0 0 7.9 15.7 23.4 30.8 17.4 1.5 1.0 0.0 0.0 Sero- Day A 470 36 4 2 3 1 0 0 0 1 group 0 7.7 0.9 0.4 0.6 0.2 0.0 0.0 0.0 0.2 C B 478 32 14 8 2 2 0 0 0 0 6.7 2.9 1.7 0.4 0.4 0.0 0.0 0.0 0.0 Day A 470 80 79 77 74 51 10 10 1 3 28 17.0 16.8 16.4 15.7 10.9 2.1 2.1 0.2 0.6 B 478 79 62 59 36 35 5 9 0 0 16.5 13.0 12.3 7.5 7.3 1.0 1.9 0.0 0.0 Sero- Day A 470 37 9 4 2 0 0 0 0 1 group 0 7.9 1.9 0.9 0.4 0.0 0.0 0.0 0.0 0.2 Y B 478 35 15 6 5 0 0 0 0 0 7.3 3.1 1.3 1.0 0.0 0.0 0.0 0.0 0.0 Day A 470 74 114 77 56 28 1 2 0 3 28 15.7 24.3 16.4 11.9 6.0 0.2 0.4 0.0 0.6 B 478 113 73 47 15 3 0 0 0 0 23.6 15.3 9.8 3.1 0.6 0.0 0.0 0.0 0.0 Sero- Day A 470 15 3 1 0 0 0 0 0 I group 0 3.2 0.6 0.2 0.0 0.0 0.0 0.0 0.0 0.2 W- B 478 11 1 2 0 0 0 0 0 0 135 135 2.3 0.2 0.4 0.0 0.0 0.0 0.0 0.0 0.0 Day A 470 85 90 77 74 37 3 2 0 3 28 18.1 19.1 16.4 15.7 7.9 0.6 0.4 0.0 0.6 B 478 109 60 39 19 12 2 1 0 0 22.8 12.6 8.2 4.0 2.5 0.4 0.2 0.0 0.0 Group A: Vaccination I Td TetraMenD Vaccination 2 Placebo Group B: Vaccination 1 Td Placebo Vaccination 2 TetraMenD Table E-13: Summary of Antibody Titers: Distribution of Tetanus and Diphtheria Titers (Per-Protocol Population)[l] Td+TetraMenD, Placebo Td+Placebo, TetraMenD Antibody Titer Test Date n/N n/N Tetanus <0.1 IU/ml Day 0 5/468 3/472 (0.6) Day 28 0/465 0/477 (0.0) >0.1 1.0 IU/ml Day 0 281/468 (60.0) 274/472 (58.1) Day 28 4/465 7/477 lU/ml Day 0 182/468 (38.9) 195/472 (41.3) Day 28 461/465 (99.1) 470/477 (98.5) Diphtheria 0.1 lU/ml Day 0 87/469 (18.6) 103/476 (21.6) Day 28 0/467 0/476 (0.0) >0.1 1.0 IU/ml Day 0 255/469 (54.4) 267/476 (56.1) Day 28 0/467 2/476 (0.4) IU/ml Day 0 127/469 (27.1) 106/476 (22.3) Day 28 467/467 (100.0) 474/476 (99.6) Related studies were conducted to access the safety and immunogenicity of MCV-4 vaccine concomitantly administered with licensed Td vaccine in healthy 10 -17 year old adolescents.

Briefly, in a multicenter randomized trial healthy 10-17 year olds (mean age 12.9 years) received TetraMenD (MCV-4) Td either concomitantly (n 509), or at separate visits one month apart (n 512). Safety assessments for the two vaccines, given separately and concomitantly, were collected on days 8 and 28 post-vaccination. Immune responses were assessed prior to and 4 weeks post-vaccination by antibody titers to diphtheria and tetanus, and serum bactericidal activity (SBA) to the meningococcal serogroups. The safety profile for subjects given Td alone was similar to that in subjects given Td TetraMenD. Concomitant administration of Td TetraMenD did not interfere with the immune response to either tetanus or diphtheria toxoids. SBA responses to the four serogroups are summarized in Table E-14 shown below.

Table E-14: SBA response to the four serogroups

O\

0- 0 (N, Serogroup SBA (GMT) Concomitant Administration Separate Administration Pre Post Pre Post (N 468) (N 466) (N 477) (N 478) A 232 11313 228 10391 C 66 5059 57 2136 Y 124 3391 115 1331 W-135 26 4195 27 1339 This study shows that the co-administration of Td and TetraMenD was safe and well tolerated in the test subjects. Concomitant administration of TetraMenD Td does not adversely affect immune responses to tetanus and diphtheria toxoids. The immune responses to serogroups C, Y, and W135 polysaccharide were enhanced when MCV-4 was coadministered with Td. The enhanced immune response observed in this study was surprising and unexpected.

Example 14 Comparison of Serum Bactericidal Assay with Baby Rabbit Complement and with Human Complement for N. Meningococcal Serogroups C, W-135 and Y A subset of serum samples from Study A, Stage 111, is used in this study to compare the results obtained with SBA-BR titers with SBA-HC titers for Serogroups C, W-135 and Y.

Subjects enrolled into this trial are at least 2 years of age but not yet 11 years of age and each is randomly assigned to one of the two vaccine groups. Approximately 5 mL of whole blood is collected from each subject at baseline (prior to vaccination) and on Day 28 post-vaccination.

Blood specimens from subjects are centrifuged within 4 hours of collection. The serum is taken off the clot, transferred into labeled cryotubes and stored in a temperature-monitored freezer at -20 0 C or colder. All samples that are used in the analysis in this report are from paired sera obtained from the first subjects enrolled in the clinical study and that are of sufficient serum volume to complete all planned testing. All samples are from 2 year old and 3 year old subjects with the exception of a single 4 year old. There are 2 subjects in the intentto-treat category. One of these is from the TetraMenD vaccine group (Day 28 post-vaccination cN sample is collected on Day 24) and one is from the Menomune® vaccine group (Day 28 postt vaccination is collected on Day 9).

Baby rabbit complement (Pel-Freez®, Clinical Systems LLC, Brown Deer, W1, product code 31038) is pre-screened for suitability in each of the serogroup specific assays. The criteria for suitability included an agreement with SBA-BR test results for a defined set of serum samples (within a 2-fold dilution) using a previously qualified lot of rabbit complement.

00 Criteria for meeting predetermined titers for a reference serum and control samples are also S used. Aliquots of 2.5 ml of the rabbit complement are stored at -70 0 C or colder until ready for Suse. Aliquots are thawed once and used or discarded.

Serum from subjects enrolled is screened for anti-meningococcal polysaccharide IgG and IgM levels by ELISA and tested in the SBA-BR for functional antibodies to identify potential sources of complement for use in the SBA-H. Criteria established for selection of a human source of complement are the following; lack of detectable antibody when assayed in the SBA-BR assay, lack of intrinsic bactericidal activity when used as the complement source in the assay, acceptable performance when used as a complement source with a panel of negative control, using sera with previous negative test results determined at an independent outside lab by Dr. Ray Borrow, and acceptable reproducibility performance with a panel of 24 samples. Exogenous complement sources used in each of the serogroup specific assays are from different subjects. No complement sources are found to work for more than one serogroup. Also, the three complement sources used in the SBA assays are from a single donor per serogroup.

Serogroup C Serum from several subjects with acceptably low ELISA values (less than 0.5 Pig/ml for both IgG and IgM) demonstrated bactericidal activity.

Serogroup Y The complement source for the serogroup Y SBA-H is selected from the subjects enrolled in the collection protocol. Serum from the source of complement displayed low-level l serogroup Y IgG and IgM antibodies by ELISA and is negative in the SBA-BR assay. Serum Sfrom the source showed no intrinsic bactericidal activity when used in the SBA.

Serogroup W-135 The complement source for the serogroup W-135 SBA-H is selected from the subjects enrolled in the collection protocol. Serum from the source of complement displayed low-level 00 Sserogroup W-135 IgG and IgM antibodies by ELISA and is negative in the SBA-BR assay.

O Serum from the source showed no intrinsic bactericidal activity when used in the SBA.

Serum Bactericidal Assays Briefly, meningococcal serogroups C, Y, and W-135 strains are obtained from the Centers for Disease Control, Atlanta, GA (CDC). Target strains of bacteria are prepared for use in the assays from freshly thawed working seed lot vials of serogroups C, Y and W-135.

Each vial is used to streak a Thayer Martin plate that is incubated overnight at 37 0 C 0.5 0

C

in 5% CO 2 The following day, isolated colonies are harvested with a sterile swab and used to inoculate the entire surface of fresh Thayer Martin plates that had been warmed to ambient temperature. Plates are incubated for 4 h at 37 0 C 0.5 0 C in 5% CO 2 to obtain a light veil of confluent bacterial growth that is harvested with sterile swabs and suspended in Dulbecco's PBS 0.1% Dextrose Buffer to a prescribed optical density (absorbance at 600 nm). A working solution with a prescribed concentration of bacteria is prepared in Dulbecco's PBS 0.1% Dextrose Buffer, maintained at ambient temperature and used within 30 minutes of preparation.

Test samples are heat-treated at 56 0 C for 30 minutes to inactivate endogenous complement. To all wells of a 96-well microtiter plate, Dulbecco's PBS 0.1% Dextrose Buffer is added, then test serum samples are dispensed in 2-fold serial dilutions across the plate leaving the final two columns of wells for complement and serum control wells.

Columns on every plate included a complement column ([column 11] serum/+complement) and a serum control column ([column 12] +serum/-complement).

Freshly thawed complement is mixed with the working concentration of bacteria and the mixture is dispensed into all but the serum control wells of the microtiter plates. Bacteria Swithout added complement are dispensed into the serum control wells. The plates are covered and placed on a plate-shaker for 1 minute then removed to a 37 0 C 0.5 0 C CO 2 incubator.

Incubation times are 90 minutes for the serogroup A assay plates and 60 minutes for the serogroups C, Y, and W-135 assay plates. After incubation, 100 PL of agarose overlay medium at 50 0 C 1IC is carefully added to all wells avoiding air bubble formation. After a 10 minute Speriod at ambient temperature with the microtiter plate lids ajar to avoid moisture formation, M the plates are covered and removed to a dry (no added humidity) 5% CO 2 incubator at 37 0 C

O

0.5 0 C for 20 4 h. After this incubation, the number of bacterial colonies per well is counted.

SThe average number of colonies per well for the complement controls wells is calculated and (C 10 divided in half to obtain the 50% survival at To.

The bactericidal titer of each unknown serum is expressed as the final reciprocal serum dilution yielding 50% killing compared with the 50% survival value at To. The starting dilution for samples in the SBA-BR is a 1:8 dilution. For the SBA-H, the starting dilution is lowered to a 1:4 dilution as described in the original assays.

A comparison of the SBA-BR procedure for the serogroup A assay described herein with the Standardized SBA procedure (CDC) and with the SBA procedure as performed at the Manchester Public Health Laboratory Services, Meningococcal Reference Unit, Manchester, UK (PHLS) is provided as Table 14-1.

Table 14-1: Serum Bactericidal Assay Methods Comparison AvP-US CDC PIHLS Frozen stock Greaves Soln w 10% Greaves Soln w 10% Glycerol Frozen in Glycerol Glycerol broth Bactericidal buffer Dulbecco's with 0.1% Dulbecco's with 0.1% Glucose Geys with 0.5% BSA Glucose Over night growth Media Thayer Martin (MR0232) Brain Heart Infusion w/ 1% Horse Serum Blood agar w/5% Horse Blood Over night Growth 37 0 C w/ 5% C02 37C w/ 5% C02 37C w/ 5% C02 Conditions Complement Baby Rabbit (Pel-Freez) Baby Rabbit (Pel-Freez) Baby Rabbit (Pel-Freez) A Strain F8238 F8238 F8238 Assay day growth Media Thayer Martin (MR0232) Brain Heart Infusion w/ 1% Horse Serum Blood agar Assay day growth 4 hours 37°C w/ 5% CO2 4 hours 37 0 C w/ 5% C02 4 hours 37°C w/ 5% C02 conditions T' targeted (per ml) 4000 CFU/ml 4000 CFU/ml 80,000 CFU/ml Initial starting dilution of 1:4 1:4 1:2 sera Serum Treatment 56C for 30 minutes 56°C for 30 minutes 56°C for 30 minutes Total volume at incubation 50 pl 50 pl 40 pl stepI Serum mixture as total 50% (25pl) 50% (25pl) 50% 2 0pl) I I Cell suspension (volume) 25% (12.5l) 25% (12 5pl) 25% (10 pI) Complement (volume) 25% (12.5pl) 25% (12 5pl) 25% (1Oal) CFU/well in r'xn mixture 50 50 800 (theor.) Final starting dilution 1 8 1 8 1:4 Serum Incubation conditions 37°C w/ 5% C02 for 90 37"C for 90 minutes 37"C w/o C02 for minutes minutes 100 pl TSB Agar overlay 100 pl TSB Noble Agar overlay added 10 pl on Agar Plates Overnight Incubation added 37 0 C w/ 5% CO2 (Tilt Method) method (in 96 well plates) 37°C w/ (in 96 well plates) 37°C CO2 T conditions 37 0 C for 90 minutes 37 0 C for 90 minutes Plated prior to 90 minute Complement Control (i e Complement Control Average) incubation Average) Overnight at 37°C w/ CO2 Endpoint Titer 50% Killing 50% Killing 50% Killing A reference serum is obtained from Dr. George Carlone, CDC (CDC donor-R21654-3430107) as lyophilized powder in vials, which are stored at 2 0 C to 8 0 C until used. When needed, vials are each rehydrated with 0.5 ml sterile water and stored at -80 0 C to -40 0 C as 100 pl working aliquots. The titer of the reference serum when reconstituted under these conditions is 1:256 1 two-fold dilution in the standardized SBA-BR for serogroups A, C, Y, and W-135.

Reference serum samples are run twice on different plates of the daily set of plates.

Group-specific rabbit antisera for serogroups A, C, Y, and W-135 are purchased from Difco as lyophilized powder in vials, that are stored at 20 to 8 0 C until used. When needed, each vial is rehydrated with I ml sterile water and stored at -80 0 C to -40°C as 50 pl aliquots for use as quality control samples in the SBA.

The results of the Serum Bactericidal Assay using baby rabbit complement (SBA-BR) provided herein for the determination of complement-mediated anti-polysaccharide bactericidal activity to Neisseria meningitidis serogroups C, Y, and W-135 in clinical serum samples is fully validated for precision, dilutability (linearity), specificity and limit of detection. The SBA-H assay (for Serogroup C) is repeated on five consecutive days with an identical set of serum samples to establish the precision of the assay.

Calculation of sensitivity and specificity of the SBA-BR Titers obtained in the SBA-BR are classified as true positive (TP) (and false positive and true negative (TN) (and false negative using the SBA-H benchmark titers of 1:4 and 1:8. Sensitivity is calculated as TP/(TP FN) and specificity is calculated as TN/(TN FP). The results of these calculations are expressed as percentages.

J SBA Titer Distribution Comparison of SBA-BR versus SBA-H The pre- and twenty-eight day post-immunization SBA titers are shown in Tables I C and 4 for serogroup C, Tables 2 and 5 for serogroup Y, and Tables 3 and 6 for serogroup W- 135. Summarized in the following subsections is an analysis of the pre- and postimmunization SBA titers comparing the results obtained for the two sources of complement (BR versus H).

Cc, Serogroup C SBA Titer Distribution Of the 101 pre-immunization serum samples, 63 are negative as defined by having a SBA-H titer of 1:4 and a SBA-BR titer 1:8. Twenty-seven of the pre-immunization samples are negative by SBA-H- 1:4) but are positive by SBA-BR The false positive rate using a SBA-BR cut off titer of< 1:8 is 30%. The false positive rate decreases at higher SBA-BR cut off titers to less than 20% at a cut off titer of 1:128, and to less than at a cut off titer of 1:512. Seven of the samples that are positive by SBA-H 1:4) are negative by SBA-BR 1:8).

In the post-immunization sera, 48 samples are negative by SBA-H, and only 11 are negative by SBA-BR. Of the 11 samples that are negative by SBA-BR, 3 are positive by SBA-H. Seventeen of 51 post-immunization samples in the conjugate group are negative by SBA-H, but positive by SBA-BR For the polysaccharide group, 23 of post-immunization samples are negative by SBA-H, but positive by SBA-BR titer In terms of positive responses in the post-immunization sera, 90 of 101 of samples are positive by SBA-BR 1:8) but only 53 of 101 are positive by SBA-H There is a notable difference in the positive response rates when comparing the SBA titers (BR versus H) obtained for the two vaccine groups. For the 51 post-immunization samples in the conjugate group, 33 of 51 are positive by SBA-H 1:4) and by SBA- BR Agreement between the SBA titers (BR versus H) improves at a SBA-BR threshold titer of 1:64 and higher. Of the 50 post-immunization samples in the polysaccharide group, 17 of 50 are positive by SBA-H 1:4) and by SBA-BR Agreement between the SBA titers (BR versus H) improves at SBA-BR threshold titer 1:512 and higher.

SSerogroup Y SBA Titer Distribution Unlike the serogroup C pre-immunization sera, only 9 of the serogroup Y preimmunization samples are negative as defined by having an SBA-H titer 1:4 and a SBA-BR titer 1:8. Fifty-two of 61 pre-immunization samples are negative by SBA-H 1:4) but positive by SBA-BR The false positive rate using a SBA-BR cut off titer of 1:8 is 00 The false positive rate decreases at higher SBA-BR cut off titers to less than 15% at a 0 cut off titer of 1:256, and less than 2% at 1:512. Two samples are positive by SBA-H 1:4) b but negative by SBA-BR 1:8).

There are no post-immunization serum samples that had a SBA-H titer 1:4 and a SBA-BR titer that is 1:8. Nineteen samples that are negative by SBA-H 1:4) are positive by SBA-BR As noted for serogroup C, there is a difference in the proportion of false negative results. In the conjugate group, 5 of 48 samples are negative by SBA-H 1:4), but positive by SBA-BR. In the polysaccharide group, 14 of 52 samples are negative by SBA-H but positive by SBA-BR.

There is good agreement between the two SBA titers (BR versus H) for positive responses in the post-immunization sera for serogroup Y. For the total set of 100 samples, all 100 post-immunization samples had SBA-BR titers 1:8, and 81 of 100 had SBA-H titers of 1:4. As noted for the SBA responses for serogroup C, there is better correlation between the SBA titers (BR versus H) in the conjugate group compared to the SBA titers (BR versus H) obtained for the polysaccharide group. Of the 48 post-immunization samples in the conjugate group, 43 are positive by SBA-H 1:4) and by SBA-BR Only 1 of 48 samples had a SBA-BR titer less than 1:32, and that sample is positive by SBA-H The agreement between the SBA titers (BR versus H) is not as good in the polysaccharide group. Only 38 of 52 had post-immunization SBA-H titers 1:4 and a SBA-BR titer Agreement between the SBA titers (BR versus H) in the postimmunization sera for the polysaccharide group improves at a SBA-BR titers of>= 1:128.

Serogroup W-135 SBA Titer Distribution For serogroup W-135, 54 of 100 are negative where both the SBA-H titer is 1:4 and the SBA-BR titer is 1:8. Of the pre-immunization samples, 27 of 81 are negative by 132 rc SBA-H 1:4) but positive by SBA-BR The false positive rate using a SBA-BR cut 0 off titer of 1:8 is 33%. The false positive rate decreases as higher SBA-BR cut off titers to less than 15% at a cut off titer of 1:128, and to less than 5% at a cut off titer of 1:256. Eleven samples are positive by SBA-H 1:4) but are negative by SBA-BR 1:8).

Three post-immunization samples are negative by SBA-H 1:4) and negative by SBA-BR Thirty-nine post-immunization samples are negative by SBA-H 1:4) but 00 are positive by SBA-BR titer In the conjugate group, 11 of 47 samples are O negative by SBA-H but positive by SBA-BR. In the polysaccharide group, 28 of 53 samples are negative by SBA-H but positive by SBA-BR.

The agreement between the post-immunization SBA-BR and SBA-H titers is comparable to serogroup C, but not as good compared to serogroup Y. As with both serogroup C and serogroup Y, there is a notable difference in the agreement between the two SBA titers (BR versus H) when comparing the two vaccine groups. The agreement between the two SBA titers (BR versus H) is better for the conjugate group compared to the polysaccharide group. In the post-immunization SBA titers for the conjugate group, 36 of 47 had an SBA-H titer of 1:4 and all are positive by SBA-BR All samples from the conjugate group had post vaccination SBA-BR titers 1:32. For the postimmunization titers for the polysaccharide group, the correlation between the two titers is not as good, only 22 of 53 had an SBA-H titer 1:4 and 50 of 53 had a SBA-BR titer 1:8.

Table 1. Comparison of the SBA-BR titer in sera positive and negative by SBA-H for serogroup C No. of sera with indicated SBA-H titer: 1/SBA-BR 28-day post-immunization Pre-immunization titer Pre-imTetraMenD Menomune Combined <4 <4 <4 <4 >=4 <8 63 7 1 0 7 3 8 3 8 4 0 1 0 1 0 2 0 16 1 1 1 0 1 1 2 1 32 2 0 5 3 1 1 6 4 64 4 0 4 6 5 2 9 8 128 3 2 3 9 8 2 11 11 256 11 1 1 3 4 3 5 6 512 1 0 0 5 1 3 1 8 1024 0 0 2 6 1 4 3 2048 1 0 0 1 I 0 1 1 4096 0 0 0 0 0 1 0 I Total 90 11 18 33 30 20 48 53 Table 2. Comparison of the SBA-BR titer in sera positive and negative by SBA-H for serogroup Y No. of sera with indicated SBA-H titer: 1/SBA-BR Pre-immunization 28dy post-mmuniztion titer TetraMenfl Menomuneo Combined 4 4 4 4 4 4 4 8 9 2 0 0 0 0 0 0 8 1 1 0 1 0 0 01 16 4 2 0 0 0 0 0 0 32 10 6 1 1 3 2 4 3 64 14 6 1 3 4 2 5 128 15 12 1 9 4 6 5 256 7 9 1 13 2 16 3 29 512 1 0 1 7 1 6 2 13 1024 0 0 0 5 0 4 0 9 2048 0 1 0 3 0 1 0 4 4096 0 0 0 1 0 1 0 2 Total 61 39 5 43 13819 8 Table 3. Comparison of the SBA-BR titer in sera positive and negative by SBA-H for serogroup W-135 No. of sera with indicated SBA-H titer: 1/SBA-BR Pr-muian28dy pot-mmunizaion titer Pr-muiain TetraMenD MenomuneR Combined 4 4 4 4 4 4 4 8 54 11 0 0 3 0 3 0 8 1 3 0 0 0 0 0 0 16 3 0 0 0 1 0 1 0 32 2 0 0 1 0 0 0 1 64 11 2 1 2 3 3 4 128 7 1 2 2 7 2 9 4 256 3 2 4 3 6 5 10 8 512 0 0 4 13 5 4 9 17 1024 0 0 0 3 5 3 5 6 2048 0 0 0 11 1 5 1 16 4096 0 0 0 1 0 0 0 Total 81 19 11 36 31 22 42 58 Table 4. Summary of distribution of serogroup C titers measured by the SBA-BR and the SBA-H No. of samples' of pre- or post-) with indicated titer by: 1/bactericidal SBA-BR SBA-H titer Pr-m.Post-imm. Post-imm.

Pr-im.Menomuneo TetraMenD Pre-imm. Menomnune~o TetraMenD (91 30(60.00) 18(35.29) 4 1 (2.00) 1 (1.96) <8 (603 10(20.00) 1 (1.96) 8 4(3.96) 1 (2.00) 1 (1.96) 0 2(4.00) 1 (1.96) 16 2(1.98) 2(4.00) 1 (1.96) 3(2.97) 2(4.00) 5(9.80) 32 2(1.98) 2(4.00) 8(15.69) 1 (0.99) 3(6.00) 3 (5.88) 64 4(3.96) 7(14.00) 10(19.61) 1 (0.99) 0 5(9.80) 128 5(4.95) 10 (20.00) 12 (23.53) 3 2 .97) 3 (6.00) 5 (9.80) 12 256 11.88j 7(14.-00) 4(7.84) 1 (0.99) 1 (2.00) 5(9.80) 512 1 (0.99) 4(8.00) 5(9.8) 0 5(10.00) 2 3.92) 1024 0_ 5(10.00) 8(15.69) 0 1 (2.00) 5(9.80) 2048 1 (0.99) 1 (2.00) 1 (1.96) 0 0 1 (1.96) 4096 1 0 1 (2.00) 1 0 1 0 1 (2.00) 0 8192 1 0 0 0 0 1 (2.00) 0 Total number of samples is 10 1.

Table 5. Summary of distribution of serogroup Y titers measured by the SBA-BR and the SBA-H No. of samples 1 of pre- or post-) with indicated titer by: 1/bactericidal SBA-BR SBA-H titer Pre-imm. Post-imm. Pr-m.Post-imm.

Menomune® TetraMenD Pr-m.Menomunew TetraMenD <4 61 14(26.92) 5(10.42) (61.00) 4 1 (1.00) 0 1 (2.08) <8 (11I. 00) 8 2(2.00) 0 1 (2.08) 3(3.00) 1 (1.92) 4(8.33) 16 6(6.00) 0 0 11 12(23.08) 2(4.17) 32 16 5(9.62) 2(4.17) 12 11 (21.15) 11 (22.92) (16.00) (12.00) 6420 6(11.54) 4(8.33) 5(5.00) 9(17.31) 5(10.42) 64 (20.00) 1827 10(19.23) 10(20.83) 6(6.00) 2 (3.85) 8 (16.67) 128 (27.00) 2616 18(34.62) 14 (29.17) 1 (1.00) 1 (1.92) 6(12.50) 256 (16.00) 512 1 (1.00) 7(13.46) 8(16.67) 0 1 (1.92) 4(8.33) 1024 0 4(7.69) 5(10.42) 0 1 (1.92) 2(4.17) 2048 1 (1.00) 1 (1.92) 3 (6.25) 0 0 0 4096 0 1 (1.92) 1 (2.08) 1 0 0 0 Total number of samples is 100.

Table 6. Summary of distribution of serogroup W-135 titers measured by the SBA-BR and the SBA-H No. of samples' of pre- or post-) with indicated titer by I/bactericidal SBA-BR SBA-H titer Pr-m.Post-imm. Pr-m.Post-imm.

Pr-m.Menom une® TetraMenD Pr-m.Menomune Q TetraMenD 81 4 _4 (4.00) 5(9.43) 6(112.77) <8 650 3(5.66) 0 (65.010 8 4(4.00) 0 0 (100 7(13.21) 2(4.26) 16 3 (3.00) 1 (1.89) 0 2(.2.00) 5 (9.43) 11 (23.40) 32 2(2.00) 0 1 (2.13) 3(3.00) 1 (1.89) 51.4 64 (130 6(11.32) 3 (6.38) 0 3 (5.66) 2(4.26) 128 8(8.00) 9(16.98) 4(8.51) 0 0 5(10.64) 256 5 (5.00) 11 (20.75) 7 (14.89) 0 0 3 (6.38) 512 0 9(16.98) 17 (36.17) 0 1 (1.89) 1 (2.13) 1024 0 8 (15.09)_ 3 (6.38 0 0 1 (2.13) 2048 0 6(11.32) 11 (23.40) 0 0 0 4096 0 0 1 (2.13) 0 -0 0 'Total number of samples is 100.

cN Sensitivity and Specificity Comparison of the SBA-BR and SBA-H Titers tb SBA-BR titers are compared to the SBA-H protective titers of 1:4 and 1:8 in performing sensitivity and specificity assessments between the two sets of titers. Both pre- and postrimmunization sera are used in this analysis. Using the SBA-H benchmark titers of 1:4 and 1:8, specificity and sensitivity are calculated for all three serogroups and are summarized in Tables 7, 9 and 10. The analysis of sensitivity and specificity are discussed in turn for each 00 serogroup.

O For serogroup C, the sensitivity is greater than 80% for SBA-BR threshold titers of 1:8, 1:16, and 1:32 relative to both a 1:4 and 1:8 SBA-H titer. However, the specificity at these O 10 SBA-BR titers is less than 60%. Specificity increased above 60% at a SBA-BR titer of 1:64, and above 70% at a SBA-BR titer of 1:128. For these latter two SBA-BR titers, the specificity begins to drop off. At a SBA-BR threshold titer of 1:64, the sensitivity is between 75 and 78%, but at a SBA-BR titer 1:128, sensitivity falls to between 62 and 65%. Specificity continues to improve at SBA-BR titers 1:64 ranging from 73% up to 83% for 1:128 and 1:256, respectively. However, sensitivity falls from 43% to less than 20%. The SBA-BR titer for serogroup C with the best balance between sensitivity and specificity falls in a range of SBA-BR titers between 1:32 and 1:128. The sensitivity and specificity results for serogroup C are found to be quite comparable to the results obtained by Santos GF, et al., 2001. Clin.

Diagn. Lab. Immunol. 8:616-623, obtained by different set of serum samples and reagents (Table With respect to Santos's results, the best balance of sensitivity and specificity is observed between the SBA-BR titers of 1:64 and 1:128 versus both the SBA-H titers of 1:4 and 1:8.

Table 7. Sensitivity and Specificity of the SBA-BR at Protective Titers in the SBA-H for Serogroup C SSBA-H titer of 1:4 SBA-H titer of 1:8 I/SBA-BR titer Sensitivity Specificity Sensitivity Specificity 8 84 51 88 52 16 84 56 88 56 32 81 58 85 58 64 75 64 78 64 128 62 73 65 73 256 42 83 43 83 512 31 95 33 1024 19 96 20 96 Table 8. Sensitivity and Specificity of the SBA-BR at Protective Titers in the SBA-H for Serogroup C as reported by Santos et al. 2001. Clin. Diagn. Lab. Immunol. 8:616-623.

A-BR t SBA-H titer of>= 1:4 SBA-H titer of>= 1:8 1/SBA-BR titer Sensitivity Specificity Sensitivity Specificity 32 85 61 91 58 64 78 73 86 68 128 69 83 78 81 256 54 87 63 88 512 41 92 49 94 For serogroup Y, sensitivity is highest for SBA-BR threshold titers ranging from 1:8 to 1:64, but, as expected, drops off at higher SBA-BR threshold titers. Specificity results for serogroup Y start out much lower compared to the results for serogroup C, and do not reach a level of greater than 50% until a threshold SBA-BR titer of 1:128. The SBA-BR titer that is best balanced for sensitivity and specificity for serogroup Y falls in a range between 1:64 and 1:256. At 1:256, the sensitivity drops off to approximately 55%, but specificity increases from the mid-30% region to approximately 82-83%.

Table 9. Sensitivity and Specificity of the SBA-BR at Protective Titers in the SBA-H for Serogroup Y SBA-H titer of 1:4 SBA-H titer of 1:8 I/SBA-BR titer Sensitivity Specificity Sensitivity Specificity 8 98 11 98 11 16 97 12 97 12 32 95 17 95 17 64 87 35 87 34 128 78 59 78 57 256 56 84 55 82 512 56 84 55 82 1024 13 100 14 100 fFor serogroup W135, the values for sensitivity follow more closely to the values obtained for serogroup C, but the overall pattern is the same for all three serogroups.

Sensitivity starts out high at a SBA-BR threshold titer of 1:8 and drops off at titers 1:128.

Likewise, specificity starts out low at a SBA-BR titer of 1:8 begins to level out at a titer of S1:256. As observed for serogroup Y, the SBA-BR with the best balance between sensitivity 00 and specificity for serogroup W135 falls in a range between 1:64 and 1:256.

STable 10. Sensitivity and Specificity of the SBA-BR at Protective Titers in the SBA-H for Serogroup W-135 A-R tite SBA-H titer of>= 1:4 SBA-H titer of>= 1:8 I/SBA-BR titer Sensitivity Specificity Sensitivity Specificity 8 86 46 87 43 16 82 48 82 44 32 82 50 82 47 64 80 52 81 49 128 71 64 73 61 256 65 77 64 72 512 52 88 50 83 1024 30 95 34 94 Table 11 summarizes the proportion of four-fold rise in SBA titers using both baby rabbit complement or human complement for serogroups C, Y, and W135 relative to the postimmunization SBA-BR titer. This analysis is performed separately on the conjugate group, TetraMenD, and the polysaccharide group, Menomune®, and both sets of analysis are included in Table 11. There are some observable differences in the four-four rise patterns comparing the bactericidal responses induced to the three serogroups and for the two vaccine groups. The response patterns are discussed in turn for each serogroup and for both vaccine groups.

Table 11. Stratified Comparative Ratios to a 4-Fold Rise in Meningococcal Polysaccharide Titers Determined by SBA-BR and SBA-H Serogroup C Serogroup Y Serogroup W-135 TetraMenD Menomune~ TetraMenD Menomune TetraMenD Menomune 1/ SBA SBA- SBA- SBA- SBA- SBA- SBA- SBA- SBA SBA- SBA- SBA SBA -BR BR H BR H BR HI BR -H BR H -BR -H titer III 0/3 0/1 0/1 0/10 1/10 0/3 0 3 8 na na na na na na 0 I/t 0/1 0/1 0/1 0/1 0/1 1 1 8 (100 na na na na na na 0/1 0/1 0/2 1/2 0/1 0 1 1 6 N a na na na na na (0% 7/8 3/8 2/2 1/2 1/2 0/2 2/5 1/ 5 0/1 32 (100%) (40% (20% (100 na na 2/6 3/3 6/6 0/6 S 9/10 6/10 6/7 2/7 1/4 0/4 2 6 0/6 3 3 0/3 6 6 0 6 64 (33% (100% (00 (0% 11/12 7/12 8/10 2/10 5/10 6/10 4/10 2/10 2/4 1/4 8/9 2/9 128 (60% (40% (20% 8 18/3 32/3 11/18 14/18 12/18 7/18 28/31 23/3 17/31 36/39 23/39 4 6/34 6 (74% (58% (94 9 1 Tota 21/5 4615 ot a 38/51 30/51 28/50 14/50 35/48 30/48 25/52 2 1 5 41/47 25/47 46 5 8/53 I I I 1 2 3 The Table shows as a ratio (and percentage) the 4-fold rise in SBA titer as determined in each assay (from paired samples pre-immunization and 28-day post-immunization) stratified by SBA-BR titer, vaccine and serogroup.

For serogroup C, there is close agreement in the four-fold rise between SBA-BR versus SBA-H for the conjugate group. Within this vaccine group, there appears to be a trend that at low post-immunization titers, e.g. 1:32 1:128, the SBA-H four-fold rise is lagging behind the four-fold rise in SBA-BR. But, at post-immunization SBA-BR titers 1:256, the number of subjects achieving a four-fold rise by SBA-H appears to be greater than the number of subjects achieving a four-fold rise by SBA-BR. This difference in the fold rises comparing the two 140

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rcl complement sources is small, and may be due to higher pre-immunization SBA-BR titers that alter the percentage of achieving a four-fold rise by SBA-BR. For the polysaccharide group, the agreement between the four-fold rise between SBA-BR versus SBA-H is not as close as it c, is for the conjugate group. Also, there is no notable trend that the four-fold rise by SBA-H becomes more sensitive at higher post-immunization SBA-BR titers than is observed for the conjugate group.

00 For serogroup Y, the agreement between the four-fold rise by SBA-H and SBA-BR is O very close for both vaccine groups. There are very few post-immunization SBA-BR titers less b than 1:32 for either vaccine group compared to the post-immunization SBA-BR titers for O 10 serogroup C. For this reason, the proportion of subjects achieving a four-fold rise in both SBA-BR and SBA-H for serogroup Y occurs at a higher post-immunization SBA-BR titer compared to serogroup C. For serogroup Y, the proportion of four-fold rise increases to at a post-immunization SBA-BR titer of 1:128 for the conjugate group, whereas for serogroup C the threshold SBA-BR titer is 1:32 for the conjugate group.

The agreement between four-fold rise by SBA-H and SBA-BR for serogroup W135 is not as close compared to the other two serogroups. As observed for serogroup Y, there are a limited number of subjects with post-immunization SBA-BR titers less than 1:32 for either vaccine group. The agreement between the four-fold rise in SBA titers (BR versus H) occurs at SBA-BR titers 1:256. As noted for serogroup C, there is a difference in the proportion of four-fold rises (BR versus H) between the two vaccine groups that is less evident for serogroup Y. The agreement between the four-fold rise in the serogroup W135 SBA titers (BR versus H) is poorest in the polysaccharide group compared to the four-fold rise in SBA titers (BR versus H) by the polysaccharide vaccine for the two other serogroups.

The Serum Bactericidal Assay with baby rabbit complement (SBA-BR) is compared to the corresponding SBA using human complement (SBA-H) for measuring titers in serum samples from 2 to 3 year old subjects vaccinated with either the licensed quadrivalent meningococcal polysaccharide vaccine (Menomune®) or an experimental quadrivalent meningococcal polysaccharide conjugate vaccine (TetraMenD). Human complement sources for serogroups C, Y, and W135 are identified and used to support this comparison in the SBA.

The SBA results from this comparative study are analyzed by two approaches. In one cK approach the SBA-BR and SBA-H- data obtained by measuring the pre- and post- Simmunization titers for both vaccine groups are pooled for analysis. In the second approach, the pre- and post-immunization titers from the two vaccine groups are analyzed separately.

One of the goals of this study is to describe the SBA-BR serum titer that best correlates to a negative SBA-H serum titer. A second goal is to determine the titer using baby rabbit complement that best correlated with a positive titer using human complement in the assay as 00 a correlate of protection for serogroup C and to extrapolate to the protective bactericidal titers O for serogroups Y and W135. Other laboratories have published results attempting to establish a protective threshold correlate for the SBA-BR for serogroup C. The results of this study will be compared to those published results. Lastly, the four-fold rise in SBA titers measured in pre- and post-immunization sera are compared for the two complement sources.

A correlation of a SBA titer to protection against disease has only been established for serogroup C. The SBA correlate of protection for serogroup C is determined using human complement in the SBA assay. For the other serogroups an assumption will be made that the SBA-H correlate of protection for serogroup C (SBA titer of 1:4) applies for the other serogroups. Defining a SBA-BR titer(s) that correlates to the SBA titer of 1:4 for serogroup C may differ between serogroups. In terms of defining a SBA-BR titer that best correlates to a negative SBA-H serum titer, a SBA-BR titer of <1:8 is compared to SBA-H titer of<l:4 in the pre- and post-immunization sera. The SBA-BR titer of 1:8 is used based in part on the results from the WHO/CDC study for comparing serogroup C SBA titers (BR versus H) and on the recent finding that a SBA-BR titer of 1:4 is linked to susceptibility to serogroup C disease in a University Outbreak in the United Kingdom (Jones, et al., J. Infect. Dis., 181:1172-1175 [2000]).

Based upon the SBA titers (BR versus H) generated in this study, the false positive rate for serogroup C using a SBA-BR cut off titer of< 1:8 is 30%. Using higher SBA-BR cut off titers improves the false positive rate as follows: at >=1:16 the false positive rate decreases to 26%, at 1:32 the false positive rate decreases to 24%, at 1:64, the false positive rate decreases to 22%, at 1:128 the false positive rate decreases to 18%, at 1:256 the false positive rate decreases to 14%, and at 1:512 the false positive rate decreases to 2%.

Increasing the SBA-BR cut off titer does serve to improve the accuracy of defining a negative titer that corresponds to a SBA-H- titer of 1:4, however the sensitivity in discriminating 142

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Cl between a positive response and a negative response is much lower when using higher SBAf BR cut off titers. The data in this report showed that sensitivity of the SBA-BR is highest (81- 84%) at cut off titers of 1:8, 1:16, or 1:32. At SBA-BR titers greater than 1:32, the sensitivity C, drops below 80%. However, specificity of the assay at cut off titers of 1:8, 1:16, and 1:32 is at a minimum, ranging from 51 to 58%. A balance is made between sensitivity, specificity and false positive rates in selecting a cut off titer that correlates to a negative titer in the human 00 complement assay. The assignment of 1:32 as the cut off titer for the SBA-BR would result in Ounnecessarily rejecting true positive responders. The results of this study indicate that a SBA- BR titer of 1:16 may be a more appropriate cut off titer. It is minimally 2-dilutions above O 10 the titer deemed protective based upon both the WHO/CDC study analysis 1:8) and the U.K. University Outbreak analysis 1:4).

Identification of the protective cut off titer in the assays for serogroups W135 and Y are derived by assuming that bactericidal antibody protection is analogous with serogroup C disease and the bactericidal titers that correspond to a negative titer in the human complement assay. For serogroup Y, the false positive rate using a SBA-BR cut off titer of 1:8 is quite high at 85% compared to 30% for serogroup C. However, as with serogroup C, increasing the SBA-BR cut off titer lowers the false positive rate. At a SBA-BR cut off titer of 1:16 the false positive rate decreases to 84%, at 1:32 the false positive rate is 75%, at 1:64 the false positive rate is 61%, at 1:128 the false positive rate is 38%, at 1:256 the false positive rate is 13%, and at 1:512 the false positive rate is Even though the false positive rate for serogroup Y starts out much higher compared to serogroup C, at cut off titers of 1:128, the false positive rates for the two serogroup assays become quite comparable. Such high cut off titers, hoarer, may overstate the threshold titers for a positive response. Based upon the sensitivity and specificity analysis, sensitivity is maximized at SBA-BR cut off titers of 1:8 to 1:32, where sensitivity ranged from 95 to 98%. However, as with serogroup C, specificity at these SBA- BR titers is correspondingly low, ranging from 11 to 18%. At the next highest SBA-BR titer, 1:64, sensitivity decreases from 95% to 88%, but the specificity sharply increases to 35%. A cut offtiter of< 1:64 in the serogroup Y assay appears to best correspond to a negative titer in the human complement assay.

For serogroup W135, the false positive rate at a SBA-BR cut off titer of 1:8 is 33%, which is similar to serogroup C. As noted for both serogroups C and Y at higher SBA-BR cut 143 C1 off titers, the false positive rate decreases to lower levels. At a SBA-BR cut off titer of 1:16 Sthe false positive rate decreases to 32%, at 1:32 the false positive rate decreases to 28%, at 1:64 the false positive rate decreases to 26%, at 1:128 the false positive rate decreases to 12%, at 1:256 the false positive rate decreases to and at 1:512 the false positive rate is minimized at Sensitivity is highest at SBA-BR titers ranging from 1:8 to 1:64 (86% to Specificity, as expected, is lowest (46% to 52%) at this range of SBA-BR titers. Even 00 though the false positive rates for serogroup W135 start out much lower compared to 0 serogroup Y, the cut off titer that best corresponds to a negative titer in the human complement assay is 1:64.

Having identified titers for the three serogroups that correspond to negative titers in the human complement assay, SBA-BR titers above these levels are analyzed for a threshold titer for consideration of a positive response. For serogroup C the threshold titer for a positive response is 1:16, for serogroup Y the threshold titer for a positive response is 1:64, and for serogroup W135 the threshold titer for a positive response is 1:64. It appears that a threshold SBA-BR titers of 1:128 for serogroup C provides good assurance that a protective titer is achieved, relative to either a SBA-H titer of 1:4 or 1:8 and correlates of efficacy SBA-H titer of 1:4 for serogroup C. This threshold titer compares well with the analysis made on the WHO/CDC data set for serogroup C, and to the data set of Santos. As noted in both of these studies, titers 1:128 are highly predictive of protection, but SBA-BR titers less than 1:128 may also be protective. For the WHO/CDC and the Santos data set, SBA-BR titers of 1:8, 1:16, 1:32, 1:64 are referred to as the equivocal titers. Since SBA-BR titers less than 1:16 for the data set presented herein for serogroup C are regarded as negative, the equivocal titers for this analysis is 1:16 to 1:64, which is a subset of the other two studies.

In all of these analyses, SBA-BR titers are being compared to SBA-H titers (1:4 or 1:8) that correlate to natural protection data that is collected in the 1960's.

More recently, efforts have been made to correlate the United Kingdom efficacy data for the monovalent C conjugates directly to a SBA-BR titer (Miller E, et al., 2002, Vaccine 20:S58-S67). In this analysis both SBA-BR titers of 1:8 and 1:128 are found to correlate well with the efficacy data that has been collected thus far for 15 to 17 year old subjects vaccinated with one dose of the monovalent C conjugates. However, when Miller and colleagues performed the same analysis for the toddler age group (12 to 30 months of age)

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N they found very good agreement between SBA-BR titers of>= 1:8 with efficacy, but at SBAtb BR titers of>= 1:128 the agreement is not as close. Miller presented additional data at the 13 t h International Pathogenic Neisseria Conference, September 1-6, 2002 in Oslo, Norway where cthe predicted efficacy of subjects achieving a SBA-BR titers 1:64, one-month post vaccination, are outside the 95% confidence interval of the observed efficacy for this age group. These data help support the notion that SBA-BR titers in the equivocal region of 1:8 to 00 1:64 may lend assurance of protection. Based upon the analysis for this study, SBA-BR titers O of 1:16 to 1:64 may likewise lend assurance of protection against serogroup C.

The serogroup Y assay at a SBA-BR titer of 1:64 has a specificity of only 35%, but as the cut off titer is increased to 1:128 and 1:256, specificity increases to 59% and 84%, respectively. A threshold titer of>= 1:256 provides good assurance of a protective titer in the human complement assay of 1:4 and 1:8. However, sensitivity and specificity are better balanced at a threshold titer of 1:128 for serogroup Y. The range of SBA-BR titers of 1:64 to 1:128 for serogroup Y represent the equivocal range of titers compared to the corresponding range of SBA-BR titers of 1:16 to 1:64 for serogroup C that have been correlated to the SBA- H protective titer.

The serogroup W-135 assay at a SBA-BR titer of 1:64, has a specificity of 52%, but as the titer is increased to 1:128 and 1:256, specificity increases to 64 and 77%, respectively. As with serogroup Y, a threshold titer of>= 1:256 provides good assurance of a protective titer in the human complement assay of 1:4 and 1:8. Like serogroup Y, sensitivity and specificity are better balanced at a threshold titer of 1:128 for serogroup W135. The range of SBA-BR titers of 1:64 to 1:128 for serogroup W135 represent the equivocal range compared to the range of SBA-BR titers of 1:16 to 1:64 for serogroup C that are correlated to the SBA-H protective titer in this study.

The four-fold rise in bactericidal titers are calculated for each serogroup and separately analyzed by vaccine group using both assays. In general, there is an indication that a higher four-fold rise in SBA titer (BR and H) is detected at higher post-immunization SBA-BR titers for all three serogroups. There are differences in the four-fold rises in SBA titers (BR vs. H) both by serogroup and by vaccine group. For serogroup C, four-fold rises in the assay with human complement appeared lower compared to titers in the assay with baby rabbit complement at low post-immunization SBA-BR titers. However at higher post-immunization 145 SBA-BR titers, the four-fold rise in titer appeared higher in the assay with human 3~ complement. This pattern seems to suggest that at low post-immunization SBA-BR titers, the assay is less sensitive with human complement than it is with baby rabbit complement, but at high post-immunization SBA-BR titers, the opposite is true, that is, the assay with human complement becomes more sensitive. This pattern is not apparent when assaying samples from the polysaccharide vaccine group. In those samples the four-fold rise in titer appeared 00 lower when using human complement in the assay. Although no explanation for this O observation between sample type is apparent, it does suggest that the assay performed with ('i human complement lacks sensitivity with serum samples containing low titers of bactericidal 1 0 activity.

For serogroup Y, there is good agreement in the four-fold rises in SBA titers in comparing the two complement sources. The four-fold rises in SBA titers (BR and H) are slightly higher for the conjugate group compared to the polysaccharide group, but the difference is not as large as noted for serogroup C.

For serogroup W135, the agreement between four-fold rise in SBA titers using human complement or baby rabbit complement in the assay for either vaccine group is not as good as compared to the results with the other two serogroup assays. The four-fold rise by SBA-BR titer is very good for both vaccine groups, but the proportion of four-fold rise titers by SBA-H is lower compared to the other two serogroups. The four-fold rise by SBA-H in the polysaccharide groups is quite low, and comparatively lower in the four-fold rise in SBA-H titers for the other two serogroups.

Four-fold rise in SBA titer using BR complement has been the benchmark for registration of the meningococcal polysaccharide vaccines. More recently, an effort has been made to link four-fold rise in SBA-BR titers to clinical efficacy from the post-registration surveillance data on the monovalent C conjugates in the United Kingdom (Borrow R, el al., 2001, Infect. Immun. 69:1568-1573). Based on this analysis the efficacy of the monovalent C conjugates in toddlers, ranging in age from 12 to 30 months, has been estimated at 88% (69 to within 16 months of the first dose. For this age group, the proportion of subjects achieving a four-fold rise in SBA-BR titer ranges from 89 to 100% following one dose of the monovalent C conjugate vaccines.

C The SBA-BR provided herein provides bactericidal titer values that are comparable to the values obtained using human complement as the source of complement in the assay for serogroup C. Therefore, these SBA-BR titers are relevant to the original studies that established the surrogate for protective immunity to serogroup C meningococcal disease and support the extrapolation of the clinical results provided herein to protection, and the SBA-BR titers for serogroup C at are comparable to those reported from other laboratories. Performance 00 of the SBA using human complement in the determination of the serogroup specific response Sto serogroup Y and serogroup W-135 capsular polysaccharides, by analogy to the serogroup C model, support the relevance of the SBA-BR for determining bactericidal titers to serogroups Y and W-135.

Example 15 Concomitant administration of TetraMenD (MenomuneTM) and Typhim Vi® in Adults This example describes the results obtained in a phase 2b modified double-blinded, safety and immunogenicity study of TetraMenD (Menactra T M given alone or concomitantly with the licensed Typhim Vi® vaccine in healthy 18 to 55 year olds in the US.

Typhim Vi e is commercially available in the United States. Each 0.5ml dose of Typhim Vi® contains 25 ltg of purified Vi polysaccharide, isotonic phosphate buffered saline, and 0.25% phenol, which is added as a preservative. The Typhim Vi e vaccine also contains residual polydimethylsiloxane or fatty acid ester antifoam. There were a total of 945 participants enrolled in this study. Briefly, study participants were randomized to either one of two treatment groups; Group A received MenactraTM and Typhim Vi e concomitantly at Visit 1 and a saline placebo 28 days later at Visit 2; Group B received Typhim Vi® and placebo at Visit I and Menactra T M 28 days later at Visit 2. A total of 469 participants were enrolled in Group A and 476 in Group B.

There were two primary immunogenicity objectives in this study. The first objective was to describe and compare the antibody responses after one dose of Typhim Vi® in each of the study groups 28 days post-vaccination. Antibody responses to Typhim Vi e in participants from Group A would be considered similar to the antibody responses to Typhim Vi e from Group B if the difference in the proportion of recipients achieving an antibody level of mg/mL from Group B minus the proportion from Group A was less than 10%. A level of> 147 N I.Oug/mL of anti Vi antibodies was chosen as the primary serologic endpoint as this antibody t level is considered protective. The second of the two primary objectives was to describe and compare the SBA antibody responses to each of the four serogroups in Menactra T M recipients 28 days post-vaccination. Participants were evenly distributed by age, sex and race between the two study groups. Sixty nine percent of participants were female with a median age of all 01 subjects being 31 years. In Group A, 432 met the criteria for inclusion in the per 00 protocol population and 439 for Group B.

1- Immunogenicity results O 10 Twenty-eight days post-vaccination with Typhim Vi® vaccine 81.6% of participants from (Nl Group A and 78.5% of participants from Group B achieved antibody levels 1.0 ug/mL. A summary of results are shown in Table 1 below.

Table 1 Percent of Participants with Typhoid Vi Antibody Titer 1.0 pg/mL on Day 28 Following Typhim Vi® Vaccination (Per-Protocol Population) Group A Group B Typhim Vi Typhim Vi Placebo Menactra anti-Vi PS (341 of 418) (328 of418) titer 1.0 81.6% 78.5% pg/mL The antibody response to Typhim Vi® when administered concomitantly with MenactraTM is similar to the corresponding response when Typhim Vi® is given alone, using two-sided Type I error rate a 0.05 and a margin of 10% (Figure These results were consistent with expectations based on the seroconversion rates reported in the literature when Typhim Vi® is given alone or together with other common traveler vaccines including polysaccharide meningococcal and Hepatitis A vaccines. Figure 1 shows the results from these studies: testing with Typhim Vi Antibody Titer 1.0 gg/mL on day 28 in adults, 95% Cl of% difference Typhim Vi Placebo, Menactra Typhim Vi Menactra, Placebo).

CI Figure 2 shows the proportion of participants achieving a 4-fold rise in SBA antibody o titer from Group A, when Menactra is given concomitantly with Typhim Vi® vaccine, was comparable to that demonstrated in Group B, when Menactra was administered one month after a Typhim Vi® vaccination (Per Protocol Population). The antibody response to MenactraTM when administered concomitantly with Typhim Vie is similar to the corresponding response when MenactraTM is given alone, using two-sided Type I error rate I S0.05 and a margin of 10% (Figure 3).

2. GMTs O 10 An observational objective was added to compare the GMT of the responses to MenactraTM in participants who were enrolled in a separate safety and immunogenicity trial in healthy adults to the GMT of Menactra recipients from both study groups in the present trial.

The same criteria used in the secondary immunogenicity hypothesis were applied to this comparison. In each case, the GMT for each serogroup in safety and immunogenicity study (MTA09) participants compared favorably with that of MenactraTM recipients from either Group A or B in this trial. In no case did the upper 97.5% confidence limit (which is equivalent to the upper two-sided 95% confidence limit) of the ratio of the GMT exceed 2 for any serogroup In addition, over 95% of participants in both study groups demonstrate antibody titers above the level of protection for all 4 serogroups (Figure 4).

Study participants were monitored for immediate reactions for 30 minutes after vaccination, and for local and systemic reactogenicity during the 7 days following vaccination.

Pre-specified adverse events include localized reactions erythema, swelling, induration, and pain) and systemic symptoms fever measured by oral temperature, headache, fatigue, chills, arthralgia, anorexia, vomiting, diarrhea, seizures, malaise, and rash), which were assessed after each vaccination. These events were recorded daily on a diary card, and also collected by study personnel through telephonic interview eight days after each vaccination. If rash was reported, the investigator was prompted to record additional details on a separate case report form. Other non-serious, unexpected adverse events were obtained by telephonic interview eight and twenty days after each vaccination. Serious adverse events were reported and recorded during the entire study duration. Studies show that the Scontaminant administration of MenactraTM with other vaccines, such as Typhim Vi®, is safe Sand does not cause significantly increased rates of serious adverse reactions.

These data demonstrate that MenactraTM is highly immunogenic in the adult population when given alone or concomitantly with the travel vaccine Typhim Vi When these data are applied to the primary hypothesis, all criteria are met.

00 Example 16 Diphtheria Toxoid Conjugate Vaccine Versus a Non-meningococcal O Control Vaccine in Children Previously vaccinated With a Monovalent Meningococcal C Conjugate Vaccine The present example describes a randomized, modified double-blind trial to evaluate the antibody responses to TetraMenD (Menactra), a tetravalent C, Y, and W-135) meningococcal diphtheria toxoid conjugate vaccine, in children 2 to 5 years of age who had received a monovalent meningococcal C conjugate vaccine at least one year before enrollment into the trial. More particularly, the present examples describes a comparison of the SBA antibody response for serogroups C in subjects receiving TetraMenD to the serogroup C antibody response in a control group of participants receiving a licensed Hib conjugate vaccine (l-liberix®), as measured by the proportion of participants with at least a 4-fold rise in serogroup C titers 28 days following vaccination. All participants had previously received at least one dose of a monovalent meningococcal C conjugate vaccine at least one year before enrollment in the trial. The control group received a Hib conjugate vaccine rather than placebo in order to establish clinical benefit for these participants.

1. Inclusion and Exclusion Criteria A participant had to meet all of the following criteria to be enrolled in the trial: I) participant was healthy, as determined by medical history and physical examination; 2) participant was 2 years to 5 years of age at the time of vaccination; 3) at least one year from primary vaccination with a monovalent meningococcal C conjugate vaccine had elapsed; and 4) parent/guardian had signed an Ethics Committee-approved informed consent form.

Potential participants were excluded from the trial for any of the following reasons: 1) had a serious chronic disease cardiac, renal, neurologic, metabolic, rheumatologic, etc.); 2) known or suspected impairment of immunologic function; 3) acute medical illness with or 150 cK1 without fever within the last 72 hours or an axillary temperature 37.5 0 C at the time of Sinclusion; 4) history of documented invasive meningococcal disease; 5) administration of immune globulin or other blood products within the last three months, or injected or oral C, corticosteroids or other immunomodulatory therapy within six weeks of the study vaccine (individuals on a tapering dose schedule of oral steroids lasting 7 days could be included in the trial as long as they had not received more than one course within the last two weeks prior 00 to enrollment); 6) antibiotic therapy within the 72 hours prior to vaccination or 72 hours prior O to drawing any blood sample; 7) received any vaccine in the 28-day period prior to enrollment, or scheduled to receive any vaccine during enrollment in the trial; 8) received a meningococcal C conjugate vaccine within one year prior to enrollment; 9) suspected or known hypersensitivity to any of the vaccine components; 10) unable to attend all of the scheduled visits or comply with the study procedures; 11) enrolled in another clinical trial; and 12) any condition which, in the opinion of the investigator, would have posed a health risk to the participant or interfered with the evaluation of the vaccine.

Prior to vaccination, antipyretics or analgesics acetaminophen, ibuprofen, etc.) were not to be given routinely. Analgesic/antipyretic use on the day of and/or prior to vaccination was documented. During the 8-day observation period following vaccination, if a participant experienced an elevated temperature or complained of pain or discomfort, a standardized dose of an antipyretic/analgesic was permitted. The concomitant use during the 8-day observation period of any other medications was documented.

2. Administered Compositions A. TetraMenD The TetraMenD composition administered in this trial is composed of four distinct meningococcal capsular polysaccharides, serogroups A, C, Y, and W 135, which are covalently attached to diphtheria toxoid protein. The vaccine was formulated in sterile, pyrogen-free, phosphate-buffered physiological saline, with no preservative. The vaccine was prepared as a clear to slightly turbid liquid in single dose (0.5 mL) pre filled syringes. The purified meningococcal capsular polysaccharide used to prepare TetraMenD was the same material used to manufacture the licensed polysaccharide vaccine, Menomune A/C/Y/W 135 at Aventis Pasteur Inc., Swiftwater, Pennsylvania, USA. The diphtheria toxoid protein used 151

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CN was a thimerosal-free intermediate taken from the U.S.-licensed process for preparing the Sintermediate of other licensed diphtheria toxoid-containing vaccines at Aventis Pasteur Inc., Swiftwater, Pennsylvania, U.S.A. Each 0.5 mL dose of TetraMenD contains 16 Vg of Smeningococcal polysaccharide antigen (4 pg of each group specific polysaccharide antigen C, Y, and W 135]), conjugated to 48 Vg of diphtheria toxoid protein. Inactive ingredients in each 0.5 mL dose of TetraMenD include 0.6 mg sodium phosphate, 4.4 mg sodium 00 Schloride, and sterile pyrogen-free water for injection.

tc, B. Conjugated Hib Vaccine (Hiberix®) 0 10 Hiberix* is provided as a lyophilized vaccine product. After reconstitution with diluent, each 0.5 mL dose of Hiberix® consists of 10 pg of purified polyribosyl-ribitolphosphate (PRP) capsular polysaccharide of Hib covalently bound to approximately 30 Pg of tetanus toxoid. H-iberix® diluent contains sodium chloride and water for injections (as sterile saline solution The Hiberix® vaccine was prepared and reconstituted prior to administration according to the manufacture's instructions.

3. Trial Design and Dose Administration A total of 103 participants were actually enrolled in this study: 52 participants received TetraMenD, and 51 received the Hiberix" conjugate vaccine. Eligible participants were randomized to receive a single injection of either TetraMenD or the Hib conjugate vaccine.

No concomitant vaccinations were administered in this trial.

Briefly, at visit 1, TetraMenD was administered as a single 0.5 mL dose intramuscularly into the deltoid of the arm of choice using a pre-filled syringe with a 25 mm, needle. Alternatively, at visit 1, Hiberix® vaccine was administered as a single mL dose intramuscularly into the deltoid of the arm of choice using a syringe with a 25 mm, needle. The site of injection was cleansed with an appropriate antiseptic prior to injection. Only the specified dose (0.5 mL) was given. Care was taken to avoid administering the injection into or near blood vessels and nerves. After aspiration, if blood or any suspicious discoloration appeared in the syringe, the vaccine was discarded, and the procedures were repeated using a new dose of vaccine administered at a different site.

(Several safety parameters were assessed during the trial. During the 30-minute period Sfollowing vaccination, each participant was monitored for immediate reactions. On the evening of vaccination and for each of the 7 subsequent days, parents/legal guardians recorded ,i the presence and severity of solicited local and systemic reactions and any other unsolicited adverse events that according to the parent/legal guardian represented a change in the health status of the participant. During the 28-day period following vaccination, all unsolicited 00 adverse events and serious adverse events were identified (by interview and chart review) and recorded.

Blood specimens (5 mL whole blood) for serologic testing were drawn from each S10 participant on Day 0 prior to vaccination (baseline) and on Day 28 post-vaccination. At the (Ni Day 0 and Day 28 time points, sera were collected and assayed for serum bactericidal activity and lgG antibodies against meningococcal serogroups A, C, Y, and W 135, as described herein.

Each blood specimen was centrifuged within 4 hours of collection. The serum was then separated into 3 cryotubes and labeled with the protocol number, participant number and initials, and sample number (V-I or The primary sample had to have at least 1.0 mL of serum. The remaining serum in some cases was split between other retention aliquots. If there was less than 1.0 mL of serum, then all of the serum was placed in the primary tube, and not split among the three cryotubes. The serum specimens were stored in a non-frost-free, temperature-monitored freezer maintained at 20'C or less.

The following Table presents a summary of participant disposition by administered composition.

Table 1 Participant Disposition (All Participants) TetraMenD Hiberix® n n All randomized 52 51 Randomized, but did not receive any vaccine 0 1 Safety population (ITT population for safety)' 52 (100.0) 50(100.0) Received vaccine at Visit 1 52(100.0) 50(100.0) Completed Visit 2 (Day 28) 50 (96.2) 47 (94.0) Did not complete Visit 2 (Day 28) 2 3 Reasons for withdrawal Adverse reaction 0 0 (0.0) Lost to follow-up 1 2(4.0) Non-compliance with protocol 0 0 (0.0) Voluntary withdrawal 1 1 Other reason 0 0 (0.0) ITT population for immunogenicity t 52 (100.0) 49 (98.0) Protocol violators excluded from the PP population for(15.4) 1 immunogenit 8(15.4) 14(28.0) immunogenicity Did not meet entry criteria 2 1 Visit out of window 3 6(12.0) Other 3 7(14.0) PP population for immunogenicity t 44 (84.6) 36 (72.0) Percentages are based on the number of participants in the safety population in each vaccine group.

SStudy populations are defined in Section Error! Reference source not found.. ITT: intent-to-treat; PP: per-protocol.

S Protocol violators are counted only once according to their first violation.

Table 2, presented below, summarizes the demographic characteristics of the participants. The baseline distributions of participants by sex, age, and race were not statistically different between the 2 administration groups (p-values: 0.326, 0.696, and 0.720, respectively). Participants ranged in age from 24 to 57 months (mean age: 37.3 months).

There were 51.5% males and 48.5% females. The majority of participants were either Caucasian or Asian Table 2 Participant Demographics (All Participants) Demographic Total TetraMenD Hiberix Characteristics N 103 N 52 N 51 Sex In 0.326 Male 53 (51.5) 24(46.2) 29(56.9) Female 50 (48.5) 28 (53.8) 22 (43.1) Age (Months) Mean Median 0.696 37.3 36.0 37.0 35.0 37.6 38.0 Table 2 Participant Demographics (All Participants) Demographic Total TetraMenD Hiberix pvalue* Characteristics N 103 N 52 N 51 SD 8.3 8.5 8.3 Range 24 -57 25-55 24-57 Race In 0.720 Caucasian 48 (46.6) 22 (42.3) 26 (51.0) Black 7 4 3 (5.9) Hispanic 1 1 0(0) Asian 43(41.7) 22(42.3) 21 (41.2) Other 4 3 1 p-values were calculated using Fisher's Exact Test for sex and race, and an F-test in an ANOVA for age.

n: number of participants in the vaccine group satisfying the condition. The denominator for the percentage is the total number of randomized participants in each vaccine group.

4. Immunologic Laboratory Methods The first criterion for evaluating immunogenicity was the SBA-BR antibody responses for serogroups A, C, Y, and W-135 at baseline (pre-vaccination) and at Day 28 after vaccination. This criteria included: 1) the proportion of participants with a 4 fold rise in SBA-BR titer from baseline to Day 28 (serogroups A, Y, and W-135); 2) the distribution of titers pre-vaccination and 28 days post-vaccination (all four serogroups); 3) the reverse cumulative distribution curves pre- and 28 days post-vaccination (all four serogroups); and 4) the GMTs pre- and 28 days post-vaccination and the geometric mean fold rises from baseline to Day 28 (all four serogroups). The second criterion for evaluating immunogenicity was the total IgG antibody responses for serogroups A, C, Y, and W-135 at baseline (pre-vaccination) and at Day 28 after vaccination measured using an indirect IgG ELISA). This included the GMCs pre-vaccination and 28 days post-vaccination and the geometric mean fold rises from baseline to Day 28. All immunologic evaluations were conducted for each serogroups A, C, Y, and W-135) separately.

The proportions of participants with a 4 fold rise in SBA-BR titers from baseline to Day 28 were calculated, as well as the 95% CI (using normal approximation) (Agresti A., Chap. 3.4 Large-Sample Confidence Intervals. In: Categorical Data Analysis. New York: John Wiley Sons; 1990. pp. 54-59) of these proportions. When computing the fold rises, baseline pre-vaccination values reported as 8 were translated to 8, while post-vaccination values reported as 8 were translated to 4 for analysis purposes.

I

C1 Reverse cumulative distribution curves were plotted at Days 0 and 28 for each vaccine oi group and serogroup. The reverse cumulative distribution curves show the percentage of ;participants with a titer greater than or equal to the given titer on the x-axis.

,i SBA-BR GMTs at Days 0 and 28 and the 95% Cls for the GMTs were calculated as log base 2 titers assuming that the titers follow the lognormal distribution (Smith Mean.

In: Armitage P, Colton T, editors. Encyclopedia of Biostatistics, Vol. Chichester, UK: John 00 SWiley Sons; 1998. pp. 2487-2488; and Ratnaparkhi Lognormal Distribution. In: O Armitage P, Colton T, eds. Encyclopedia of Biostatistics, Vol. Chichester John 7- Wiley Sons; 1998. pp. 2349-2351). When computing GMTs, any value reported as 8 was O 10 converted to 4 for analysis purposes. The geometric means of the fold rises in SBA-BR titers from Day 0 to Day 28 and the 95% Cls for the geometric means were calculated.

IgG GMCs at Days 0 and 28 and the 95% Cls for the GMCs were calculated as log base 2 concentrations assuming that the concentrations follow the lognormal distribution.

A. Anti-Meningococcal Antibody Determination by a Serum Bactericidal Assay Using Baby Rabbit Complement The SBA-BR is an in vitro method using baby rabbit (BR) complement that measures the antibody-mediated, complement-dependent killing of target bacteria for the purpose of measuring the immunological response to the capsular polysaccharides of N. meningitidis serogroups A, C, Y, or W-135.

Functional antibody to meningococcal serogroups A, C, Y, and W-135 was measured by the Clinical Immunology Platform, Swiftwater, PA, USA, with a serum bactericidal assay using BR complement. Briefly, serogroup specific bacterial seeds were streaked onto Thayer- Martin plates and incubated overnight. Isolated colonies from the overnight plates were harvested and the bacteria were swabbed onto Thayer-Martin plates. These plates were then incubated to obtain a light veil of confluent bacterial growth. Sera were heat-treated according to inactivate endogenous complement.

Dulbecco's phosphate-buffered saline (PBS) was dispensed into the wells of a 96-well microtiter plate. Heat-inactivated serum samples, reference antisera, or serogroup-specific antibodies (as controls), were then dispensed and diluted twofold across the plate. There was also a serum control and complement control associated with each sample. The serogroup- 156 cK, specific meningococcal bacteria along with baby rabbit complement and the serum dilutions were allowed to incubate.

Bacterial cells were harvested from the Thayer-Martin plate and suspended in buffer.

A working dilution of bacterial cells was prepared and mixed with an equal volume of complement. This bacterial suspension was then dispensed into the 96-well microtiter plates, covered, and placed on a plate shaker for 1 min. The plates were removed to a 37°C CO 2 00 incubator and incubated for 90 min for serogroup A and 60 min for serogroups C, Y, and W- 135.

After incubation, an agar overlay was added to each well. The plates were covered and incubated overnight at 37°C with 5% CO 2 The next day the bacterial colonies in the wells were counted. The bactericidal titer of each unknown serum was expressed as the final reciprocal serum dilution yielding 50% killing compared with the TO colony-forming units (cfu) of the mean of the complement control wells. The serogroup-specific antibody controls were used to identify the serogroup being tested. The limit of detection for this assay was a titer of eight.

The reference serum was obtained from the CDC (donor R21654-3430107) and was stored at -70 0 C. Commercially available serogroup-specific antibodies for A, C, Y, and W- 135 were used as quality controls. The serogroup-specific antibodies were received refrigerated at 5 0 C and were rehydrated with ImL sterile water, aliquoted, and stored at -70 0

C.

B. Anti-Meningococcal IgG Antibody Determination In general, the ELISA method uses specific serum antibodies to bind a target antigen attached to a solid substrate. A secondary antibody, raised against human IgG, is added to complete the antigen-antibody-antibody complex. This secondary antibody is conjugated to an enzyme that catalyses a chromogenic reaction after addition of an appropriate substrate.

The ELISA reader's software plots the optical density (OD) and constructs a standard curve for the reference serum from which the test serum antibody titer can be calculated, with assigned unitage using a 4-parameter logistic curve (4PL) method.

Total IgG antibody activity for anti-meningococcal antibody to serogroups A, C, Y, and W-135 was measured by Clinical Immunology Platform, Swiftwater, PA, USA, using an indirect ELISA. In brief, clinical, control, or reference sera were incubated on microtiter 157 Splates precoated with excess serogroup-specific antigen (coating adsorption was enhanced 3 with methylated human serum albumin). The plates were then incubated at room temperature with a peroxidase-labeled mouse anti-human IgG specific antibody. Tetramethylbenzidine (TMB) was added to generate a chromogenic product. Plates were monitored at 650 nm until the top reference wells reached an OD in the range between 0.6 and 0.7. Once the range was attained, sulfuric acid stopping solution was added to each well. The resulting OD was read 00 Son an ELISA plate reader. The anti-meningococcal polysaccharide IgG level of the test sera 0 was calculated through comparison to reference sera (Lot CDC 1992) with an assigned value using a 4-parameter logistic (4PL) curve method. Results were reported in mg/ml.

0 10 The primary reference serum was a commercially available pooled human antiserum: CDC Lot 1992 (current NIBSC code 99/706). The primary reference, received in lyophilized state, was stored at 2 0 C to 8 0 C upon receipt.

The serum used for quality control serum was Aventis Pasteur human serum pool AvP Lot 100B189A. Vials were stored at -80°C to -40°C. Working aliquots were stored at to-10 0

C.

Statistical Analysis The statistical analysis was performed with SAS® software version 8.2. In general, all data collected are presented in summary tables and/or data listings. Categorical variables were presented by frequency distributions (number and proportion [with its 95% Cl] of participants in each category). Continuous variables were presented by summary statistics (number of observations mean, standard deviation median, and minimum and maximum values).

The trial had at least an 85% power to reject the hypothesis that the true difference between the proportions of participants with a 4 fold rise in serogroup C antibody titer in the TetraMenD group and in the Hib conjugate vaccine group is 5 50%. The calculation was based on a one-sided test with a type I error rate of 0.05 and on the assumption that the true proportion is at least 85% in the TetraMenD group and less than 10% in the Hib conjugate vaccine group.

The primary endpoint is the proportion of participants with at least a 4-fold rise in 3 antibody titers to serogroup C from baseline to Day 28 after vaccination in the TetraMenD group compared to the corresponding proportion in the control group.

In support of the primary hypothesis, a one-sided 95% confidence interval was constructed around the observed proportion in the TetraMenD group minus the corresponding proportion in the control group. The primary hypothesis was to be supported by the data if the lower 00 limit of the one-sided 95% confidence interval of the difference was 0.50. This was O equivalent to testing the null hypothesis that the proportion of participants with a 4 fold rise in antibody titers to serogroup C in the TetraMenD group minus the corresponding proportion O 10 in the control group was 0.50 (Ho: PTetraMenD Piberix 0.50), versus the alternative hypothesis that the difference is 0.50 (HI: pTetraMenD PHibrix 0.50), using a one-sided test with a type I error rate of 0.05. If the primary null hypothesis was rejected, it would be concluded that the proportion of participants with a 4 fold rise in antibody titers to serogroup C in the TetraMenD group was more than 50% higher than the proportion in the control group.

A participant was eligible for the per-protocol population for immunogenicity if the participant: 1) met the above-mentioned criteria for inclusion in the trial; 2) received the assigned vaccine; and 3) had Blood Sample 1 drawn on Day 0 (before vaccination) and Blood Sample 2 at least 28 but not more than 35 days after vaccination.

However, for analysis purposes, participants without any valid serology results were excluded from the per-protocol analyses. In this context, "valid" refers to the evaluability of the sera and/or results. Participants excluded from the per-protocol immunogenicity analyses include those for whom sera was lost or for whom an insufficient quantity of serum was available for analysis.

Participants with protocol violations that did not impact their immunologic response or their evaluability for immunogenicity were included in the per-protocol population. The perprotocol population was used for all immunogenicity analyses.

The intent-to-treat population for immunogenicity consisted of all participants who received one injection of either TetraMenD or the Hib conjugate vaccine, and had at least one valid serology result needed for analysis. The same immunogenicity analyses were performed on the per-protocol and intent-to-treat populations.

C,1 The safety population consisted of all participants who received one injection of either TetraMenD or the -lib conjugate vaccine and had any safety information available.

The safety analysis was performed using the safety population. If the safety data were not available from any given participant, the data from that particular participant's reaction were reported as missing.

00 6. Immunogenicity Results SSummary Table 3 shows the number and percentage of participants with a 4 fold rise in SBA BR antibody titers from baseline to Day 28 for serogroups A, C, Y, and W 135.

Twenty-eight days after vaccination, the majority of TetraMenD recipients experienced a 4 fold rise in SBA BR antibody titers from baseline, while a minority of Hiberix® recipients achieved this result.

Table 3 Number and Percentage of Participants with a 4-Fold Rise in SBA-BR Antibody Titers From Baseline to Day 28 (Per-Protocol Population) TetraMenD Hiberixe Serogroup 95% CI n/N* 95% C! n/N* %t 95% CI

A

C

Y

43/44 97.73 87.98, 99.94 11/36 30.56 16.35, 48.11 41/44 93.18 81.34, 98.57 2/36 5.56 0.68, 18.66 35/44 79.55 64.70, 90.20 1/36 2.78 0.07, 14.53 W-135 43/44 97.73 87.98, 99.94 8/36 22.22 10.12, 39.15 n: number of participants with a 4-fold rise from baseline titer.

N: number of participants with valid serology data.

t n/N expressed as a percentage.

Table 4 shows the number and percentage of participants with a 4 fold rise in SBA- BR antibody titers from baseline to Day 28 for serogroup C. This percentage is much higher in the TetraMenD group (93.18%) than in the -liberix® group The difference in percentages (P-retraMenD Plibenx®) is 87.63%, and the lower limit of the one-sided confidence interval of the difference is 78.77%, which is above 50%. Based on these results, it can be concluded that the percentage of per-protocol participants in the TetraMenD group with a 4 fold rise in SBA BR titers for serogroup C minus the corresponding percentage in the Hiberix® group is greater than 50%, using a one-sided test with a type I error rate of 0.05.

Table 4 Number and Percentage of Participants with a 4-Fold Rise in Serogroup C SBA-BR Antibody Titers from Baseline to Day 28, Testing of the Primary Hypothesis (Per-protocol Population) TetraMenD Hiberix® Lower Limit of the Difference One-sided Serogroup C n/N* Pi 1 n/N* P2 (PI P 2 confidence interval of the difference 41/44 93.18 2/36 5.56 87.63 78.77 n: number of participants with a 4-fold rise in serogroup C SBA-BR titer from baseline to Day 28.

N: total number of participants with valid serology data for serogroup C.

t pl: percentage of participants with a 4-fold rise in serogroup C SBA-BR titer from baseline to Day 28 in the TetraMenD group.

P2: percentage of participants with a 4-fold rise in serogroup C SBA-BR titer from baseline to Day 28 in the Hiberix® group.

Table 5 shows the SBA-BR GMTs at baseline and on Day 28 after vaccination, as well as the geometric mean fold rises in SBA BR titers from baseline to Day 28. In the TetraMenD group, there was a significant increase in SBA-BR antibody titers between Day 0 and Day 28; the geometric mean fold rises in SBA-BR titers were 70.34 for serogroup A, 136.33 for serogroup C, 16.51 for serogroup Y, and 159.59 for serogroup W 135. In the Hiberix® group, there was no or little increase in SBA-BR antibody titers between Day 0 and Day 28.

2007203489 27 Aug 2007 Table 5 SBA-BR Geometric Mean Titers Baseline to Day 28 (Per-Protocol Population) at Baseline and Day 28, and Geometric Mean Fold Rises in SBA-BR Titers from TetraMenD Hiberix® Serogroup Parameter Bleed GMT or Geometric GMT or Geometric Mean Fold Rise' CI Mean Fold Riset 95% Cl GMT Day 0 44 136.33 69.39, 267.84 36 99.66 47.66, 208.40 A Day 28 44 11404.16 7383.63, 17613.96 36 199.31 93.14, 426.52 Geometric Mean Fold Rise t Day 28 44 70.34 34.46, 143.61 36 1.65 0.97 2.80 GMT Day 0 44 76.11 36.57, 158.41 36 26.91 12.49, 57.97 C _Day 28 44 12534.67 8407.56, 18687.71 36 22.63 10.50, 48.76 Geometric Mean Fold Rise t Day 28 44 136.33 61.66, 301.43 36 0.57 0.40,0.82 GMT Day 0 44 236.61 133.11,420.60 36 266.05 151.05,468.59 Y Day 28 44 4031.98 2399.10, 6776.24 36 298.63 155.64, 572.99 Geome t ric Mean Fold Rise Day 28 44 16.51 8.57, 31.80 36 1.06 0.76, 1.47 GMT Day 0 44 26.07 13.30, 51.13 36 24.44 11.76, 50.79 Day 28 44 5978.03 3852.54, 9276.19 36 32.00 16.33, 62.71 W-135 Geometric Mean Fold Day 28 44 159.59 84.77, 300.43 36 0.91 0.56, 1.48 Riset N: number of participants with valid serology data.

Geometric mean fold rise geometric mean of [titer at Day 28/Titer at Day 0] IJ Table 6 shows the frequency distribution of baseline and Day 28 SBA-BR antibody titers for each serogroup.

00

O-

tm, 2007203489 27 Aug 2007 Table 6 Distribution of SBA-BR Antibody Titers on Day 0 and Day 28 (Per-Protocol Population) SGroup SBA Titers Sero- Bleed NT: 44 n group NH:36* <8 8 16 32 64 128 256 512 1024 2048 4096 8192 16384 32768 65536 >65536 TetraMenD 20) 0(0.0) 0(0.0) 0(0.0) 3(6.8) 2(4.5) 8(18.2) 9(20.5) 8(18.2) 3(6.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) DayO Hiberix 10 0(0.0) 0(0.0) 1(2.8) 1(2.8) 4(11.1) 7(19.4) 7(19.4) 5(13.9) 1(2.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) A (27.8) 28 TetraMenD 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(2.3) 1(2.3) 8(18.2) 4(9.1) 7(15.9) 10 5(11.4) 5(11.4) 3(6.8) Da 28 (22.7) Hiberix 8(222) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(2.8) 8(22.2) 8(22.2) 6(16.7) 4(11.1) 1(2.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) TetraMenD 23 3(6.8) 1(2.3) 2(4.5) 2(4.5) 7(15.9) 4(9.1) 4 4(9.1) 3(6.8) 1 0(0.0) 1(2.3) 0(0.0) 0(0.0) 0(0.0) Day 0 Hiberix 0(0.0) 0(0.0) 1(2.8) 1(2.8) 3(8.3) 3(8.3) 6(16.7) 1(2.8) 1(2.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) C (55.6 TetraMenD 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 2(4.5) 6(13.6) 5(11.4) 6(13.6) 10 7(15.9) 6(13.6) 2(4.5) Day 28 Hiberix 1(2.8) 2(5.6) 1(2.8) 1(2.8) 0(0.0) 5(13.9) 1(2.8) 4(11.1) 1(2.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) (55.6) 1 1 TetraMenD 2(4.5) 1(2.3) 3(6.8) 2(4.5) 4(9.1) 7(15.9) 5(11.4) 8(18.2) 4(9.1) 5(11.4) 1 2(4.5) 0(0.0) 0(0.0) 0(0.0) 0(0.0) Day 0 11 Hiberix" 3(8.3) 0(0.0) 0(0.0) 1(2.8) 2(5.6) 3(8.3) 8(22.2) 5(13.9) 0(0.0) 3(8.3) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) (30.6) V 12 5 TetraMenD 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(23) 0(0.0) 3(6.8) 2(4.5) 3(6.8) (273) 6(13.6) 6(13.6) (11.4 2(4.5) 1(2.3) 3(6.8) Day 28 Hiberix 4(11.1) 0(0.0) 0(0.0) 0(0.0) 2(5.6) 6(16.7) 2(5.6) (333) 4(11.1) 4(11.1) 0(0.0) 2(5.6) 0(0.0) 0(0.0) 0(0.0) 0(0.0) (33.3 TetraMenD 23 1(2.3) 0(0.0) 3(6.8) 2(4.5) 2(4.5) 5(11.4) 5(11.4) 1(2.3) 2(4.5) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) Day 0 Hiberix 19 1(2.8) 0(0.0) 1(2.8) 2(5.6) 4(11.1) 6(16.7) 0(0.0) 2(5.6) 0(0.0) 1(2.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) W-13 (52.8) 10 TetraMenD 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(2.3) 2(4.5) 3(6.8) 8(18.2) 8(182) 10 4(9.1) 5(11.4) 0(0.0) 3(6.8) Day 28 Hiberix (417) 0(0.0) 0(0.0) 3(8.3) (19) 5(13.9) 6(16.7) 0(0.0) 1(2.8) 0(0.0) 1(2.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) NT: number of TetraMenD recipients with valid serology data at the indicated timepoints; NH: number of Hiberix recipients with valid serology data at the indicated timepoints.

n: number of participants with the indicated titer; n/N expressed as a percentage.

N Figures 5, 6, 7, and 8, respectively, present the Day 0 and Day 28 reverse cumulative distribution curves of SBA BR antibody titers for serogroups A, C, Y, and W 135.

Table 7 shows the IgG GMCs at baseline and on Day 28 after vaccination as well as the r^.

CI geometric mean fold rises in IgG concentrations from baseline to Day 28. In the TetraMenD group, there was an increase in IgG antibody titers between Day 0 and Day 28; the geometric mean fold rises in IgG titers were 26.74 for serogroup A, 15.86 for serogroup C, 12.13 for 00 serogroup Y, and 22.00 for serogroup W-135. In the Hiberix® group, there was no or little increase in IgG antibody titers between Day 0 and Day 28.

1 0' 2007203489 27 Aug 2007 Table 7 IgG Geometric Mean Concentrations at Baseline and Day 28, and Geometric Mean Fold Rises in IgG Concentrations from Baseline to Day 28 (Per-Protocol Population) TetraMenD Hiberix® Serogroup Parameter Bleed GMC or Geometric GMC or Geometric N* t 95% CI N* 95% CI N Mean Fold Rise 95 Mean Fold Rise t 95% GMC Day 0 44 0.82 0.58,1.17 36 0.63 0.45, 0.88 GMC (Fg/mL) A Day 28 44 25.75 18.41, 36.00 35 0.69 0.50,0.94 Geometric Mean Fold Rise t Day 28 44 26.74 18.94, 37.76 35 0.89 0.78, 1.01 Day 0 44 0.44 0.33,0.59 36 0.43 0.34, 0.55 GMC (pg/mL) C G mti Day 28 44 9.56 6.53, 14.00 35 0.41 0.33, 0.52 Geometric Mean Fold Rise* Day 28 44 15.86 11.11, 22.62 35 0.74 0.66, 0.82 GMC g/mL) Day 0 44 0.45 0.37, 0.55 36 0.33 0.28, 0.40 Y Day 28 44 7.83 5.24, 11.71 35 0.35 0.28, 0.44 Geometric Mean FoldRise Day 28 44 12.13 8.31, 17.73 35 0.63 0.54, 0.74 Day 0 44 0.33 0.26, 0.42 36 0.23 0.18, 0.30 GMC (pg/mL) W-135 Day 28 44 9.02 6.33, 12.84 35 0.20 0.16, 0.26 Geometric Mean Fold Rise t Day 28 44 22.00 1 5 5 7 3 1 0 8 35 0.62 0.56, 0.69 N: number of participants with valid serology data. The quantity of serum remaining from the sample provided by Participant 355 (Hiberix group) at Visit 2 was not sufficient to perform the Day 28 IgG testing.

t Geometric mean fold rise geometric mean of [(concentration at Day 28)/(concentration at Day 0)] (C Immunogenicity Summary SPrior to vaccination, the SBA and IgG antibody levels were similar in both the TetraMenD and the Hib conjugate vaccine groups. Twenty-eight days after TetraMenD administration, there was a marked increase in SBA and IgG antibody levels for all the serogroups contained in the composition (Tables 5 and In particular, the serogroup C responses were consistent with a memory response to that antigen. For each serogroup, the Sproportion of TetraMenD recipients achieving a 4 fold rise in antibody titers (Table 3) was similar to that reported in a large trial in 2- to 10-year-olds who received a single dose of the investigational vaccine. (Pichichero M. el al., A Comparative Trial of the Safety and Immunogenicity of Quadrivalent C, Y, W-135) Meningococcal Diphtheria Conjugate Vaccine (MCV-4) Versus Quadrivalent Polysaccharide Vaccine (Menomune®, PS) in Children 2-10 Years of Age. Abstracts of the 43rd Annual ICAAC, Chicago, Illinois. American Society for Microbiology. p. 286). This proportion was 79.6% for serogroup Y, compared with 97.7% for serogroup A, 93.2% for serogroup C, and 97.7% for serogroup W-135 (Table The highest pre-vaccination antibody levels were observed for serogroup Y (Table This may be due to natural exposure to this serogroup, which may be more common than previously believed. Higher circulating antibody levels reflect recent natural exposure and may reduce the proportion of vaccine recipients achieving a 4-fold rise in antibody levels. Higher prevaccination antibody levels were also observed for serogroup A, compared to serogroups C or W-135. This is probably the result of intermittent exposure over a prolonged period of time to several naturally occurring cross-reacting antigens.

There was no significant difference between the pre- and post-meningococcal antibody responses in the control group who received the Hib vaccine. The difference between the TetraMenD and Hib vaccine groups in the proportion of participants with a 4 fold rise in SBA-BR titers was 50% for each of the four serogroups (Table In the control group, the proportion of participants achieving a 4-fold rise in SBA-BR titers was higher for serogroups A and W-135 than for serogroup C or Y. This may have resulted from natural exposure to serogroup-specific antigens circulating in this population during the conduct of the study. A large proportion of the participants were recruited from the Muslim community in London, where clinical cases of serogroup W-135 meningococcal disease have been identified. This increased risk has been associated with pilgrimage to the Hajj. Although assay results are 167 N known to differ between laboratories, a similar trend was observed in the SBA and IgG antibody responses following the administration of a booster dose of a serogroup C meningococcal conjugate vaccine in children who were primed in infancy with a monovalent Cconjugate vaccine. (McVernon et al., Safety and immunogenicity of meningococcus serogroup C conjugate vaccine administered as a primary or booster vaccination to healthy four-year-old children, Ped. Infect. Dis. 21(8):747-753 [2002]).

In the TetraMenD group, the magnitude of the post-vaccination GMTs to all four serogroups (Table 5) demonstrates that the vaccine is highly immunogenic in this population.

All TetraMenD recipients achieved levels of antibody consistent with protection. (Balmer P.

and Borrow Serologic correlates of protection for evaluating the response to meningococcal vaccines, Expert Rev. Vaccines. 3(1):77-87 [2004]).

7. Overall Safety Profile Table 8 presents an overall summary of participants with immediate reactions within minutes, solicited local and systemic reactions within 7 days after vaccination, and unsolicited adverse events and serious adverse events during the entire study period (Days 0-28 after vaccination).

Table 8 Overall Participant Safety Profile (Safety Population) TetraMenD Hiberix® Type of Adverse Events n/N* n/N* Immediate Reactions (within 30 minutes) 0/52 0.0 0/50 0.0 Solicited Local Reactions (Days 0-7) 27/51 52.9 24/49 49.0 Solicited Systemic Reactions (Days 0-7) 35/52 67.3 25/49 51.0 Unsolicited Adverse Events (Days 0-28) 29/51 56.9 31/47 66.0 Serious Adverse Events (Days 0-28) 1/52 1.9 0/50 0.0 n: number of participants reporting at least one event in this category; N: total number of participants who submitted safety information at each of these time points.

Safety Summary The following safety data were reported during the trial: immediate reactions within minutes of vaccination, solicited local and systemic reactions within the first 7 days postvaccination, and all unsolicited adverse events in the 28-day period after vaccination.

c No immediate reactions were observed within 30 minutes after vaccination. The b frequency of solicited local reactions was similar in both vaccine groups. In both vaccine groups, most solicited local reactions were reported as mild and resolved within 3 days (median duration). Irritability and drowsiness were the most common systemic complaints. One TetraMenD recipient experienced a febrile convulsion within 24 hours of receiving the vaccine.

He also was noted to have cough with upper respiratory symptoms. This participant had a 00 history of febrile convulsions associated with respiratory infections. This event was reported as Cn) O an SAE possibly related to vaccination. No other SAEs were reported during the study period.

Unsolicited adverse events were common and their frequency similar in both vaccine groups. Most of them were common medical complaints and conditions that were reported as unrelated to vaccination. Overall, TetraMenD was well tolerated and had a safety profile similar to that of the licensed Hib conjugate vaccine used as a control.

Claims (16)

1. A method for administering an immunogenic composition to a subject comprising, a) providing: i) a subject, wherein said subject has been vaccinated with a dose of a O 0 monovalent meningococcal conjugate vaccine; ii) a tetravalent meningococcal polysaccharide protein conjugate composition; and 0 10 b) administering said tetravalent meningococcal polysaccharide protein conjugate composition to said subject at least twelve months after said subject has been vaccinated with said monovalent meningococcal conjugate vaccine.

2. A method for administering an immunogenic composition to a subject comprising, a) providing: i) a subject, wherein said subject has been vaccinated with a dose of a monovalent meningococcal conjugate vaccine; ii) a tetravalent meningococcal polysaccharide protein conjugate vaccine; and b) administering said tetravalent meningococcal polysaccharide protein conjugate vaccine to said subject at least twelve months after said subject has been vaccinated with said monovalent meningococcal conjugate vaccine.

3. A method for administering an immunogenic composition to a subject comprising, a) providing: i) a subject, wherein said subject has been vaccinated with a protective antigen against N. meningitidis serogroup C; ii) a tetravalent meningococcal polysaccharide protein conjugate vaccine; and t",I Sb) administering said tetravalent meningococcal polysaccharide protein conjugate vaccine to said subject at least twelve months after said subject has been vaccinated NC with said monovalent meningococcal conjugate vaccine. 00

4. A method for administering an immunogenic composition to a subject comprising, C a) providing: i) a subject, wherein said subject has been vaccinated with a monovalent N. N 10 meningitidis serogroup C meningococcal vaccine; ii) a tetravalent meningococcal polysaccharide protein conjugate composition; and b) administering said tetravalent meningococcal polysaccharide protein conjugate vaccine to said subject at least twelve months after said subject has been vaccinated with said monovalent meningococcal conjugate vaccine.

A method of preventing meningococcal disease in a subject comprising administering to said subject a therapeutically effective dose of a tetravalent meningococcal conjugate vaccine at least twelve months after said subject has been vaccinated with a monovalent meningococcal conjugate vaccine.

6. A method of preventing meningococcal disease in a subject comprising administering to said subject a therapeutically effective dose of a tetravalent meningococcal conjugate vaccine at least twelve months after said subject has been vaccinated with monovalent N. meningitidis serogroup C meningococcal vaccine.

7. A method of stimulating an immunologic response in a subject comprising administering to said subject a therapeutically effective dose of a tetravalent meningococcal conjugate vaccine at least twelve months after said subject has been vaccinated with a monovalent meningococcal conjugate vaccine.

8. A method of inducing an immune response in a subject against one or more serogroups of N. meningitidis bacteria, the method comprising administering to said Cl subject a therapeutically effective dose of a tetravalent meningococcal conjugate vaccine at least twelve months after said subject has been vaccinated with a monovalent 00 meningococcal conjugate vaccine. (N

9. The method of claim 8, wherein said tetravalent meningococcal conjugate Svaccine is formulated as 0.5ml of a sterile liquid in a single use syringe. C The method of claim 10, wherein 0.5ml said tetravalent meningococcal conjugate vaccine comprises from about 0.01 yg to about

10 A/g of purified capsular polysaccharides from each of serogroups A, C, Y, and W-135 of N. meningitidis.

11. The method of claim 11, wherein 0.5ml said tetravalent meningococcal conjugate vaccine comprises about 4 pg of purified capsular polysaccharides from each of serogroups A, C, Y, and W-135 of N. meningitidis.

12. The method of claim 11, wherein 0.5ml said tetravalent meningococcal conjugate vaccine comprises from about 0.05 Ag to about 5 Ag of purified capsular polysaccharides from serogroups A, C, Y, and W-135 of N. meningitidis.

13. Use of a tetravalent meningococcal conjugate vaccine for the manufacture of a medicament for preventing meningococcal disease in a subject wherein said medicament is administered to the subject in a therapeutically effective dose at least twelve months after said subject has been vaccinated with a monovalent meningococcal vaccine.

14. Use of a tetravalent meningococcal conjugate vaccine for the manufacture of a medicament for preventing meningococcal disease in a subject wherein said medicament is administered to the subject in a therapeutically effective dose at least twelve months 00 C-O after said subject has been vaccinated with a monovalent N. meningitidis serogroup C meningococcal vaccine.

Use of a tetravalent meningococcal conjugate vaccine for the manufacture of a medicament for stimulating an immune response in a subject wherein said medicament is administered to the subject in a therapeutically effective dose at least twelve months after said subject has been vaccinated with a monovalent meningococcal vaccine.

16. Use of a tetravalent meningococcal conjugate vaccine for the manufacture of a medicament for stimulating an immune response in a subject wherein said medicament is administered to the subject in a therapeutically effective dose at least twelve months after said subject has been vaccinated with a monovalent N. meningitidis serogroup C meningococcal vaccine. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON Dated 24 August 2007 Sanofi Pasteur, Inc.