WO2009108168A2 - Gold nanoparticle glycoconjugates for generating igg specific for the carbohydrate - Google Patents

Abstract

A composition can include a gold nanoparticle and a peptide and an oligosaccharide bound to the gold nanoparticle. The oligosaccharide can be an antigen, so that the composition can be used to induce an immunogenic response.

Description

GOLD NANOPARTICLE GLYCOCONJUGATES FOR GENERATING IGG SPECIFIC

FOR THE CARBOHYDRATE

[0001] This application claims the benefit of U.S. Provisional Application No.

61/083,773, filed July 25, 2008, the specification of which is hereby incorporated by reference, and claims the benefit of U.S. Provisional Application No. 60/987,224, filed November 12, 2008, the specification of which is hereby incorporated by reference.

[0002] Aspects of this invention were made with U.S. government support provided by

National Science Foundation (NSF) grant CHE0511219478. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Bacterial infections remain major killers of infants and children, particularly in developing countries. Several million children die each year due to such infections, with the most important pathogens being Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis, Salmonella entericus subspecies typhi, Staphylococcus aureus, and diarrhea-causing organisms such as Shigella, Salmonella and Vibrio cholerae. Each of these pathogens possesses a cell surface capsular polysaccharide (CPS) or lipopolysaccharide(LPS)/lipooligosaccharide(LOS), or both, which helps the pathogen to establish infection. Antibodies directed against theses molecules can prevent disease. [0004] Neisseria meningitidis is the leading cause of bacterial meningitis and is a worldwide health problem, with serogroup B strains producing the majority of disease in developed countries. Vaccines able to prevent serogroup B strains have been difficult to develop, due to the fact that the capsule of these strains is poorly immunogenic, and shares chemical structures with host tissues. Therefore, the development of vaccines to prevent disease caused by this serogroup has focused on subcapsular antigens, and several vaccines have been developed and tested in large-scale efficacy trials. No protection has been demonstrated in children less than 2 years old, and group B vaccines have only been successful in preventing clonal outbreaks. Bactericidal antibody directed against meningococcal LOS is generated after natural infection, and the presence of these antibodies appears to correlate with disease protection. However, the use of complex carbohydrates derived from LPS or LOS for use as a vaccine is problematic, because they have significant toxicity associated with them, due to their chemical linkage to lipid A. For example, LOS produced by N. meningitidis has been implicated in the successful immune response to natural infection; however, its use in vaccines has been contraindicated due to its high toxicity. Vaccination using derivatives containing the complex carbohydrates generates poor immune responses, even though antibody to these carbohydrates mediates high levels of opsonic killing activity in vitro, which can be a strong correlate of an effective immune mediator.

[0005] Pathogens can express cell surface capsular polysaccharide (CPS), LPS)ZLOS, or both, resulting in a number of different serotypes or serogroups. These sugar-containing molecules are important in establishing a variety of bacterial-host cell interactions, and individuals containing antibody directed against them are immune to the disease causing agent (for a recent review, see Weintraub, A. 2003. Immunology of bacterial polysaccharide antigens. Carbohydr Res. 338:2539- 2547.). Carbohydrates are major virulence factors found on the surface of bacteria, and antibody directed against these components confers protection against a variety of diseases. CPS vaccines work, and vaccines of this type are licensed and used in a number of countries. However, this approach has several severe limitations, and developing vaccines based on polysaccharides is difficult. Carbohydrate-induced immune responses are T cell-independent. Without the involvement of T cells they do not induce immunological memory, avidity maturation and isotype switching do not occur, and the antibodies induced, largely IgM and IgG2 are not good activators of complement (see, Lortan, J. E., A. S. Kaniuk, and M. A. Monteil. 1993. Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults. Clin. Exp. Immunol. 91:54-57; and Musher, D. M., M. J. Luchi, D. A. Watson, R. Hamilton, and R. E. Baugbn. 1990. Pneumococcal polysaccharide vaccine in young adults and older bronchitics: determination of IgG responses by ELISA and the effect of adsorption of serum with non-type-specific cell wall polysaccharide. J. Infect. Dis. 161:728- 735.). Therefore, vaccines of this type fail to induce immune responses in infants below the age of about two years, and repeated vaccination does not lead to increased antibody levels. [0006] No vaccine is licensed for Francisella tularensis, which causes tularemia — a zoonotic disease that affects a range of animals, including rabbits, hares and rodents — and is widely feared as being a likely biological weapon or bioterrorism agent. F. tularensis is one of the most virulent microorganisms known. Humans can be infected with as few as 10 cfu of type A F. tularensis. This is also the lethal dose for various experimental animals when the pathogen is administered IV, and this dose is assumed to be the same for humans. F. tularensis can be spread by aerosol to initiate respiratory infection (see Ericsson, M., A. Tarnvik, K. Kuoppa, G. Sandstrom, and A. Sjostedt. 1994. Increased synthesis of DnaK, GroEL, and GroES homologs by Francisella tularensis LVS in response to heat and hydrogen peroxide. Infect Immun 62:178-83.). Because of the low infective dose and ability to be disseminated by aerosol, various governments developed F. tularensis as a biowarfare agent (see, Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medical and public health management. Jama 285:2763-73.). Although a live attenuated strain of F. tularensis (strain LVS, derived from the multiple passage of a fully virulent strain of F. tularensis) has been used to immunize millions of people and numerous studies have shown the effectiveness of the LVS vaccine in humans (see, Oyston, P. C, and J. E. Quarry. 2005. Tularemia vaccine: past, present and future. Antonie Van Leeuwenhoek 87:277-281.), there are limitations associated with the use of this vaccine and, for these reasons, the US IND status of the LVS vaccine was recently revoked.

SUMMARY OF THE INVENTION

[0007] In an embodiment according to the invention, an immunogenic composition includes an antigenic saccharide or glycoconjugate molecule, an immunogenic peptide, and a carrier particle. The antigenic saccharide or glycopeptide molecule can be bound to the carrier particle, and the immunogenic peptide can be bound to the carrier particle. The antigenic saccharide or glycoconjugate molecule of the immunogenic composition can include, for example, a glycoprotein, a lipopolysaccharide (LPS), a lipooligosaccharide (LOS), a capsular polysaccharide, a detoxified oligosaccharide (OS), or lacto-N-neotetraose. The immunogenic peptide can bind to, for example, a protein, an antigen, a leukocyte antigen, or a human leukocyte antigen (HLA) class II molecule. The immunogenic peptide of the immunogenic composition can include, for example, a pan DR helper T cell epitope or a pan DR binding oligopeptide. [0008] The carrier particle of the immunogenic composition can include a material selected from the group consisting of a metal, a noble metal, gold, a metal oxide, silica, titania, a non-metal, a metalloid, a carbon fϊillerene, or combinations. The carrier particle of the immunogenic composition can include a nanoparticle that has a diameter of from about 1 nm to about 1000 nm, a diameter of less than or equal to about 100 nm, or a diameter of greater than about 100 nm. The carrier particle of the immunogenic composition can be substantially biologically inert.

[0009] The immunogenic composition can have a ratio of antigenic saccharide or glycoconjugate molecules to immunogenic peptides bound to the carrier particle in the range of from about 20:1 to about 1 :20. For example, the immunogenic composition can have a ratio of antigenic saccharide or glycoconjugate molecules to immunogenic peptides bound to the carrier particle of about 1 :1. For example, the total number of antigenic saccharide or glycoconjugate molecules and immunogenic peptides bound to the carrier particle can be in a range of from about 2 to about 100. For example, one antigenic saccharide or glycoconjugate molecule and one immunogenic peptide can be bound to the carrier particle.

[0010] A method according to the invention for producing an immunogenic response includes administering an immunogenic composition to a human, mammal, vertebrate, or animal, so that the immunogenic response is produced in the human, mammal, vertebrate, or animal. For example, the immunogenic response can be immunity of the human, mammal, vertebrate, or animal to infection by a pathogen or bacterium.

[0011] A method according to the invention for treating a subject in need of induction of an immunogenic response includes administering an immunogenic composition to a subject to induce the immunogenic response in the subject. The method of treating can include vaccinating the subject.

[0012] A method according to the invention for making an immunogenic composition can include the following. A gold chloride, for example, gold (I) chloride (AuCl) or gold (III) chloride (AuCb), can be reduced in aqueous solution with citrate ion to produce gold nanoparticles. A glycoconjugate solution of a lipooligosaccharide disulfide (LOS-disulfide) and a PADRE-disulfide can be formed and reacted with the gold nanoparticles to produce PADRE- lipooligosaccharide disulfide functionalized nanoparticles. The PADRE-lipooligosaccharide functionalized nanoparticles can be isolated and rinsed to remove unreacted PADRE- lipooligosaccharide disulfide and citrate ion. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 presents a mechanism for the formation of isoxazoline 3.

[0014] Figure 2 presents a mechanism for the synthesis of glucopyranosyl isoxazonline 3.

[0015] Figure 3 presents a synthetic scheme for gold nanoparticle surface functionalization. X and Y are same or different biomolecules and represent targeting moieties of the substrate. The attachment of a molar mixture of symmetric or asymmetric disulfides of varying content can be achieved on the surface.

[0016] Figure 4 presents AFM images of self-assembled monolayers (SAM) on gold.

The panels present: (A) an AFM image of a SAM from β-glucose thiol conjugate; and (B) an AFM image of a SAM from α-glucose thiol conjugate.

[0017] Figure 5 presents a TEM image of concanavalin A mediated aggregation of glucose-coated Au nanoparticles. The bar represents ~ 10 nm.

[0018] Figure 6 presents images from fluorescent microscopy. Lactose-labeled gold nanoparticles specifically interact with N. gonorrhoeae. Gonococci were incubated with gold- labeled nanoparticles and visualized by fluorescent microscopy. Interaction of gold particles results in. luminescence. The images show the amount of luminescence detected from: (1) N. gonorrhoeae; (2) N. gonorrhoeae with uncoated gold nanoparticles; (3) N. gonorrhoeae with glucosylated nanoparticles; and (4) N. gonorrhoeae with lactosylated nanoparticles. [0019] Figure 7 presents the results of multiplicity of infection (MOI) in vitro challenges on the production of cytokines by human monocytes. The cytokine profile of primary human monocytes (106) after an 18-hour challenge with killed N. gonorrhoeae, strain F62ΔlgtD is shown. TNF α, IL-I β and IL-8 levels as measured by ELISA are shown. (A) 10 bacteria/monocyte (B) 10 monocytes^acteria.

[0020] Figure 8 presents the susceptibility of various gonococcal strains to killing by

NHS.

[0021] Figure 9 presents a FACS-Scan profile of B-cells for the measurement of B7-1 and B7-2 expression. Fluorescence was measured as described in the text. F62 is a gonococcal strain that expresses the lacto-N-neotetraose LOS and an LOS structure containing an additional sugar. F62ΔrfaK LOS only contains LipidA, KDO and heptose. The light line represents unstimulated controls while the dark line represents fluorescence after stimulation. [0022] Figure 10 (at left) presents a wavescan of purified LOS. The purity of the extracted LOS is determined by reading absorbance over a spectrum of wavelengths. The results shown indicate that the sample is free of DNA and protein contamination. [0023] Figure 10 (at right) presents an image of an SDS-PAGE gel of LOS. The right lane illustrates that genetically modified F62ΔlgtD produces only one lacto-N-neotetraose LOS. [0024] Figure 11 presents a scheme for the synthesis of LOS-thiol.

[0025] Figure 12 presents a scheme for the synthesis of PADRE-LOS-disulfide.

[0026] Figure 13 presents a cartoon showing a procedure for making TRIAD vaccine particles.

[0027] Figure 14A presents ELISA results of when mouse 791 was injected with purified LOS and ELISA was conducted with 1:5000 dilution of anti-mouse IgG. [0028] Figure 14B presents ELISA results of when mouse 791 was injected with purified

LOS and ELISA was conducted with 1:5000 dilution of anti-mouse IgM. [0029] Figure 14C presents ELISA results of when mouse 799 was injected with the vaccine construct, Au-OS-PADRE, and ELISA was conducted with 1 :5000 dilution of anti- mouse IgG.

[0030] Figure 14D presents ELISA results of when mouse 799 was injected with the vaccine construct, Au-OS-PADRE, and ELISA was conducted with 1:5000 dilution of anti- mouse IgM.

DETAILED DESCRIPTION

[0031] Embodiments of the invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention. All references cited herein are incorporated by reference as if each had been individually incorporated.

[0032] N. meningitidis is a significant cause of bacterial meningitis and septicemia.

Meningococci are divided into groups on the basis of their chemically distinct capsular polysaccharides but only organisms belonging to one of five serogroups, A, B, C, Y or Wl 35, cause significant disease (see, Cartwright, K. A. V. 1995. Meningococcal disease., p. 1 15- 146. 1« K. A. V. Cartwright (ed.), Meningococcal carriage and disease, vol. 5. Wiley, Chichester.). Group A organisms cause widespread epidemic disease in the so-called "meningitis belt" countries, whereas the other four groups are responsible for endemic disease and localized outbreaks world-wide (see, Jodar, L., I. M. Feavers, D. Salisbury, and D. M. Granoff. 2002. Development of vaccines against meningococcal disease. Lancet 359:1499- 1508.). Disease caused by group C isolates occurs primarily in infants although outbreaks caused by group C isolates among students and military recruits have contributed to an elevated incidence of meningococcal disease in teenagers and young adults. Glycoconjugate vaccines, in which a cell surface carbohydrate from a microorganism is covalently attached to an appropriate carrier protein can be an effective means for generating a protective immune response for the prevention of a wide range of diseases. An ideal vaccine would offer comprehensive protection against all five of the pathogenic serogroups, but its development has faced major obstacles related to the immunobiology of the capsular antigens. Bivalent (A and C) and tetravalent (A, C, Y and W135) polysaccharide vaccines have been widely available since the early 1970s (see, Frasch, C. E. 1995 Meningococcal disease, p. 245-284. In K. A. V. Cartwright (ed.), Meningococcal vaccines: past, present and future,, vol. 10. Wiley, Chichester.). Studies carried out during the 1960s confirmed the essential role of antibody dependent complement mediated lysis of the meningococcus as the principal immunological mechanism of protection (see, Goldschneider, L, E. C. Gotschlich, and M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129:1307-1326.; and Goldschneider, L, E. C. Gotschlich, and M. S. Artenstein. 1969. Human immunity to the meningococcus. II. Development of natural immunity. J. Exp. Med. 129:1327-1348.). The polysaccharide vaccines elicit good bactericidal antibody responses in immunologically mature individuals and have been used effectively to manage epidemics and localized outbreaks. However, vaccines based upon plain polysaccharides have important drawbacks: their immunogenicity is age-related and they fail to elicit a booster response upon re-injection. [0033] The current meningococcal vaccine contains capsular polysaccharides from a limited number of meningococcal serogroups including A, C, Y and W 135. The quadrivalent meningococcal polysaccharide vaccine is not routinely administered but has been recommended for the control of meningococcal serogroup C outbreaks. Several controlled field trials have been performed in adults and estimate the vaccine efficacy of the serogroup C meningococcal vaccine between 86% and 91% (see, Broker, M. 2003. Development of new vaccines against meningococcal disease. Arzneimittelforschung 53:805-813.; and Gold, R. 1979. Polysaccharide meningococcal vaccines-current status. Hosp Pract. 14:41-48.). The T-cell independent nature of this vaccine limits its utility as a worldwide strategy for meningococcal disease prevention. Meningitis and septicaemia due to N. meningitidis serogroup B remain an important health problem in developed and developing countries (see, Rosenstein, Ν. E., B. A. Perkins, D. S. Stephens, T. Popovic, and J. M. Hughes. 2001. Meningococcal disease. Ν Engl J Med 344:1378-1388.), causing approximately 30% of meningococcal disease in the United States (see, Rosenstein, Ν. E., B. A. Perkins, D. S. Stephens, L. Lelkowitz, M. L. Cartter, R. Danila, P. Cieslak, K. A. Shutt, T. Popovic, A. Schuchat, L. H. Harrison, and A. L. Reingold. 1999. The changing epidemiology of meningococcal disease in the United States, 1992-1996. J Infect Dis 180: 1894-1901.). Efforts to develop a N. meningitidis group B vaccine have been hampered by poor immunogenicity of the polysaccharide capsule, which cross-reacts with host polysialic acid, and the danger of eliciting autoantibodies. Current vaccines against the meningococcus have high cost, depend on refrigeration for transport, and induce a T-cell independent immune response.

[0034] Meningococcal LOS is a critical virulence factor in N. meningitidis infections and is involved in many aspects of pathogenesis, including the colonization of the human nasopharynx, survival after bloodstream invasion, and the inflammation associated with the morbidity and mortality of meningococcemia and meningitis. Meningococcal LOS, which is a component of serogroup B meningococcal vaccines currently in clinical trials, has been proposed as a candidate for a new generation of meningococcal vaccines (see, Kahler, C. M., and D. S. Stephens. 1998. Genetic basis for biosynthesis, structure, and function of meningococcal lipooligosaccharide (endotoxin). Crit Rev Microbiol 24:281-334,), which would protect against disease caused by all serogroups. However the challenge is to develop an effective means of delivering this immunogen such that it elicits the appropriate immune response.

[0035] The LOS structure that seems to be important in meningococcal disease is identical to that expressed by a gonococcal mutant that we have made, N. gonorrhoeae F62ΔlgtD. This strain represents an ideal strain for isolating LOS because it only produces a single LOS component, and for analyzing the properties of antibody raised against it, because it fails to make a capsule, which can interfere with antibody binding, and its sensitivity to antibody/complement mediated killing is well known.

[0036] Without being bound by theory, the poor antigenicity of complex carbohydrates may reside in the fact that these sugars are not presented to the immune system in a natural configuration. Complex carbohydrates are soluble in water, while native LPS forms micelles. Therefore, attaching carbohydrates to carrier particles, such as gold nanoparticles, can alter the solubility of the carbohydrate, and change its presentation to the immune system. By coupling this glycoconjugate with a peptide that can induce a helper T-cell response, the immune response generated can be T-cell dependent and hence involve immunological memory. This can allow for the development of vaccines that artificially stimulate the production of antibodies that are known to be responsible for protection/recovery of many diseases for which no current effective vaccines exist (i.e. Group B meningococcal disease).

[0037] Glycoconjugate vaccines, in which a cell surface carbohydrate from a microorganism is covalently attached to an appropriate carrier protein, can be effective in generating protective immune responses to prevent a wide range of diseases. However, the use of large protein carriers can create difficulties in terms of reproducibility of the conjugation reactions and of chemical characterization of the resulting complex immunogens. These difficulties can complicate large-scale production and may endanger vaccine effectiveness and/or practical feasibility.

[0038] Developing efficient adjuvants for human vaccines is difficult. An ideal adjuvant would elicit broad and sustained immune responses at systemic or mucosal surfaces. Conventional approaches in the past have been largely empirical. Selection of an adjuvant can be based on the balance between toxicity and adjuvanticity, first in an animal model and then in clinical trials. Colloidal gold can enhance specific and nonspecific immune response in laboratory animals, for example, rabbits, rats, and mice, immunized with antigens of various nature. Application of colloidal gold can increase nonspecific immune responses as well, for example, lysozyme concentration in the blood, activity of complement system proteins, and phagocytic and bactericidal activities. Immunization of animals with colloidal gold conjugates with haptens as well as complete antigens can induce formation of highly active antibodies without using other antigens such as complete Freund's adjuvant. Antigen quantities needed for animal immunizations with colloidal gold can be one order of magnitude lower as compared to complete Freund's adjuvant immunizations. Thus, colloidal gold may possess significant adjuvant properties (see Dykman, L. A., M. V. Sumaroka, S. A. Staroverov, I. S. Zaitseva, and V. A. Bogatyrev. 2004. Immunogenic properties of the colloidal gold. Izv Akad Nauk Ser Biol. 1.). Gold possesses unique properties that can be exploited for functional ization, purification or application (see, Tkachenko, A., H. Xie, S. Franzen, and D. L. Feldheim. 2005. Assembly and characterization of biomolecule-gold nanoparticle conjugates and their use in intracellular imaging. Methods MoI Biol. 303:85-99.). The use of gold is advantageous because it is inert in biological media and shows no in vivo toxicity (see, Paciotti, G. F., L. Myer, D. Weinreich, D. Goia, N. Pavel, R. E. McLaughlin, and L. Tamarkin. 2004. Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery. Drug Deliv 11:169-183.). Gold particles display several features that make them well suited for biomedical applications including straightforward synthesis, stability and facile ability to incorporate secondary tags such as peptides targeted to specific cell types to afford selectivity. Antigens attached to microspheres given orally to mice can stimulate an immune response and give rise to protective immunity (see, Brayden, D. J., and A. W. Baird. 2001. Microparticle vaccine approaches to stimulate mucosal immunisation. Microbes. Infect. 3:867-876.). Current data suggests that nanoparticles can be used to facilitate the transport of antigens across the nasal epithelium, thus leading to efficient antigen presentation to the immune system. These particles may also serve as an adjuvant, because it has been shown that the size and surface properties of the nanoparticle influence the efficiency of the immune responses (see, Koping-Hoggard, M., A. Sanchez, and M. J. Alonso. 2005. Nanoparticles as carriers for nasal vaccine delivery. Expert Rev Vaccines 4:185-196.). Conjugation of the antigen to Au has been shown to induce responses that were significantly higher (2- to 10-fold) than those elicited by other bead types/sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A) (see, Fifis, T., A. Gamvrellis, B. Crimeen-Irwin, G. A. Pietersz, J. Li, P. L. Mottram, I. F. McKenzie, and M. Plebanski. 2004. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol 173:3148- 3154.).

[0039] Vaccinating can refer to any method known to persons of skill in the art, including injections, intramucosal administration, ingestion, in a suitable regimen of single administration, prime-boost administration, and other suitable techniques of eliciting an immunogenic response.

[0040] The functionalization of nanoparticle surfaces with biomolecules such as DNA and proteins can be used to form conjugates that combine the properties of both materials, that is, the physicochemical properties of the nanoparticle and the biomolecular function of the surface-attached entities (see, Medvedev, A. E., T. Flo, R. R. Ingalls, D. T. Golenbock, G. Teti, S. N. Vogel, and T. Espevik. 1998. Involvement of CD14 and complement receptors CR3 and CR4 in nuclear factor-kappaB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J Immunol 160:4535-42.). Such nanoparticles can be integrated into appropriate vaccines.

[0041] Low molecular weight OSs can be immunogenic when conjugated onto protein carriers (see, Pozsgay, V. 2000. Oligosaccharide-protein conjugates as vaccine candidates against bacteria, vol. 56. Academic Press, San Diego.), for example, such neoglycoproteins can be immunogenic in mice (see, Alonso de Velasco, E., A. F. Verheul, G. H. Veeneman, L. J. Gomes, J. H. van Boom, J. Veithoef, and H. Snippe. 1993 Protein-conjugated synthetic di- and trisaccharides of pneumococcal type 17F exhibit a different immunogenicity and antigenicity than tetrasaccharide. Vaccine 11:1429-1436.; and Mawas, F., J. Niggemann, C. Jones, M. J. Corbel, J. P. Kamerling, and J. F. Vliegenthart. 2002. Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM197. Infect Immun 70:5107-5114.). Conjugates of a synthetic tetrasaccharide corresponding to the repeating unit of the pneumococcal polysaccharide (serogroup 14), conjugated to CRM197 (a nontoxic, immunologically cross- reacting mutant protein of diphtheria toxin (see, Mekada, E., and T. Uchida. 1985. Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain. J. Biol. Chem. 260:12148-12153.)) can induce anti-pneumococcal polysaccharide antibodies when injected subcutaneously into mice. Oligosaccharides as short as four sugars can induce a protective response (see, Mawas, F., J. Niggemann, C. Jones, M. J. Corbel, J. P. Kamerling, and J. F. Vliegenthart. 2002. Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM197. Infect Immun 70:5107-5114.). This approach can be used to generate vaccines against S. flexneri (see, Wright, K., C. Guerreiro, I. Laurent, F. Baleux, and L. A. Mulard. 2004. Preparation of synthetic glycoconjugates as potential vaccines against Shigella flexneri serotype 2a disease. Org Biomol Chem. 2:1518-1527.).

[0042] Helper T cell responses play an important role in the induction of both humoral and cellular immune responses and can be a component of prophylactic and immunotherapeutic vaccines. Pan DR helper T cell epitope (PADRE) can augment the potency of vaccines designed to stimulate a cellular immune response (see, Alexander, J., M. F. del Guercio, B. Frame, A. Maewal, A. Sette, M. H. Nabm, and M. J. Newman. 2004. Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE). Vaccine 22:2362- 2367.), for example, by stimulating T-cells. To overcome the limitations associated with the extensive polymorphism of human leucocyte antigen (HLA) molecules, strategies targeting an efficient T-helper contribution in humans, T-helper peptides, such as PADRE, were engineered based on their capacity to bind to a large number of HLA class II molecules (see, Alexander, J., J. Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal, K. Snoke, H. M. Serra, R. T. Kubo, and A. Sette. 1994 Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity 1:751-761.). PADRE raised a T-cell dependent response in mice when evaluated as a carrier for carbohydrate antigens, such as the lacto-N-fucopentose II from the O-specific polysaccharide of S. typhimurium (see, Alexander, J., M. F. del Guercio, A. Maewal, L. Qiao, J. Fikes, R. W. Chesnut, J. Paulson, D. R. Bundle, S. DeFrees, and A. Sette. 2000. Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. J Immunol. 164: 1625-1633.) and various S. pneumoniae polysaccharide antigens (see, Alexander, J., M. F. del Guercio, B. Frame, A. Maewal, A. Sette, M. H. Nahm, and M. J. Newman. 2004. Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE). Vaccine 22:2362-2367.). The optimization of helper T cell function by use of synthetic epitopes such as PADRE or pathogen-derived, broadly crossreactive epitopes can improve the efficaciousness of vaccines.

[0043] This application presents a new vaccine, TRIAD, that can protect humans from disease. TRIAD is made by the chemical coupling of a peptide that possesses a T-cell helper epitope (abbreviated PADRE) with a short oligosaccharide (OS), coupled on a gold nanoparticle. For example, a TRIAD vaccine that includes the short oligosaccharide (OS) found on meningococcal LOS can protect humans against group B meningococcal disease. The TRIAD vaccine can induce a T-cell dependent antibody response directed against the OS, the antibodies produced can be long lived, the host can acquire a memory response, and the antibody can be bactericidal.

[0044] In order to assess the efficacy of this new TRIAD vaccine (detoxified oligosaccharide coupled to gold coupled to PADRE, abbreviated herein as OS-Au-PADRE, the nature of the immune response it elicits can be compared to that obtained via a series of control vaccines. The following is a list of the vaccines to which the immunogenicity of TRIAD can be compared: 1) detoxified OS; 2) detoxified OS coupled to gold; 3) detoxified OS mixed with Alum; 4) detoxified OS mixed with Freund's incomplete adjuvant; 5) detoxified OS in the presence of Alum; 6) detoxified OS coupled with PADRE (OSPADRE); 7) purified LOS; 8) OS-Au-PADRE; 9); OS-Au-PADRE in the presence of Alum and 10) OS-Au-PADRE mixed with Freund's incomplete adjuvant.

[0045] We expect that OS-Au-PADRE mixed with Freund's incomplete adjuvant will generate a robust immune response, providing a "best case" scenario for antibody induction. We have included in our vaccination regimen OS-Au-PADRE in the presence of Alum. By comparing the amount of antibody made with this vaccination regimen to that obtained without Alum, and by comparing the amount of antibody induced with the OS-PADRE combination, we will be able to determine if gold is providing any adjuvant effects.

[0046] Vaccines that lack Padre will allow us to determine the immunogenicity of the

OS in the absence of T-cell help. While data obtained with detoxified OS coupled directly with PADRE (OS-PADRE) to that obtained with OS-Au-PADRE will allow us to determine if gold possesses adjuvant properties, more revealing will be the analysis of the nature of the antibodies expressed (see below for methods to be employed). If a vaccine is invoking an antibody response mediated via T-cell help, we would expect the antibody profile that is generated to contain significant levels of IgG2, indicating that isotype maturation has occurred. Carbohydrates typically induce IgM and or IgG3, demonstrating the lack of T-cell involvement. Therefore, If PADRE is stimulating T-CeIIs, we expect vaccines containing this immunogen to produce antibody isotypes that are reflective of this help.

[0047] In addition to producing vaccine particles, such as OS-Au-PADRE, in which the carrier particle is a nanoparticle, vaccine particles can be produced in which the carrier particle is larger. For example, the carrier particle can be 75 nm in diameter instead of 15 nm in diameter. For example, the carrier particle can be on the order of 1 μm or greater in diameter instead of on the order of nanometers. For example, the carrier particle can have a diameter of less than or equal to about 100 nm or a diameter of greater than about 100 nm. A lower bound on the size of a carrier particle may be that it have a size sufficiently great, so that at least one antigenic saccharide or glycoconjugate molecule and at least one immunogenic peptide (molecule) can be attached to the carrier particle. An upper bound on the size of a carrier particle may be that its physical size does not present a health or safety risk to the organism to which it is administered.

[0048] A carrier (vaccine) particle can have as few as a single attached antigenic saccharide or glycoconjugate (e.g., OS) and as few as a single attached immunogenic peptide (e.g., PADRE). Alternatively, the valency may be greater, that is, the carrier particle can bear more than one attached antigenic saccharide or glycoconjugate and/or attached immunogenic peptide molecule. The ratio of the number of attached antigenic saccharide or glycoconjugate molecules to the number of attached immunogenic peptide molecules can be 1:1 or other than 1 :1. For example, the ratio of the number of attached antigenic saccharide or glycoconjugate molecules to the number of attached immunogenic peptide molecules can be in the range of from about 20:1 to about 1:20, from about 10:1 to about 1 :10, from about 5:1 to about 1:5, or from about 2:1 to about 1:2. That is, this technology has the ability to prepare gold nanoparticles with virtually any ratio of oligosaccharide conjugate to peptide conjugate, so that the ratio is not limited to 1:1. For example, the ratio of attached antigenic saccharide or glycoconjugate (e.g., OS) to attached immunogenic peptide (e.g., PADRE) molecules on a carrier particle can be varied by mixing solutions of antigenic saccharide or glycoconjugate conjugate (e.g., OS-disulfide) and immunogenic peptide - conjugate (e.g., PADRE-disulfide) in the desired ratio and treating the carrier particle with the mixture. For example, by altering the ratio of LOS conjugate to PADRE conjugate, any desired ratio of LOS:PADRE on the carrier particle can be prepared, e.g., for the purpose of maximizing an immune response in an organism to which the immunogenic composition, e.g., LOS-OS-carrier particle, is administered.

[0049] This application describes linking oligosaccharides to synthetic protein epitopes , via a gold nanoparticle carrier, that involve T-cells in antibody development. This configuration can retain all of desired vaccine qualities, in terms of immunogenicity, but offer distinct advantages in terms of manufacturing and chemical characterization because of small size and defined chemical nature of the molecules. For example, the chemical coupling of a small peptide that possesses a T-cell helper epitope with a short oligosaccharide found on meningococcal LOS (coupled on a gold nanoparticle) can induce a T-cell dependent antibody response directed against the carbohydrate moiety. The antibody generated can be bactericidal. [0050] A TRIAD glycoconjugate that we tested contains an oligosaccharide derived from N. gonorrhoeae F62ΔlgtD and a peptide that possesses the ability to bind to a large number of HLA class II molecules, chemically conjugated to a gold nanoparticle. This vaccine possessed one oligosaccharide (OS) and one peptide on each gold nanoparticle. However, the ratio of peptide to OS could be varied to further direct the desired immune response. Immunization of mice with OS-Au-PADRE produced no observable adverse effects in C57 BL6 mice. A series of biweekly or triweekly immunizations were performed. Blood was periodically collected and assayed by ELISA for the presence of antibody. The ELISA data demonstrated that significant levels of antibody were elicited after a single immunization, with the predominant isotype expressed being IgG; IgM levels were minimal and comparable to those elicited by LOS, used to vaccinate control groups of mice. Subsequent immunizations did not result in an increase of anticarbohydrate titer. Therefore, the TRIAD vaccine is capable of eliciting a potent IgG response that is directed against polysaccharide, and a single dose of TRIAD vaccine is sufficient to generate a potent antibody response.

[0051] In an embodiment, an immunogenic composition, a TRIAD vaccine, includes an an antigenic saccharide or glycoconjugate, an immunogenic peptide, and a carrier particle. The antigenic saccharide or glycoconjugate can be bound to the carrier particle, and the immunogenic peptide can be bound to the carrier particle. A glycoconjugate can include, for example, a glycoprotein, a glycopeptide, a peptidoglycan, a glycolipid, or a lipopolysaccharide. The immunogenic protein can be capable of binding to a protein, an antigen, or a leukocyte antigen, such as a human leukocyte antigen (HLA) class II molecule. For example, the immunogenic peptide comprises a pan DR binding oligopeptide, such as described in U.S. Patent Number 6,413,935, which is hereby incorporated by reference.

[0052] The carrier particle can include, for example, a metal. The metal can be a noble metal, such as ruthenium, rhodium, palladium, osmium, iridium, platinum, or gold, silver, a base metal, such as copper, iron, nickel, or zinc, or another metal. The carrier particle can include, for example, a nonmetal, such as carbon, sulfur, or selenium, a metalloid, such as silicon or boron. The carrier particle can include, for example, a metal oxide, such as titania, or a metalloid oxide, such as silica. The carrier particle can include mixtures and/or compounds of metals, nonmetals, and metalloids, such as alloys and oxides. The carrier particle can have crystalline structure, amorphous structure, or combination of crystalline and amorphous structures. The carrier particle can have a homogeneous or heterogeneous distribution of elements. For example, the carrier particle can have a core-shell or onion skin structure. For example, the carrier particle can include a carbon fullerene or a diamondoid or adamantane structure. The carrier particle can have a spherical, nonspherical, regular, or irregular shape. The carrier particle can have a size, for example, a diameter of from about 1 nm to about 1000 nm. For example, the carrier particle can be a nanoparticle with a diameter of less than or equal to about 100 nm. For example, the carrier particle can have a diameter for greater than about 100 nm.

[0053] The carrier particle can be substantially biologically inert. For example, the carrier particle can react minimally or not at all with chemicals in a living organism. For example, the carrier particle can induce no or a minimal biological or chemical response in a living organism. For example, the carrier particle may be nontoxic to living organism in general or to a specific living organism or class of living organisms. "Substantially biologically inert" embraces a range of interactions of a carrier particle with a living organism, from having no effect at all on the living organism to having an effect on the living organism, but allowing the living organism to function normally, e.g., carry out metabolism and cell division. [0054] The immunogenic composition can be administered to a living organism to produce or induce an immunogenic response in the organism. For example, the immunogenic composition can be administered to an animal, a vertebrate, a jawed vertebrate, a mammal, or a human to produce an immunogenic response. The immunogenic response can be minor or substantial. The immunogenic response can have associated biological effects, such as an increase production of antibodies. The immunogenic response can include the aquisition of immunity by the living organism to which the immunogenic composition has been administered with respect to a pathogen, such as a parasite, a fungus, a bacterium, a virus, a viroid, or a microscopic organism, constituting vaccination of the living organism, sensitization of the living organism to an antigen, or a lesser immunogenic response. Such administration of the immunogenic composition to a living organism or subject can be part of a course of treatment of the living organism or subject. For example, the immunogenic composition can be administered prophylactically, as in vaccinating a subject against a pathogen, or therapeutically.

EXAMPLE 1: Synthesis of Saccharide Azides

[0055] α- and β-Glucopyranosyl (and glycosyl) azides can be stereospecifically synthesized from α- or β-glycopyranosyl chlorides, respectively (see, Damkaci, F., and P. DeShong. 2003. Stereoselective Synthesis of α- and β-Glycosylamide Derivatives from Glycopyranosyl Azides via Isoxazoline Intermediates. J. Am. Chem. Soc 125:4408-4409). Treatment of either the α-azide [1] or β-azide [2] with Ph₃P in refluxing 1,2-dichloroethane in the presence of 4 molecular sieves for 15 h gave isoxazoline [3] (see Fig. 1). Formation of isoxazoline [3] from either azide can be explained by the mechanism shown in Fig. 2 involving anomerization of the intermediate phosphorimines [4] and [5]. Isoxazoline formation from [4] cannot occur due to strain in the resulting product. Accordingly, epimerization followed by cyclization gave exclusively α-isoxazoline [3]. Monitoring of the reaction mixture by ¹H NMR showed that isoxazoline [3] was the only glucosyl derivative observed in the NMR spectrum following the disappearance of starting material. The resulting isoxazoline derivatives can subsequently undergo coupling with thiopyridyl esters to provide bioconjugates bearing N- linked glycopeptide-like functionality. This chemistry has been extended to a variety of monosaccharide, disaccharide, trisaccharide, and complex polysaccharide derivatives. Thus, complex carbohydrate derivatives that are required for coupling to gold labeled nanoparticles can be produced.

[0056] Oligosaccharide conjugates can be attached to either gold surfaces or gold nanoparticles via thiols and disulfide linkages. The valency of this addition can be modified by a number of methods. The approach is illustrated in Fig. 3. The size of the gold nanoparticle and the valency of each added conjugate can be controlled.

EXAMPLE 2: Self Assembled Monolayers of Oligosaccharides on Gold Surfaces

[0057] Figure 4 shows oligosaccharide conjugates attached to gold. Oligosaccharide coupling on gold surfaces was used to form self assembled monolayers (SAMs). Surface characterization techniques such as Atomic Force Microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier Transform Reflectance Infrared spectroscopy (FT-IR) of two- dimensional gold films show that the density of surface coverage on a gold surface depends on the nature of the oligosaccharide and the stereochemistry of its anomeric linkage to the conjugating function. In the example shown in Fig. 4, carbohydrate (glucopyranosyl) conjugates were attached to a gold surface via a thiol tether. As shown in Fig. 4, a SAM formed from the α- glucose thiol conjugate has "holes" in the SAM as compared to its stereochemical β-glucose counterpart. This demonstrates that functional ized nanoparticles can be synthesized with control over the coating density. Thiol conjugates of di- and trisaccharides show similar behavior on gold films.

EXAMPLE 3: Agglutination of Labeled Nanoparticles by Concanavalin A

[0058] Glucose, lactose, or galactose were derivatized and added to a gold nanoparticle.

These "labeled" nanoparticles were tested for their ability to be agglutinated by the lectin concanavalin A (ConA), a lectin that reacts with non-reducing β-D-glucose and/or β-D- mannose. Upon successful attachment of glucose to gold nanoparticles, the particles are expected to be specifically agglutinated in the presence of ConA. Figure 5 shows a transmission electron micrograph (TEM) of concanavalin A aggregated nanoparticles that were prepared by treating glucosylated gold nanoparticles with Con A. No aggregation of nanoparticles coated with lactose or galactose was observed when ConA was added. However, when lactose coated nanoparticles were treated with β-galactosidase, the particles agglutinated, indicating that the terminal galactosyl residue of lactose had been removed and exposing glucosyl residue to binding by Con A. This aggregation phenomenon can be employed to monitor surface functionalization chemistry. For example, a range of commercially available lectins or antibodies can be used to demonstrate that surface functionalization with more complex carbohydrate conjugates has been accomplished.

[0059] The aggregation reaction can be monitored visually, for example, observing a clear solution turn turbid. A more sensitive monitoring technique can employ dynamic light scattering (DLS) in which not only can the extent of aggregation be measured, but the size of the aggregates can also be measured. The rate of aggregation can be monitored in real time. DLS is useful for monitoring aggregation, because it is both fast and sensitive.

[0060] Non-aggregated gold nanoparticles (which are not luminescent), when arranged is a close spatial orientation, i.e., bound to the surface of a cell via receptor-mediated binding, become highly luminescent. This phenomenon is observed for very small gold particles that are highly aggregated. For example, the aggregates shown in Fig. 5 are highly luminescent.

EXAMPLE 4: Binding of Lactose-Coated Gold Nanoparticles to Gonococci

[0061] In the natural course of infection, gonococci are capable of binding to a variety of glycosylated proteins on the surface of endothelial cells. Opa, an opacity associated protein, can bind to structures that contain a terminal lactose. Various glycosyl-labeled nanoparticles were incubated with Opa-expressing gonococci. Figure 6 shows that lactose-coated gold nanoparticles bound to the surface of gonococci. By contrast, uncoated or glucose-labeled nanoparticles did not bind to the surface of gonococci.

EXAMPLE 5: Effect of Size of Bacterium Inoculum of N. gonorrhoeae On Cytokine Response

[0062] The effects of different sizes of bacterial inoculum of N. gonorrhoeae on inducing a cytokine response in primary human monocytes was examined. Monocytes were collected from the blood of healthy human volunteers, purified from other blood cells, and then incubated with gonococci. The data indicated that a low MOI (multiplicity of infection) challenge (1 bacterial cell per 10 monocytes) resulted in substantial production of IL-8 and other chemokines/cytokines, in the absence of significant TΝF α production. High MOI challenge (10 bacterial cells per monocyte) produced comparable levels of IL8 (as seen in the Low MOI challenge), but now produced significant levels of TΝFα (see, Fig. 7). Induction of IL8 expression was found to not be mediated by an autocrine response as pretreatment of monocytes with antibody directed against TΝFα or IL- lβ and a low MOI of gonococci did not result in any change in IL8 expression. Microarray analysis of cytokine expression by human monocytes revealed dose-dependent differences in the production of the chemokines/cytokines GRO, MCP- 1, and IL-6. Thus, host responses to gonococcal challenge can be analyzed.

EXAMPLE 6: Serum Sensitivity of Gonococcal Strains

[0063] The serum sensitivity of a variety of gonococcal strains was analyzed (see, Fig.

8). Serum sensitivity varied among the strains and was dependent on the LOS structure expressed by these strains. MUG 102 was quantitatively the most resistant to ΝHS killing. MUG303 was the most sensitive to ΝHS killing. Thus, the bactericidal activity of serum can be analyzed.

EXAMPLE 7: Flow Cytometry Analysis

[0064] The ability of microbes to elicit an adaptive immune response depends on the extent to which they are recognized by host lymphocytes to induce the expression of costimulatory ligands on host antigen presenting cells. Two costimulatory ligands, B7-1 and B7-2, are required for the induction of a primary immune response. An experiment was conducted to determine whether N. gonorrhoeae LOS was able to stimulate the expression of these costimulatory ligands. Murine splenic B-cells were purified and cultured with 10 μg of purified gonococcal LOS. After 48 hrs incubation, cells were harvested, washed, stained with fluorescein-coupled anti-B7-l or B7-2 antibodies and analyzed using a FACScaliburTM (Becton-Dickinson). The data presented in Fig. 9 show that a truncated form of gonococcal LOS (isolated from a cell mutant in rfaK; contains only lipid A and heptose) increases the expression of both B7-1 and B7-2, whereas cells that express wild type LOS stimulate significantly less B7-1 and B7-2. However, LOS was able to induce expression of both B7-1 and B7-2 on the surface of mouse B lymphocytes. B7-2 was expressed at a higher level than B7-1. These populations of B-cells were sorted and it was found that cells that failed to respond to F62 LOS stimulation remained responsive to other stimuli. Thus, flow cytometry analysis and cell sorting can be used to study various aspects of gonococcal pathogenesis. Similar methods can be used to isolate specific populations of mouse cells and analyze their response to antigens in vitro.

EXAMPLE 8: Production of Gold Νanoparticles

[0065] Gold nanoparticles with a mean diameter of 15 nm (size distribution of 12-18 nm) are prepared by reduction of AuCl in the presence of citrate (see, Hone, D. C, A. H. Haines, and D. A. Russell. 2003. Rapid, Quantitative Colorimetric Detection of a Lectin Using Mannose-Stabilized Gold Νanoparticles. Langmuir 19:7141-7144.). The method can be modified by changing the concentration of reagents to produce gold particles of diameters 15- 73 nm in incremental fashion. For example, treatment of 100 ml aqueous solutions of AuCl (0.05% by weight) with 0.5 mL aqueous citrate solution (10% by weight) at room temperature in the absence of additional ligands gives gold nanoparticles of 73 nm diameter (53-93 nm distribution as measured by TEM and UV). If twice the amount of citrate solutions is added, the resulting nanoparticles have a diameter of 28 nm. Addition of additional citrate will produce smaller particles, however, nanoparticles with a diameter of 15 nm are the smallest particles that can be prepared by citrate reduction in the absence of additional ligands.

[0066] Some parameters of the procedure for making citrate-capped gold nanoparticles with 16 nm as a mean diameter are presented in the following. HAuCI₄

mean diameter = 16 nm

An aqueous solution of sodium citrate dihydrate (2.00 mL, 0.340 M) was added into a boiling solution OfHAuCl₄ (100 mL, 1.47 mM in H₂O). In a few seconds, the color of the boiling solution changed from a light yellow to a deep red, indicating the formation of gold nanospheres. The reaction mixture was refluxed for 20 min and then cooled at room temperature. The particle solution was stored at 4 ⁰C.

EXAMPLE 9: Functionalization of Gold Nanoparticles

[0067] Surface functionalisation of gold nanoparticles with glycosyl conjugates (e.g., oligosaccharide (OS)) (see, Fig. 3) was achieved by suspending the citrate-coated nanoparticles in an aqueous solution of the appropriate bioconjugate at room temperature for approximately 1 hour. The resulting surface functionalised nanoparticles were isolated, for example, centrifϊigation of the aqueous solution can be used, followed by washing repeatedly with water to remove unreacted bioconjugate (e.g., oligosaccharide (OS)) and citrate (displaced from the surface). The displacement of citrate is rapid as measured by a variety of surface characterization methods including surface reflectance FT-IR and X-ray photoelectron spectroscopy (XPS). The resulting glycosyl functionalised nanoparticles have been fully characterized by standard chemical and surface analytical techniques, including ¹H NMR spectroscopy, surface reflectance FT-IR, XPS, and dynamic light scattering (DLS). Nanoparticles coated with glucose can also be aggregated by using ConA (vide supra), demonstrating that glucose is present on the surface of the nanoparticle. Using this methodology, glycosyl conjugates of glucose, mannose, galactose, N-acetylglucosamine, N- acetylgalactosamine, lactose, cellobiose, chitobiose, and maltotriose have been prepared. The monoclonal antibody 1B2 is able to bind to terminal lactosamines. In an embodiment, vaccines have a terminal lactosamine, therefore, this binding/agglutinating ability of this antibody can be used to monitor the binding of oligosaccharide to the gold nanoparticles. EXAMPLE 10: One Step Functionalization of Gold Nanoparticles

[0068] The diameter of gold nanoparticles can be controlled more conveniently if the citrate reduction is performed in the presence of thiol conjugates. In the presence of thiol (or disulfide) conjugate ligands such as shown in Fig. 3, the diameter of the nanoparticles produced can be controlled from 5-50 nm by controlling the concentration of reagents. This method for preparing nanoparticles has two major advantages over the known methodology: first, the concentration range required to control the diameter of the particles is more narrow (0.05-0.2 M) and, thus, high dilution techniques are not required. More importantly, under these reaction conditions, the resulting gold nanoparticles have the glycosyl conjugate attached to the nanoparticle, thus avoiding the additional surface functionalisation step. [0069] Employing this methodology, gram quantities of the glucosyl and lactosyl- functionalised nanoparticles were synthesized with diameters of 5 and 15 nm, respectively. Thus, milligram quantities of the OS-PADRE conjugates required for the studies outlined below can be synthesized.

EXAMPLE 11: Valency of Functionalized Gold Nanoparticles

[0070] The valency of the nanoparticles can be determined readily by analysis of the

XPS data derived from the sample. Based on studies with self-assembled monolayers, the surface coverage of the sugar can be accurately determined from the ratio of S/Au measured by XPS. For glucose thiol conjugate SAMs on gold, the S/Au ratio of 0.045 (values uncorrected for the relative response of S and Au nuclei) confirmed that the conjugates were close packed and were separated by ca. 0.5 nm. The model for close packed structures on films is a cylinder extending away from the surface. Glucose fiinctionalised nanoparticles gave almost identical XPS values confirming that the nanoparticles had virtually identical surface coverage as the SAMs. Accordingly, the nanoparticles are highly fiinctionalised with glycosyl residues.

EXAMPLE 12: Isolation and Detoxification of LOS

[0071] The detoxified OS derived from N. gonorrhoeae, used as a carbohydrate antigen, can generate a short-lived protective immune response. N. gonorrhoeae F62ΔlgtD is a strain that has been genetically modified to produce a single lipooligosaccharide (LOS) (the lacto-N- neotetraose LOS or L7 immunotype). This LOS is expressed by almost all meningococcal strains. LOS was obtained from N. gonorrhoeae F62ΔlgtD after DOC treatment as described by Tsai et al. (see, Tsai, C. M., C. E. Frasch, and L. F. Mocca. 1981. Five structural classes of major outer membrane proteins in Neisseria meningitidis. J Bacterid 146:69-78.). In brief, LOS was isolated by two successive extractions using 4OmM Tris-HCl, pH 8.5, containing 1% DOC and 4mM EDTA. LOS samples were incubated in the presence of DΝase, RΝase, and proteinase K. The extracted LOS was further purified on Sephacryl HR S-300 with 0.5% DOC and precipitated at -20 ⁰C with 4 vol. ethanol and 0.25 M NaCl. Purified LOS was treated with anhydrous hydrazine with stirring at 37 ⁰C for 1 h to prepare O-deacylated LPS (LPS-OH). The reaction was cooled in an ice bath, and gradually cold acetone (-70 ⁰C, 5 vols.) was added to destroy excess hydrazine. The precipitated LOS-OH was isolated by centrifugation. For conjugate production, LPS-OH was further purified by column chromatography on a Sephadex G-50 column (2.5 cm x 50 cm) with distilled water as the eluent. Fractions were collected, lyophilized and examined by ¹H ΝMR. Fractions giving a resolved ¹H ΝMR spectra were combined and lyophilized. In order to isolate approximately 100 mg of purified LOS for constructing the vaccine, about 100 L of N. gonorrhoeae must be processed. [0072] Alternatively, LOS can be obtained from N. gonorrhoeae F621gtD, a strain genetically modified to produce only the lacto-N-neotetraose LOS (L7 immunotype), by using the hot-phenol/water extraction methods of Westphal and Jann (see, Westphal, O. and Jann, K., 1965. Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Meth Carbohydr Chem 5, 83-91.), with modifications. To collect approximately 50 mg of LOS/LPS, 16 L of Neisseria were grown in broth overnight. Cells were pelleted at 9,000 rpm and re-suspended in 68⁰C HPLC water. An equal volume of 68°C phenol was added and the solution was periodically vortexed for 30 min while remaining hot. The mixture was cooled to 4°C by placing on ice for 10 minutes, and then centrifuged at 3,000 rpm for 30 minutes; the aqueous layer was collected. The remaining phenol layer was extracted with HPLC water, and the aqueous layers were combined. Crude LOS/LPS was precipitated by adding two volumes of cold acetone, sodium acetate (200 mg / 100 mL), and storing at -8O⁰C overnight. After thawing on ice, crude LOS/LPS was centrifuged and the pellet was re-suspended in HPLC water. DΝAse and RΝAse (1000 units each) were added and after a 1 hr bench top incubation, Protinase K was added for an additional hour. Hot phenol-water extractions were performed again and after acetone precipitation, LOS/LPS was dialyzed against several changes of HPLC water and analyzed for purity using the Wavescan program on the spectrometer (see Fig. 10). That is, the purity of the LOS preparations was determined by measuring the absorbance of the solution. Contaminating proteins have an absorbance at a wavelength of 280 nm, and contaminating DNA will absorb at 260 and 280 nm. LOS/LPS underwent lyophilization and was re-scanned for purity, then re-suspended in a minimal amount of HPLC water, and the process repeated until a clean product was obtained. Figure 10 shows no absorbance peaks at 260 and 280 nm, indicating that the LOS was pure.

[0073] Figure 10 also presents an SDS-PAGE gel. N. gonorrhoeae strain F62 produces two LOS components that have been structurally characterized (see, Yamasaki R, Bacon BE, Νasholds W, Schneider H, Griffiss JM. Structural determination of oligosaccharides derived from lipooligosaccharide of Neisseria gonorrhoeae F62 by chemical, enzymatic, and two- dimensional ΝMR methods. Biochemistry. 1991 30:10566-75.). This strain was genetically modified, so that it only expresses a single LOS. The control lane on the left of the gel shows the two LOS components made by F62. By contrast, the lane on the right of the gel shows that the mutated N. gonorrhoeae F62ΔlgtD only makes a single lacto-Ν-neotetraose LOS. [0074] In an alternative embodiment, an antigenic saccharide or glycoconjugate, such as

LOS, for use in the immunogenic composition can be produced through chemical synthesis in the laboratory rather than by extraction from microorganisms. An immunogenic peptide, such as PADRE, for use in the immunogenic composition can be produced through chemical synthesis in the laboratory.

EXAMPLE 13: Dephosphorylation of LOS-OH

[0075] To disaggregate LPS-OH, it was dissolved in NH₄HCO₃ and incubated with alkaline phosphatase. The reaction mixture was incubated at 54 ⁰C overnight and stirred. Enzymatic activity was neutralized by boiling the the mixture; the denatured enzyme was removed by centrifugation. Dephosphorylated LOS-OH (LOS-OH-de-P) was fractionated on a Sephadex G-50 column. Alternative methods for dephosphorylating LOS-OH are known to those of skill in the art. Individual fractions were examined by lyophilization and IH NMR. Fractions giving a resolved IH NMR spectra were combined and lyophilized.

EXAMPLE 14: Synthesis of LOS-Thiol Conjugates for Attachment to Gold Nanoparticles

[0076] LOS-thiol was synthesized by a procedure for conjugating oligosaccharides via glycosyl azide intermediates (see, Figs. 1, 2, and 11) (see, Damkaci, F., and P. DeShong. 2003. Stereoselective Synthesis of a- and b-Glycosylamide Derivatives from Glycopyranosyl Azides via Isoxazoline Intermediates. J. Am. Chem. Soc 125:4408-4409; and Soli, E. D., and P. DeShong. 1999 Recent Developments in Glycosyl Azide Preparation via Hypervalent Silicates. J. Org. Chem. 64:9724-9726.). Exhaustive acetylation of LOS-OH with acetyl chloride in pyridine, for example, at room temperature for 6-12 h, provided the peracetylated LOS derivative. Removal of the pyridine and excess acetyl chloride by lyophilization gave a crude product that was passed through a short column of silica gel with methylene chloride to remove impurities. Treatment of peracetylated LOS with TMS azide and SnCl₄ in 1.2- dichloroethane at 0⁰C for 1 h gave LOS-azide. The only bond that will react with TMS-azide under these conditions is the glycosyl acetate at the reducing end of the oligosaccharide chain. Purification of the LOS-azide by HPLC provided the purified azide derivative. [0077] Treatment of LOS azide with triphenylphosphine in 1,2-dichloroethane at 5O⁰C for 6 hours provided the LOS-isoxazoline analog (see, Fig. 11). After evaporation of the solvent at high vacuum, treatment of the crude LOS-isoxazoline with the thiopyridyl ester of 6- thioacetylcaproic acid and FeCl₃ in DMF at room temperature provided the peracetylated LOS- thiol conjugate. Dilution with water followed by HPLC purification gave the peracetylated conjugate. The acetyl ester functionalities were removed by repeated treatment with sodium methoxide in methanol at room temperature for 4h. After each treatment, the reaction mixture was evaporated to dryness and analyzed by ¹H NMR analysis to determine the number of acetyl groups remaining. When all groups have been removed, the product, LOS-thiol was exposed to air to give the disulfide and then purified by reverse phase HPLC (C 18, acetonitrile-water). Alternative methods useful for the synthesis of LOS-thiol conjugates are known to those of skill in the art.

EXAMPLE 15: Synthesis of Glucosyl Conjugate

[0078] Peracetylated β-glucopyranosyl azide (2). To a solution of β-D-glucose pentaacetate (1) (10.0 g, 25.6 mmol) in anhydrous CH₂Cl₂ was added trimethylsilyl azide (4.40 mL, 33.3 mmol), followed by 1.0 M solution of SnCl₄ (12.8 mL, 12.8 mmol). The resulting solution was stirred at room temperature for 24 h under a nitrogen atmosphere. The reaction mixture was diluted with CH₂Cl₂, washed with sat. aq. NaHCO₃, H₂O, dried over MgSO₄, filtered, and concentrated in vacuo. Purification by recrystallization (hexane/CH₂Cl₂) afforded 9.1O g (95%) of β-glucose azide 2 as a white solid.

[0079] 5-Bromopentanoyl chloride (3). Thionyl chloride (6.05 mL, 82.9 mmol) was added to a flask containing 5-bromovaleric acid (5.00 g, 27.6 mmol). The reaction mixture was refluxed for 2.5 h and concentrated in vacuo. Purification by vacuum distillation at 112 - 114 ⁰C (26 mmHg) afforded 4.80 g (87%) of acid chloride 3 as a colorless oil.

[0080] Acetylated β-glucose-5-bromopentanamide 4. To a solution of β-glucose azide 2

(1.00 g, 2.68 mmol) in anhydrous CH₂Cl₂ was added diisopropylethylamine (0.930 mL, 5.36 mmol), followed by 1.0 M solution of PMe₃ (2.95 mL, 2.95 mmol). The reaction mixture was stirred at room temperature for 30 min and then added acid chloride 3 (1.10 g, 5.36 mmol). After being stirred 24 h, the reaction mixture was diluted with CH₂Cl₂ and washed with brine, dried over MgSO₄, filtered, and concentrated in vacuo. Purification by column chromatography (hexane/EtOAc, 1/1) afforded 0.630 g (46%) of β-glucose-5-bromopentanamide 4 as a yellow gum.

[0081 ] Acetylated β-glucose-5-thioacetyl pentanamide 5. To a solution of acetylated β- glucose-5-bromopentanamide 4 (0.610 g, 1.20 mmol) in anhydrous DMF was added potassium thioacetate (0.177 g, 1.55 mmol). The reaction mixture was stirred at room temperature for 20 h and then diluted with EtOAc (100 mL). After being washed with H₂O, the organic layer was dried over MgSO₄ and concentrated in vacuo. Purification by column chromatography (hexane/EtOAc, 1/1) afforded 0.580 g (96%) of β-glucose-5-thioacetyl pentanamide 5 a s a yellow gum.

[0082] β-Glucose disulfide 6. To a solution of β-glucose-5-thioacetyl pentanamide 5

(0.54 g, 1.1 mmol) in MeOH was added 0.2 M solution of sodium methoxide (7.0 mL, 1.4 mmol). After being stirred for 24 h under a nitrogen atmosphere, the reaction mixture was exposed to air for an additional 24 h to complete the oxidation of the thiol group to the disulfide. The reaction mixture was neutralized with Amberlite ER- 120 resin. Without further purification, 0.25 g (80%) of β-glucose disulfide 6 as a light yellow solid was obtained. The ¹H NMR of the crude product indicated high purity.

EXAMPLE 16: Construction of Glycoconjugates

[0083] Cystamine HCl is coupled to OS by carbodiimide-mediated condensation with

EDC and Sulfo-NHS. OS (60 mg) is dissolved in distilled water (10 mg/ml) and cooled to 4°C. Cystamine HCl and Sulfo-NHS are added as solids to concentrations of 1.0 M and 8 mM, respectively. The pH is adjusted to 4.8 with 0.2 M HCl, and EDC is added as a solid to a concentration of 0.1 M. The reaction mixture is stirred at pH 4.8 for 3 h. The solution is adjusted to pH 7.0 and applied to a Sephadex G-25 column (diameter = 2.8 cm, length = 49 cm) equilibrated in distilled water. The eluent is assayed for sugar and amino group content. The first peak containing amino group and sugar-positive fractions (OS coupled to OS by the spacer molecule) and the second peak containing sugar- and strong amino group-positive fractions (OS containing the spacer molecule) are pooled and lyophilized.

EXAMPLE 17: Synthesis of the PADRE-LOS Disulfide for Attachment to Gold Nanoparticles

[0084] The mixed disulfide of PADRE and LOS (see Fig. 12, see also Fig. 3, X=PADRE, y=LOS) was prepared by the classic disulfide exchange reaction. In this reaction, LOS-thiol (as the thiolate anion) was allowed to undergo disulfide exchange with the disulfide of PADRE. LOS-thiolate was prepared by treatment of LOS-disulfide with sodium borohydride in water at room temperature for 5 m. Addition of PADRE-disulfide to the thiolate anion mixture allowed for disulfide exchange to occur. Depending on the rate of exchange, a statistical mixture of "homo" disulfides (LOS-disulfide and PADRE-disulfide) is formed along with the "hetero" PADRE-LOS-disulfide. The mixture of homo- and hetero-disulfide products was purified by HPLC. The course of the exchange reaction was monitored by HPLC. Alternative methods useful for the synthesis of PADRE-LOS disulfide conjugates are known to those of skill in the art.

EXAMPLE 18: Conjugation of PADRE Glycoconjugates

[0085] The PADRE peptide aKXVAA WTLKAAaZC (X 5 L-cyclohexylalanine, Z-5- aminocaproic acid) was prepared according to standard solid phase F-moc peptide synthesis procedures (standard one-letter abbreviations for the amino acids are used). Glycoconjugates were generated by first preparing the corresponding glycosylamine derivative of LOS-OH-de-P. A solution of LOS-OH-de-P was resuspended in tetrahydrofuran and cooled to 4°C. Saturated NaHCO₃ solution was added, and the glycosylamine was exhaustively acylated with 6- bromocaproyl chloride. After 10 m, an additional portion of 6-bromocaproyl chloride was added with additional stirring at 0⁰C for 2 h, and then at room temperature for 2 h. Water was added to the reaction mixture, and the solution was acidified to pH 5 by addition of 1.0 M HCl. This solution was extracted with diethyl ether, and the separated aqueous phase was concentrated and lyophilized, producing acylated oligosaccharide PADRE conjugate. Alternative methods useful for the conjugation of PADRE glycoconjugates are known to those of skill in the art.

EXAMPLE 19: Synthesis of PADRE Conjugate for Attachment to Gold Nanoparticles

[0086] After the final amino acid has been added in synthesizing the PADRE peptide and the protecting agent removed with pyrrolidine, the resulting N-terminal amino acid was reacted with 6-bromocaproyl chloride in anhydrous DMF at room temperature for Ih. This N- capping procedure was followed immediately and without purification by treatment of the resulting product with sodium sulfide at room temperature, which gave the thiol-PADRE peptide precursor. Water was added to the reaction mixture, and the solution was acidified to pH 5 by addition of 0.01 M HCl. This solution was extracted with diethyl ether. Exposure of the crude thiol in the aqueous phase to air, for example, for 1-2 h, yielded the disulfide derivative (see Fig. 3, X=V=PADRE). Purification of the PADRE disulfide was accomplished by lyophilization of the pH 5 aqueous phase to dryness, followed by HPLC (C 18 column, acetonitrile-water). Alternative methods useful for the synthesis of PADRE conjugates are known to those of skill in the art.

EXAMPLE 20: Preparation of Lipoic Acid Tethered PADRE Peptide Conjugate

Ala-Lys-cyclohθxylalanine-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-Ala-Cys

Structure of PADRE peptide.

Preparation of PADRE peptide conjugate.

[0087] N-Hydroxy succinimidyl lipoic ester 8. A solution of lipoic acid 6 (2.62 mmol,

0.540 g), N-hydroxy succinimide 7 (2.88 mmol, 0.330 g), l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (2.88 mmol, 0.550 g) in anhydrous DMF was stirred at room temperature for 4 h under a nitrogen atmosphere. The solution was diluted with EtOAc and washed with sat. aq. NaHCO₃ and H₂O. The organic layer was dried over MgSO₄, filtered off, and concentrated in vacuo. Purification by recrystalization (EtOAc/hexane) afforded 0.768 g (97%) of lipoic ester 8 as light yellow crystals.

[0088] Lipoic acid tethered PADRE peptide conjugate 9: To a solution of PADRE peptide (1.45 mg, 0.913 μmol) in 4 mL of HEPES buffer (0.1 M, pH = 7.4) was added a solution of lipoate ester 8 (0.415 mg, 1.37 μmol) in 4.0 mL of acetone. The resulting mixture was stirred at room temperature for 24 h and then concentrated in vacuo. To a dried crude solid was added 6.0 mL of water, vortexed, and centrifuged. The supernatant was collected with a total volume of 12 mL water. The crude conjugate 9 solution was used for functionalization of gold nanospheres without further purification.

EXAMPLE 21: Preparation of Functionalized Gold Nanoparticles

[0089] Gold nanoparticles coated with LOS, PADRE, or both LOS and PADRE can be prepared by either treating 15 nm citrate coated gold nanoparticles with LOS-disulfide, PADRE- disulfide, a mixture of LOS-disulfide and PADRE-disulfide, or the mixed disulfide of PADRE- LOS (Fig. 12), respectively, in aqueous solution for 1 h at room temperature. The particles were purified by centrifugation, and resuspended in water repeatedly. Characterization of the particles by TEM, surface reflectance FT-IR spectroscopy, and XPS was performed to confirm that the particles were coated with the appropriate ligands. Alternative methods useful for the preparation of functionalized gold nanoparticles are known to those of skill in the art. A procedure for producing a TRIAD OS-Au-PADRE particle is illustrated by the cartoon of Fig. 13.

EXAMPLE 22: Preparation of Functionalized Carrier Particles

[0090] An antigen, such as LOS, and an immunogenic peptide, such as PADRE, can be bound to a carrier particle that contains an element or elements other than or in addition to gold. For example, the carrier particle can include silica. In such a case, an antigen, such as LOS, can be bound to the silica carrier particle through a siloxane attachment group instead of through a thiol or disulfide group. Such a silica carrier particle can be produced, for example, by the reaction of a compound such as tetraethoxysilane and water. The silica carrier particle can be reacted with a siloxane or siloxane derivative such as alkyltriethoxysilane to make a carrier particle with a functionalized silica surface. That is, an attachment group is selected to complement the surface chemistry of the carrier particle and allow the attachment of a biofunctional group such as a saccharide, glycoconjugate, or peptide.

EXAMPLE 23: Results of In-Vivo Testing (Animal Trials)

[0091] 10 female C57bL/6J mice that were 6 weeks old were obtained from The Jackson Laboratory. After an acclimation period the mice were ear tagged and 200 ul of pre-injection sera was obtained. Mice were immunized intraperitonealy with either 10 ug of purified LOS alone, or 8.5 ug of conjugated vaccine (Au-OS-PADRE). On day 21 and 42, mice were boosted with an equivalent amount of vaccine or oligosaccharide and blood samples were taken. Sera was recovered from all mice on day 51 in a terminal bleed. For obtaining ELISA data mice were immunized on days 0 (red squares), 21 (blue circles), and 42 (green triangles). They were bled on each of those days as well as a terminal bleed on day 52 (orange diamonds). Two fold dilutions of sera were tested starting from a 1 :50 stock.

[0092] Enzyme-Linked Immunosorbent Assays (ELISAs) were performed to determine the amount of antibody elicited by the vaccine. N. gonorrhoeae strain F62ΔlgtD was used as the capture antigen and was coated onto a polystyrene microtiter plate. After blocking all of the unbound sites on the plastic with bovine serum albumin, various dilutions of mouse sera were added and incubated at room temperature overnight. Unbound antibodies and other serum components were removed by washing at least 5 times with phosphate buffered saline. A secondary antibody (goat anti- mouse IgG or goat antimouse IgM, conjugated to horse radish peroxidase) was added to various wells, and allowed to incubate at room temperature for at least 1 hour. Unbound antibody was removed by washing at least 5 times with phosphate buffered saline. To determine how much antibody was present in the sera, an aliquot of 3,5,3',5'- tetramethylbenzidine (TMB) was added to each well, and the plate was incubated until significant color was observed in some wells. The optical density of each well was determined and the data were plotted as observed absorbance against the dilution of antisera. The antibody titer of a sera was determined as the highest dilution of antibody that gave a two fold rise in absorbance.

[0093] Figure 14A shows results of when mouse 791 was injected with purified LOS and

ELISA was conducted with 1 :5000 dilution of anti-mouse IgG. Figure 14B shows results of when mouse 791 was injected with purified LOS and ELISA was conducted with 1 :5000 dilution of anti-mouse IgM. Figure 14C shows results of when mouse 799 was injected with the vaccine construct, Au-OS-PADRE, and ELISA was conducted with 1 :5000 dilution of anti- mouse IgG. Figure 14D shows results of when mouse 799 was injected with the vaccine construct, Au-OS-PADRE, and ELISA was conducted with 1 :5000 dilution of anti-mouse IgM. [0094] Figures 14A and 14B indicate that the administration of LOS alone does not stimulate a strong immunogenic response. [0095] The data represented by the squares in Figs. 14A-14D represent the control in the experiment and demonstrate the amount of preexisting antibody found in the mouse that is able to bind to the ELISA plate. Increases in absorbance over background indicate that the sera has an increase in the amount of antibody specific for N. gonorrhoeae LOS. In order to interpret the data in the graph and determine the antibody titer, one has to account for the dilutions of sera used in the ELISA experiment. Because the starting concentration of sera used was a 1/50 dilution, the x-axis number of 1 indicates the absorbance obtained when a 1/50 dilution of mouse sera was used. A reading obtained at 0.1 indicates what would have been observed had the original sera been diluted 1/500. In the data for the mouse shown in figure 14C, what is seen is that the dilution that gives rise to a two-fold increase over background is around a 1/64 dilution of the 1/50 dilution, giving an observed titer of about 1/3200 +/- 1 dilution. All three post-immunization curves (circles, triangles, and diamonds) in Fig. 14C are about the same, and this indicates that the multiple injections of vaccine did not result in increased amounts of antibody. From the data in Fig. 14C, we concluded that the vaccine generated a significant IgG response. The data in Fig. 14D is an analysis of the same sera, using antibody specific for mouse IgM. There is not much difference over the prebleed antibody levels, indicating that the vaccine did not generate much anti IgM.

[0096] Immunization of mice with Au-OS-PADRE was repeated with a total of 8 mice, and the data obtained for all mice were similar to the data seen in Figs. 3C and 3D. Some variation is seen with the amount of IgM that is elicited (some mice produce a slightly elevated amount of IgM). AU mice produced anti IgG titers in the range of 1/2000. [0097] We have demonstrated our ability to produce nanoparticle constructs containing

LOS and PADRE. We have shown that a single dose of the immunogenic composition TRIAD (OS-Au-PADRE) injected intraperitonealy induces a robust antibody response against carbohydrate without adverse effects. The generated response is mostly IgG and reaches a maximum titer with one dose of the vaccine. From these data, we have concluded that we have devised a safe and effective way of generating large IgG titers against carbohydrate with a small dose of vaccine.

EXAMPLE 24: Analysis of Bactericidal Properties of the Antibody

[0098] A bactericidal assay can be performed. The assays can be carried out in 96-well polystyrene plates. For example, N. gonorrhoeae strain F62ΔlgtD can be grown overnight on agar. Colonies can be inoculated into broth culture, and incubated until the cells reach midlog phase (-Klett = 100, which equals ~1 x 10⁸ cfu/ml). Two-fold dilutions of heat inactivated murine polyclonal antisera can be made directly in the plate (20 μ I/well, final volume). Freshly thawed baby rabbit complement can be added (20 μL) to each well, followed by 10 μL of a dilution of bacteria that can give a final concentration of 2,500 CFU/well. The plate can be incubated with gentle shaking at 37 ⁰C for 1 h. The content of each well can be plated onto agar, and the plates can be incubated overnight at 37 ⁰C, 5% CO₂. The number of CFU on each plate can be determined, and the percent of killing can be calculated relative to the mean values of control wells that contained no antisera (cfu control - cfu Ab/cfu control) x 100. The bactericidal antibody titers can be expressed as Iog2 of the final dilution that gives at least 50% killing of the inoculum.

EXAMPLE 25: F. Tularensis Vaccine

[0099] Our TRIAD vaccine can also be used to produce a vaccine against Francisella tularensis, a pathogen which causes tularemia. G old nanoparticles functionalized with an oligosaccharide derived from F. tularensis and an immunogenic peptide can be used.

[00100] The TRIAD compositions of the invention, comprising particle, antigen, and peptide, may be formulated into a pharmaceutical composition comprising a suitable carrier. The pharmaceutical composition is acceptable for introduction into an animal, e.g. a vertebrate, mammal, and human. The pharmaceutical composition has minimal side effects in view of the desired therapeutic immunogenic effect, taking into consideration typical pharmacokinetic characteristics including absorption, digestion, metabolism, excretion, and toxicity. The size and content of the carrier particles is consistent with such pharmacokinetic requirements.

[00101] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

REFERENCES

Alexander, J., M. F. del Guercio, B. Frame, A. Maewal, A. Sette, M. H. Nahm, and M. J. Newman. 2004. Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T- lymphocyte epitope (PADRE). Vaccine 22:2362-2367.

Alexander, J., M. F. del Guercio, A. Maewal, L. Qiao, J. Fikes, R. W. Chesnut, J. Paulson, D. R. Bundle, S. DeFrees, and A. Sette. 2000. Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. J Immunol. 164: 1625-1633.

Alexander, J., J. Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal, K. Snoke, H. M. Serra, R. T. Ku bo, and A. Sette. 1994 Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity 1:751-761.

Alonso de Velasco, E., A. F. Verbeul, G. H. Veeneman, L. J. Gomes, J. H. van Boom, J. Veithoef, and H. Snippe. 1993 Protein-conjugated synthetic di- and trisaccharides of pneumococcal type 17F exhibit a different immunogenicity and antigenicity than tetrasaccharide. Vaccine 11:1429-1436.

Anderson, P. W., M. E. Pichichero, R. A. Insel, R. Betts, E. R, and D. H. Smith. 1986. Vaccines consisting of periodate-cleaved oligosaccharides from the capsule of Haemophilus influenzae type b coupled to a protein carrier: structural and temporal requirements for priming in the human infant. J Immunol 137:1181-1186.

Asian, K., J. R. Lakowicz, and C. D. Geddes. 2004. Nanogold-plasmon-resonance- based glucose sensing. Anal. Biochem. 330:145-155.

Bendayan, M. 2000. A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology. Biotech. Histochem. 75:203-242.

Brayden, D. J. 2001. Oral vaccination in man using antigens in particles: current status. Eur J Pharm Sci 14:183-189.

Brayden, D. J., and A. W. Baird. 2001. Microparticle vaccine approaches to stimulate mucosal immunisation. Microbes. Infect. 3:867-876.

Broker, M. 2003. Development of new vaccines against meningococcal disease. Arzneimittelforschung 53:805-813.

Burke, D. S. 1977. Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J. Infect. Dis. 135:55-60.

Cartwright, K. A. V. 1995. Meningococcal disease., p. 115-146. IM K. A. V. Cartwright (ed.), Meningococcal carriage and disease, vol. 5. Wiley, Chichester.

Cherwonogrodzky, J. W., M. H. Knodel, and M. R. Spence. 1994. Increased encapsulation and virulence of Francisella tularensis live vaccine strain (LVS) by subculturing on synthetic medium. Vaccine 12:773-775.

Chhibber, S., M. Rani, and Y. Vanashree. 2005. Immunoprotective potential of polysaccharide-tetanus toxoid conjugate in Klebsiella pneumoniae induced lobar pneumonia in rats. Indian J Exp Biol. 43:40-45.

Damkaci, F., and P. DeShong. 2003. Stereoselective Synthesis of a- and b- Glycosylamide Derivatives from Glycopyranosyl Azides via Isoxazoline Intermediates. J. Am. Chem. Soc 125:4408-4409.

Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medical and public health management. Jama 285:2763-73.

Diks, S. H., D. J. Richel, and M. P. Peppelenbosch. 2004. LPS signal transduction: the picture is becoming more complex. Curr Top Med Chem 4:1115-26.

Donnelly, J. J., R. R. Deck, and M. A. Liu. 1990. Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine. J Immunol 145:3071-9.

Dreisbach, V. C, S. Cowley, and K. L. Elkins. 2000. Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and gamma interferon. Infect. Immun. 68: 1988-1996.

Dykman, L. A., M. V. Sumaroka, S. A. Staroverov, I. S. Zaitseva, and V. A. Bogatyrev. 2004. Immunogenic properties of the colloidal gold. Izv Akad Nauk Ser Biol. 1.

Ericsson, M., A. Tarnvik, K. Kuoppa, G. Sandstrom, and A. Sjostedt. 1994. Increased synthesis of DnaK, GroEL, and GroES homologs by Francisella tularensis LVS in response to heat and hydrogen peroxide. Infect Immun 62:178-83.

Fifis, T., A. Gamvrellis, B. Crimeen-Irwin, G. A. Pietersz, J. Li, P. L. Mottram, I. F. McKenzie, and M. Plebanski. 2004. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol 173:3148-3154.

Frasch, C. E. 1995 Meningococcal disease, p. 245-284. In K. A. V. Cartwright (ed.), Meningococcal vaccines: past, present and future,, vol. 10. Wiley, Chichester.

Fulop, M., P. Mastroeni, M. Green, and R. W. Titball. 2001. Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis. Vaccine 19:4465-4472.

Gold, R. 1979. Polysaccharide meningococcal vaccines-current status. Hosp Pract. 14:41-48.

Goldschneider, L, E. C. Gotschlich, and M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129: 1307-1326.

Goldschneider, I., E. C. Gotschlich, and M. S. Artenstein. 1969. Human immunity to the meningococcus. II. Development of natural immunity. J. Exp. Med. 129:1327-1348.

Hone, D. C, A. H. Haines, and D. A. Russell. 2003. Rapid, Quantitative Colorimetric Detection of a Lectin Using Mannose-Stabilized Gold Nanoparticles. Langmuir 19:7141-7144.

Jodar, L., I. M. Feavers, D. Salisbury, and D. M. Granoff. 2002. Development of vaccines against meningococcal disease. Lancet 359:1499-1508.

Kahler, C. M., and D. S. Stephens. 1998. Genetic basis for biosynthesis, structure, and function of meningococcal lipooligosaccharide (endotoxin). Crit Rev Microbiol 24:281-334.

Kieffer, T. L., S. Cowley, F. E. Nano, and K. L. Elkins. 2003. Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS, and contributes to F. novicida murine pathogenesis. Microbes Infect. 5:397-403.

Koping-Hoggard, M., A. Sanchez, and M. J. Alonso. 2005. Nanoparticles as carriers for nasal vaccine delivery. Expert Rev Vaccines 4:185-196.

Kreuter, J. 1996. Nanoparticles and microparticles for drug and vaccine delivery. J Anat. 189:503- 505-

Lortan, J. E., A. S. Kaniuk, and M. A. Monteil. 1993. Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults. Clin. Exp. Immunol. 91:54-57.

Mawas, F., J. Niggemann, C. Jones, M. J. Corbel, J. P. Kamerling, and J. F. Vliegenthart. 2002. Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM 197. Infect Immun 70:5107-51 14. Medintz, I. L., H. T. Uyeda, E. R. Goldman, and H. Mattoussi. 2005. Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4:435-446.

Medvedev, A. E., T. Flo, R. R. Ingalls, D. T. Golenbock, G. Teti, S. N. Vogel, and T. Espevik. 1998. Involvement of CD 14 and complement receptors CR3 and CR4 in nuclear factor-kappaB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J Immunol 160:4535-42.

Mekada, E., and T. Uchida. 1985. Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain. J. Biol. Chem. 260:12148-12153.

Musher, D. M., M. J. Luchi, D. A. Watson, R. Hamilton, and R. E. Baughn. 1990. Pneumococcal polysaccharide vaccine in young adults and older bronchitics: determination of IgG responses by ELISA and the effect of adsorption of serum with non-type-specific cell wall polysaccharide. J. Infect. Dis. 161:728-735.

Oyston, P. C, and J. E. Quarry. 2005. Tularemia vaccine: past, present and future. Antonie Van Leeuwenhoek 87:277-281.

Paciotti, G. F., L. Myer, D. Weinreich, D. Goia, N. Pavel, R. E. McLaughlin, and L. Tamarkin. 2004. Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery. Drug Deliv 11:169-183.

Pozsgay, V. 2000. Oligosaccharide-protein conjugates as vaccine candidates against bacteria, vol. 56. Academic Press, San Diego.

Rosenstein, N. E., B. A. Perkins, D. S. Stephens, L. Lefkowitz, M. L. Cartter, R. Danila, P. Cieslak, K. A. Shutt, T. Popovic, A. Schuchat, L. H. Harrison, and A. L. Reingold. 1999. The changing epidemiology of meningococcal disease in the United States, 1992-1996. J Infect Dis 180:1894-1901.

Rosenstein, N. E., B. A. Perkins, D. S. Stephens, T. Popovic, and J. M. Hughes. 2001. Meningococcal disease. N Engl J Med 344: 1378-1388.

Rothstein, E. P., D. V. Madore, and S. S. Long. 1991. Antibody persistence four years after primary immunization of infants and toddlers with Haemophilus influenzae type b CRM197 conjugate vaccine. J Pediatr 119.:655-657.

Sandstrom, G., A. Sjostedt, T. Johansson, K. Kuoppa, and J. C. Williams. 1992. Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS. FEMS Microbiol. Immunol. 5:201-210.

Soli, E. D., and P. DeShong. 1999 Recent Developments in Glycosyl Azide Preparation via Hypervalent Silicates. J. Org. Chem. 64:9724-9726.

Tai, J. Y., P. P. VeIIa, A. A. McLean, A. F. Woodhour, W. J. McAleer, A. Sha, C. Dennis-Sykes, and M. R. Hillerman. 1987. Haemophilus influenzae type b polysaccharide- protein conjugate vaccine. Proc Soc Exp Biol Med 184:: 154-161.

Tarnvik, A. 1989. Nature of protective immunity to Francisella tularensis. Rev. Infect. Dis. 11:440-451.

Thornton, D. J., D. F. Holmes, J. K. Sheehan, and I. Carlstedt. 1989. Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes. Anal Biochem 182.:160-164.

Tkachenko, A., H. Xie, S. Franzen, and D. L. Feldheim. 2005. Assembly and characterization of biomolecule-gold nanoparticle conjugates and their use in intracellular imaging. Methods MoI Biol. 303:85-99.

Tsai, C. M., C. E. Frasch, and L. F. Mocca. 1981. Five structural classes of major outer membrane proteins in Neisseria meningitidis. J Bacterid 146:69-78.

Waag, D. M., K. T. McKee, Jr., G. Sandstrom, L. L. Pratt, C. R. Bolt, M. J. England, G. O. Nelson, and J. C. Williams. 1995. Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis. Clin. Diagn. Lab Immunol. 2:143-148.

Weintraub, A. 2003. Immunology of bacterial polysaccharide antigens. Carbohydr Res. 338:2539- 2547.

Westphal, O., K. Jann, and K. Himmelspach. 1983. Chemistry and immunochemistry of bacterial lipopolysaccharides as cell wall antigens and endotoxins. Prog. Allergy 33:9-39.

Wright, K., C. Guerreiro, I. Laurent, F. Baleux, and L. A. Mulard. 2004. Preparation of synthetic glycoconjugates as potential vaccines against Shigella flexneri serotype 2a disease. Org Biomol Chem. 2:1518-1527.

Claims

WE CLAIM:

1. An immunogenic composition, comprising: an antigenic saccharide or glycoconjugate molecule; an immunogenic peptide; and a carrier particle, wherein the antigenic molecule and the immunogenic peptide are bound to the carrier particle.

2. The immunogenic composition of claim 1, wherein the antigenic molecule is a glycoprotein.

3. The immunogenic composition of claim 1, wherein the antigenic molecule is selected from the group consisting a lipopolysaccharide (LPS), a lipooligosaccharide (LOS), or a capsular polysaccharide.

4. The immunogenic composition of claim 1, wherein the antigenic molecule comprises a detoxified oligosaccharide (OS).

5. The immunogenic composition of claim 1, wherein the antigenic molecule comprises lacto-N-neotetraose.

6. The immunogenic composition of claim 1, wherein the immunogenic peptide binds to a protein, an antigen, or a leukocyte antigen.

7. The immunogenic composition of claim 1, wherein the immunogenic peptide binds to a human leukocyte antigen (HLA) class II molecule.

8. The immunogenic composition of claim 1, wherein the immunogenic peptide comprises a pan DR helper T cell epitope.

9. The immunogenic composition of claim 1, wherein the immunogenic peptide comprises a pan DR binding oligopeptide.

10. The immunogenic composition of claim 1, wherein the carrier particle comprises a material selected from the group consisting of a metal and a noble metal.

11. The immunogenic composition of claim 1, wherein the carrier particle comprises gold.

12. The immunogenic composition of claim 1, wherein the carrier particle comprises a metal oxide.

13. The immunogenic composition of claim 1, wherein the carrier particle comprises a material selected from the group consisting of silica, titania, and combinations.

14. The immunogenic composition of claim 1, wherein the carrier particle comprises a non- metal or metalloid.

15. The immunogenic composition of claim 1, wherein the carrier particle comprises a carbon fullerene.

16. The immunogenic composition of claim 1, wherein the carrier particle is a nanoparticle having a diameter of from about 1 nm to about 1000 nm.

17. The immunogenic composition of claim 1, wherein the carrier particle is a nanoparticle having a diameter of less than or equal to about 100 nm.

18. The immunogenic composition of claim 1, wherein the carrier particle has a diameter of greater than about 100 nm.

19. The immunogenic composition of claim 1, wherein the carrier particle is substantially biologically inert.

20. The immunogenic composition of claim 1, wherein the ratio of antigenic molecules to immunogenic peptides bound to the carrier particle is from about 20:1 to about 1 :20.

21. The immunogenic composition of claim 1, wherein the ratio of antigenic molecules to immunogenic peptides bound to the carrier particle is about 1 :1.

22. The immunogenic composition of claim 1, wherein the total number of antigenic molecules and immunogenic peptides bound to the carrier particle is from about 2 to about 100.

23. The immunogenic composition of claim 1, wherein one antigenic molecule is bound to the carrier particle and wherein one immunogenic peptide molecule is bound to the carrier particle.

24. A pharmaceutical composition comprising the immunogenic composition of claim 1 and a pharmaceutically acceptable carrier.

25. A method of producing an immunogenic response, comprising: administering an immunogenic composition to an animal, so that the immunogenic response is produced in the animal, wherein the immunogenic composition comprises an antigenic saccharide or glycoconjugate molecule, an immunogenic peptide, and a carrier particle and wherein the antigenic molecule and the immunogenic peptide are bound to the carrier particle.

26. The method of claim 25, wherein the immunogenic response produced is immunity of the animal to infection by a pathogen.

27. The method of claim 25, wherein the immunogenic response produced is immunity of the animal to infection by a bacterium.

28. A method of treating a subject in need of induction of an immunogenic response, comprising: administering an immunogenic composition to a subject to induce the immunogenic response in the subject, wherein the immunogenic composition comprises an antigenic saccharide or glycoconjugate molecule, an immunogenic peptide, and a carrier particle and wherein the antigenic molecule and the immunogenic peptide are bound to the carrier particle.

29. The method of claim 28, wherein the method of treating comprises vaccinating the subject.

30. A method of making an immunogenic composition comprising, reducing gold chloride in aqueous solution with citrate ion to produce gold nanoparticles; forming a glycoconjugate solution of a lipooligosaccharide disulfide (LOS- disulfide) and a PADRE-disulfide; reacting the gold nanoparticles with the lipooligosaccharide disulfide and PADRE-disulfide solution to produce PADRE-lipooligosaccharide disulfide functional ized nanoparticles; and isolating and rinsing the PADRE-lipooligosaccharide functional ized nanoparticles to remove unreacted PADRE-lipooligosaccharide disulfide and citrate ion.

31. The method of claim 30, wherein the gold chloride is selected from the group consisting of gold (I) chloride (AuCl) and gold (III) chloride (AuCl₃).

32. The method of claim 30, further comprising obtaining a lipooligosaccharide (LOS) from N. gonorrhoeae cells; purifying the lipooligosaccharide obtained; acetylating the lipooligosaccharide with acetyl chloride in pyridine to produce a peracetylated lipooligosaccharide; reacting the peracetylated lipooligosaccharide with trimethylsilyl (TMS) azide and tin (IV) chloride (SnCl₄) in 1,2-dichloroethane to produce a lipooligosaccharide azide; reacting the lipooligosaccharide azide with triphenylphosphine to produce a lipooligosaccharide isoxazoline; reacting the lipooligosaccharide isoxazoline with thiopyridyl ester of 6- thioacetylcaproic acide and iron (III) chloride (FeCl₃) to produce a peracetylated lipooligosaccharide thiol conjugate; reacting the peracetylated lipooligosaccharide thiol conjugate with sodium methoxide to produce a lipooligosaccharide thiol (LOS-thiol); and reacting the lipooligosaccharide thiol with air to produce a lipooligosaccharide disulfide (LOS-disulfide).

33. The method of claim 30, further comprising synthesizing a PADRE peptide; reacting the PADRE peptide with 6-bromocaproyl chloride to form a first product; reacting the first product with sodium sulfide to produce a thiol-PADRE peptide precursor; adding water and acid to the thiol-PADRE peptide precursor to form a thiol- PADRE peptide; and reacting the thiol-PADRE peptide with air to produce a PADRE disulfide.