INJECTABLE VACCINES AGAINST MULTIPLE MENINGOCOCCAL SEROGROUPS

Abstract

An injectable immunogenic composition comprising capsular saccharides from at least two of serogroups A, C, W135 and Y of Neisseria meningitidis, wherein said capsular saccharides are conjugated to carrier protein(s) and/or are oligosaccharides, and wherein (i) the composition comprises <50 g meningococcal saccharide per dose, and/or (ii) the composition further comprises an antigen from one or more of: (a) serogroup B N.meningitidis; (b) Haemophilus influenzae type B; and/or (c) Streptococcus pneumoniae. Saccharide antigens in the compositions are generally conjugated to a carrier.

Claims

1-25. (canceled)

26: An injectable immunogenic composition comprising capsular saccharides from at least three serogroups A, C, W135 and Y of Neisseria meningitidis, wherein the capsular saccharides are oligosaccharides and are conjugated to one or more carrier proteins, and wherein the composition comprises less than 50 ug meningococcal saccharide per dose.

27: An injectable immunogenic composition comprising capsular saccharides from at least three serogroups A, C, W135 and Y of Neisseria meningitidis, wherein: the capsular saccharides are oligosaccharides and are conjugated to one or more carrier proteins, and wherein the composition further comprises an antigen from one or more of (a) serogroup B Neisseria meningitidis; (b) Haemophilus influenza type B; and (c) Streptococcus pneumoniae.

28: The composition of claim 26 or claim 27, comprising antigens that protect against serogroups C, W135 and Y of Neisseria meningitidis.

29: The composition of claim 28, further comprising an antigen that protects against serogroup A of Neisseria meningitidis.

30: The composition of claim 29, wherein the antigen that protects against serogroup A of Neisseria meningitidis is a modified serogroup A capsular saccharide in which one or more hydroxyl groups have been replaced with blocking groups.

31: The composition of claim 28, further comprising an antigen that protects against Haemophilus influenza type B.

32: The composition of claim 28, further comprising one or more antigens that protect against serogroup B of Neisseria meningitidis.

33: The composition of claim 32, where the one or more antigens can induce, after administration to a subject, a bacterial antibody response in that subject against two or more of hypervirulent lineages A4, ET5 and lineage 3 of Neisseria meningitidis serogroup B.

34: The composition of claim 32, wherein the one or more antigens comprise one or more of (a) a NadA protein in oligomeric form; (b) a 741 protein; (c) a 936 protein; (d) a 953 protein; and (e) a 287 protein.

35: The composition of claim 34, wherein the one or more antigens comprise (a) a NadA protein in oligomeric form; (b) a 741 protein; (c) a 936 protein; (d) a 953 protein; and (e) a 287 protein.

36: The composition of claim 34, comprising a first polypeptide comprising amino acid sequence SEQ ID NO:2, a second polypeptide comprising amino acid sequence SEQ ID NO: 7 and a third polypeptide comprising amino acid sequence SEQ ID NO: 8.

37: The composition of claim 28 further comprising one or more antigens that protect against Streptococcus pneumoniae.

38: The composition of claim 37, wherein the one or more antigens comprise a capsular saccharide from Streptococcus pneumoniae.

39: The composition of claim 38, comprising capsular saccharides from between 5 and 11 different serotypes of Streptococcus pneumoniae.

40: The composition of claim 37, wherein the one or more antigens comprise polypeptides from Streptococcus pneumoniae.

41: The composition of claim 40, wherein the composition comprises one or more of the Streptococcus pneumoniae antigens PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128 and Sp130.

42: The composition of claim 28, wherein saccharide antigens in the composition are conjugated to carrier proteins.

43: The composition of claim 42, wherein saccharide antigens in the composition are oligosaccharides conjugated to CRM197 protein, protein D of Haemophilus influenza, tetanus toxoid or diphtheria toxoid.

44: The composition of claim 42, wherein the conjugates have a saccharide:protein ratio (w/w) between 1:5 and 5:1.

45: The composition of claim 28 comprising less than 25 ug meningococcal saccharide per dose.

46: The composition of claim 28, further comprising an aluminum salt adjuvant.

47: A kit comprising lyophilized capsular saccharides from Neisseria meningitidis serogroup A and liquid capsular saccharides from Neisseria meningitidis serogroup C, W135 and Y

48: The kit of claim 47, wherein the capsular saccharides from Neisseria meningitidis serogroup A are modified serogroup A capsular saccharides in which one or more hydroxyl groups have been replaced with blocking groups.

49: A method of preparing the composition of claim 28 comprising mixing lyophilized capsular saccharides from Neisseria meningitidis serogroup A with liquid capsular saccharides from Neisseria meningitidis serogroup C, W135 and Y.

50: The method of claim 49, wherein the capsular saccharides from Neisseria meningitidis serogroup A are modified serogroup A capsular saccharides in which one or more hydroxyl groups have been replaced with blocking groups.

51: A method of generating an immune response in a human comprising injecting the human with the composition of claim 28.

52: A method of generating an immune response in a human comprising injecting the human with capsular conjugated oligosaccharides from serogroups A, C, W135 and Y of Neisseria meningitidis and one or more antigens chosen from serogroup B Neisseria meningitidis; Haemophilus influenza type B; and Streptococcus pneumoniae.

Description

MODES FOR CARRYING OUT THE INVENTION

[0220] 1. Meningococcal Saccharide Compositions for Human Intramuscular Administration

[0221] Oligosaccharide conjugates from MenC, MenW135, MenY and, optionally, MenA were prepared as disclosed in reference 7. These were used to prepare individual 0.5 ml doses of the following six compositions (amounts per 0.5 ml dose):

TABLE-US-00005 Component A* B C* D* E* F Serogroup A oligosaccharide- g 10 0 10 5 2.5 0 CRM.sub.197 conjugate Serogroup C oligosaccharide- g 10 10 5 5 2.5 10 CRM.sub.197 conjugate Serogroup W135 oligosaccharide- g 10 10 5 5 2.5 0 CRM.sub.197 conjugate Serogroup Y oligosaccharide- g 10 10 5 5 2.5 0 CRM.sub.197 conjugate Aluminium phosphate adjuvant mg 0.3 Sodium chloride mg 4.5 Mannitol mg 7.5 Sodium phosphate monobasic (pH 7.6) mg 0.69 Potassium dihydrogen phosphate mg 0.34 Tween 80 mg 0.025 *The serogroup A component was in lyophilised form and was diluted with a CWY liquid composition to give the final ACWY composition.

[0222] These vaccines are administered by intramuscular injection in the thigh region to toddlers aged 12-16 months, either in a single dose (which is effective for Menjugate in children >12 months) or with a second injection 4 weeks later. Serum BCA and IgG can be compared pre-vaccination and post-vaccination (e.g at 4 weeks, and then at 8 weeks if two doses are received).

[0223] 2. Two-Vial Composition

[0224] Conjugates for human use were prepared in two separate vials. Vial 1 contained a lyophilised powder of MenA conjugate, with sucrose and potassium dihydrogen phosphate. Vial 2 contained the MenC, MenW135 and MenY conjugates, with sodium chloride, polysorbate 80, sodium phosphate buffer, and an optional aluminium phosphate adjuvant, which is present in suspension. Prior to use, vial 1 is reconstituted with 0.6 ml liquid from vial 2, to give 0.5 ml available for administration.

[0225] Three doses were prepared. In reconstituted form, vaccines contained antigens as follows:

TABLE-US-00006 Component Quantity per 0.5 ml dose Serogroup A conjugate 10 g saccharide + 12.5-33 g CRM.sub.197 OR 5 g saccharide + 6.25-16.5 g CRM.sub.197 OR 2.5 g saccharide + 3.125-8.25 g CRM.sub.197 Serogroup C conjugate 10 g saccharide + 12.5-25 g CRM.sub.197 OR 5 g saccharide + 6.25-12.5 g CRM.sub.197 OR 2.5 g saccharide + 3.125-6.25 g CRM.sub.197 Serogroup W135 10 g saccharide + 6.6-20 g CRM.sub.197 conjugate OR 5 g saccharide + 3.3-10 g CRM.sub.197 OR 2.5 g saccharide + 1.65-5 g CRM.sub.197 Serogroup Y conjugate 10 g saccharide + 6.6-20 g CRM.sub.197 OR 5 g saccharide + 3.3-10 g CRM.sub.197 OR 2.5 g saccharide + 1.65-5 g CRM.sub.197

[0226] In reconstituted form, vaccines contained other components as follows:

TABLE-US-00007 Component Quantity per 0.5 ml dose Aluminium phosphate adjuvant 0.3 mg as Al.sup.3+ zero Sodium dihydrogen phosphate 1 mM 2.5 mM Disodium hydrogen phosphate dihydrate 9 mM 7.5 mM Sodium phosphate buffer 10 mM Potassium dihydrogen phosphate 5 mM Tween 80 (surfactant) 0.025 mg Sodium chloride (tonicity) 4.5 mg Sucrose (lyophilisation & tonicity) 12.5 mg Water for injection To final volume

[0227] Six vaccines were thus availablethree different doses (10, 20 or 40 g total saccharide), each with or without aluminium phosphate adjuvant.

[0228] The adjuvanted vaccine with the highest saccharide dose was administered to healthy human subjects aged 18-45. For comparison, control subjects received either (a) the vial 1 (reconstituted in buffer) and vial 2 products in different arms at the same time, or (b) Mencevax. Each patient group contains 30 people.

[0229] Blood was collected before and 28 days after vaccination to evaluate the immune response and to collect laboratory safety parameters (complete blood count, blood chemistry analyses, liver and renal function tests and urinalysis). The vaccine was well tolerated, with no unexpected adverse reactions. No significant abnormal changes in laboratory parameters occurred during the study.

[0230] Serogroup A, C, W-135, Y specific SBA and IgG (measured by ELISA) were determined in the serum samples. SBA titres were expressed as the reciprocal of the final serum dilution giving 50% killing at 60 minutes. For IgG measurement, a modified ELISA was performed to assay high avidity antibodies. For the detection of functional antibodies, SBAs with two different exogenous sources of complement were used: a baby rabbit complement source and a human complement source.

[0231] The high avidity IgG results were as follows (mean GMC (g/mL) with 95% confidence intervals):

TABLE-US-00008 Vaccine Group Serogroup Serum ACWY A + CWY Mencevax A Pre 0.67 (0.3-1.2) 0.85 (0.4-1.5) 0.45 (0.2-0.8) Post 10 (6.6-16) 14 (8.8-22) 9.8 (6.2-15) C Pre 0.21 (0.1-0.3) 0.13 (0.07-0.2) 0.16 (0.1-0.2) Post 7.7 (4.7-13) 5.2 (3.19-8.46) 8.5 (5.2-14) W-135 Pre 0.21 (0.1-0.3) 0.2 (0.1-0.3) 0.29 (0.19-0.4) Post 12 (6.5-21) 9 (5.5-18) 6.7 (3.7-12) Y Pre 0.35 (0.2-0.5) 0.31 (0.1-0.5) 0.57 (0.3-0.9) Post 18 (12-29) 21 (13-33) 20 (12-31)

[0232] The SBA results were as follows (% responders and mean GMT, both with 95% CI):

TABLE-US-00009 Rabbit Complement SBA Human Complement SBA Sero- Titres Titres group Vaccine Group 1:128 (%) GMT 1:4 (%) GMT A ACWY 93 (78-99) 989 (558-1754) 90 (73-98) 42 (23-76) A + CWY 97 (83-100) 2566 (1448-4549) 97 (83-100) 66 (36-119) MENCEVAX 100 (88-100) 3132 (1767-5552) 83 (65-94) 28 (15-50) C ACWY 97 (82-100) 4480 (2455-8176) 100 (88-100) 213 (106-427) A + CWY 100 (88-100) 3794 (2100-6855) 100 (88-100) 162 (80-325) MENCEVAX 93 (78-99) 3829 (2119-6918) 100 (88-100) 223 (111-448) W-135 ACWY 100 (88-100) 10343 (5988-17865) 100 (88-100) 248 (123-500) A + CWY 100 (88-100) 10376 (6007-17923) 93 (78-99) 142 (71-287) MENCEVAX 100 (88-100) 6795 (3934-11737) 97 (83-100) 99 (49-199) Y ACWY 100 (88-100) 22075 (14689-33175) 100 (88-100) 263 (151-457) A + CWY 100 (88-100) 24034 (15993-36120) 100 (88-100) 162 (194-588) MENCEVAX 100 (88-100) 14630 (9735-21987) 100 (88-100) 198 (114-344)

[0233] For each serogroup and in each vaccine group (ACWY, A+CWY and MENCEVAX control) the high avidity ELISA anti-capsular IgG GMCs and the SBA GMT measured with both rabbit and human complement assay, increased after injection. At day 29 after vaccine injection, the percentage of subjects with human complement SBA titers 1:4 for each serogroup ranged between 90%-100% in the conjugate vaccines and between 83%-100% in the control group. Using the rabbit complement source, the percentage of subjects with SBA titers 1:128 for each serogroup ranged between 93%-100% for the conjugate vaccines and between 90%-100% for the control group.

[0234] Overall, immune responses (GMC and GMT) were better in the conjugate groups than in the MENCEVAX control group. The improvement was particularly seen for serogroup W-135. The conjugate vaccines of the invention are thus safe, well tolerated and induce functional immune responses equal to or better than those observed following immunisation with a licensed tetravalent polysaccharide vaccine.

[0235] 3. Use of Modified MenA Saccharide

[0236] Capsular polysaccharide was purified from MenA and was hydrolysed to give MenA oligosaccharide. The polysaccharide (2 g) was hydrolyzed at 50 C. in 50 mM sodium acetate buffer, pH 4.75, at a polysaccharide concentration of 10 mg/mL for about 4 hours [73]. After hydrolysis, the solution was dried by rotary evaporation.

[0237] The oligosaccharide was activated using the following reaction scheme:

##STR00003##

[0238] The oligosaccharide was dissolved in DMSO to give a saccharide concentration of 10 mg/mL. According to a molar ratio of oligosaccharide:CDI being 1:20, 21.262 g of CDI was then added and the reaction mixture stirred for 16 hours at room temperature. The resulting MenA-CDI compound was purified by selective precipitation in a 80:20 (v/v) acetone:DMSO mixture followed by centrifugation. The efficiency of the activation reaction was calculated to be about 67.9% by determining the ratio of free imidazole to bonded imidazole.

[0239] In the second reaction step, the MenA-CDI oligosaccharide was solubilised in DMSO at a saccharide concentration of about 10 mg/mL. According to a molar ratio of MenA-CDI unit:DMA being 1:100, 36.288 g of 99% dimethylamine hydrochloride (i.e. R.sup.1 & R.sup.2=Me) was added and the reaction mixture stirred for 16 hours at room temperature. The reaction product was freeze-dried and re-solubilised in 10 mg/mL water solution.

[0240] To remove the low molecular weight reaction reagent (in particular the dimethylamine (DMA)) from the oligosaccharide preparation, a dialysis step was performed through a 3.5 kDa MWCO membrane (Spectra/Por). Four dialysis steps were carried out: (i) 16 hours against 2 L of 1 M sodium chloride (dialysis factor 1:20), (ii) 16 hours against 2 L of 0.5 M sodium chloride (dialysis factor 1:20), (iii) and (iv) 16 hours against 2 L of WFI (dialysis factor 1:20). To improve the purification a diafiltration step was also performed through a 1 kDa MWCO membrane (Centricon)

[0241] The purified MenA-CDI-DMA product was buffered at pH 6.5 in 25 mM L-histidine (Fluka)

[0242] For preparing conjugates of the modified MenA saccharide (MenA-CDI-DMA), the overall process was as follows: [0243] hydrolysis of the polysaccharide to give oligosaccharide fragments [0244] sizing of the oligosaccharide fragments [0245] reductive amination of terminal aldehyde groups on the sized oligosaccharides [0246] protection of terminal NH.sub.2 groups by Fmoc group before the CDI reaction [0247] intrinsic de-protection of NH.sub.2 groups during the DMA reaction [0248] activation of terminal NH.sub.2 groups by SIDEA (N-hydroxysuccinimide adipic acid) [0249] covalent attachment to CRM.sub.197 protein.

[0250] The modified MenA oligosaccharide conjugate was much more resistant to hydrolysis than its natural counterpart at elevated temperatures. After 28 days at 37 C., for instance, the percentage of released saccharide is 6.4% for the modified oligosaccharide vs. 23.5% for the natural antigen. Moreover, the titres induced by the modified oligosaccharides are not significantly lower than those obtained using the native sugar structures.

[0251] The modified MenA conjugate is combined with MenC, MenW135 and MenY conjugates as a substitute for the conjugate of unmodified oligosaccharide.

[0252] 4. Addition of MenB Antigens

[0253] Prior to reconstitution of the lyophilised MenA conjugate as described above, MenB antigens G287-953 (SEQ ID NO: 7), 936-G741 (SEQ ID NO: 8) and NadA (SEQ ID NO: 2) are added to the highest-dose liquid C-W135-Y mixture to give a final concentration of 20 g/dose of each of the three polypeptides. The reconstituted vaccine thus contains the following antigens:

TABLE-US-00010 Component Quantity per 0.5 ml dose Serogroup A conjugate 10 g saccharide + 12.5-33 g CRM.sub.197 Serogroup C conjugate 10 g saccharide + 12.5-25 g CRM.sub.197 Serogroup W135 conjugate 10 g saccharide + 6.6-20 g CRM.sub.197 Serogroup Y conjugate 10 g saccharide + 6.6-20 g CRM.sub.197 G287-953 20 g polypeptide 936-G741 20 g polypeptide NadA 20 g polypeptide

[0254] 5. Addition of Hib Antigen

[0255] Lyophilised HbOC conjugate is mixed with the lyophilised MenA conjugate and both are reconstituted together by liquid C-W135-Y mixture to give following vaccine:

TABLE-US-00011 Component Quantity per 0.5 ml dose Serogroup A conjugate 10 g saccharide + 12.5-33 g CRM.sub.197 Serogroup C conjugate 10 g saccharide + 12.5-25 g CRM.sub.197 Serogroup W135 conjugate 10 g saccharide + 6.6-20 g CRM.sub.197 Serogroup Y conjugate 10 g saccharide + 6.6-20 g CRM.sub.197 HbOC Hib conjugate 10 g saccharide + 2-5 g CRM.sub.197

[0256] 6. Addition of Pneumococcal Antigens

[0257] Prior to reconstitution of the lyophilised MenA conjugate as described above, pneumococcal conjugate antigens are added to the medium-dose liquid C-W135-Y mixture to give a final concentration of 2 g/dose of each of the serotypes (double for serotype 6B). The reconstituted vaccine thus contains the following antigens:

TABLE-US-00012 Component Quantity per 0.5 ml dose Serogroup A conjugate 5 g saccharide + 6.25-16.5 g CRM.sub.197 Serogroup C conjugate 5 g saccharide + 6.25-12.5 g CRM.sub.197 Serogroup W135 conjugate 5 g saccharide + 3.3-10 g CRM.sub.197 Serogroup Y conjugate 5 g saccharide + 3.3-10 g CRM.sub.197 Pneumococcus serotype 4 2 g saccharide + 2.5 g CRM.sub.197 conjugate Pneumococcus serotype 9V 2 g saccharide + 2.5 g CRM.sub.197 conjugate Pneumococcus serotype 14 2 g saccharide + 2.5 g CRM.sub.197 conjugate Pneumococcus serotype 18C 2 g saccharide + 2.5 g CRM.sub.197 conjugate Pneumococcus serotype 19F 2 g saccharide + 2.5 g CRM.sub.197 conjugate Pneumococcus serotype 23F 2 g saccharide + 2.5 g CRM.sub.197 conjugate Pneumococcus serotype 6B 4 g saccharide + 5 g CRM.sub.197 conjugate

[0258] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

The Contents of which are Hereby Incorporated in Full

[0259] [1] Chapter 28 of Vaccines (Plotkin & Orenstein) 3rd edition (1999) ISBN 0-7216-7443-7. [0260] [2] Armand et al. (1982) J. Biol. Stand. 10:335-339. [0261] [3] Cadoz et al. (1985) Vaccine 3:340-342. [0262] [4] MMWR (1997) 46(RR-5) 1-10. [0263] [5] Baldaic et al. (1983) Infect. Immun. 42:599-604. [0264] [6] WO02/058737. [0265] [7] WO03/007985. [0266] [8] Rennels et al. (2002) Pediatr Infect Dis J 21:978-979. [0267] [9] Campbell et al. (2002) J Infect Dis 186:1848-1851. [0268] [10] Costantino et al. (1992) Vaccine 10:691-698. [0269] [11] Jones (2001) Curr Opin Investig Drugs 2:47-49. [0270] [12] WO02/00249. [0271] [13] WO97/28273. [0272] [14] Lieberman et al. (1996) JAMA 275:1499-1503. [0273] [15] Frash (1990) p. 123-145 of Advances in Biotechnological Processes vol. 13 (eds. Mizrahi & Van Wezel) [0274] [16] Inzana (1987) Infect. Immun. 55:1573-1579. [0275] [17] Kandil et al. (1997) Glycoconj J 14:13-17. [0276] [18] Berkin et al. (2002) Chemistry 8:4424-4433. [0277] [19] WO03/080678. [0278] [20] WO98/08543. [0279] [21] Tettelin et al. (2000) Science 287:1809-1815. [0280] [22] Pizza et al. (2000) Science 287:1816-1820. [0281] [23] WO99/24578. [0282] [24] WO99/36544. [0283] [25] WO99/57280. [0284] [26] WO00/22430. [0285] [27] WO00/66791. [0286] [28] WO00/66741. [0287] [29] WO01/64920. [0288] [30] WO01/64922. [0289] [31] WO03/020756. [0290] [32] UK patent applications 0223741.0, 0305831.0 & 0309115.4, and international application PCT/IB03/04848. [0291] [33] Comanducci et al. (2002) J. Exp. Med. 195:1445-1454. [0292] [34] WO03/010194. [0293] [35] Parkhill et al. (2000) Nature 404:502-506. [0294] [36] International patent application PCT/IB03/06320. [0295] [37] WO03/063766. [0296] [38] Masignani et al. (2003) J Exp Med 197:789-799. [0297] [39] http://neisseria.org/nm/typing/mlst/ [0298] [40] Pettersson et al. (1994) Microb Pathog 17(6):395-408. [0299] [41] Maiden et al. (1998) PNAS USA 95:3140-3145. [0300] [42] Welsch et al. (2002) Thirteenth International Pathogenic Neisseria Conference, Norwegian Institute of Public Health, Oslo, Norway; Sep. 1-6, 2002. Genome-derived antigen (GNA) 2132 elicits protective serum antibodies to groups B and C Neisseria meningitidis strains. [0301] [43] Santos et al. (2002) Thirteenth International Pathogenic Neisseria Conference, Norwegian Institute of Public Health, Oslo, Norway; Sep. 1-6, 2002. Serum bactericidal responses in rhesus macaques immunized with novel vaccines containing recombinant proteins derived from the genome of N. meningitidis. [0302] [44] Lindberg (1999) Vaccine 17 Suppl 2:S28-36. [0303] [45] Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168. [0304] [46] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-33, vii. [0305] [47] Goldblatt (1998) J. Med. Microbiol. 47:563-567. [0306] [48] European patent 0477508. [0307] [49] U.S. Pat. No. 5,306,492. [0308] [50] WO98/42721. [0309] [51] Dick et al. in Conjugate Vaccines (eds. Cruse et al.) Karger, Basel, 1989, 10:48-114. [0310] [52] Hermanson Bioconjugate Techniques, Academic Press, San Diego (1996) ISBN: 0123423368. [0311] [53] Kanra et al. (1999) The Turkish Journal of Paediatrics 42:421-427. [0312] [54] Ravenscroft et al. (2000) Dev Biol (Basel) 103: 35-47. [0313] [55] WO97/00697. [0314] [56] WO96/37222; U.S. Pat. No. 6,333,036. [0315] [57] Watson (2000) Pediatr Infect Dis J 19:331-332. [0316] [58] Rubin (2000) Pediatr Clin North Am 47:269-285, v. [0317] [59] Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207. [0318] [60] Zielen et al. (2000) Infect. Immun. 68:1435-1440. [0319] [61] Darkes & Plosker (2002) Paediatr Drugs 4:609-630. [0320] [62] Tettelin et al. (2001) Science 293:498-506. [0321] [63] Hoskins et al (2001) J Bacteriol 183:5709-5717. [0322] [64] Rappuoli (2000) Curr Opin Microbiol 3:445-450 [0323] [65] Rappuoli (2001) Vaccine 19:2688-2691. [0324] [66] Masignani et al. (2002) Expert Opin Biol Ther 2:895-905. [0325] [67] Mora et al. (2003) Drug Discov Today 8:459-464. [0326] [68] Wizemann et al. (2001) Infect Immun 69:1593-1598. [0327] [69] Rigden et al. (2003) Crit Rev Biochem Mol Biol 38:143-168. [0328] [70] WO02/22167. [0329] [71] Nilsson & Svensson (1979) Carbohydrate Research 69: 292-296) [0330] [72] Ravenscroft et al. (1999) Vaccine 17:2802-2816. [0331] [73] Costantino et al. (1999) Vaccine 17:1251-1263. [0332] [74] Ramsay et al. (2001) Lancet 357(9251):195-196. [0333] [75] Anonymous (January 2002) Research Disclosure, 453077. [0334] [76] Anderson (1983) Infect Immun 39(1):233-238. [0335] [77] Anderson et al. (1985) J Clin Invest 76(1):52-59. [0336] [78] EP-A-0372501. [0337] [79] EP-A-0378881. [0338] [80] EP-A-0427347. [0339] [81] WO93/17712 [0340] [82] WO94/03208. [0341] [83] WO98/58668. [0342] [84] EP-A-0471177. [0343] [85] WO91/01146 [0344] [86] Falugi et al. (2001) Eur J Immunol 31:3816-3824. [0345] [87] EP-A-0594610. [0346] [88] WO00/56360. [0347] [89] WO02/091998. [0348] [90] WO01/72337 [0349] [91] WO00/61761. [0350] [92] WO99/42130 [0351] [93] WO96/40242 [0352] [94] Lees et al. (1996) Vaccine 14:190-198. [0353] [95] WO95/08348. [0354] [96] U.S. Pat. No. 4,882,317 [0355] [97] U.S. Pat. No. 4,695,624 [0356] [98] Porro et al. (1985) Mol Immunol 22:907-919. [0357] [99] EP-A-0208375 [0358] [100] WO00/10599 [0359] [101] Gever et al. Med. Microbiol. Immunol, 165: 171-288 (1979). [0360] [102] U.S. Pat. No. 4,057,685. [0361] [103] U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700. [0362] [104] U.S. Pat. No. 4,459,286. [0363] [105] U.S. Pat. No. 4,965,338 [0364] [106] U.S. Pat. No. 4,663,160. [0365] [107] U.S. Pat. No. 4,761,283 [0366] [108] U.S. Pat. No. 4,356,170 [0367] [109] Lei et al. (2000) Dev Biol (Basel) 103:259-264. [0368] [110] WO00/38711; U.S. Pat. No. 6,146,902. [0369] [111] WO03/009869. [0370] [112] Vaccine Design . . . (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum. [0371] [113] WO00/23105. [0372] [114] U.S. Pat. No. 5,057,540. [0373] [115] WO96/33739. [0374] [116] EP-A-0109942. [0375] [117] WO96/11711. [0376] [118] WO00/07621. [0377] [119] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271. [0378] [120] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32:321-338. [0379] [121] Niikura et al. (2002) Virology 293:273-280. [0380] [122] Lenz et al. (2001) J Immunol 166:5346-5355. [0381] [123] Pinto et al. (2003) J Infect Dis 188:327-338. [0382] [124] Gerber et al. (2001) Virol 75:4752-4760. [0383] [125] WO03/024480 [0384] [126] WO03/024481 [0385] [127] Gluck et al. (2002) Vaccine 20:B10-B16. [0386] [128] EP-A-0689454. [0387] [129] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278. [0388] [130] Evans et al. (2003) Expert Rev Vaccines 2:219-229. [0389] [131] Meraldi et al. (2003) Vaccine 21:2485-2491. [0390] [132] Pajak et al. (2003) Vaccine 21:836-842. [0391] [133] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400. [0392] [134] WO02/26757. [0393] [135] WO99/62923. [0394] [136] Krieg (2003) Nature Medicine 9:831-835. [0395] [137] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185. [0396] [138] WO98/40100. [0397] [139] U.S. Pat. No. 6,207,646. [0398] [140] U.S. Pat. No. 6,239,116. [0399] [141] U.S. Pat. No. 6,429,199. [0400] [142] Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658. [0401] [143] Blackwell et al. (2003) J Immunol 170:4061-4068. [0402] [144] Krieg (2002) Trends Immunol 23:64-65. [0403] [145] WO01/95935. [0404] [146] Kandimalla et al. (2003) BBRC 306:948-953. [0405] [147] Bhagat et al. (2003) BBRC 300:853-861. [0406] [148] WO03/035836. [0407] [149] WO95/17211. [0408] [150] WO98/42375. [0409] [151] Beignon et al. (2002) Infect Immun 70:3012-3019. [0410] [152] Pizza et al. (2001) Vaccine 19:2534-2541. [0411] [153] Pizza et al. (2000) Int J Med Microbiol 290:455-461. [0412] [154] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313. [0413] [155] Ryan et al. (1999) Infect Immun 67:6270-6280. [0414] [156] Partidos et al. (1999) Immunol Lett 67:209-216. [0415] [157] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293. [0416] [158] Pine et al. (2002) J Control Release 85:263-270. [0417] [159] Domenighini et al. (1995) Mol Microbiol 15:1165-1167. [0418] [160] WO99/40936. [0419] [161] WO99/44636. [0420] [162] Singh et al] (2001) J Cont Release 70:267-276. [0421] [163] WO99/27960. [0422] [164] U.S. Pat. No. 6,090,406 [0423] [165] U.S. Pat. No. 5,916,588 [0424] [166] EP-A-0626169. [0425] [167] WO99/52549. [0426] [168] WO01/21207. [0427] [169] WO01/21152. [0428] [170] Andrianov et al. (1998) Biomaterials 19:109-115. [0429] [171] Payne et al. (1998) Adv Drug Delivery Review 31:185-196. [0430] [172] Stanley (2002) Clin Exp Dermatol 27:571-577. [0431] [173] Jones (2003) Curr Opin Investig Drugs 4:214-218. [0432] [174] WO96/14086. [0433] [175] Vaccines (eds. Plotkin & Mortimer), 1988. ISBN: 0-7216-1946-0 [0434] [176] Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355. [0435] [177] Rappuoli et al. (1991) TIBTECH 9:232-238. [0436] [178] Bell (2000) Pediatr Infect Dis J 19:1187-1188. [0437] [179] Iwarson (1995) APMIS 103:321-326. [0438] [180] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80. [0439] [181] WO93/24148. [0440] [182] WO02/09643. [0441] [183] Katial et al. (2002) Infect Immun 70:702-707. [0442] [184] WO01/52885. [0443] [185] European patent 0301992. [0444] [186] Bjune et al. (1991) Lancet 338(8775):1093-1096. [0445] [187] Fukasawa et al. (1999) Vaccine 17:2951-2958. [0446] [188] WO02/09746. [0447] [189] Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333. [0448] [190] WO01/09350. [0449] [191] European patent 0449958. [0450] [192] EP-A-0996712. [0451] [193] EP-A-0680512. [0452] [194] WO02/062378. [0453] [195] WO99/59625. [0454] [196] U.S. Pat. No. 6,180,111. [0455] [197] WO01/34642. [0456] [198] WO03/051379. [0457] [199] U.S. Pat. No. 6,558,677 [0458] [200] PCT/IB03/04293. [0459] [201] WO02/062380. [0460] [202] WO00/25811. [0461] [203] Peeters et al. (1996) Vaccine 14:1008-1015. [0462] [204] Vermont et al. (2003) Infect Immun 71:1650-1655. [0463] [205] Sutter et al. (2000) Pediatr Clin North Am 47:287-308. [0464] [206] Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126. [0465] [207] Charalambous & Feavers (2001) J Med Microbiol 50:937-939. [0466] [208] Westerink (2001) Int Rev Immunol 20:251-261. [0467] [209] Grothaus et al. (2000) Vaccine 18:1253-1263. [0468] [210] Paoletti et al. (2001) Vaccine 19:2118-2126. [0469] [211] WO00/56365. [0470] [212] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th ed ISBN: 0683306472 [0471] [213] WO01/30390.