Immunogenic composition

Abstract

The invention provides an immunogenic composition comprising: a) a conjugate that is a capsular saccharide from GBS serotype Ia conjugated to a carrier protein; b) a conjugate that is a capsular saccharide from GBS serotype Ib conjugated to a carrier protein; c) a conjugate that is a capsular saccharide from GBS serotype III conjugated to a carrier protein; d) a conjugate that is a capsular saccharide from GBS serotype II conjugated to a carrier protein; and e) a conjugate that is a capsular saccharide from GBS serotype V conjugated to a carrier protein.

Claims

1. An immunogenic composition comprising: a) a conjugate that is a capsular saccharide from Streptococcus agalactiae (GBS) serotype Ia conjugated to a carrier protein; b) a conjugate that is a capsular saccharide from GBS serotype Ib conjugated to a carrier protein; c) a conjugate that is a capsular saccharide from GBS serotype III conjugated to a carrier protein; d) a conjugate that is a capsular saccharide from GBS serotype II conjugated to a carrier protein; and e) a conjugate that is a capsular saccharide from GBS serotype V conjugated to a carrier protein, wherein the capsular saccharide from GBS serotype V has an N-acetyl-neuraminic acid content of greater than 75% compared to the N-acetyl-neuraminic acid content of a native GBS serotype V capsular saccharide, and the capsular saccharide from GBS serotype III has an N-acetyl-neuraminic acid content of between 74% and 39% compared to the N-acetyl-neuraminic acid content of a native GBS serotype III capsular saccharide.

2. The immunogenic composition according to claim 1, wherein each GBS capsular saccharide is present at an amount from 1 to 30 g per unit dose.

3. The immunogenic composition according to claim 2, wherein the ratio of the masses of the GBS serotype Ia, Ib, II, V and III capsular saccharides is 1:1:1:1:1.

4. The immunogenic composition according to claim 1, wherein the composition is for administration in a single dose.

5. The immunogenic composition according to claim 1, wherein the immunogenic composition does not contain an aluminium salt adjuvant.

6. The immunogenic composition according to claim 1, wherein the immunogenic composition does not contain any adjuvant.

7. The immunogenic composition according to claim 1, wherein the carrier proteins in a), b), c), d) and e) are diphtheria toxoid, tetanus toxoid or CRM197.

8. The immunogenic composition according to claim 7, wherein the carrier proteins in a), b), c), d) and e) are CRM197.

9. The immunogenic composition according to claim 1, wherein the capsular saccharide from GBS serotype Ia has a molecular weight (MW) in the range of 150-300 kDa; the capsular saccharide from GBS serotype Ib has a MW in the range of 150-300 kDa; the capsular saccharide from GBS serotype III has a MW in the range of 50-200 kDa; the capsular saccharide from GBS serotype II has a MW in the range of 150-300 kDa; and the capsular saccharide from GBS serotype V has a MW in the range of 150-300 kDa.

10. The immunogenic composition according to claim 1, wherein the conjugate that is a capsular saccharide from GBS serotype Ia conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 1:1 to 1:2; the conjugate that is a capsular saccharide from GBS serotype Ib conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 1:1 to 1:2; the conjugate that is a capsular saccharide from GBS serotype II conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 1:1 to 1:2; the conjugate that is a capsular saccharide from GBS serotype V conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 1:1 to 1:2; and the conjugate that is a capsular saccharide from GBS serotype III conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 3:1 to 1:1.

11. The immunogenic composition according to claim 1, wherein the composition is for administration intramuscularly.

12. The immunogenic composition according to claim 1, wherein the composition is an injectable liquid solution or suspension.

13. The immunogenic composition according to claim 1, wherein the composition is lyophilised.

14. The immunogenic composition according to claim 1, wherein the composition is a vaccine.

15. The immunogenic composition according to claim 1, wherein the composition is for administration to a human.

16. The immunogenic composition according to claim 1, wherein the composition is for administration to humans selected from females of child-bearing age, pregnant females and elderly patients.

17. The immunogenic composition according to claim 14, wherein the composition is for the prevention and/or treatment of a disease caused by S. agalactiae.

18. The immunogenic composition according to claim 17, wherein the disease is neonatal sepsis, bacteremia, neonatal pneumonia, neonatal meningitis, endometritis, osteomyelitis or septic arthritis.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the survival of mice (% protection) in a neonatal challenge model. Maternal immunisation was carried out with (A) GBS Ia, (B) GBS Ib, (C) GBS III, (D) GBS II and (E) GBS V, each conjugated to CRM197. Mice were challenged with the corresponding antigen.

(2) FIG. 2 shows the OPK titers against (A) GBS Ia, (B) GBS Ib, (C) GBS III, (D) GBS II and (E) GBS, each conjugated to CRM197. Mice were immunised with the corresponding antigen.

(3) FIG. 3 shows IgG titers (FIG. 3A) and OPK titers (FIG. 3B) against GBS V. Mice were immunised with (A) Alum, (B) CRM conjugated GBS V, (C) CRM conjugated GBS V+trivalent vaccine (CRM conjugated GBS Ia, Ib and III), and (D) CRM conjugated GBS V+CRM conjugated GBS II trivalent vaccine (CRM conjugated GBS Ia, Ib, and III).

(4) FIG. 4 shows IgG titers (FIG. 4A) and OPK titers (FIG. 4B) against GBS II. Mice were immunised with (A) Alum, (B) CRM conjugated GBS II, (C) CRM conjugated GBS II+trivalent vaccine (CRM conjugated GBS Ia, Ib and III), and (D) CRM conjugated GBS II+CRM conjugated GBS V+trivalent vaccine (CRM conjugated GBS Ia, Ib and III).

(5) FIGS. 5A and 5B show IgG titers (5A) against GBS Ia, Ib, II, III and V and OPK titers (5B) against strains 515 Ia, H36B, COH1, 5401, CJB111. Mice were immunised with (A) trivalent vaccine: CRM conjugated GBS Ia, Ib and III and (B) pentavalent vaccine: CRM conjugated GBS Ia, Ib, II, III and V.

(6) FIG. 6 shows IgG titers against GBS V. Mice were immunised with (A) PBS/Alum, (B) CRM conjugated GBS V, (C) CRM conjugated GBS V+trivalent vaccine (CRM197 conjugated GBS Ia, Ib and III), (D) CRM conjugated GBS II and V+trivalent vaccine (CRM197 conjugated Ia, Ib, III), (E) TT conjugated GBS V, (F) TT conjugated GBS V+trivalent vaccine (CRM197 conjugated GBS Ia, Ib and III), (G) TT conjugated GBS II and V+trivalent vaccine (CRM197 conjugated Ia, Ib, III).

(7) FIG. 7 shows IgG titers against GBS II. Mice were immunised with (A) PBS/Alum, (B) CRM conjugated GBS II, (C) CRM conjugated GBS II+trivalent vaccine (CRM197 conjugated GBS Ia, Ib and III), (D) CRM conjugated GBS II and V+trivalent vaccine (CRM197 conjugated Ia. Ib, III), (E) TT conjugated GBS II, (F) TT conjugated GBS II+trivalent vaccine (CRM197 conjugated GBS Ia, Ib and III), (G) TT conjugated GBS II and V+trivalent vaccine (CRM197 conjugated Ia, Ib, III).

(8) FIG. 8 shows potency analysis of samples with different sialic acid content from CRM-Ia, results from immunization with 2 doses of 1 g of conjugates/mouse. A) OPKA titers, mean value from three protocols is represented by a horizontal bar. B): Cumulative percent survival % of challenged pups from the three protocols. C): IVRP Potency values of administered lots.

(9) FIG. 9 shows potency analysis of desialylated CRM-Ib samples (results from immunization with 1 g of conjugates/mouse). Panels A shows OPKA titers (pooled sera) in mice immunized with 2 doses of desialylated Ia-CRM197 samples formulated with Alum. Data from Preparation #1 are shown in triangles and from Preparation #2 are shown in circles; percent SA in the two preparations is indicated in the X axis. Horizontal bars show the mean of ELISA GMTs and OPKA titers. B): Percent survival (mean of the three protocols) of challenged pups. C) IVRP Potency values of Preparation.

(10) FIG. 10 shows potency analysis of desialylated CRM-III samples (results from immunization with 0.2 g of conjugates/mouse). Panels A shows OPKA titers in sera from mice immunized with two 0.2 g doses of Ia-CRM197 desialylated samples from Preparation #1 formulated in Alum (A) Circles represent the IgG titers from each single mouse, horizontal bars show the mean of ELISA GMTs. (b) Circles represent the OPKA titers of pooled sera from the same protocol. Panel B shows the percent survival of challenged pups from immunized female mice.

MODES FOR CARRYING OUT THE INVENTION

(11) Vaccines

(12) GBS monovalent vaccines used in Studies 1-4 are all conjugated to CRM197.

(13) The GBS trivalent vaccine used in all studies is composed of capsular polysaccharides derived from serotypes: Ia, Ib and III, each conjugated to CRM197.

(14) Study 1

(15) The level of protection of mice in a neonatal challenge model (Maione et al., Science 1 Jul. 2005: vol. 309 no. 5731 pp. 148-150) was investigated. Three doses of GBS monovalent vaccines (1 g antigen) adjuvanted with aluminium salt (400 g) were given to the mothers. The GBS monovalent vaccines tested were GBS Ia, Ib, 11, 111 and V, each conjugated to CRM197. The results are shown in FIG. 1.

(16) OPK assays were also carried out, and the results are shown in FIG. 2. A viable approach to assess a vaccine's efficacy is to use a surrogate of protection which in the case of GBS is the opsonizing activity of serum antibody. The opsonophagocytosis killing assay measures the ability of serum antibody to opsonize GBS for killing by effector cells in the presence of complement. Generally, there is good correlation between ELISA IgG Abs and OPK titers. It can be seen that protection and OPA levels by GBS II and GBS V are comparable to those by GBS Ia, Ib and III.

(17) Study 2

(18) Mice were immunised with three doses of mono- or multivalent vaccines (1 g of each antigen) adjvuanted with aluminium salt (400 Mg). The vaccines tested were (1) CBS V, (2) a tetravalent vaccine containing GBS V and the GBS trivalent vaccine, and (3) a pentavalent vaccine containing GBS II, GBV and the GBS trivalent. Mice were immunised with aluminium salt alone as a negative control. ELISA and OPK assays (using type V CJB111 strain) were carried out (repeated twice).

(19) The results of these experiments are shown in FIG. 3. It can be seen that no significant immune interference was observed between CBS V and the other antigens.

(20) The immunised mice were challenged with GBS V, and the results are provided in Table 1.

(21) TABLE-US-00001 TABLE 1 Results of challenge against a GBS type V strain (type V CJB111). Challenge with GBS V Antigens Protected\Treated % Protection PBS 39/100 39 CRM-V 108/119 91 CRM-Ia/Ib/III 32/102 31 CRM-Ia/Ib/III + CRM-V 60/70 86 CRM-Ia/Ib/III + CRM-II + CRMV-V 89/95 94
Study 3

(22) Mice were immunised with three doses of mono- or multivalent vaccines (1 g of each antigen) adjvuanted with aluminium salt (400 g). The vaccines tested were (1) GBS II, (2) a tetravalent vaccine containing GBS II and the GBS trivalent vaccine, and (3) a pentavalent vaccine containing GBS II, GBV and the GBS trivalent vaccine. Mice were immunised with aluminium salt alone as a negative control. ELISA and OPK assays (using type II 5401 strain) were carried out (repeated twice).

(23) The results are shown in FIG. 4. No significant interference was observed between GBS II and the other antigens.

(24) The immunised mice were challenged with GBS II, and the results are provided in Table 2.

(25) TABLE-US-00002 TABLE 2 Results of challenge against GBS type II strain (type II 5401). Challenge with GBS II Antigens Protected\Treated % Protection PBS 37/120 31 CRM-II 55/80 69 CRM-Ia/Ib/III 15/100 15 CRM-Ia/Ib/III + CRM-II 72/99 73 CRM-Ia/Ib/III + CRM-II + CRM-V 62/129 48
Study 4

(26) Mice were immunised with three doses of tri- or pentavalent vaccines (1 g of each antigen) adjvuanted with aluminium salt (400 g). The vaccines tested were (1) GBS II, (2) the GBS trivalent vaccine (CRM197 conjugated GBS Ia, Ib and III), (3) the GBS tetravalent vaccine (CRM197 conjugated GBS Ia, Ib, II and III or CRM197 conjugated GBS Ia, Ib, and III+TT conjugated GBS II) and (4) a pentavalent vaccine containing (CRM197 conjugated GBS Ia, Ib, II, III and V or CRM197 conjugated GBS Ia, Ib, and III+TT conjugated GBS II and V). Mice were immunised with aluminium salt alone as a negative control. ELISA and OPK assays (using type II 5401 strain) were carried out (repeated twice).

(27) The immunised mice were challenged with CBS II, and the results are provided in Table 3.

(28) TABLE-US-00003 TABLE 3 Results of challenge against GBS type II strain (type II 5401). Challenge with GBS II Antigens Protected\Treated % Protection PBS 19/60 31% CRM-II 23/30 77% CRM-Ia/Ib/III 8/30 26% CRM-Ia/Ib/III + CRM-II 40/50 80% CRM-Ia/Ib/III + CRM-II + CRM-V 32/77 41% CRM-Ia/Ib/III + TT-II 62/77 80% CRM-Ia/Ib/III + TT-II + TT-V 75/77 97%
Study 5

(29) Mice were immunised with three doses of tri- or pentavalent vaccines (1 g of each antigen) adjvuanted with aluminium salt (400 g). ELISA and OPK assays (using type II 5401 strain) were carried out (repeated twice).

(30) The results are shown in FIGS. 5A and 5B. No significant interference was observed between the tri- and the pentavalent vaccines.

(31) Immunogenicity and protection of the conjugates Ia, Ib and III are not affected by the addition of PS-II and V. Comparable ELISA and OPK titers against each PS in the pentavalent formulation.

(32) Study 6

(33) The immunogenicity of GBS saccharides conjugated to different carrier proteins was investigated.

(34) Mice were immunized with compositions containing GBS II and/or V conjugated to CRM197 (CRM) or tetanus toxoid (TT). The antibody titre against GBS V was analysed by ELISA. The results are shown in FIGS. 6 and 7. Mice were immunized with PBS/aluminium salt only as negative controls.

(35) It can be seen that CRM and TT conjugates both provide significant immune responses against the corresponding antigen. For GBS II, no significant immune interference was observed between GBS II and the other antigens in the multivalent vaccines, irrespective of whether it is conjugated to CRM or TT. Similarly for GBS V, no significant immune interference was observed between GBS V and the other antigens in the multivalent vaccines, irrespective of whether it is conjugated to CRM or TT.

(36) Study 7

(37) To investigate the contribution of the sialic acid component, Streptococcus agalactiae capsular polysaccharide antigens Ia, Ib and II conjugated to CRM197 were tested. Vaccine lots with different sialic acid content were prepared and used to immunize mice and determine IgG titers, functional activity of induced antibodies and elicited protection against GBS in a mouse maternal immunization/neonatal challenge model. Monovalent lots of Polysaccharide-CRM197 conjugates with different sialic acid content, from 100% to <5%, were produced by treatment of native conjugates in mild acidic conditions (e.g. pH 4.75 at 80 C. for different incubation times). Potency was evaluated by IVRP. Sera from mice immunized with desialylated lots were analyzed by ELISA and Osonophagocytosis Assay (OPKA) in order to test vaccine immunogenicity and functional activity of induced antibodies, respectively. Survival experiments were also conducted to study protection in pups born to female mice receiving the vaccines.

(38) Briefly, GBS conjugates were desialylated by treatment with deuterated sodium acetate. Sialic acid content was monitored by NMR technology. For all preparations, 3 mg of conjugate (determined by total saccharide content) were dried under vacuum (Genevac mod. EZ-2 Plus) and dissolved in 3 mL of deuterated (D2OAldrich 151882-100G Lot #STBC04462V) 50 mM sodium acetate at pH 4.75 (Sigma S80750-500G Lot#050M0213V); the mixture was incubated at 80 C. at different times and aliquots of the obtained preparations were characterized as follows: (1) 1H NMR analysis to estimate the ratio of bound/free sialic acid content, (2) Estimation of saccharide content based on Galactose (Gal) determination, (3) Determination of protein content, (4) SDS-PAGE analysis and (5) Capillary Electrophoresis (data not shown).

(39) Monovalent lots of Polysaccharide-CRM197 conjugates with different SA from 100% (native untreated material) to 0% were formulated in Alum and administered to groups of 5 week-old CD1 female mice by intraperitoneal (i.p.) injection on days 1 and 21. After the second immunization on day 21, females were mated and pups were challenged with a LD90 dose of GBS of the same serotype (GBS 090 for serotype Ia, GBS H36b for Ib and GBS M781 for serotype III). Mortality was recorded daily for 3 days after challenge. Vaccine immunogenicity was tested by analyzing sera collected 2 weeks after the last vaccine injection for the presence of antibodies to each of the serotype specific antigens (Ia, Ib and III). Specific IgG antibody titers against each polysaccharide were quantified by ELISA. Functional antibody activity was evaluated on pools of sera from animals receiving the same vaccine using the opsonophagocytosis assay (OPKA) and by neonatal challenge.

(40) For Polysaccharide Ia-CRM197, IgG titers in animals immunized with 1 g vaccine doses were similar for all samples except for <5%, which resulted in about 4 fold statistically significant reduction compared to native polysaccharide (P=0.0067, Mann Whitney test). A decrease in antibody functional activity could be appreciated at 19% and <5% (OPKA killing) and <5% sialic content (survival). Concerning IVRP results, potency values of samples with sialic acid content equal or below 33% decreased by more than 10 fold (FIG. 8).

(41) For Polysaccharide Ib-CRM197, samples containing less than 5% SA induced lower ELISA titers, OPKA titers and survival compared to samples with higher sialic content (FIG. 9).

(42) For Polysaccharide III-CRM197, IgG GMTs to PS III were significantly higher in animals who received samples containing less than 66% SA compared to those immunized with samples with higher SA content (P<0.01 Mann-Whitney test). The levels of functional antibodies were similar in animals receiving 1 g doses of native and desialylated samples. However, a difference was appreciable in animals receiving 0.2 g doses of conjugates, with a drastic reduction in OPKA titers in animals receiving samples with 39% SA and lower survival rates under 24% SA (FIG. 10).

(43) Study 8

(44) To investigate the contribution of the sialic acid component, Streptococcus agalactiae capsular polysaccharide antigen V conjugated to CRM197 was tested. Vaccine lots with differing sialic acid content were prepared and used to immunize mice. Monovalent lots of Polysaccharide-CRM197 conjugates with different sialic acid content, from 100% to 25%, were produced by treatment of native conjugates in mild acidic conditions (e.g. pH 4.75 at 80 C. for different incubation times).

(45) TABLE-US-00004 PSV-CRM197 Vaccine % NeuNAc % Protection (CJB111) Lot 1 + Alum 100 66 Lot 2 + Alum 100 81 Lot 3 + Alum 100 69 Lot 4 + Alum 100 73 Lot 5 + Alum 75 45 Lot 6 + Alum 50 22 Lot 7 + Alum 50 36 Lot 8 + Alum 50 46 Lot 9 + Alum 25 38 Lot 10 + Alum 25 17 Lot 11 + Alum 25 10 Control (Alum only) 11 Control 2 (Alum only) 2

(46) Removal of NeuNAc appears to impact protection following vaccination in mice. These experiments suggest that polysaccharide type V conjugates wherein content of NeuNAc is greater than 75% are useful.

(47) TABLE-US-00005 % Protection Crosslinking CRM197:PSV % NeuNAc (CJB111) Level 0.25:1.0 0 100 LOW 0.5:1.0 0 79 | 0.5:1.0 0 48 | 1.0:1.0 0 32 | 1.0:1.0 0 46 | 1.5:1.0 0 42 | 1.5:1.0 0 43 | 2.0:1.0 0 13 | 3.0:1.0 0 37 | 4.0:1.0 0 31 Control (Alum only) 0 5 HIGH

(48) The immunogenicity of Polysaccharide V-CRM197 desialated polysaccharide conjugates is inversely proportional to its cross-linking level. Thus, when an immunogenic composition comprises polysaccharide Type V, particularly desialylated polysaccharide, conjugates with lower protein:polysaccharide crosslinking levels may be beneficial.

(49) 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

(50) [1] Paoletti et al. (1990) J Biol Chem 265:18278-83. [2] Wessels et al. (1990) J Clin Invest 86:1428-33. [3] Paoletti et al. (1992) Infect Immun 60:4009-14. [4] Paoletti et al. (1992) J Clin Invest 89:203-9. [5] Wessels et al. (1987) Proc Natl Acad Sci USA 84:9170-4. [6] Wang et al. (2003) Vaccine 21:1112-7. [7] Wessels et al. (1993) Infect Immun 61:4760-6 [8] Wessels et al. (1995) J Infect Dis 171:879-84. [9] Baker et al. (2004) J Infect Dis 189:1103-12. [10] Paoletti & Kasper (2003) Expert Opin Biol Ther 3:975-84. [11] WO2012/035519 [12] Lewis et al. (2004) PNAS USA 101:11123-8. [13] WO2006/050341 [14] Guttormsen et al. (2008) Proc Natl Acad Sci USA. 105(15):5903-8. Epub 2008 Mar. 31. [15] WO96/40795 [16] Michon et al. (2006) Clin Vaccine Immunol. 2006 August; 13(8):936-43. [17] U.S. Pat. No. 6,027,733 & 6274144. [18] www.polymer.de [19] Wessels et al. (1989) Infect Immun 57:1089-94. [20] WO2006/082527. [21] US patent application U.S. 61/008,941, entitled FERMENTATION PROCESSES FOR CULTIVATING STREPTOCOCCI AND PURIFICATION PROCESSES FOR OBTAINING CPS THEREFROM filed on 20 Dec. 2007 and international patent application WO 2009/081276. [22] Ramsay et al. (2001) Lancet 357(9251):195-196. [23] Lindberg (1999) Vaccine 17 Suppl 2:S28-36. [24] Buttery & Moxon (2000) JR Coil Physicians Lond 34:163-68. [25] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-33, vii. [26] Goldblatt (1998) J. Med. Microbiol. 47:563-7. [27] European patent 0477508. [28] U.S. Pat. No. 5,306,492. [29] WO98/42721. [30] Dick et al. in Conjugate Vaccines (eds. Cruse et al.) Karger, Basel, 1989, 10:48-114. [31] Hermanson Bioconjugae Techniques, Academic Press, San Diego (1996) ISBN: 0123423368. [32] U.S. Pat. No. 4,356,170. [33] WO2006/082530. [34] WO2005/000346 [35] Anonymous (January 2002) Research Disclosure, 453077. [36] Anderson (1983) Infect Immun 39(1):233-238. [37] Anderson et al. (1985) J Clin Invest 76(1):52-59. [38] EP-A-0372501. [39] EP-A-0378881. [40] EP-A-0427347 [41] WO93/17712 [42] WO94/03208. [43] WO98/58668. [44] EP-A-0471177. [45] WO91/01146 [46] Falugi et al. (2001) Eur J Immunol 31:3816-24. [47] Baraldo et al. (2004) Infect Immun 72:4884-87. [48] EP-A-0594610. [49] WO00/56360. [50] WO02/091998. [51] Kuo et al. (1995) Infect Immun 63:2706-13. [52] WO01/72337 [53] WO00/61761. [54] WO00/33882 [55] WO99/42130. [56] WO2004/011027. [57] WO96/40242. [58] Lei et al. (2000) Dev Biol (Basel) 103:259-264. [59] WO00/38711; U.S. Pat. No. 6,146,902. [60] International patent application PCT/IB2008/02690, CONJUGATE PURIFICATION, claiming priority from GB-0713880.3 (NOVARTIS AG), published as WO 2009/010877. [61] Paoletti et al. (2001) Vaccine 19:2118-2126. [62] WO00/56365. [63] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition. ISBN: 0683306472. [64] Paoletti (2001) Vaccine 19(15-16):2118-26. [65] WO03/009869. [66] Almeida & Alpar (1996) J. Drug Targeting 3:455-467. [67] Agarwal & Mishra (1999) Indian J Exp Biol 37:6-16. [68] WO00/53221. [69] Jakobscn et al. (2002) Infect Immun 70:1443-1452. [70] Bergquist et al. (1998) APMIS 106:800-806. [71] Baudner et al. (2002) Infect Immun 70:4785-4790. [72] Ugozzoli et al. (2002) J Infect Dis 186:1358-1361. [73] U.S. Pat. No. 6,355,271. [74] WO00/23105. [75] Vaccine Design . . . (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum. [76] WO90/14837. [77] Podda (2001) Vaccine 19:2673-80. [78] Frey et al. (2003) Vaccine 21:4234-7. [79] U.S. Pat. No. 6,299,884. [80] U.S. Pat. No. 6,451,325. [81] U.S. Pat. No. 5,057,540. [82] WO96/33739. [83] EP-A-0109942. [84] WO96/11711. [85] WO00/07621. [86] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271. [87] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32:321-338. [88] Niikura et al. (2002) Virology 293:273-280. [89] Lenz et al. (2001) J Immunol 166:5346-5355. [90] Pinto et al. (2003) J Infect Dis 188:327-338. [91] Gerber et al. (2001) Virol 75:4752-4760. [92] WO03/024480 [93] WO03/024481 [94] Gluck et al. (2002) Vaccine 20:B 10-B 16. [95] EP-A-0689454. [96] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278. [97] Evans et al. (2003) Expert Rev Vaccines 2:219-229. [98] Meraldi et al. (2003) Vaccine 21:2485-2491. [99] Pajak et al. (2003) Vaccine 21:836-842. [100] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400. [101] WO02/26757. [102] WO99/62923. [103] Krieg (2003) Nature Medicine 9:831-835. [104] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185. [105] WO98/40100. [106] U.S. Pat. No. 6,207,646. [107] U.S. Pat. No. 6,239,116. [108] U.S. Pat. No. 6,429,199. [109] Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658. [110] Blackwell et al. (2003) J Immunol 170:4061-4068. [111] Krieg (2002) Trends Immunol 23:64-65. [112] WO01/95935. [113] Kandimalla et al. (2003) BBRC 306:948-953. [114] Bhagat et al. (2003) BBRC 300:853-861. [115] WO03/035836. [116] WO95/17211. [117] WO98/42375. [118] Beignon et al. (2002) Infect Immun 70:3012-3019. [119] Pizza et al. (2001) Vaccine 19:2534-2541. [120] Pizza et al. (2000) Int J Med Microbiol 290:455-461. [121] Scharton-Kersten et al. (2000) Infect Inmmun 68:5306-5313. [122] Ryan et al. (1999) Infjct Immun 67:6270-6280. [123] Partidos et al. (1999) Immunol Lett 67:209-216. [124] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293. [125] Pine et al. (2002) J Control Release 85:263-270. [126] Domenighini et al. (1995) Mol Micnobiol 15:1165-1167. [127] WO99/40936. [128] WO99/44636. [129] Singh et al] (2001) J Cont Release 70:267-276. [130] WO99/27960. [131] U.S. Pat. No. 6,090,406 [132] U.S. Pat. No. 5,916,588 [133] EP-A-0626169. [134] WO99/52549. [135] WO01/21207. [136] WO01/21152. [137] Andrianov et al. (1998) Biomaterials 19:109-115. [138] Payne et al. (1998) Adv Drug Delivey Review 31:185-196. [139] Stanley (2002) Clin Exp Dermnatol 27:571-577. [140] Jones (2003) Curr Opin Investig Drugs 4:214-218. [141] WO04/60308 [142] WO04/64759. [143] WO99/11241. [144] WO94/00153. [145] WO98/57659. [146] European patent applications 0835318, 0735898 and 0761231. [147] Hennings et al. (2001) J Infect Dis. 183(7): 1138-42. Epub 2001 Mar. 1. [148] Lin et al. (2001). Inject Dis. 184(8):1022-8. [149] Lin et al. (2004) J Infect Dis. 190(5):928-34 [150] Glezen & Alpers (1999) Clin. Infect. Dis. 28:219-224 [151] Madoff et al. (1994) J Clin Invest 94:286-92. [152] Paoletti et al. (1994) Infect Immun 62:3236-43.