Enhancement of the Immunogenicity of Synthetic Carbohydrates by Conjugation to Virosomes: A Leishmaniasis Vaccine Candidate Xinyu Liu†, Sibylle Siegrist‡, Mario Amacker§, Rinaldo Zurbriggen§, Gerd Pluschke‡, and Peter H. Seeberger†,* †Laboratorium für Organische Chemie, ETH Zürich, Wolfgang-Pauli Strasse 10, 8093 Zürich, Switzerland, ‡Swiss Tropical
Institute, Socinstrasse 57, 4002 Basel, Switzerland, and §Pevion Biotech Ltd., Rehhagstrasse 79, 3018 Bern, Switzerland
V
accinations based on defined anti gens aim to elicit a specific immune response against a molecule expressed by the pathogen to be eliminated by the immune system. In addition to pro teins, carbohydrate antigens are becoming increasingly important as target structures, and vaccines based on capsular polysacch arides extracted from Haemophilus influenzae type B, Neisseria meningitidis, and Streptococcus pneumoniae are already introduced into routine immunization schedules (1–4 ). Since polysaccharides are often very heterogeneous and difficult to isolate from their natural sources, it is expected that recent improvements in carbohydrate synthesis technologies (5 ) will lead to the development of new synthetic carbohydrate vaccines (6, 7 ). Unconjugated polysaccharide antigens typically elicit only T-cell independent short-lived and low-affinity IgM antibody responses, and the immunogenicity of smaller synthetic oligosaccharides tends to be even weaker. Therefore synthetic carbohydrate vac cine design is critically dependent on the development of an antigen delivery platform that promotes the generation of T-cell dependent immune responses against oligosaccharides. Stimulation of B‑cells should be associated with the development of long-term memory, Ig affinity maturation, and isotype class switching to IgG. The T-cell independent properties of carbohydrate antigens can be overcome by conjugation to a carrier protein. In addition, the immuno genicity has to be enhanced by delivery www.acschemicalbiolog y.o rg
of the conjugates with an immunological adjuvant. Alum has remained the dominant adjuvant for human vaccines, since many other candidate adjuvants have shown unsuitable properties including reacto genicity, toxicity, instability, or high costs. Since alum has a relatively poor adjuvant effect on polysaccharide antigens (8 ), the use of immunostimulating reconstituted influenza virosomes (IRIVs) may constitute an attractive alternative integrated carrier and adjuvant platform for synthetic oligo saccharides. Potential advantages of IRIVs for subunit vaccine antigen delivery include their fusogenic activity, serial display of the molecularly defined antigens on the surface of the virus-like particles, and an excellent safety record both in animals and humans (9 ). Two IRIV-based vaccines against hepatitis A and influenza are currently on the market (10 ), and it has been shown that IRIVs represent an excellent delivery system for small synthetic peptides (11 ). We have evaluated IRIVs for the first time as a synthetic carbohydrate antigen delivery system as part of an ongoing leishmania vaccine development program. The results suggest that IRIVs represent a general platform for oligosaccharide vaccines, and details are reported here. Kala azar, now known as visceral leish maniasis, threatens more than 350 million people worldwide and kills 60,000 annu ally. Transmitted by the bite of the female phlebotomine sandfly, protozoan para sites of the genus Leishmania cause the tropical disease that is still treated with old
A B S T R A C T Novel virosomal formulations of a synthetic oligosaccharide were prepared and evaluated as vaccine candidates against leishmaniasis. A lipophosphoglycan-related synthetic tetrasaccharide antigen was conjugated to a phospholipid and to the influenza virus coat protein hemagglutinin. These glycan conjugates were embedded into the lipid membrane of reconstituted influenza virus virosomes. The virosomal formulations elicited both IgM and IgG anti-glycan antibodies in mice, indicating an antibody isotype class switch to IgG. The antisera cross-reacted in vitro with the corresponding natural carbohydrate antigens expressed by leishmania cells. These findings support the concept of using virosomes as universal antigen delivery platform for synthetic carbohydrate vaccines.
*To whom correspondence should be addressed. Email:
[email protected]. ethz.ch.
Received for review February 25, 2006 and accepted April 4, 2006 Published online April 21, 2006 10.1021/cb600086b CCC: $33.50 © 2006 by American Chemical Society
VOL.1 NO. 3 • ACS CHEMICAL BIOLO GY
161
HO OH HO
O O P OH HO
O O OH
O O
HO OH O
HO HO OH O HO HO OH
P O O-
O
HO HO
O OH
O HO
OH OH O
O
O OH
OH HO
O
HO
n
OH
O
HO
HO HO
O O OH
O
Phosphoglycan
HO O O
O OH
O O P OH O OH O O
OH
HO
OH O
OH OH OH
Cap Tetrasaccharide
OH
HO HO
O
O NH2
HO OH
O
OH
O O P O OH
OH
OR1
R1 = C24H49 or C26H53
GPI Anchor Figure 1. General structure of the leishmania lipophosphoglycan (LPG).
antimony-based drugs that are expensive and associated with significant side effects. Despite of many efforts, no effective vaccine for any form of leishmaniasis has emerged yet (World Health Organization, State of the Art of New Vaccines: Research and Development, http://www.who.int/vaccine_ research/documents/new_vaccines/en/). The lipophosphoglycans (LPGs) on the cell surface of leishmania parasites are an important virulence factor and essential for the survival and infectivity of the parasites. These cell surface glycoconjugates are therefore specific markers such as those on many cancer cells, bacteria, viruses, and parasites (12, 13 ). Disease-specific carbo hydrates have attracted the attention of immunologists as antigens for the creation of novel vaccines in recent years as the methods for the synthesis of complex oligo saccharides have significantly improved (14, 15 ). Vaccines based on synthetic oligo saccharide antigens against bacterial infec tions, HIV, and malaria, as well as cancers including breast and prostate cancers, are at different stages of preclinical and clinical development (16–22 ). A unique, structur ally well-defined capping tetrasaccharide that has been implicated as crucial for the invasion of the parasite into macrophages terminates the LPGs of leishmania parasites (Figure 1) (23 ). This tetrasaccharide was the focal point of our efforts to create a leishmaniasis vaccine candidate. The tetrasaccharide epitope, equipped with a short PEG-linker terminated by a thiol group at the reducing end, was synthesized in a linear fashion (24–26 ). The thiol serves as a handle for conjugation to different carriers (27 ). The chemical synthesis of the tetrasaccharide epitope started with the installation of thiol-based linker 1 on the carbohydrate moiety (Figure 2). The coupling of 1 with readily available mannose building block 4 gave mannoside 5 in 92% yield (see 162
VO L .1 N O. 3 • 1 6 1 –164 • 2006
Supporting Information). Selective opening amine (PE) with 4‑maleimidobutyric acid of 4,6-O-benzylidene afforded mannoside 6 sulfo-N-succinimidyl ester (sulfo-GMBS). in 63% yield. Union of galactosyl phosphate The in situ reduced glycan 11 was mixed 2 (28 ) with 6 and subsequent cleavage of with 12 to generate the lipid conjugate 13. the acetyl ester in the presence of a pivalo The conjugate was formulated into IRIVs ate using dilute HCl generated in situ under as described (29 ), by resuspending it in nonaqueous conditions gave disaccharide 7 octaethyleneglycol (OEG) and combin in 76% yield. The disaccharide was further ing it with egg phosphatidylcholine (PC), decorated with mannose by glycosylation surface glycoproteins, and phospholipids using mannosyl trichloroacetimidate 3 of inactivated influenza A/Singapore/6/86 to furnish trisaccharide 8 in 69% yield. (H1N1). PE-Glycan loaded IRIVs were formed Selective removal of acetate afforded 9 by detergent removal. For the prepara in 90% yield. Placement of the terminal tion of protein conjugates, keyhole limpet mannose proved far more challenging as an hemocyanin (KLH) or solubilized influenza excess (3 equiv) of 3 was needed to afford hemagglutinin (HA) was activated with the protected tetrasaccharide 10 in good sulfo-GMBS to result in maleimide-activated yield. The conditions of dissolving metal carrier proteins that were mixed with the reductions ensured the complete removal reduced glycan 11. While the resulting of all protecting groups before purification glycan-HA conjugate 14 was resuspended by Sephadex column chromatography and in OEG to prepare HA-glycan loaded IRIVs, dialysis furnished the pure tetrasaccharide the glycan-KLH conjugate 15 was used to 11 in 81% yield. Predominantly, the dimer coat ELISA plates with the glycan 11. of 11 was obtained as indicated by NMR Glycan-PE 13 and glycan-HA 14 loaded and HR-MS analysis (see Supporting IRIVs were used to immunize groups of OBn Information). OAc OBn OBn O BnO O O For immuno BnO HO O O P OBu = ROH BnO CCl3 O SBn O OPiv logical studies, OBu 2 1 3 NH the disulfide in glycan 11 was first OAc O BnO OBn Ph OBn R1O O OAc O OH reduced in situ (i) O (iii) O O BnO R 2O O BnO BnO with tricarboxy OPiv BnO O CCl3 OR OR 4 NH ethylphosphine 7 5: R ,R = PhCH< (ii) 6: R = Bn, R = H OH (TCEP) and then OH O OBn HO conjugated either OR3 HO O HO to a phospholipid BnO O O (iv) BnO OBn BnO (vii) HO or to a carrier pro BnO HO O O HO OH O O O HO BnO O tein (Scheme 1). O OPiv BnO O HO OR MaleimideHO OH 8: R = Ac (v) O O 11 9: R = H O SH activated (vi) 10: R = 2-O-Ac-3,4,6-tri-O-Bn-α-D-mannosyl phospholipid 12 Figure 2. Synthesis of the thiol-tethered LPG tetrasaccharide epitope. was prepared (i) 1, TBSOTf (cat.), Et2O/CH2Cl2, 0 °C, 92%; (ii) Et3SiH, TfOH, 4 Å MS, by combining CH2Cl2, –78 °C, 63%; (iii) 2, TMSOTf, CH2Cl2, –40 °C; then AcCl in THF/ 1-oleoyl-3-palmi MeOH, 0 °C to rt, 76% 2 steps; (iv) 3, TMSOTf (cat.), Et O/CH Cl , 0 °C, 2 2 2 toyl-sn-glycero-2- 69%; (v) AcCl in THF/MeOH, 0 °C to RT, 90%; (vi) 3, TMSOTf (cat.), Et2O/ phosphoethanol CH2Cl2, 0 °C, 53%; (vii) Na, NH3, THF/MeOH, –78 °C, 81%. 1 1
2
2
3 3
3
Liu E t A l .
w w w. a c s c h e m i ca l biology.org
O O
O
N
N H
O
N
TCEP
O
12
O
O
Glycan11
O
O O P O O-
HA
O
O
O
N
KLH
C15H31
7
C8H17
Glycan
Glycan
Lipid
S
HA
14
N O
S 13
N
O
O
O
Glycan
S
KLH
15
Scheme 1. Chemical conjugation of LPG tetrasaccharide for immunological studies
BALB/c mice intramuscularly. After preimmunization with the IRIV-based influenza vaccine Inflexal Berna (Berna Biotech) the mice received at 3-week intervals three doses of IRIV containing 50 µg of glycan conjugated either to PE or to HA. Priming of mice with influenza antigen enhances the antibody response against IRIV-associated antigens (30 ). When the generation of glycan-specific antibodies was analyzed by ELISA using KLH-glycan coated microtiter plates, both IgG and IgM response was observed. IgG isotyping indicated that IgG1 contributed dominantly to the antibody elicited, with only negligible amounts of IgG3 detected (ELISA data not shown). These data provided solid evidence that the observed IgG antibody response is T-cell dependent. Cross-reactivity with natural carbohydrate antigens produced by leishmania cells was analyzed by immunofluorescence staining of axenic amastigote Leishmania donovani (MHOM-ET-67/L82) parasites. The in vitro cultivated parasites were washed, sus pended in PBS containing 1% bovine serum albumin, and fixed on diagnostic micro scope slides for at least 60 min at room temperature using a 4% paraformaldehyde fixing solution containing 0.1% Triton X-100. After being washed, slides were incubated with serial dilutions of mouse sera for 2 h at room temperature in a humid chamber. After being washed, slides were incubated for 60 min with Cy3-conjugated AffiniPure F(ab’)2 fragment goat-anti-mouse IgG, Fcγ fragment specific antibodies for immunoflu www.acschemicalbiolog y.o rg
orescence staining and 1 µg mL–1 Hoechst dye 33258 for DNA staining. After being washed, the slides were dried, mounted with mounting solution, covered with a cover slide, and assessed by fluorescence microscopy. Although LPG expression is downregulated in axenic amastocygotes (31 ), staining by parasite cross-reactive IgG was observed with sera of all mice immu nized with glycan-loaded IRIV. Whereas glycan-HA loaded IRIVs induced an ELISA titer higher than that of glycan-PE loaded IRIVs, both formulations elicited comparable titers of parasite cross-reactive IgG in the
immunofluorescence analysis (Figure 3, panel a). No staining was observed with preimmune sera and after three immunizations of mice with IRIV that were not loaded with glycan 8 (Figure 3, panel b). The anti-glycan antisera stained also parasites in liver sec tions from leishmania-infected hamsters (data not shown). These assays demonstrate that the new vaccine candidate is highly immunogenic and elicits IgG antibodies that recognize leishmania parasites. Challenge studies in animals are the next step en route to a leishmaniasis vaccine. In conclusion, IRIVs represent a viable antigen delivery system suitable to induce T‑cell dependent antibody responses against oligosaccharide antigens. Our results with two IRIV-based formulations of a leishmanial tetrasaccharide indicate that IRIVs have a great potential for the design of safe and effective synthetic carbohydrate vaccines. Thus, IRIVs present an attractive alternative to currently used carbohydrate vaccine formulations and should find appli cation for diseases including bacterial, viral, and parasitic infections and cancer.
Figure 3. Generation of Leishmania donovani crossreactive IgG by immunization with glycan 11 loaded IRIVs. a) Immunofluorescence staining of parasites by serial dilution (1:100 to 1:1600) of serum from mice immunized three times with HA-glycan (left panel) or PE-glycan (right panel) loaded IRIV. b) Lack of immunofluorescence staining (left panel) of parasites by pre-immune sera (dilution 1:100) and by serum (dilution 1:100) of a mouse immunized three times with unloaded IRIV. Presence of parasites on the microscopic slides was demonstrated by staining of parasite DNA with Hoechst dye 33258 (right panel). Typical results are shown. VOL.1 NO. 3 • 161—164 • 2 0 0 6
163
METHODS
Preparation of Lipid–Glycan Conjugate 13. PE (4 mg) was dissolved in a mixture of CHCl3/ MeOH (500 µL, 9:1), and triethylamine (5 µL) was added. This solution was mixed with GMBS (3 mg) and incubated for 2 h at 30 °C using a thermomixer. Leishmania glycan 11 (2 mg) was dissolved in OEG-PBS (0.5 mL, 100 mM), and TECP solution (5 µL, 0.5 mM) added. The solution was incubated for 5 min at 30 °C and pipetted into the activated PE solution. The mixture was then further incubated for 3 h at 30 °C. The resulting solution was stored at 4 °C for immunization study. Preparation of KLH–Glycan Conjugate 15. Sulfo-GMBS (2 mg) was mixed with 1 mL of KLH solution and incubated for 3 h at 30 °C using a thermomixer. Leishmania glycan (2 mg) was dissolved in PBS (0.2 mL), and TECP solution (5 µL, 0.5mM) was added. The solution was incubated for 5 min at 30 °C and pipetted into the activated KLH solution. The mixture was then further incubated for 3 h at 30 °C. The resulting solution was stored at 4 °C for immunization studies. HA–glycan conjugate was prepared in an analogous fashion. Virosome Formulation. Glycan-IRIVs were pre pared by the method described previously (29 ). Briefly, 32 mg of egg PC (phosphatidylcholine), 8 mg of PE, and 2 mg of the glycan-PE conjugate were dissolved in 3 mL of PBS, 100 mM OEG (OEG-PBS). Next, 4 mg HA of inactivated influenza A/Singapore virus was centrifuged at 100 000g for 1 h at 4 °C, and the pellet was dissolved in 1 mL of OEG-PBS. The detergent-solubilized phospho lipids and viruses were mixed and sonicated for 1 min. This mixture was centrifuged at 100 000g for 1 h at 20 °C, and the supernatant was sterile filtered (0.22 µm). Virosomes were then formed by detergent removal using 1.25 g of wet SM2 BioBeads (BioRad, Glattbrugg, Switzerland) for 1 h at room temperature with shaking and three times for 30 min with 625 mg of SM2 Bio-Beads each. ELISA. For enzyme-linked immunosorbent assay (ELISA) analyses, Polysorp plates were coated overnight at 4 °C with 100 µL of a 10 µg mL–1 solution of KLH-glycan conjugate in PBS (pH 7.4). Wells were then blocked with 5% milk powder in PBS for 2 h at 37 °C, followed by three washes with PBS containing 0.05% Tween 20. Plates were then incubated with serial dilutions of the mouse sera in PBS containing 0.05% Tween 20 and 0.5% milk powder for 2 h at 37 °C. After washing, plates were incubated with either goat anti-mouse Ig-HRP (dilution 1:1000), goat anti-mouse IgG-HRP (dilution 1:2000), or goat anti-mouse IgM-HRP (dilution 1:2000), or for IgG isotypings, rabbit anti-mouse IgG1-HRP (dilution 1:2000) and goat anti-mouse IgG3-HRP (dilution 1:1000), respectively, for 1 h at 37 °C. After the plates were washed, O-phenylendiamine substrate was added, the plates were incubated in the dark at room temperature until the colori metric reaction had progressed sufficiently, the reaction was stopped by addition of 100 µL of 1 M H2SO4, and optical densities (OD) were read at 492 nm on a Spectra Max Plus. Endpoint titer is calculated as the reciprocal of the last serum dilution with the ODtest sera ≥ 2 × ODnegativ serum. A strong signal is considered with endpoint titers greater than 5000.
164
VO L .1 N O. 3 • 1 6 1 –164 • 2006
Acknowledgment: Financial support from the Swiss National Science Foundation and ETH Zürich are gratefully acknowledged. We thank S. Rosenfellner for technical assistance and R. Brun for support with the cultivation of leishmania. Supporting Information Available: This material is available free of charge via the Internet.
REFERENCES
1. Jennings, H. J., and Pon, R. A. (1996) Polysacch arides and glycoconjugates as human vaccines, in Polysaccharides in Medicinal Applications (Dumitriu, S., Ed.) pp 443–479, Marcel Dekker, New York. 2. Robbins, J. B., Schneerson, R., Szu, S. C., and Pozsgay, V. (1999) Bacterial polysaccharide-protein conjugate vaccines, Pure Appl. Chem. 71, 745–754. 3. Moreau, M., and Schulz, D. (2000) Polysaccharide based vaccines for the prevention of pneumococcal infections, J. Carbohydr. Chem. 19, 419–434. 4. Weintraub, A. (2003) Immunology of bacterial polysaccharide antigens, Carbohydr. Res. 338, 2539–2547. 5. Ernst, B., Hart, G. W., and Sinay, P. (2000) Carbohydrates in Chemistry and Biology, Part I: Chemistry of Saccharides, Volume 1: Chemical Synthesis of Glycosides and Glycomimetics, WileyVCH, Weinheim. 6. Roy, R. (2004) New trends in carbohydrate-based vaccines, Drug Discovery Today: Technol. 1, 327–336. 7. Borman, S. (2004) Carbohydrate vaccines, Chem. Eng. News 82, 31–35. 8. Lindblad, E. B. (1995) Aluminium adjuvants, in The Theory and Practical Application of Adjuvants (Stewart-Tull, D. E. S., Ed.) pp 21–35, John Wiley & Sons, Chichester, U.K. 9. Zurbriggen, R. (2003) Immunostimulating reconsti tuted influenza virosomes, Vaccine 21, 921–924. 10. Westerfeld, N., and Zurbriggen, R. (2005) Peptides delivered by immunostimulating reconstituted influenza virosomes, J. Pept. Sci. 11, 707–712. 11. Pfeiffer, B., Peduzzi, E., Moehle, K., Zurbriggen, R., Gluck, R., Pluschke, G., and Robinson, J. A. (2003) A virosome-mimotope approach to synthetic vaccine design and optimization: Synthesis, conformation, and immune recognition of a potential malariavaccine candidate, Angew. Chem., Int. Ed. 42, 2368–2371. 12. Varki, A. (1993) Biological roles of oligosaccha rides–All of the theories are correct, Glycobiology 3, 97–130. 13. Dwek, R. A. (1996) Glycobiology: Toward under standing the function of sugars, Chem. Rev. 96, 683–720. 14. Koeller, K. M., and Wong, C. H. (2000) Synthesis of complex carbohydrates and glycoconjugates: Enzyme-based and programmable one-pot strate gies, Chem. Rev. 100, 4465–4493. 15. Seeberger, P. H., and Werz, D. B. (2005) Automated synthesis of oligosaccharides as a basis for drug discovery, Nat. Rev. Drug Discovery 4, 751–763. 16. Danishefsky, S. J., and Allen, J. R. (2000) From the laboratory to the clinic: A retrospective on fully synthetic carbohydrate-based anticancer vaccines, Angew. Chem., Int. Ed. 39, 836–863. 17. Dudkin, V. Y., Orlova, M., Geng, X. D., Mandal, M., Olson, W. C., and Danishefsky, S. J. (2004) Toward fully synthetic carbohydrate-based HIV antigen design: On the critical role of bivalency, J. Am. Chem. Soc. 126, 9560–9562. Liu E t A l .
18. Adams, E. W., Ratner, D. M., Bokesch, H. R., McMahon, J. B., O’Keefe, B. R., and Seeberger, P. H. (2004) Oligosaccharide and glycoprotein microar rays as tools in HIV glycobiology: Glycan-depen dent gp120/protein interactions, Chem. Biol. 11, 875–881. 19. Lee, H. K., Scanlan, C. N., Huang, C. Y., Chang, A. Y., Calarese, D. A., Dwek, R. A., Rudd, P. M., Burton, D. R., Wilson, I. A., and Wong, C.-H. (2004) Reactivity-based one-pot synthesis of oligo mannoses: Defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody, Angew. Chem., Int. Ed. 43, 1000–1003. 20. Wang, L. X., Ni, J. H., Singh, S., and Li, H. G. (2004) Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: Implications for HIV-1 vaccine design, Chem. Biol. 11, 127–134. 21. Verez-Bencomo, V., Fernandez-Santana, V., Hardy, E., Toledo, M. E., Rodriguez, M. C., Heynngnezz, L., Rodriguez, A., Baly, A., Herrera, L., Izquierdo, M., Villar, A., Valdes, Y., Cosme, K., Deler, M. L., Montane, M., Garcia, E., Ramose, A., Aguilar, A., Medina, E., Torano, G., Sosa, I., Hernandez, I., Martinez, R., Muzachio, A., Carmenates, A., Costa, L., Cardoso, F., Campa, C., Diaz, M., and Roy, R. (2004) A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b, Science 305, 522–525. 22. Schofield, L., Hewitt, M. C., Evans, K., Siomos, M. A., and Seeberger, P. H. (2002) Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria, Nature 418, 785–789. 23. Descoteaux, A., and Turco, S.J. (2002). Functional aspects of the Leishmania donovani lipophos phoglycan during macrophage infection. Microbes Infect. 4, 975–981. 24. Arasappan, A., and FraserReid, B. (1996) N‑Pentenyl glycoside methodology in the stere oselective construction of the tetrasaccharyl cap portion of Leishmania lipophosphoglycan, J. Org. Chem. 61, 2401–2406. 25. Upreti, M., Ruhela, D., and Vishwakarma, R. A. (2000) Synthesis of the tetrasaccharide cap domain of the antigenic lipophosphoglycan of Leishmania donovani parasite, Tetrahedron 56, 6577–6584. 26. Hewitt, M. C., and Seeberger, P. H. (2001) Solution and solid-support synthesis of a potential leish maniasis carbohydrate vaccine, J. Org. Chem. 66, 4233–4243. 27. Ratner, D. M., Adams, E. W., Su, J., O’Keefe, B. R., Mrksich, M., and Seeberger, P. H. (2004) Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides, ChemBioChem 5, 379–382. 28. Plante, O. J., Andrade, R. B., and Seeberger, P. H. (1999) Synthesis and use of glycosyl phosphates as glycosyl donors, Org. Lett. 1, 211–214. 29. Zurbriggen, R., Novak-Hofer, I., Seelig, A., and Gluck, R. (2000) IRIV-adjuvanted hepatitis A vaccine: in vivo absorption and biophysical charac terization, Prog. Lipid Res. 39, 3–18. 30. Poltl-Frank, F., Zurbriggen, R., Helg, A., Stuart, F., Robinson, J., Gluck, R., and Pluschke, G. (1999) Use of reconstituted influenza virus virosomes as an immunopotentiating delivery system for a peptidebased vaccine, Clin. Exp. Immunol. 117, 496–503. 31. Gupta, N., Goyal, N., and Rastogi, A. K. (2001) In vitro cultivation and characterization of axenic amastigotes of Leishmania, Trends Parasitol. 17, 150–153.
w w w. a c s c h e m i ca l biology.org
