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Humoral responses following immunization with Leishmania infantum (ex. Oklahoma): A comparison of adjuvant efficacy in the antibody responses of Balb-C mice
Int. J. lmmunopharmac,, Vol. 14, No. 5, pp. 767-772, 1992. Printed in Great Britain.
0192-0561/92 $5.00 + .00 Pergamon Press Ltd. ~) 1992 International Society for Immunopharmacology.
HUMORAL RESPONSES FOLLOWING IMMUNIZATION WITH L E I S H M A N I A I N F A N T U M (EX. OKLAHOMA): A COMPARISON OF ADJUVANT EFFICACY IN THE ANTIBODY RESPONSES OF BALB-C MICE R. M.
LASAROW, *f
D. L.
WILLIAMS ~
and J. H. THEIS*
*Department of Medical Microbiology and Immunology, University of California, Davis, School of Medicine, Davis, CA 95616; *Department of Physiology, Tulane University, School of Medicine, New Orleans, Louisiana, U.S.A. (Received 20 September 1991 and in final form 10 January 1992)
Abstract -- Adjuvants are commonly used in immunization protocols for the purpose of augmenting
immune responses to antigens. The antigenic profile of Leishmania infantum (ex. Oklahoma) is described and the efficacies of three adjuvants delivered coincidentally with killed promastigotes are compared, by measuring relative serologic responses of Balb-C mice to specific antigenic determinants. Western blotting techniques were employed to visualize humoral responses to isolated antigens; serologic profiles were compared and contrasted. Four immunization protocols utilizing Freund's complete adjuvant, glucan adjuvant, lipovant adjuvant or phosphate-buffered saline, in conjunction with killed L. infantum (ex. Oklahoma) promastigotes were executed in parallel. All groups receiving adjuvant protocols developed enhanced serologic responsiveness. Similar profiles were observed in mice treated with glucan or lipovant. Animals receiving promastigotes in Freund's complete adjuvant also developed strong humoral responses, binding cross-reactive epitopes not recognized by other groups. Our findings indicate that glucan and lipovant present effective adjuvant alternatives, to Freund's complete adjuvant and may be of value in immunization against visceral leishmaniasis.
Numerous adjuvants have been utilized recently in the immunization of test animals with Leishmania sp. The adjuvant preparations include Corynebacterium parvum (Jaffe, Rachan,.:a & Sarfstein, 1990), Freund's complete adjuvant (Champsi & McMahonPratt, 1988), aluminum hydroxide (Jarecki-Black, Hallman, James & Glassman, 1988), muramyl dipeptide (Ogunkolade, Voldoukin, Frommel, Davoust, Rhodes-Feuillette & Monjour, 1988), and glucan (Obaid, Ahmad, Khan, Mahdi & Khanna, 1989). Complete or partial host resistance to subsequent infectious challenge of visceral leishmania organisms has been reported following sub-unit/adjuvant immunization protocols consisting of specific purified antigens (Jardim, Alexander, Teh, Ou & Olafson, 1990), partially purified and fractionated antigen preparations in combination with adjuvants (Ogunkolade et al., 1988), and inactivated or killed promastigotes delivered
with adjuvants (Scott, Pearce, Natovitz & Sher, 1987a). In 1980, an outbreak of canine visceral leishmaniasis was detected in an Oklahoma dog kennel (Anderson, Buckner, Glems & McVean, 1980). The isolated organism represented the first autochthonous occurrence of a visceral species of Leishmania in the United States, and it has been maintained in vitro for 6 yr. The organism has been typed and identified as Leishmania infantum. Studies have been conducted in our laboratory to determine the antigenic profile of the organism and the membrane association of certain epitopes. We have examined the immune responses of mice to killed preparations of L. infantum (ex. Oklahoma) delivered in concert with three different adjuvant preparations: CFA (a mycobacterium- oil emulsion), particulate/3-1, 3 glucan (a potent potentiator of ceilmediated immunity), and lipovant (Reynolds,
tAuthor to whom correspondence should be addressed. 767
R. M. LASAROWet al.
768
Harrington, Crabbs, Peters & Diluzio, 1980) (a lipid emulsion of peanut oil, glycerol, and lecithin). Humoral responses to L. infantum were compared and contrasted.
EXPERIMENTAL
PROCEDURES
Test animals. Specific pathogen-flee Balb-C mice were obtained from Matsunaga Tayman Scientific, San Diego, CA. Adjuvants. Freund's complete adjuvant (Difco, Detroit, MI), lipovant (Accurate Chemical & Scientific, Westbury, NY), and /3-1, 3 glucan (Accurate Chemical & Scientific) were used in this study. Biotinylated IgG and IgM heavy chainspecific antisera and peroxidase conjugated streptavidin were purchased from Zymed Laboratories, San Francisco, CA. Culture of organ&ms. Pure cultures of L. infantum (ex. Oklahoma) promastigotes were maintained in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 15°/0 fetal bovine serum, penicillin (100 IU/ml), and 0.1070 streptomycin. Cultures were maintained at room temperature in Falcon 75 cm 2 tissue culture flasks. Cultures were diluted 10-fold when a density of 10v organisms/ml was reached. Cultured organisms were washed three times in PBS (pH 7.2) prior to their use in electrophoretic and immunologic studies. Preparation of ant&era. Eight-week-old Balb-C mice were immunized intraperitoneally with 107 organisms in Freuad's complete adjuvant, PBS (pH 7.2), 20 mg lipovant, 1 mg /3-1, 3 glucan, or Freund's complete adjuvant alone. Total immunization volumes were 0.1 ml. Prior to immunization, the organisms were washed in PBS and killed by freezing and thawing three times to disrupt the membranes. Animals were given intraperitoneal booster injections of 2 × 106 organisms in PBS 4 and 8 weeks following the primary immunizations in order to increase their antibody titers. The mice were bled at 2, 4, 6, 8, 12 and 16 weeks after the initial immunizations. Serum was separated from clotted blood by centrifugation and stored at - 3 0 ° C . Pooled sera were diluted 1 : 50 or 1 : 100 prior to blot development. SDS-PAGE and Western blot electrophoresis transfer. Whole-organism homogenates were electrophoretically separated in denaturing acrylamide gels (Laemmli, 1970). Organisms were washed three
times in PBS, diluted to a concentration of 108 in 2.5 ml of sample buffer, boiled for 1 rain and aliquoted for electrophoretic and immunogenic procedures. Sample buffer was made in a twice concentration and contained 0.125 M T r i s - C1, 4°70 SDS, 20070 glycerol, 0.1 070 bromphenol blue tracking dye, and 10°70 2-mercaptoethanol in double-distilled water at a pH of 6.8. The samples were diluted 1 : 1 with the twice sample buffer prior to boiling. Molecular weight standards were diluted in sample buffer and loaded into reference wells at a concentration of 10/A/horizontal cm. Ten microliters of standard yielded 1.6/ag of protein per band. Electrophoretic separations were conducted using 0.75 mm gels with a concentration of 10070 acrylamide. Solubilized antigen was overlaid upon 5070 acrylamide stacking gels at a concentration of 20 gl per horizontal cm. Gels were electrophoresed at 15 mA and transferred electrophoretically to nitrocellulose paper (Towbin, Staehelin & Gordon, 1979) using a constant current of 200 mA (23.5 mA per cm) for 2 h. The efficiency of transfer was determined by staining the gels for residual protein following the transfer procedure. Blot and immunoblot development. Membranes were stained with India ink to visualize all transferred protein bands (Hancock & Tsang, 1983). Nitrocellulose strips were stained for 12 h with an India ink staining solution (0.1°70 v/v India ink, 5.0°7o v/v Tween 20, in T r i s - C l - b u f f e r e d saline (TBS), pH 7.5). TBS was composed of 0.02 M T r i s - C l and 0.5 M sodium chloride in doubledistilled water. The pattern of a n t i g e n - a n t i b o d y complexes was revealed by sequential incubation with pooled Balb-C antisera ( 1 : 5 0 or 1 : 1 0 0 dilution), biotinylated goat anti lgG heavy chainspecific immunoglobulin, peroxidase-conjugated streptavidin, and a final incubation with a substrate of activated 4-chloronapthol. Determination of membrane-associated protein components. Membrane-associated protein components were identified by biotinylating intact viable organisms (Hurley, Finkelstein & Hoist, 1985). Leishmania infantum (ex. Oklahoma) promastigotes were washed three times with PBS (pH 7.2), diluted to a concentration of 108 promastigotes in 10 ml of PBS and combined with 200/A of an N H S - biotin solution (20 mg per ml in DMSO). The promastigotes were incubated with agitation for 10 rain and washed three times with PBS (pH 7.2). Viability was assessed at above 95o70 by motility observation. The organisms were boiled for 3 min in 2 ml of sample buffer, separated into 100 tal aliquots and frozen at -30°C.
Adjuvant Efficacy Following Leishmania Immunization
769
M W x 103
M W x 103
,
iiii ,i ,iiiiiiii!iiii!,ili
97
J.
40
~~
29
"=P
20 i~iiiiiiiiiii
14 ~ .
117--,66---.45-
1
2
3
4
5
Fig. 1. Adjuvant efficacy. Lanes 1 - 5 contain electrophoretically separated proteins of L. infantum (ex. Oklahoma). Antiserum in lane 1 was raised against promastigotes with Freund's complete adjuvant. Antiserum in lane 2 was raised against promastigotes with glucan. Antiserum in lane 3 was raised against promastigotes with lipovant. Antiserum in lane 4 was raised against promastigotes with phosphatebuffered saline. Antiserum in lane 5 was raised against Freund's complete adjuvant alone. Antisera were obtained 16 weeks post-primary immunization. All antisera were pooled from five immunized animals and were diluted 1 : 50 prior to blot development.
2
3
4
Fig. 2. Presumptive membrane association of unique Leishmania antigens. Specific leishmanial antigenic proteins are visualized in lane 2 by immunoblot development with pooled serum from mice immunized with promastigotes and Freund's complete adjuvant. The serum was collected 16 weeks post-primary immunization and was diluted 1 : 5 0 prior to blot development. In lane 3, membrane-associated Leishmania proteins was specifically biotinylated (the use of viable organisms precluded the labeling of internal proteins), and following electrophoretic separation, they were visualized by incubation with peroxidase-conjugated streptavidin and exposure to 4-chloronapthol. Comparison of unique leishmanial antigenic bands with membrane-associated proteins led to a presumptive membrane association of 110, 100, 72, 66, 52 and 46 kDa antigens. Lane 1 illustrates molecular weight standards and lane 4 illustrates the entire complement of leishmanial proteins, both internal and membrane-associated.
770
R . M . LASAROW et al.
i.W.
~
~
~'II7K • ~" 66K
~
~-45K "~29K
WK 2
4
6
8
12
16
Fig. 3. Temporal responses to L. infantum (ex. Oklahoma) antigens. IgG antibody was detected as early as 2 weeks postprimary immunization. The antigenic profile was completely visible by week 12. The test serum was a pool of five antisera from Balb-C mice immunized with promastigotes in Freund's complete ,adjuvant and was diluted 1 : 100 prior to blot development.
Adjuvant Efficacy Following Leishmania Immunization The biotinylated proteins were run parallel with non-biotinylated promastigote proteins at a concentration of 8/al per horizontal cm on 3-27°70 gradient SDS-denaturing acrylamide gels. The nonbiotinylated sample was reacted against mouse antiL. infantum (ex. Oklahoma) antiserum and developed as described above. The biotinylated proteins were developed directly with peroxidaseconjugated streptavidin and 4-chloronapthol. RESULTS Mice immunized with L. infantum (ex. Oklahoma) in Freund's complete adjuvant (FCA) responded to proteins of the following molecular weights in kilodaltons: kDa 117, 115, 112, 110, 100, 89, 72, 66, 52, 50, 46, 45, 43, 40, 39, 35, 34, 30 and 27.5 (Fig. 1). Mice immunized with promastigotes in conjunction with glucan or lipovant responded with similar serologic profiles except that bands were weaker at 115, 110, 100, 89 and 72 kDa. Immune responses to the 34 kDa protein were completely absent. Investigation into the membrane association of leishmanial antigens was undertaken by electrophoretically comparing the relative molecular weights of discrete antigens, visualized by immunoblot development, with those of biotinylated promastigote membrane proteins (see Fig. 2). Molecular weight homology was observed at 110, 100, 72, 66, 52 and 46 kDa. Biotinylated membrane bands were too numerous to count, easily exceeding one hundred discrete proteins. Prornastigote lysates yielded still higher numbers of bands, so thick in some areas as to make visual discrimination impossible. Adjuvant efficacy was determined by comparing antisera from animals receiving primary immunizations of promastigotes with Freund's complete adjuvant, glucan adjuvant, lipovant adjuvant, or phosphate-buffered saline (Fig. 1). Glucan, lipovant, and Freund's complete adjuvant enhanced responses to antigens with molecular weights between 46 and 117 kDa. It was further observed that Freund's complete adjuvant facilitated the production of antibodies directed to a 34 kDa membraneassociated component of both L. infantum (ex. Oklahoma). This response was not observed with other immunization protocols, or in sera from animals immunized with Freund's complete adjuvant alone. The assessment of temporal antibody responses to promastigote antigens emulsified in Freund's adjuvant revealed that immunized mice elaborate IgG as early as 2 weeks post-primary immunization,
771
binding with antigenic proteins of 66, 61 and 48 kDa. By 4 weeks post-primary immunization, 117, 115, 64, 52, and 46 kDa antigens were bound. The number of antigenic proteins bound by pooled antisera increased in number and intensity of binding over time. Maximum response was reached by the 12th week post-primary immunization. No increase in antibody response was noted by week 16 (see Fig. 3). DISCUSSION The significance of cellular immune responses to Leishmania infections have been well-documented and directly related to pathogenesis (DeRossell, Bray & Alexander, 1987). Humoral responses, however, have presented a conundrum, in that infected hosts may respond polyclonally to both specific and nonspecific leishmanial antigens and may not necessarily correlate with or be required for disease resistance (Scott et al., 1987a). Recent reports of Leishmaniaspecific antigens and their protective efficacy in subunit vaccines (Jardim et al., 1990), have prompted numerous investigations concerning appropriate adjuvant delivery systems for Leishmania sub-unit vaccines. Several protective immunization mechanisms have been hypothesized including the upregulation of CD4 + cell proliferation (Jardim et al., 1990), increased secretion of IL-3 (Kahl, Scott, Lelchuk, Gregoriadis & Liew, 1989), and the upregulation macrophage-activating factor (Scott, Pearce, Natovitz & Sher, 1987b). Our findings indicate that both lipovant and glucan are effective adjuvant alternatives to Freund's complete adjuvant. Both lipovant and glucan enhance immune responses to promastigote membrane-associated antigens with molecular weights of 110, 100, 72, 66 and 52 kDa. Assuming that membrane-associated antigens provide the best immunization targets, all three adjuvants effectively appear to upregulate the humoral immune response to Leishmania promastigotes. Freund's complete adjuvant, however, has the attendant problem of severe granuloma induction (Mishell & Shiigi, 1980) and is, therefore, not a viable adjuvant for use in humans or a number of other species. Recent reports of Jaffe (Jaffe et al., 1990), have shown that Balb-C mice, immunized with a specific 72 kDa protein delivered in concert with Corynebacterium parvum adjuvant show increased resistance to L. donovani challenge. Both lipovant and glucan adjuvants augmented the responses of our animals to the 72 kDa protein (Fig. 1) and may prove to be effective alternative adjuvants in immunization against Leishmania infections.
772
R. M. LASAROWet al. REFERENCES
ANDERSON, D. C., BUCKNER,R. G., GLEMS, B. L. & MACVEAN,D. W. (1980). Endemic canine leishmaniasis. Vet. Path., 17, 94 - 96. CHAMPSI, J. & MCMAHON-PRATT, D. (1988). Membrane glycoprotein M-2 protects against Leishmania amazonensis infection. Infect. Immun., 56, 3272-3279. DEROSSELL, R. A., BRAY, R. S. & ALEXANDER,J. (1987). The correlation between delayed hypersensitivity, lymphocyte to activation and protective immunity in experimental murine leishmaniasis. Parasite Immun., 9, 105- 115. HANCOCK, K. & TSANG, V. C. W. (1983). India ink staining of proteins on nitrocellulose paper. Analyt. Biochem., 133, 157- 162. HURLEY, W. L., FINKELSTEIN, E. & HOLST, B. D. (1985). Identification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique. J. Immun. MYth., 85, 195- 202. JAFEE, C. L., RACHAMIM,N. & SARESTEIN,R. (1990). Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis. J. Immun., 144, 699- 706. JARDIM, A., ALEXANDER,J., TEH, H. S., Ou, D. W. & OLAFSON, R. W. (1990). Immunoprotective Leishmania major synthetic T cell epitopes. J. exp. Med., 172, 645- 648. JARECKI-BLACK,J. C., HALLMAN,K. L., JAMES,E. R. & GLASSMAN,A. B. (1988). Resistance against Leishmania donovani induced with an aluminium hydroxide vaccine. Ann. clin. Lab. Sci., 18, 72-77. KAHL, L. P., SCOTT, C. A., LELCHUK,R., GREGORIADIS, G. & LIEW, F. Y. (1989). Vaccination against murine cutaneous leishmaniasis by using Leishmania major antigen/liposomes. Optimization and assessment of the requirement of intravenous immunization. J. Immun., 142, 4441- 4449. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680 - 685. MISHELL, B. B. & SHIIGI, S. M. (1980). In Selected Methods in Cellular Immunology, p. 467. W. H. Freeman, San Francisco. OBAID, K. A., AHMAD, S., KHAN, H. M., MAHDI, A. A. & KHANNA, R. (1989). Protective effect of L. donovani antigens using glucan as an adjuvant. Int. J. Immunopharmac., 11, 229-235. OGUNKOLADE, B. W., VOULDOUKIN, I., FROMMEL, D., DAVOUST, B., RHODES-FEUILLETTE, A. & MONJOUR, L. (1988). Immunization of dogs with a Leishmania infantum-derived vaccine. Vet. Parasit., 28, 33 - 4 1 . REYNOLDS, J. A., HARRINGTON,D. G., CRABBS,C. L., PETERS, C. J. & DILUZIO, N. R. (1980). Adjuvant activity of a noval metabolizable lipid emulsion with inactivated viral vaccines. Infect. hnmun., 28, 937- 943. SCOTT, P., PEARCE,E., NATOVlTZ,P. & SHER, A. (1987a). Vaccination against cutaneous leishmaniasis in a murine model. 1. Induction of protective immunity with a soluble extract of promastigotes. J. Immun., 139, 221 -227. SCOTT, P., PEARCE,E., NATOVITZ,P. & SHER, A. (1987b). Vaccination against cutaneous leishmaniasis in a murine model. 11. Immunologic properties of protective and nonprotective sub fractions of soluble promastigote extract. J. Immun., 139, 3118-3125. TOWBIN, H., STAEHELIN, T. & GORDON, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. HatH. Acad. Sci. U.S.A., 76, 4350-4354.
0192-0561/92 $5.00 + .00 Pergamon Press Ltd. ~) 1992 International Society for Immunopharmacology.
HUMORAL RESPONSES FOLLOWING IMMUNIZATION WITH L E I S H M A N I A I N F A N T U M (EX. OKLAHOMA): A COMPARISON OF ADJUVANT EFFICACY IN THE ANTIBODY RESPONSES OF BALB-C MICE R. M.
LASAROW, *f
D. L.
WILLIAMS ~
and J. H. THEIS*
*Department of Medical Microbiology and Immunology, University of California, Davis, School of Medicine, Davis, CA 95616; *Department of Physiology, Tulane University, School of Medicine, New Orleans, Louisiana, U.S.A. (Received 20 September 1991 and in final form 10 January 1992)
Abstract -- Adjuvants are commonly used in immunization protocols for the purpose of augmenting
immune responses to antigens. The antigenic profile of Leishmania infantum (ex. Oklahoma) is described and the efficacies of three adjuvants delivered coincidentally with killed promastigotes are compared, by measuring relative serologic responses of Balb-C mice to specific antigenic determinants. Western blotting techniques were employed to visualize humoral responses to isolated antigens; serologic profiles were compared and contrasted. Four immunization protocols utilizing Freund's complete adjuvant, glucan adjuvant, lipovant adjuvant or phosphate-buffered saline, in conjunction with killed L. infantum (ex. Oklahoma) promastigotes were executed in parallel. All groups receiving adjuvant protocols developed enhanced serologic responsiveness. Similar profiles were observed in mice treated with glucan or lipovant. Animals receiving promastigotes in Freund's complete adjuvant also developed strong humoral responses, binding cross-reactive epitopes not recognized by other groups. Our findings indicate that glucan and lipovant present effective adjuvant alternatives, to Freund's complete adjuvant and may be of value in immunization against visceral leishmaniasis.
Numerous adjuvants have been utilized recently in the immunization of test animals with Leishmania sp. The adjuvant preparations include Corynebacterium parvum (Jaffe, Rachan,.:a & Sarfstein, 1990), Freund's complete adjuvant (Champsi & McMahonPratt, 1988), aluminum hydroxide (Jarecki-Black, Hallman, James & Glassman, 1988), muramyl dipeptide (Ogunkolade, Voldoukin, Frommel, Davoust, Rhodes-Feuillette & Monjour, 1988), and glucan (Obaid, Ahmad, Khan, Mahdi & Khanna, 1989). Complete or partial host resistance to subsequent infectious challenge of visceral leishmania organisms has been reported following sub-unit/adjuvant immunization protocols consisting of specific purified antigens (Jardim, Alexander, Teh, Ou & Olafson, 1990), partially purified and fractionated antigen preparations in combination with adjuvants (Ogunkolade et al., 1988), and inactivated or killed promastigotes delivered
with adjuvants (Scott, Pearce, Natovitz & Sher, 1987a). In 1980, an outbreak of canine visceral leishmaniasis was detected in an Oklahoma dog kennel (Anderson, Buckner, Glems & McVean, 1980). The isolated organism represented the first autochthonous occurrence of a visceral species of Leishmania in the United States, and it has been maintained in vitro for 6 yr. The organism has been typed and identified as Leishmania infantum. Studies have been conducted in our laboratory to determine the antigenic profile of the organism and the membrane association of certain epitopes. We have examined the immune responses of mice to killed preparations of L. infantum (ex. Oklahoma) delivered in concert with three different adjuvant preparations: CFA (a mycobacterium- oil emulsion), particulate/3-1, 3 glucan (a potent potentiator of ceilmediated immunity), and lipovant (Reynolds,
tAuthor to whom correspondence should be addressed. 767
R. M. LASAROWet al.
768
Harrington, Crabbs, Peters & Diluzio, 1980) (a lipid emulsion of peanut oil, glycerol, and lecithin). Humoral responses to L. infantum were compared and contrasted.
EXPERIMENTAL
PROCEDURES
Test animals. Specific pathogen-flee Balb-C mice were obtained from Matsunaga Tayman Scientific, San Diego, CA. Adjuvants. Freund's complete adjuvant (Difco, Detroit, MI), lipovant (Accurate Chemical & Scientific, Westbury, NY), and /3-1, 3 glucan (Accurate Chemical & Scientific) were used in this study. Biotinylated IgG and IgM heavy chainspecific antisera and peroxidase conjugated streptavidin were purchased from Zymed Laboratories, San Francisco, CA. Culture of organ&ms. Pure cultures of L. infantum (ex. Oklahoma) promastigotes were maintained in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 15°/0 fetal bovine serum, penicillin (100 IU/ml), and 0.1070 streptomycin. Cultures were maintained at room temperature in Falcon 75 cm 2 tissue culture flasks. Cultures were diluted 10-fold when a density of 10v organisms/ml was reached. Cultured organisms were washed three times in PBS (pH 7.2) prior to their use in electrophoretic and immunologic studies. Preparation of ant&era. Eight-week-old Balb-C mice were immunized intraperitoneally with 107 organisms in Freuad's complete adjuvant, PBS (pH 7.2), 20 mg lipovant, 1 mg /3-1, 3 glucan, or Freund's complete adjuvant alone. Total immunization volumes were 0.1 ml. Prior to immunization, the organisms were washed in PBS and killed by freezing and thawing three times to disrupt the membranes. Animals were given intraperitoneal booster injections of 2 × 106 organisms in PBS 4 and 8 weeks following the primary immunizations in order to increase their antibody titers. The mice were bled at 2, 4, 6, 8, 12 and 16 weeks after the initial immunizations. Serum was separated from clotted blood by centrifugation and stored at - 3 0 ° C . Pooled sera were diluted 1 : 50 or 1 : 100 prior to blot development. SDS-PAGE and Western blot electrophoresis transfer. Whole-organism homogenates were electrophoretically separated in denaturing acrylamide gels (Laemmli, 1970). Organisms were washed three
times in PBS, diluted to a concentration of 108 in 2.5 ml of sample buffer, boiled for 1 rain and aliquoted for electrophoretic and immunogenic procedures. Sample buffer was made in a twice concentration and contained 0.125 M T r i s - C1, 4°70 SDS, 20070 glycerol, 0.1 070 bromphenol blue tracking dye, and 10°70 2-mercaptoethanol in double-distilled water at a pH of 6.8. The samples were diluted 1 : 1 with the twice sample buffer prior to boiling. Molecular weight standards were diluted in sample buffer and loaded into reference wells at a concentration of 10/A/horizontal cm. Ten microliters of standard yielded 1.6/ag of protein per band. Electrophoretic separations were conducted using 0.75 mm gels with a concentration of 10070 acrylamide. Solubilized antigen was overlaid upon 5070 acrylamide stacking gels at a concentration of 20 gl per horizontal cm. Gels were electrophoresed at 15 mA and transferred electrophoretically to nitrocellulose paper (Towbin, Staehelin & Gordon, 1979) using a constant current of 200 mA (23.5 mA per cm) for 2 h. The efficiency of transfer was determined by staining the gels for residual protein following the transfer procedure. Blot and immunoblot development. Membranes were stained with India ink to visualize all transferred protein bands (Hancock & Tsang, 1983). Nitrocellulose strips were stained for 12 h with an India ink staining solution (0.1°70 v/v India ink, 5.0°7o v/v Tween 20, in T r i s - C l - b u f f e r e d saline (TBS), pH 7.5). TBS was composed of 0.02 M T r i s - C l and 0.5 M sodium chloride in doubledistilled water. The pattern of a n t i g e n - a n t i b o d y complexes was revealed by sequential incubation with pooled Balb-C antisera ( 1 : 5 0 or 1 : 1 0 0 dilution), biotinylated goat anti lgG heavy chainspecific immunoglobulin, peroxidase-conjugated streptavidin, and a final incubation with a substrate of activated 4-chloronapthol. Determination of membrane-associated protein components. Membrane-associated protein components were identified by biotinylating intact viable organisms (Hurley, Finkelstein & Hoist, 1985). Leishmania infantum (ex. Oklahoma) promastigotes were washed three times with PBS (pH 7.2), diluted to a concentration of 108 promastigotes in 10 ml of PBS and combined with 200/A of an N H S - biotin solution (20 mg per ml in DMSO). The promastigotes were incubated with agitation for 10 rain and washed three times with PBS (pH 7.2). Viability was assessed at above 95o70 by motility observation. The organisms were boiled for 3 min in 2 ml of sample buffer, separated into 100 tal aliquots and frozen at -30°C.
Adjuvant Efficacy Following Leishmania Immunization
769
M W x 103
M W x 103
,
iiii ,i ,iiiiiiii!iiii!,ili
97
J.
40
~~
29
"=P
20 i~iiiiiiiiiii
14 ~ .
117--,66---.45-
1
2
3
4
5
Fig. 1. Adjuvant efficacy. Lanes 1 - 5 contain electrophoretically separated proteins of L. infantum (ex. Oklahoma). Antiserum in lane 1 was raised against promastigotes with Freund's complete adjuvant. Antiserum in lane 2 was raised against promastigotes with glucan. Antiserum in lane 3 was raised against promastigotes with lipovant. Antiserum in lane 4 was raised against promastigotes with phosphatebuffered saline. Antiserum in lane 5 was raised against Freund's complete adjuvant alone. Antisera were obtained 16 weeks post-primary immunization. All antisera were pooled from five immunized animals and were diluted 1 : 50 prior to blot development.
2
3
4
Fig. 2. Presumptive membrane association of unique Leishmania antigens. Specific leishmanial antigenic proteins are visualized in lane 2 by immunoblot development with pooled serum from mice immunized with promastigotes and Freund's complete adjuvant. The serum was collected 16 weeks post-primary immunization and was diluted 1 : 5 0 prior to blot development. In lane 3, membrane-associated Leishmania proteins was specifically biotinylated (the use of viable organisms precluded the labeling of internal proteins), and following electrophoretic separation, they were visualized by incubation with peroxidase-conjugated streptavidin and exposure to 4-chloronapthol. Comparison of unique leishmanial antigenic bands with membrane-associated proteins led to a presumptive membrane association of 110, 100, 72, 66, 52 and 46 kDa antigens. Lane 1 illustrates molecular weight standards and lane 4 illustrates the entire complement of leishmanial proteins, both internal and membrane-associated.
770
R . M . LASAROW et al.
i.W.
~
~
~'II7K • ~" 66K
~
~-45K "~29K
WK 2
4
6
8
12
16
Fig. 3. Temporal responses to L. infantum (ex. Oklahoma) antigens. IgG antibody was detected as early as 2 weeks postprimary immunization. The antigenic profile was completely visible by week 12. The test serum was a pool of five antisera from Balb-C mice immunized with promastigotes in Freund's complete ,adjuvant and was diluted 1 : 100 prior to blot development.
Adjuvant Efficacy Following Leishmania Immunization The biotinylated proteins were run parallel with non-biotinylated promastigote proteins at a concentration of 8/al per horizontal cm on 3-27°70 gradient SDS-denaturing acrylamide gels. The nonbiotinylated sample was reacted against mouse antiL. infantum (ex. Oklahoma) antiserum and developed as described above. The biotinylated proteins were developed directly with peroxidaseconjugated streptavidin and 4-chloronapthol. RESULTS Mice immunized with L. infantum (ex. Oklahoma) in Freund's complete adjuvant (FCA) responded to proteins of the following molecular weights in kilodaltons: kDa 117, 115, 112, 110, 100, 89, 72, 66, 52, 50, 46, 45, 43, 40, 39, 35, 34, 30 and 27.5 (Fig. 1). Mice immunized with promastigotes in conjunction with glucan or lipovant responded with similar serologic profiles except that bands were weaker at 115, 110, 100, 89 and 72 kDa. Immune responses to the 34 kDa protein were completely absent. Investigation into the membrane association of leishmanial antigens was undertaken by electrophoretically comparing the relative molecular weights of discrete antigens, visualized by immunoblot development, with those of biotinylated promastigote membrane proteins (see Fig. 2). Molecular weight homology was observed at 110, 100, 72, 66, 52 and 46 kDa. Biotinylated membrane bands were too numerous to count, easily exceeding one hundred discrete proteins. Prornastigote lysates yielded still higher numbers of bands, so thick in some areas as to make visual discrimination impossible. Adjuvant efficacy was determined by comparing antisera from animals receiving primary immunizations of promastigotes with Freund's complete adjuvant, glucan adjuvant, lipovant adjuvant, or phosphate-buffered saline (Fig. 1). Glucan, lipovant, and Freund's complete adjuvant enhanced responses to antigens with molecular weights between 46 and 117 kDa. It was further observed that Freund's complete adjuvant facilitated the production of antibodies directed to a 34 kDa membraneassociated component of both L. infantum (ex. Oklahoma). This response was not observed with other immunization protocols, or in sera from animals immunized with Freund's complete adjuvant alone. The assessment of temporal antibody responses to promastigote antigens emulsified in Freund's adjuvant revealed that immunized mice elaborate IgG as early as 2 weeks post-primary immunization,
771
binding with antigenic proteins of 66, 61 and 48 kDa. By 4 weeks post-primary immunization, 117, 115, 64, 52, and 46 kDa antigens were bound. The number of antigenic proteins bound by pooled antisera increased in number and intensity of binding over time. Maximum response was reached by the 12th week post-primary immunization. No increase in antibody response was noted by week 16 (see Fig. 3). DISCUSSION The significance of cellular immune responses to Leishmania infections have been well-documented and directly related to pathogenesis (DeRossell, Bray & Alexander, 1987). Humoral responses, however, have presented a conundrum, in that infected hosts may respond polyclonally to both specific and nonspecific leishmanial antigens and may not necessarily correlate with or be required for disease resistance (Scott et al., 1987a). Recent reports of Leishmaniaspecific antigens and their protective efficacy in subunit vaccines (Jardim et al., 1990), have prompted numerous investigations concerning appropriate adjuvant delivery systems for Leishmania sub-unit vaccines. Several protective immunization mechanisms have been hypothesized including the upregulation of CD4 + cell proliferation (Jardim et al., 1990), increased secretion of IL-3 (Kahl, Scott, Lelchuk, Gregoriadis & Liew, 1989), and the upregulation macrophage-activating factor (Scott, Pearce, Natovitz & Sher, 1987b). Our findings indicate that both lipovant and glucan are effective adjuvant alternatives to Freund's complete adjuvant. Both lipovant and glucan enhance immune responses to promastigote membrane-associated antigens with molecular weights of 110, 100, 72, 66 and 52 kDa. Assuming that membrane-associated antigens provide the best immunization targets, all three adjuvants effectively appear to upregulate the humoral immune response to Leishmania promastigotes. Freund's complete adjuvant, however, has the attendant problem of severe granuloma induction (Mishell & Shiigi, 1980) and is, therefore, not a viable adjuvant for use in humans or a number of other species. Recent reports of Jaffe (Jaffe et al., 1990), have shown that Balb-C mice, immunized with a specific 72 kDa protein delivered in concert with Corynebacterium parvum adjuvant show increased resistance to L. donovani challenge. Both lipovant and glucan adjuvants augmented the responses of our animals to the 72 kDa protein (Fig. 1) and may prove to be effective alternative adjuvants in immunization against Leishmania infections.
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R. M. LASAROWet al. REFERENCES
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