Immunology 1992 77 123-128
Liposome potentiation of humoral immune response to lipopolysaccharide and O-polysaccharide antigens of Brucella abortus J. P. WONG, J. W. CHERWONOGRODZKY, V. L. DI NINNO, L. L. STADNYK & M. H. KNODEL Biomedical Defence Section, Defence Research Establishment Suffield, Ralston, Alberta, Canada
Acceptedfor publication 23 April 1992
SUMMARY Liposomes were evaluated for their effectiveness as vaccine carriers in the potentiation of the mouse humoral response to the lipopolysaccharide (LPS) and O-polysaccharide (OPS) antigens of Brucella abortus. LPS and OPS were extracted from a pathogenic strain of B. abortus and were encapsulated within multilamellar vesicles. Groups of mice, immunized with liposome-encapsulated and free LPS or OPS, were bled weekly and the specific IgM and IgG levels in the sera were determined by an indirect fluorogenic enzyme-linked immunosorbent assay. Humoral responses to these antigens were found to be dose-dependent. Mice immunized with LPS and OPS encapsulated within liposomes were found to have significantly higher IgG levels than mice immunized with free LPS and OPS. In addition, the antibody levels in mice that were immunized with liposome-encapsulated LPS and OPS were more sustained and remained at elevated levels-even after 5 weeks post immunization. As expected, OPS was found to be less immunogenic than LPS, but multiple injections of OPS encapsulated within liposomes greatly improved the immunogenicity. These results indicate that the humoral response to LPS and OPS of B. abortus can be enhanced when these antigens are encapsulated within liposomes. INTRODUCTION Brucella abortus, the causative agent of brucellosis, can cause spontaneous abortions in cattle and persistent undulant fever in humans. At present, there are no effective vaccines against brucellosis, and indeed, the live attentuated strains used to immunize cattle do not offer absolute protection, ' may revert to pathogenic forms,2 and are pathogenic for humans.3 Purified antigens from B. abortus have been tested and results indicate that the protection is due to the O-polysaccharide (OPS) of smooth lipopolysaccharide (LPS).4 However, OPS by itself is weakly or non-immunogenic and to improve its immunogenicity, OPS must either be conjugated to another component (e.g. protein) or form part of a larger molecule (e.g. LPS). Subcellular vaccines using B. abortus OPS covalently bound to porin have recently been reported but the observed protection was partial and temporary.5 Porin by itself may cause a marked inflammatory response when injected intradermally6 and as noted above does not confer significant protection. The poor immunogenicity of OPS can potentially be overcome by utilizing liposomes as carriers. Liposomes are lipid vesicles which are compatible with cell membranes, readily biodegradable in the host and non-toxic. The encapsulation of
LPS and OPS in these artificial membranes may, therefore, eliminate the side-effects observed when using whole bacteria for immunization and, at the same time, enhance the inherent immunogenicity of these antigens. In this study, we have evaluated liposomes for their effectiveness as vaccine carriers for the potentiation of the mouse humoral response to LPS and OPS antigens.
MATERIALS AND METHODS Animals All mice used in this study were 15-16 g BALB/c females purchased from Charles River Ltd (St Constant, Quebec, Canada). Upon arrival at DRES, the mice were placed in quarantine in the vivarium for 1 week and were housed and cared for in a manner consistent with the guidelines set by the Canadian Council on Animal Care. Chemicals
Phosphatidylcholine, from egg yolk, phosphatidylserine, from bovine brain, and cholesterol used in the preparations of liposomes were purchased from Sigma Chemical Company (St Louis, MO). Immunoassay Alkaline phosphatase-labelled goat anti-mouse IgG and antiIgM conjugates were purchased from Sigma Chemical Com-
Correspondence: J. P. Wong, Biomedical Defence Section, Defence Research Estabishment Suffield, Box 4000, Medicine Hat, Alberta, T IA 8K6, Canada.
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pany. Fluorogenic enzyme-linked immunosorbent assays (FELISA) used in this study for the titrations of antibody in sera were performed on MillititerTm - HA plates using the MillititerTM filtration system (Millipore Corporation, Bedford, MA.). LPS and OPS purification LPS and OPS from B. abortus strain 413 were purified as described previously.7 Briefly, killed cells were first washed with ethanol, acetone then diethyl ether, then dried under vacuum and resuspended in saline. Crude LPS was removed by a hot phenol-water extraction (phenol layer) and the phenol was removed by methanol-acetate washes followed by dialysis. Final purification of LPS was achieved by RNase, DNase and proteinase-K digestion followed by ultracentrifugation. The gelatinous pellet was LPS. OPS was isolated by autoclaving the cells in 10% NaCl and 2% acetic acid. The OPS was precipitated from the supernatant by methanol-acetate, dialysed, digested with enzymes, extracted with hot phenol-water and then further purified with column chromatography. Both LPS and OPS preparations contained less than 1 % protein.
Preparation of liposomes Liposomes were prepared by a modification of the freeze-drying method of Kirby & Gregoriadis.8 Negatively charged liposomes were prepared using phosphatidylcholine:cholesterol:phosphatidylserine in a molar ratio of 7:2: 1. Briefly, a total of 20 2 ymol oflipids in 1 ml ofchloroform:methanol (2: 1) was dried to a thin film on the bottom of a screw-capped tube by heating at 45° in a heating block. Throughout this procedure, the content of the tube was purged whenever possible with a gentle stream of dry nitrogen. The lipids were further dried for 30 min in a vacuum chamber to remove remaining organic solvent. The dried lipid film was then rehydrated with 1 ml distilled water and the contents were vigorously vortexed, followed by heating at 45°. The vortexing-heating cycles were repeated between 15 and 25 times, under dry nitrogen, until the dried lipids were completely dislodged from the sides of the tube (required approx. 20 min to achieve). The LPS or OPS to be encapsulated, which had been dissolved in 2400 pl of sterile phosphate-buffered saline (150 mm NaCl and 150 mm NaH2PO4, pH 7-3), was then added to the tube. The lipid-antigen mixture was gently vortexed for approximately 20 seconds and was then frozen rapidly in a dry-icemethanol mixture. The sample was then freeze-dried overnight in a lyophilizer (Virtis Company Inc., Gardiner, NY). The freeze-dried mixture was reconstituted in 100 pl of 100 mM HEPES buffer in normal saline, pH 6-7. The reconstituted liposomes were then vortexed for 2-3 min and left undisturbed at room temperature for I hr. The liposomes were washed with 8 ml of HEPES buffer, then ultracentrifuged at 125,000 g for 30 min at 4°. The pellet was washed with HEPES buffer and ultracentrifuged for another 30 min. The final pellet was then reconstituted in 1 ml of HEPES buffer. The pooled supernatant from these washes was assayed for LPS by an indirect FELISA, described below, to determine the amount of LPS not encapsulated into the liposomes. Entrapment efficiency of LPS, defined as the percentage ratio of encapsulated LPS (total LPS added to lipid mixture minus amount of unencapsulated LPS in supernatant) to total LPS added, was found to be approximately 45% + 4%. Attempts to determine similar entrapment efficiency or to quantitate OPS by FELISA was not successful as OPS has a very low binding capacity to solid-phase supports such as
nitrocellulose and polystyrene. However, previous studies with '25I-IgG and Newcastle disease virus protein have shown that the freeze-drying method produced multilamellar vesicles encapsulating approximately 45% of materials in the aqueous phase (data not shown). All doses of LPS and OPS in liposomes used in subsequent immunizations have been corrected using this 45% factor in entrapment efficiency. Immunization of mice BALB/c mice were immunized with 1, 10, or 100 pg LPS, OPS, liposome-encapsulated LPS (LIP-LPS) or liposome-encapsulated OPS (LIP-OPS), as indicated. Groups of three mice were injected either with LPS, OPS, LIP-LPS or LIP-OPS at two intramuscular and two subcutaneous sites (thigh and back, respectively). In all immunizations with liposomes, each mouse was injected with liposomes made from 1 pmol lipid and containing 1, 10 or 100 pg LPS or OPS. The mice were given either single or multiple doses (one primary and two booster injections at weeks 0, 1 and 5) and were bled weekly from the tail vein.
Antibody and LPS assay Specific IgG and IgM levels against LPS and OPS in serum samples from the weekly bleedings were assayed by an indirect FELISA, as described by Fulton et al.,9 with the following modifications. The wells ofmicrotitre plates were coated with 50 p1 of B. abortus LPS (20 pg/ml of 0 05 M carbonate-bicarbonate buffer, pH 9-6). Specific antibody titres against OPS were also determined using LPS as the detecting antigen as OPS did not bind efficiently to the solid phase. After the blocking step, serum samples were diluted serially in blocking buffer before being added to the wells. The specific IgG and IgM levels were detected by alkaline phosphatase-labelled anti-mouse IgM or IgG conjugates. The indirect FELISA for the quantitation of LPS was essentially the same as that described for the antibody assay but with the following modifications. After the blocking step, a purified rabbit anti-Brucella abortus LPS antibody (IgG), diluted 1:200 in blocking buffer, was added to the wells. The antibody-enzyme conjugate used was alkaline phosphataselabelled anti-rabbit IgG, diluted 1:500 in blocking buffer. Statistical analysis Statistical differences in antibody titres between mice immunized with non-encapsulated and liposome-encapsulated antigens were analysed by one-way analysis of variance, and were performed using the multivariate general linear hypothesis module of the Systat Computer Software Program from Systat, Inc. (Evanston, IL). RESULTS Effect of antigen dose on level of humoral immune response The mouse humoral responses to single and multiple immunizations with LPS, LPS-LIP, OPS and OPS-LIP were dosedependent. The greater the amounts of immunogens administered, the higher the antibody responses observed. A typical dose-response relationship of antibody level with increasing amounts of antigens is shown in the IgG level of mice immunized with liposomes containing 1, 10, or 100 pg LPS (Fig.
125
Humoral immune responses to B. abortus antigens * t*
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Week Figure 1. The effect of dose of LIP-LPS on the level of antibody (IgG) response to B. abortus LPS. Groups of three mice were immunized intramuscularly and subcutaneously with LIP-LPS containing 1, 10, and 100 pg LPS per mouse. The mice were bled weekly and the antibody titres in the serum samples were titrated by indirect FELISA. Arrows indicate times of injections. * Antibody titre significantly different from that of mice immunized with pg LPS (P < 005). t Antibody titre significantly different from that of mice immunized with 10 pg LPS (P < 005).
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Figure 3. IgG (a) and IgM (b) responses to multiple immunizations with 100 pg LPS or LIP-LPS containing 00 pg LPS. Arrows indicate times of injection. * Antibody titre significantly different from that of mice immunized with free antigen (P
Liposome potentiation of humoral immune response to lipopolysaccharide and O-polysaccharide antigens of Brucella abortus J. P. WONG, J. W. CHERWONOGRODZKY, V. L. DI NINNO, L. L. STADNYK & M. H. KNODEL Biomedical Defence Section, Defence Research Establishment Suffield, Ralston, Alberta, Canada
Acceptedfor publication 23 April 1992
SUMMARY Liposomes were evaluated for their effectiveness as vaccine carriers in the potentiation of the mouse humoral response to the lipopolysaccharide (LPS) and O-polysaccharide (OPS) antigens of Brucella abortus. LPS and OPS were extracted from a pathogenic strain of B. abortus and were encapsulated within multilamellar vesicles. Groups of mice, immunized with liposome-encapsulated and free LPS or OPS, were bled weekly and the specific IgM and IgG levels in the sera were determined by an indirect fluorogenic enzyme-linked immunosorbent assay. Humoral responses to these antigens were found to be dose-dependent. Mice immunized with LPS and OPS encapsulated within liposomes were found to have significantly higher IgG levels than mice immunized with free LPS and OPS. In addition, the antibody levels in mice that were immunized with liposome-encapsulated LPS and OPS were more sustained and remained at elevated levels-even after 5 weeks post immunization. As expected, OPS was found to be less immunogenic than LPS, but multiple injections of OPS encapsulated within liposomes greatly improved the immunogenicity. These results indicate that the humoral response to LPS and OPS of B. abortus can be enhanced when these antigens are encapsulated within liposomes. INTRODUCTION Brucella abortus, the causative agent of brucellosis, can cause spontaneous abortions in cattle and persistent undulant fever in humans. At present, there are no effective vaccines against brucellosis, and indeed, the live attentuated strains used to immunize cattle do not offer absolute protection, ' may revert to pathogenic forms,2 and are pathogenic for humans.3 Purified antigens from B. abortus have been tested and results indicate that the protection is due to the O-polysaccharide (OPS) of smooth lipopolysaccharide (LPS).4 However, OPS by itself is weakly or non-immunogenic and to improve its immunogenicity, OPS must either be conjugated to another component (e.g. protein) or form part of a larger molecule (e.g. LPS). Subcellular vaccines using B. abortus OPS covalently bound to porin have recently been reported but the observed protection was partial and temporary.5 Porin by itself may cause a marked inflammatory response when injected intradermally6 and as noted above does not confer significant protection. The poor immunogenicity of OPS can potentially be overcome by utilizing liposomes as carriers. Liposomes are lipid vesicles which are compatible with cell membranes, readily biodegradable in the host and non-toxic. The encapsulation of
LPS and OPS in these artificial membranes may, therefore, eliminate the side-effects observed when using whole bacteria for immunization and, at the same time, enhance the inherent immunogenicity of these antigens. In this study, we have evaluated liposomes for their effectiveness as vaccine carriers for the potentiation of the mouse humoral response to LPS and OPS antigens.
MATERIALS AND METHODS Animals All mice used in this study were 15-16 g BALB/c females purchased from Charles River Ltd (St Constant, Quebec, Canada). Upon arrival at DRES, the mice were placed in quarantine in the vivarium for 1 week and were housed and cared for in a manner consistent with the guidelines set by the Canadian Council on Animal Care. Chemicals
Phosphatidylcholine, from egg yolk, phosphatidylserine, from bovine brain, and cholesterol used in the preparations of liposomes were purchased from Sigma Chemical Company (St Louis, MO). Immunoassay Alkaline phosphatase-labelled goat anti-mouse IgG and antiIgM conjugates were purchased from Sigma Chemical Com-
Correspondence: J. P. Wong, Biomedical Defence Section, Defence Research Estabishment Suffield, Box 4000, Medicine Hat, Alberta, T IA 8K6, Canada.
123
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J. P. Wong et al.
pany. Fluorogenic enzyme-linked immunosorbent assays (FELISA) used in this study for the titrations of antibody in sera were performed on MillititerTm - HA plates using the MillititerTM filtration system (Millipore Corporation, Bedford, MA.). LPS and OPS purification LPS and OPS from B. abortus strain 413 were purified as described previously.7 Briefly, killed cells were first washed with ethanol, acetone then diethyl ether, then dried under vacuum and resuspended in saline. Crude LPS was removed by a hot phenol-water extraction (phenol layer) and the phenol was removed by methanol-acetate washes followed by dialysis. Final purification of LPS was achieved by RNase, DNase and proteinase-K digestion followed by ultracentrifugation. The gelatinous pellet was LPS. OPS was isolated by autoclaving the cells in 10% NaCl and 2% acetic acid. The OPS was precipitated from the supernatant by methanol-acetate, dialysed, digested with enzymes, extracted with hot phenol-water and then further purified with column chromatography. Both LPS and OPS preparations contained less than 1 % protein.
Preparation of liposomes Liposomes were prepared by a modification of the freeze-drying method of Kirby & Gregoriadis.8 Negatively charged liposomes were prepared using phosphatidylcholine:cholesterol:phosphatidylserine in a molar ratio of 7:2: 1. Briefly, a total of 20 2 ymol oflipids in 1 ml ofchloroform:methanol (2: 1) was dried to a thin film on the bottom of a screw-capped tube by heating at 45° in a heating block. Throughout this procedure, the content of the tube was purged whenever possible with a gentle stream of dry nitrogen. The lipids were further dried for 30 min in a vacuum chamber to remove remaining organic solvent. The dried lipid film was then rehydrated with 1 ml distilled water and the contents were vigorously vortexed, followed by heating at 45°. The vortexing-heating cycles were repeated between 15 and 25 times, under dry nitrogen, until the dried lipids were completely dislodged from the sides of the tube (required approx. 20 min to achieve). The LPS or OPS to be encapsulated, which had been dissolved in 2400 pl of sterile phosphate-buffered saline (150 mm NaCl and 150 mm NaH2PO4, pH 7-3), was then added to the tube. The lipid-antigen mixture was gently vortexed for approximately 20 seconds and was then frozen rapidly in a dry-icemethanol mixture. The sample was then freeze-dried overnight in a lyophilizer (Virtis Company Inc., Gardiner, NY). The freeze-dried mixture was reconstituted in 100 pl of 100 mM HEPES buffer in normal saline, pH 6-7. The reconstituted liposomes were then vortexed for 2-3 min and left undisturbed at room temperature for I hr. The liposomes were washed with 8 ml of HEPES buffer, then ultracentrifuged at 125,000 g for 30 min at 4°. The pellet was washed with HEPES buffer and ultracentrifuged for another 30 min. The final pellet was then reconstituted in 1 ml of HEPES buffer. The pooled supernatant from these washes was assayed for LPS by an indirect FELISA, described below, to determine the amount of LPS not encapsulated into the liposomes. Entrapment efficiency of LPS, defined as the percentage ratio of encapsulated LPS (total LPS added to lipid mixture minus amount of unencapsulated LPS in supernatant) to total LPS added, was found to be approximately 45% + 4%. Attempts to determine similar entrapment efficiency or to quantitate OPS by FELISA was not successful as OPS has a very low binding capacity to solid-phase supports such as
nitrocellulose and polystyrene. However, previous studies with '25I-IgG and Newcastle disease virus protein have shown that the freeze-drying method produced multilamellar vesicles encapsulating approximately 45% of materials in the aqueous phase (data not shown). All doses of LPS and OPS in liposomes used in subsequent immunizations have been corrected using this 45% factor in entrapment efficiency. Immunization of mice BALB/c mice were immunized with 1, 10, or 100 pg LPS, OPS, liposome-encapsulated LPS (LIP-LPS) or liposome-encapsulated OPS (LIP-OPS), as indicated. Groups of three mice were injected either with LPS, OPS, LIP-LPS or LIP-OPS at two intramuscular and two subcutaneous sites (thigh and back, respectively). In all immunizations with liposomes, each mouse was injected with liposomes made from 1 pmol lipid and containing 1, 10 or 100 pg LPS or OPS. The mice were given either single or multiple doses (one primary and two booster injections at weeks 0, 1 and 5) and were bled weekly from the tail vein.
Antibody and LPS assay Specific IgG and IgM levels against LPS and OPS in serum samples from the weekly bleedings were assayed by an indirect FELISA, as described by Fulton et al.,9 with the following modifications. The wells ofmicrotitre plates were coated with 50 p1 of B. abortus LPS (20 pg/ml of 0 05 M carbonate-bicarbonate buffer, pH 9-6). Specific antibody titres against OPS were also determined using LPS as the detecting antigen as OPS did not bind efficiently to the solid phase. After the blocking step, serum samples were diluted serially in blocking buffer before being added to the wells. The specific IgG and IgM levels were detected by alkaline phosphatase-labelled anti-mouse IgM or IgG conjugates. The indirect FELISA for the quantitation of LPS was essentially the same as that described for the antibody assay but with the following modifications. After the blocking step, a purified rabbit anti-Brucella abortus LPS antibody (IgG), diluted 1:200 in blocking buffer, was added to the wells. The antibody-enzyme conjugate used was alkaline phosphataselabelled anti-rabbit IgG, diluted 1:500 in blocking buffer. Statistical analysis Statistical differences in antibody titres between mice immunized with non-encapsulated and liposome-encapsulated antigens were analysed by one-way analysis of variance, and were performed using the multivariate general linear hypothesis module of the Systat Computer Software Program from Systat, Inc. (Evanston, IL). RESULTS Effect of antigen dose on level of humoral immune response The mouse humoral responses to single and multiple immunizations with LPS, LPS-LIP, OPS and OPS-LIP were dosedependent. The greater the amounts of immunogens administered, the higher the antibody responses observed. A typical dose-response relationship of antibody level with increasing amounts of antigens is shown in the IgG level of mice immunized with liposomes containing 1, 10, or 100 pg LPS (Fig.
125
Humoral immune responses to B. abortus antigens * t*
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Week Figure 1. The effect of dose of LIP-LPS on the level of antibody (IgG) response to B. abortus LPS. Groups of three mice were immunized intramuscularly and subcutaneously with LIP-LPS containing 1, 10, and 100 pg LPS per mouse. The mice were bled weekly and the antibody titres in the serum samples were titrated by indirect FELISA. Arrows indicate times of injections. * Antibody titre significantly different from that of mice immunized with pg LPS (P < 005). t Antibody titre significantly different from that of mice immunized with 10 pg LPS (P < 005).
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Figure 3. IgG (a) and IgM (b) responses to multiple immunizations with 100 pg LPS or LIP-LPS containing 00 pg LPS. Arrows indicate times of injection. * Antibody titre significantly different from that of mice immunized with free antigen (P
