Abstract
Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.
Development of an effective vaccine against brucellosis for human use is now more important due to the possibility that brucella could be used as a biowarfare agent. The Centers for Disease Control and Prevention has designated Brucella a class B bioterrorist threat agent (6, 27). At present, there is no vaccine licensed for use against brucellosis in humans. Several live attenuated brucella vaccines have been tried in humans, but none was found to be satisfactory. A derivative of Brucella abortus strain 19, designated 19-BA, was developed and used in the former Soviet Union and was shown to be protective in humans (11). However, when tested in United States volunteers, this vaccine was found to be insufficiently attenuated (29).
A live attenuated Brucella melitensis strain Rev1 vaccine used for immunization of goats and sheep was found to be unsuitable for human use (24). It has been reported that two veterinarians who were vaccinating sheep with Rev1 became infected with this organism (3). More recently, a genetically defined, live, attenuated purEK mutant of B. melitensis strain 16M was developed and tested in mice (9). This mutant strain, strain WR201, was found to protect against disseminated infection of spleens and livers of immunized mice and slightly enhanced the clearance of bacteria from the lungs (16). Further testing of this vaccine strain in nonhuman primates showed that it is highly attenuated and protective against aerosol challenge with strain 16M. However, one colony of WR201 was recovered from the testis of one of four immunized monkeys, and one colony was recovered from a lymph node of another monkey at 8 weeks postchallenge (15). A number of genetically defined mutants that are attenuated for growth in macrophages or in animal models have been developed recently, but their suitability for human use has not been evaluated (10, 12, 20, 26, 33).
There have been a number of studies to show the protective effect of brucella subunit vaccines in animal models. Winter et al. showed that a single vaccination with a complex consisting of porin and smooth lipopolysaccharide from B. abortus strain 2308 provided significant protection against challenge with the same strain, equivalent to the protection achieved by vaccination with live attenuated strain 19 (34). Jacques et al. showed that mice immunized with a Brucella O-polysaccharide—bovine serum albumin conjugate were protected against challenge with B. melitensis strain H38 (18). Brucella O-polysaccharide-specific monoclonal antibodies were shown to provide protection against challenge with B. melitensis and B. abortus smooth strains (7, 8). The protection demonstrated in the studies described above was defined as a 1- to 2-log reduction in the intensity of infection of spleens and livers after intravenous or intraperitoneal challenge of mice.
Several brucella proteins have been tested as vaccines to provide protection against brucellosis. Immunization of mice with recombinant B. abortus L7/L12 ribosomal protein fused to maltose binding protein has been shown to provide 0.84- to 1.21-log CFU reductions in spleen infection for brucellosis (23). Overexpression of Cu/Zn superoxide dismutase protein in B. abortus strain RB51 has been shown to induce cell-mediated immunity to Cu/Zn superoxide dismutase and to enhance the protective activity of strain RB51 in mice (32). Brucella periplasmic binding protein P39 used with CpG oligodeoxynucleotide as an adjuvant has been shown to elicit a Th1-type immune response and to provide protection to immunized mice challenged with virulent B. abortus strain 544 (1).
We developed a vaccine composed of purified B. melitensis lipopolysaccharide (LPS) as a noncovalent complex with meningococcal group B outer membrane protein (GBOMP). Neisseria meningitidis GBOMP has been shown to enhance the immunogenicity of peptide (19) and polysaccharide vaccines (28). We have shown that intranasal (i.n.) immunization of mice with this LPS-GBOMP vaccine elicited both humoral and mucosal antibody responses in both mice and guinea pigs (31). We have also shown that intranasal immunization of mice with this vaccine provided significant protection against disseminated infection of spleens and livers when the animals were challenged intranasally with a 100% infectious dose of virulent B. melitensis strain 16M (2) but did not provide protection against infection of the lungs. We define protection as complete clearance of bacteria from the organs studied. Since intranasal immunization did not provide protection against infection of the lungs, we wanted to test if changing the route of immunization provided enhanced protection. In the present study we compared the protective efficacies of purified LPS and LPS-GBOMP vaccine delivered by intranasal and subcutaneous (s.c.) immunization of mice against intranasal challenge with virulent B. melitensis strain 16M.
MATERIALS AND METHODS
Preparation of purified LPS.
B. melitensis strain 16M was obtained from Gerhardt Schurig (Virginia Polytechnic Institute and State University, Blacksburg) and was grown in brucella broth at 37°C for 48 h in a biosafety level 3 laboratory. The LPS was extracted from killed cells and purified by a modification of the method of Bundle et al. (5). Briefly, cells from a 10-liter culture were suspended in 1 liter of extraction buffer (0.05 M Tris-HCl-1% sodium chloride [pH 7.2] containing 2% [wt/vol] phenol), stirred at room temperature for 24 h, and then kept at 5°C for 5 days, during which the suspension was checked for sterility. If the suspension was sterile, it was taken into a biosafety level 2 laboratory for further processing. A sterile suspension was centrifuged at 10,000 × g for 30 min. The supernatant was dialyzed extensively against running tap water until it was free of phenol, concentrated by ultrafiltration on a PM-10 membrane (Amicon Corporation, Boston, MA) to one-half the original volume, and centrifuged at 105,000 × g for 16 h. The crude LPS pellet was resuspended in water and freeze-dried. The dry LPS was extracted twice with chloroform-methanol (2:1) and then partitioned between chloroform and water. The water phase was lyophilized to obtain partially purified LPS, which was digested with DNase, RNase, and proteinase K, and was centrifuged at 105,000 × g for 16 h. Resultant pellets were resuspended in water and lyophilized to obtain purified LPS.
General procedures.
BALB/c mice (6 to 8 weeks old) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All animals were given food and water ad libitum and kept in microisolator cages in laminar flow racks under conditions consisting of 12 h of light and 12 h of darkness in biosafety level 3 facilities. Phosphatase-labeled goat anti-mouse immunoglobulins were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Meningococcal GBOMP was prepared as described previously (35). Purified LPS-GBOMP noncovalent complex vaccine was prepared by the method described previously (2).
Immunization and challenge of mice.
Groups of female BALB/c mice (Harlan Sprague-Dawley) were immunized either i.n. or s.c. with either purified B. melitensis LPS or LPS-GBOMP noncovalent complex vaccine. Control mice were immunized with sterile saline subcutaneously. The procedure used for intranasal immunization of mice has been described previously (2). Briefly, 25 μl of vaccine containing 10 μg LPS or 10 μg LPS and 7.5 μg GBOMP for the LPS-GBOMP complex vaccine in sterile saline was administered dropwise into the nostrils of anesthetized mice with a micropipette. Two doses of vaccine were given 4 weeks apart. For subcutaneous immunization, mice were given 10 μg vaccine in 200 μl sterile saline under the right hind thigh, using a 1-ml syringe fitted with a 25-gauge needle. A second dose of vaccine was given 4 weeks after the first dose. Blood was collected from five euthanized mice in each group 4 weeks after the first dose of vaccine and 4 weeks after the second dose of vaccine. Sera were collected and stored at −20°C until they were analyzed for antibody by an enzyme-linked immunosorbent assay (ELISA).
Groups of immunized mice (15 to 20 mice in each group) were challenged intranasally 4 weeks after the second dose of vaccine with 104 CFU of B. melitensis 16M suspended in 30 μl phosphate-buffered saline (PBS) (0.01 M sodium phosphate, 0.14 M sodium chloride; pH 7.5) as described previously (21). Blood, spleens, lungs, and livers were aseptically collected from anesthetized mice 8 weeks postchallenge. The numbers of Brucella CFU in organs were determined by dilution and culture on brucella agar as described previously (17). Serum was separated and stored at −20°C until it was used.
ELISA.
The ELISA was performed in 96-well flat-bottom polystyrene microtiter plates (Costar, Cambridge, MA) by using a slight modification of the method of Engvall and Perlmann (13). Briefly, wells were coated with purified B. melitensis LPS at a concentration of 10 μg/ml in PBS-azide (0.01 M sodium phosphate, 0.14 M sodium chloride, 0.02% sodium azide; pH 7.5) by adding 100 μl to each well and incubating the plates at 37°C for 3 h. The wells were washed three times with PBS-azide using an automatic plate washer (Labsystems Well wash4 Mk2; Fisher Scientific, Pittsburgh, PA). The wells were blocked by adding 100 μl blocking buffer (1% casein in PBS-azide) and incubating the plates at 37°C for 1 h. The wells were washed three times with PBS-azide between the steps. Serial twofold dilutions of primary antibodies (100 μl) were made on the plates, and the plates were kept at room temperature (25°C) for 16 h. Then the plates were incubated with phosphatase-labeled goat anti-mouse antibodies (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at a concentration of 1 μg/ml (100 μl per well) for 16 h at room temperature. The substrate used was p-nitrophenyl phosphate disodium (Sigma Chemical Co., St. Louis, Mo.) at a concentration of 1 mg/ml, and it was incubated for 30 min at room temperature. The action of enzyme was stopped by adding 50 μl of 3.0 M sodium hydroxide solution to all wells. Absorbance at 405 nm was determined with a plate reader (Labsystems Multiskan MCC/340; Fisher Scientific, Pittsburg, PA). The titer, expressed in optical density (OD) units, was obtained by multiplying the reciprocal dilution of the serum by the OD (A405) at that dilution. OD values between 0.5 and 1.0 U (which were in the linear part of the OD-dilution curve) were used for calculation of OD values.
Statistical methods.
Antibody titers of groups of mice were expressed as means ± standard deviations. The intensities of bacterial infection in organs were expressed as the mean log CFU ± standard deviation per infected organ. The lower limit of detection of infection of organs was 2 CFU. The significance of differences in ELISA titers and in log CFU per infected organ was determined by Student's t test. The significance of differences in percentages of infected spleens, livers, and lungs of immunized mice and control mice was determined by Fisher's exact test.
RESULTS
Immune response in mice.
Subcutaneous immunization of mice with B. melitensis LPS induced antibody titers at both 4 weeks after the first dose of vaccine and 4 weeks after the second dose of vaccine (P < 0.05) that were significantly higher than the titers after intranasal immunization (Table 1). B. melitensis LPS-GBOMP vaccine given subcutaneously resulted in an antibody titer at 4 weeks after the first dose of vaccine that was fivefold higher than the titer obtained with the same vaccine given intranasally (P < 0.05). However, 4 weeks after the second dose of vaccine there was no significant difference in the antibody responses to this vaccine for the two routes of immunization (Table 1). There was a substantial decrease in immunoglobulin G (IgG) antibody at both 1 week and 8 weeks postchallenge. Similar results were obtained in two other experiments (data not shown). Control mice immunized with sterile saline did not show any anti-LPS antibody up to 1 week postchallenge but showed high anti-LPS antibody titers at 8 weeks postchallenge.
TABLE 1.
Anti-B. melitensis LPS IgG ELISA titers of mouse seraa
| Group | Vaccine | Anti-B. melitensis LPS IgG ELISA titer (mean ± SD)
|
|||
|---|---|---|---|---|---|
| 4 wk after first dose | 4 wk after second dose | 1 wk after challenge | 8 wk after challenge | ||
| 1 | B. melitensis LPS, s.c. | 2,680 ± 985 | 14,853 ± 5,996 | 6,309 ± 2,298 | 5,790 ± 9,950 |
| 2 | B. melitensis LPS, i.n. | 821 ± 371 | 2,671 ± 1,064 | 2,052 ± 1,659 | 2,142 ± 1,837 |
| 3 | B. melitensis LPS-GBOMP, s.c. | 13,967 ± 3,161 | 84,512 ± 55,263 | 27,392 ± 5,768 | 14,259 ± 9,741 |
| 4 | B. melitensis LPS-GBOMP, i.n. | 2,875 ± 1,389 | 72,253 ± 50,047 | 19,498 ± 20,638 | 12,160 ± 9,215 |
| 5 | Saline control | <3 | <4 | <4 | 22,609 ± 23,838 |
Mice were immunized either s.c. or i.n. Two doses of vaccine were given 4 weeks apart. Mice were challenged 4 weeks after the second dose intranasally with 104 CFU B. melitensis 16M. Sera were collected from five mice of each group at 4 weeks after the dose, 4 weeks after the second dose, and 1 week after challenge and from 18 to 20 mice of each group at 8 weeks after challenge. The data are expressed in OD units.
Anti-B. melitensis LPS IgM antibody titers are shown in Table 2. The group receiving B. melitensis LPS-GBOMP subcutaneously had the highest IgM titer, followed by the group that received LPS alone subcutaneously. There was no significant difference in the anti-LPS IgM antibody responses in mice immunized s.c. with B. melitensis LPS and mice immunized i.n. with B. melitensis LPS. At 4 weeks after the first dose of vaccine, B. melitensis LPS-GBOMP vaccine given s.c. produced a significantly higher antibody titer than the same vaccine given i.n. (P < 0.05). The titers were not significantly different 4 weeks after the second dose of vaccine. The anti-LPS IgG subtype titers of mouse sera at 4 weeks after the first dose of vaccine are shown in Table 3. The predominant IgG subtype for B. melitensis LPS given either s.c. or i.n. was IgG3. However, LPS-GBOMP vaccine elicited predominantly IgG1 and IgG3 subtypes when either route of immunization was used.
TABLE 2.
Anti-B. melitensis LPS IgM ELISA titers of mouse seraa
| Group | Vaccine | Anti-B. melitensis LPS IgM ELISA titer (mean ± SD)
|
|||
|---|---|---|---|---|---|
| 4 wk after first dose | 4 wk after second dose | 1 wk after challenge | 8 wk after challenge | ||
| 1 | B. melitensis LPS, s.c. | 368 ± 255 | 386 ± 82 | 299 ± 128 | 200 ± 103 |
| 2 | B. melitensis LPS, i.n. | 112 ± 37 | 132 ± 96 | 116 ± 86 | 91 ± 63 |
| 3 | B. melitensis LPS-GBOMP, s.c. | 794 ± 220 | 449 ± 104 | 280 ± 91 | 162 ± 63 |
| 4 | B. melitensis LPS-GBOMP, i.n. | 96 ± 51 | 166 ± 82 | 165 ± 93 | 107 ± 78 |
| 5 | Saline control | <4 | <2 | <3 | 166 ± 168 |
Mice were immunized either s.c. or i.n. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally 4 weeks after the second dose. Sera were collected from five mice of each group at 4 weeks after the dose, 4 weeks after the second dose, and 1 week after challenge and from 18 to 20 mice of each group at 8 weeks after challenge. The data are expressed in OD units.
TABLE 3.
Anti-B. melitensis LPS IgG subtype ELISA titers of mouse sera at 4 weeks after the first dose of vaccinea
| Group | Vaccine | Anti-B. melitensis LPS IgG subtype ELISA titer (mean ± SD)
|
|||
|---|---|---|---|---|---|
| IgG1 | IgG2a | IgG2b | IgG3 | ||
| 1 | B. melitensis LPS, s.c. | 579 ± 347 | 324 ± 162 | 263 ± 229 | 2,381 ± 622 |
| 2 | B. melitensis LPS, i.n. | 109 ± 82 | 92 ± 59 | 40 ± 23 | 960 ± 456 |
| 3 | B. melitensis LPS-GBOMP, s.c. | 6,031 ± 2,888 | 852 ± 137 | 677 ± 548 | 2,883 ± 1,024 |
| 4 | B. melitensis LPS-GBOMP, i.n. | 1,643 ± 742 | 138 ± 73 | 128 ± 110 | 915 ± 292 |
| 5 | Saline control, s.c. | <1 | <1 | <1 | <3 |
Mice were immunized either s.c. or i.n. Two doses of vaccine were given 4 weeks apart. Sera were collected from five mice of each group at 4 weeks after the first dose. The titers are expressed in OD units.
Anti-LPS IgG subtype antibody responses at 4 weeks after the second dose of vaccine are shown in Table 4. The anti-LPS IgG1 titers following immunization with B. melitensis LPS-GBOMP either s.c. or i.n. were significantly higher than the titers after administration of the LPS alone by either route of immunization (P < 0.05). However, there was no significant difference in anti-LPS IgG3 antibodies.
TABLE 4.
Anti-B. melitensis LPS IgG subtype ELISA titers of mouse sera at 4 weeks after the second dose of vaccinea
| Group | Vaccine | Anti-B. melitensis LPS IgG subtype ELISA titer (mean ± SD)
|
|||
|---|---|---|---|---|---|
| IgG1 | IgG2a | IgG2b | IgG3 | ||
| 1 | B. melitensis LPS, s.c. | 1,745 ± 518 | 786 ± 687 | 1,229 ± 1,306 | 17,226 ± 9,025 |
| 2 | B. melitensis LPS, i.n. | 384 ± 417 | 161 ± 78 | 168 ± 79 | 3,795 ± 1,759 |
| 3 | B. melitensis LPS-GBOMP, s.c. | 42,157 ± 31,266 | 4,018 ± 4,673 | 2,448 ± 3,146 | 29,062 ± 20,273 |
| 4 | B. melitensis LPS-GBOMP, i.n. | 51,492 ± 35,946 | 1,576 ± 1,102 | 2,032 ± 2,021 | 9,573 ± 6,656 |
| 5 | Saline control, s.c. | <2 | <2 | <3 | <3 |
Mice were immunized either s.c. or i.n. Two doses of vaccine were given 4 weeks apart. Sera were collected from five mice of each group at 4 weeks after the second dose. The titers are expressed in OD units.
Protection of mice after challenge.
The protective efficacy of vaccines was measured by determining the clearance of the challenge strain from lungs, livers, and spleens of mice 8 weeks postchallenge (Table 5). Both the B. melitensis LPS and B. melitensis LPS-GBOMP vaccines, given either subcutaneously or intranasally, provided significant protection from disseminated infection for spleens and livers of immunized mice compared to control mice (Table 5). The greatest protection was provided by subcutaneous immunization with B. melitensis LPS-GBOMP vaccine. This group of immunized mice also showed significant clearance of bacteria from the lungs compared to control mice. The lungs of 41 of 64 mice (64%) were cleared of infection, compared to the lungs of 15 of 64 (23%) control mice (P < 0.001). Similarly, the livers of 56 of 64 (87%) immunized mice were cleared of infection, compared to the livers of 12 of 64 (18%) control mice (P < 0.001), and the spleens of 37 of 64 (57%) immunized mice were cleared of infection, compared to the spleens of 2 of 64 (3%) control mice (P < 0.001).
TABLE 5.
Clearance of B. melitensis 16M from lungs, livers, and spleens of micea
| Group | Vaccine | No. of organs cleared/total no. (%)
|
||
|---|---|---|---|---|
| Lungs | Livers | Spleens | ||
| 1 | B. melitensis LPS, s.c.b | 27/64 (42)d | 48/64 (75)e | 39/64 (60)e |
| 2 | B. melitensis LPS, i.n.c | 15/43 (35)f | 31/43 (72)g | 24/43 (56)e |
| 3 | B. melitensis LPS-GBOMP, s.c.b | 41/64 (64)e | 56/64 (87)e | 37/64 (57)e |
| 4 | B. melitensis LPS-GBOMP, i.n.c | 12/45 (26)f | 34/45 (75)e | 27/45 (60)e |
| 5 | Saline control, s.c.b | 15/64 (23) | 12/64 (18) | 2/64 (3) |
Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally 4 weeks after the second dose of vaccine with 104 CFU of B. melitensis 16M. Organs were harvested 8 weeks after challenge and cultured on brucella agar to determine CFU counts. The limit of detection was 2 CFU per organ.
The results are cumulative data from three experiments.
The results are cumulative data from two experiments.
P < 0.05 for a comparison with the saline control, as determined by two-tailed Fisher's exact test.
P < 0.001 for a comparison with the saline control, as determined by two-tailed Fisher's exact test.
Not significantly different from the saline control, as determined by two-tailed Fisher's exact test.
P < 0.01 for a comparison with the saline control, as determined by two-tailed Fisher's exact test.
The mean log numbers of CFU for lungs, livers, and spleens of infected mice are shown in Table 6. The mean log number of CFU for spleens of mice immunized with the B. melitensis LPS-GBOMP vaccine subcutaneously (1.65 ± 0.86) was significantly lower than the mean log number of CFU (3.57 ± 0.81) for control mice (P < 0.01). The log numbers of CFU for spleens of all groups of immunized mice were significantly lower than the log numbers of CFU for spleens of control mice.
TABLE 6.
Log numbers of CFU in lungs, livers, and spleens of mice 8 weeks after challengea
| Group | Vaccine | Log CFU (mean ± SD)
|
||
|---|---|---|---|---|
| Lungs | Livers | Spleens | ||
| 1 | B. melitensis LPS, s.c. | 1.73 ± 0.87 | 1.79 ± 0.28 | 2.31 ± 1.08 |
| 2 | B. melitensis LPS, i.n. | 1.57 ± 0.69 | 1.91 ± 0.42 | 2.35 ± 0.63 |
| 3 | B. melitensis LPS-GBOMP, s.c. | 1.97 ± 0.63 | 1.55 ± 0.62 | 1.65 ± 0.86 |
| 4 | B. melitensis LPS-GBOMP, i.n. | 2.06 ± 0.80 | 1.69 ± 0.40 | 2.23 ± 1.27 |
| 5 | Saline control | 2.04 ± 0.67 | 1.86 ± 0.79 | 3.57 ± 0.81 |
Mice were immunized either s.c. or i.n. Two doses of vaccine were given 4 weeks apart. Mice were challenged with 104 CFU B. melitensis 16M 4 weeks after the second dose of vaccine. Lungs, livers, and spleens of mice were collected 8 weeks after challenge. Serial dilutions of organ homogenates were plated on brucella agar, and CFU were counted after 3 to 5 days of incubation. The limit of detection was 2 CFU per organ. The data are data from one representative experiment (n = 20).
DISCUSSION
We have previously shown that intranasal immunization of mice with B. melitensis LPS-GBOMP noncovalent complex vaccine provided significant protection against disseminated infection of spleens and livers of immunized mice, but this route of immunization failed to provide enhanced clearance of bacteria from the lungs (2). In the present work we compared the protective efficacies of subcutaneous immunization and intranasal immunization of mice with purified B. melitensis LPS and LPS-GBOMP noncovalent complex vaccines. As shown in Table 5, subcutaneous immunization of mice with LPS-GBOMP noncovalent complex vaccine provided significant protection from infection of lungs, livers, and spleens (P < 0.001). This protection was superior to the protection provided by intranasal immunization of mice with the same vaccine, where there was no significant clearance of bacteria from the lungs of immunized mice compared to control mice. Subcutaneous immunization of mice with purified LPS also provided significant protection against infection of spleens, livers, and lungs of mice. However, only 42% of immunized mice had bacteria cleared from their lungs, compared to 64% of mice immunized with the LPS-GBOMP vaccine. Intranasal immunization of mice with purified LPS failed to provide significant protection against infection of the lungs. It is interesting that there was no significant difference in the protective efficacies of the LPS and LPS-GBOMP vaccines given either subcutaneously or intranasally in terms of clearance of bacteria from the spleens or livers of immunized mice (Table 5). The only significant difference was the difference in the clearance of bacteria from the lungs of mice. These results show that only subcutaneous immunization with the LPS-GBOMP vaccine provided superior protection against infection of lungs (P < 0.001).
There were differences in the IgG subtype responses with the LPS-GBOMP vaccine given either subcutaneously or intranasally. The IgG3 subtype titer at 4 weeks after the second dose of vaccine was 37% of the total response when subcutaneous immunization was used, whereas this titer was 15% of the total subtype response when intranasal immunization was used (Table 4). The IgG2a subtype titer was also slightly higher (5.1% of the total) after subcutaneous immunization than after intranasal immunization (2.4%). TH1 cells induce antigen-specific B cells to secrete IgG2a, TH2 cells induce the cells to secrete IgG1, and both types of TH cells induce the secretion of IgM and IgG3 (30). The GBOMP is also known to increase expression of costimulatory molecules on murine B cells and to enhance antibody responses to polysaccharide antigens by a CD40-CD40L-mediated mechanism (25).
In a previous study with a burned mouse model, we showed that immunization of mice with Escherichia coli J5DLPS-GBOMP vaccine induced production of larger amounts of IgG2a when intramuscular immunization was used than when subcutaneous immunization was used (22). It will be of interest to test the brucella subunit vaccines using intramuscular immunization.
B. abortus LPS has been shown to be 10,000-fold less pyrogenic than E. coli LPS in eliciting fever in rabbits and 268-fold less potent in killing d-galactosamine-sensitized mice (14). We found that purified B. melitensis LPS is 2,500-fold less pyrogenic than E. coli LPS in the rabbit pyrogen test and 1,500-fold less potent in eliciting tumor necrosis factor alpha release from human monocytes (A. K. Bhattacharjee, H. Kong, and D. L. Hoover, unpublished results). In addition, the GBOMP used in our vaccine formulation has been used previously in human trials and was found to be safe (4). Therefore, purified B. melitensis LPS-GBOMP noncovalent complex vaccine may be a safe and promising candidate for further development as a vaccine against brucellosis for human use.
Acknowledgments
This work was supported by a grant from the Department of Defense, Medical Research and Materiel Command, RAD IV.
We thank Lynnette Young, Adrien Ravizee, Hugh Wylie, Mark Marohn, and Andrew LaClair for their superb technical assistance.
The opinions and assertions in this paper are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
This research was conducted in compliance with the Animal Welfare Act and other federal regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publications, 1996 edition. All animal use protocols were reviewed and approved by the Institutional Animal Care and Use Committee.
Editor: D. L. Burns
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