Nonviable Burkholderia mallei Induces a Mixed Th1- and Th2-Like Cytokine Response in BALB/c Mice

Abstract

Nonviable cell preparations of Burkholderia mallei, the causative agent of glanders, were evaluated as potential vaccine candidates in a BALB/c murine model. Three different B. mallei cell preparations plus Alhydrogel were evaluated: a heat-killed preparation, an irradiation-inactivated preparation, and a preparation of a capsule-negative mutant strain which had been irradiation inactivated. BALB/c mice were vaccinated twice with the different B. mallei preparations, and spleens and sera were collected to determine their cellular and humoral immune responses. All three bacterial cell preparations had essentially the same results in two cellular immune response assays. In a splenocyte proliferation assay, the amount of cell proliferation in response to the homologous immunogen, concanavalin A, or lipopolysaccharide was similar for all the cell preparations. Also, splenocytes from the inoculated mice expressed interleukin 2 (IL-2), gamma interferon, and small amounts of IL-4 and IL-5, and more IL-10 cytokine in the presence of the homologous antigen. When the immunoglobulin subclasses from these mice were examined, they all produced higher levels of IgG1 than IgG2a subclasses. The higher ratio of IgG1 to IgG2a was not due to the amount of the immunogen or the adjuvant (Alhydrogel) used in the BALB/c mice. The cell preparations did not protect the vaccinated mice from a live challenge (>300 50% lethal doses). Our results suggest that in BALB/c mice, a mixed T-helper-cell-like response to nonviable B. mallei is obtained, as demonstrated by a Th1- and Th2-like cytokine response and a Th2-like subclass immunoglobulin response. This may be the reason for the inability of the B. mallei cells that were examined as candidate vaccines to protect the mice from a live challenge.

Glanders is primarily an infection of solipeds (horses, mules, and donkeys) and is caused by Burkholderia mallei, a gram-negative, nonmotile, oxidase-positive bacillus (7, 19). Studies by Loeffler and Schuetz in 1882 were the first to identify B. mallei as the etiologic agent of glanders (2). The glanders organism was recently classified as Pseudomonas mallei, but then it was reclassified with the closely related species Burkholderia pseudomallei and Burkholderia thailandensis based on their 16S rRNA homology (3, 20). Glanders in the past was prevalent worldwide because of the wide use of solipeds, but as the result of a general decrease in their use for transportation and as work animals, and stringent health enforcement procedures, the incidence has decreased. Glanders is now found primarily in Asia, Africa, and the Middle East. Infection of solipeds by B. mallei may be presented in two general forms: (i) a chronic form found primarily in horses and (ii) an acute form found primarily in donkeys and mules. The chronic form of the disease may be presented separately or simultaneously as a pulmonary disease, an upper respiratory disease, or a cutaneous disease (farcy). Symptoms for the acute form of the disease include the presence of high temperature, depression, shortness of breath, diarrhea, and rapid weight loss (1). Death may occur after a few weeks with an acute infection, whereas the chronic form of glanders may last for years and may end in death. The disease is transmitted through close contact with diseased solipeds or by soliped products through the skin or nasal or mucosal passages. B. mallei infection in humans may also be either a chronic or acute disease.

In the United States, the last two reported cases of B. mallei infection were the result of laboratory exposure to the microorganism (5, 12). From these two reports, it appears that the routes of infection were either inhalation of an aerosolized cell suspension or direct contact with the microorganism. In the earlier report, sulfadiazine administered intravenously was the treatment of choice (12), while in the later report, rapid improvement of the patient occurred after treatment with imipenem and doxycycline (5). Currently, there is no animal or human vaccine against infection with B. mallei. Because of its highly infectious and debilitating nature, B. mallei is considered to be a potential biological weapon (4). With this prospect as a possibility, we are interested in developing a vaccine for B. mallei infection. In this study, we report the results of our initial experiments on the cellular and humoral immune response to nonviable B. mallei cells in BALB/c mice.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and antigen preparation.

B. mallei ATCC 23344 (hereafter in this work identified as GB15.1-2) was serially passaged three times through Syrian hamsters (spleen suspensions) and then stored as a glycerol stock (50% glycerol in 1× phosphate-buffered saline [PBS] [1.7 mM KH₂PO₄; 5 mM Na₂HPO₄; 150 mM NaCl, pH 7.4]) at −70°C. For antigen preparation, a suspension of the glycerol stock was inoculated into 4% glycerol-1% tryptone (Difco, Becton Dickinson, Sparks, Md.) broth (GTB) and grown overnight at 37°C with shaking (250 rpm). After 20 to 24 h of growth, the cells were pelleted by centrifugation and suspended in sterile Hanks' balanced salt solution. The pellet was appropriately diluted to the desired optical density (OD) at 660 nm. Cell concentration was determined by comparison with a standard curve (weight [in milligrams per milliliter] versus OD at 660 nm). Heat-killed cells (HKGB15) were prepared by heating the cells at 65°C for 30 min. Irradiation-inactivated cells (IRGB15) were prepared by exposure to 2.1 megarads of gamma radiation. A capsule-negative mutant of B. mallei GB15.1-2 (CapGB15) was created by DeShazer et al. (designated as “DD3008” in their report [9]), and cells were prepared and irradiation inactivated by gamma radiation (2.1 megarads). Sterility of all inactivated cell preparations was verified by plating on 4% glycerol-1% tryptone (Difco)-1.5% agar (Difco) plates.

Mice, vaccinations, and challenges.

Female, 6- to 8-week-old, BALB/c mice obtained from the National Cancer Institute (Frederick, Md.) were used in all studies. Mice, four to five per group, were vaccinated subcutaneously with 100 μg (unless stated differently) of nonviable B. mallei cell preparation mixed with 100 μg of Alhydrogel (Superfos Biosector G/S, Vedbaek, Denmark) in a total volume of 0.2 ml. Unless stated differently, mice were inoculated twice, 21 days apart. Three weeks after the second boost, mice were anesthetized for serum collection and killed by cervical dislocation, and their spleens were removed and cultured as described below. In challenge studies, mice were injected intraperitoneally (i.p.) with 0.5 ml of B. mallei (at concentrations stated in the Tables) 3 to 4 weeks after the second vaccination and then observed for up to 21 days for survival.

All animals used in this research project were cared for and used humanely according to the following policies: the Public Health Service Policy on Humane Care and Use of Animals (16a); Guide for the Care and Use of Laboratory Animals (16b); and the U.S. Government Principles for Utilization and Care of Vertebrate Animal Used in Testing, Research, and Training (1985). All U.S. Army Medical Research Institute of Infectious Diseases animal facilities and the animal program are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All animal use was approved by the Institutional Animal Care and Use Committee and conducted in accordance with federal Animal Welfare Act regulations.

Spleen cell cultures.

Spleen cell cultures were prepared from vaccinated mice 21 days after the second vaccination, essentially as described by Jankovic et al. (13). Individual spleens or two spleens from each group were combined per sample, and cell suspensions were prepared after lysis of the erythrocytes (red blood cell lysing buffer; Sigma, St. Louis, Mo.) and adjusted to 2 × 10⁶ to 10 × 10⁶cells/ml in RPMI medium (BioWhittaker, Walkersville, Md.) containing 10% fetal calf serum (HyClone, Logan, Utah, or BioWhittaker), 25 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, nonessential amino acids, 50 μM 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 μg/ml).

Proliferation assay.

To evaluate the extent of spleen cell proliferation in the presence of specific antigens, 2 × 10⁶ splenocytes/ml from vaccinated mice were incubated in Costar (no. 3596) 96-well, flat-bottom dishes (Corning Inc., Corning, N.Y.) in a final volume of 200 μl of RPMI culture medium. The antigens and mitogens were prepared in RPMI culture medium and used at the indicated final concentration: HKGB15, IRGB15, or CapGB15, 10 μg/ml; concanavalin A (ConA) (Sigma), 5 μg/ml; Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) (Sigma), 1 μg/ml. After incubation of the cultures at 37°C for approximately 48 h in the presence of 5% CO₂, [³H]thymidine ([³H]TdR) at a specific activity of 2 Ci/mmol (1.0 μCi/well; Amersham Life Sciences Inc., Arlington Heights, Ill.) was added to each well and cultures were incubated overnight. Cells were harvested after 18 h with an automated cell harvester to determine the amount of [³H]TdR incorporated. Duplicate cell cultures were made for each set of spleens, and triplicate samples for each cell culture were used to determine the extent of cell proliferation. The results are reported as the mean for the duplicate cell cultures of the amount of [³H]TdR incorporated ± 1 standard deviation (SD).

Cytokine assays.

Expression of cytokines was measured in the supernatants of stimulated spleen cell cultures prepared from vaccinated mice. Spleen cells prepared as described above were grown in Costar (no. 3524) 24-well culture dishes (Corning Inc.) at a concentration of 5 × 10⁶/ml in a final volume of 1.0 ml. The same stimulatory antigens (HKGB15, IRGB15, CapIRGB15, ConA, and LPS) were used at the same concentrations (per milliliter) as used in the proliferation assays, and cell cultures in duplicate were incubated at 37°C with 5% CO₂ for 20 to 72 h. In initial studies, we detected the expression of interleukin 2 (IL-2), IL-10, IL-12, and gamma interferon (IFN-γ) after 20 h of incubation. There was some increase in the amount of these cytokines after 48 h but also an increase in the background. Because of this we are reporting the amount of these cytokines expressed after approximately 20 h. Little IL-4 and IL-5 were expressed under the same conditions after 20 to 72 h of incubation. For these later cytokines we are reporting the amount expressed after 20 or 48 h of incubation. We determined the amount of cytokine expressed by a cytokine capture, enzyme-linked immunosorbent assay (ELISA) (OptEIA ELISA Sets) performed as recommended by the manufacturer (BD Pharmingen, San Diego, Calif.) with Immulon 2 HB, 96-well, round-bottom plates (Dynex Technologies, Chantilly, Va.). The plates were read at 450 nm with 570 nm as a reference wavelength. Samples were run in triplicate, compared to a standard curve, and results were reported as the mean concentration (in picograms per milliliter) ± 1 SD. The sensitivity of the cytokine ELISAs was approximately 7.5 to 15 pg/ml.

Antibody assays.

Immunoglobulin (Ig) class (IgG, IgM, and IgA) and subclass (IgG1, IgG2a, IgG2b, and IgG3) titers in vaccinated mice were determined by an ELISA performed in 96-well, Immulon 2 HB, round-bottom plates (Dynex Technologies). B. mallei cells (HKGB15, IRGB15, or CapIRGB15), used as antigens, were diluted in 0.1 M carbonate buffer, pH 9.5, to a concentration of 10 μg/ml, and 50 μl of diluted cells was placed into wells. Plates were stored overnight at 4°C. The plates were washed with washing solution (1× PBS, 0.05% Tween 20), and incubated with 100 μl of blocking solution (1× PBS, 1% bovine serum albumin, 0.05% Tween 20) for 1 h at 37°C. Twofold dilutions of mouse sera were made with blocking solution in triplicate, and plates were incubated for 1 h at 37°C. After the plates were washed, 50 μl of 1/5,000-diluted anti-Ig-horseradish peroxidase conjugate (anti-Ig class or subclass conjugate) obtained from Southern Biotechnology Associates, Inc. (Birmingham, Ala.) was added to each well, and plates were incubated for 1 h at 37°C. After the plates were washed, 50 μl of a buffered hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine solution (BD Pharmingen) was added to each well, and plates were incubated for 15 min at room temperature. The reaction was stopped with 25 μl of 2 N sulfuric acid, and the amount of bound antibody was determined colorimetrically by reading at 450 nm with a reference filter (570 nm). The results are reported as the reciprocal of the highest dilution giving a mean OD of at least 0.1 (which was at least twice the background) ± 1 SD. The titer of antibody (anti-Ig class or subclass) in mice vaccinated with the adjuvant only was usually less than or equal to 50.

Statistics.

All assays were performed in triplicate, and results were reported as the mean ± SD. Statistics were determined by using InStat version 3.0, which uses the Mann-Whitney, nonparametric method for comparing two unpaired groups, and two-tailed P values were reported. In the challenge studies, comparisons between tests were done by the Fisher exact test using SAS version 8.0, with statistical significance considered to be indicated by a P value of <0.05.

RESULTS

Proliferation of stimulated spleen cells obtained from BALB/c mice vaccinated with B. mallei.

We examined the extent of cell proliferation of stimulated spleen cells obtained from mice vaccinated with HKGB15, IRGB15, or CapGB15 cell preparations of B. mallei or with the adjuvant only. With the homologous immunogen as the stimulating antigen, we obtained a 21- to 30-fold increase in the amount of [³H]TdR incorporated for each of the spleen cell cultures from the three different groups of vaccinated mice compared to the amount of [³H]TdR incorporated by the same cells with medium only (Table 1). At the same time, we saw a 1.5- to 1.6-fold increase (P < 0.01) in the incorporation of label with the homologous antigen from splenocytes from vaccinated mice over splenocytes obtained from mice that received adjuvant only. As stimulating-antigen controls, we used ConA and LPS and obtained an increase of more than 188- and 56-fold, respectively, in the amount of [³H]TdR incorporated over that observed in cells incubated with medium only, although these results were either the same or 1.2-fold greater than that obtained with the control cells. Thus, there was specific stimulation of the splenocytes with the homologous antigen, and the extent of proliferation of the immune cells from the three different groups of B. mallei-vaccinated mice did not differ appreciably.

TABLE 1.

Proliferation of splenocytes obtained from B. mallei-vaccinated BALB/c mice

Group vaccinateda	[3H]thymidine incorporated (cpm)b in:
	Medium	Medium with stimulating antigen
		Cellc	ConA	LPS
HKGB15	742 ± 159	16,922 ± 1,213*	139,369 ± 13,197	52,163 ± 1,155
IRGB15	542 ± 459	16,215 ± 3,163*	137,576 ± 6,070	46,263 ± 3,338
CapGB15	752 ± 164	15,962 ± 4,474*	141,252 ± 23,944	42,178 ± 2,759
Adjuvant	554 ± 129	10,214 ± 1,297 (HK)	114,759 ± 12,263	42,428 ± 376
Adjuvant	554 ± 129	12,071 ± 1,096 (IR)	114,759 ± 12,263	42,428 ± 376
Adjuvant	554 ± 129	9,362 ± 1,120 (Cap)	114,759 ± 12,263	42,428 ± 376

^aThere were four mice per group, and spleens were processed as described in Materials and Methods.

^bResults are means ± SDs. *, P < 0.01 for comparison with the homologous adjuvant control.

^c“Cell” refers to the homologous stimulating antigen, except for mice treated with adjuvant only. Abbreviations in parentheses represent stimulating antigens: HK, heat-killed GB15; IR, irradiation-inactivated GB15; Cap, capsule-negative GB15.

Th1-like and Th2-like expression of cytokines by stimulated spleen cells from B. mallei-vaccinated mice.

To determine the type of cytokines expressed from the spleen cells obtained from the vaccinated mice when incubated in the presence of the homologous immunogen, HKGB15, IRGB15, and CapGB15, cells were used to restimulate splenocyte cell cultures for 20 to 72 h. We found that the expression of IL-2 and IFN-γ could be detected at the earliest time points (18 to 20 h) in the presence of the three B. mallei cell antigens (P < 0.01), but little IL-12 could be detected (Table 2). At the same, we found large amounts of IL-2 and smaller amounts of IFN-γ expressed by splenocytes in the presence of ConA, except in both cases it was the same or just a little more than that expressed in the control cells. LPS stimulated the expression of large amounts of IFN-γ, but not IL-2 or IL-12; however, the amount of IFN-γ expressed was essentially similar to that amount expressed by control splenocytes.

TABLE 2.

Expression of Th1-like cytokines from stimulated splenocytes obtained from B. mallei-vaccinated BALB/c mice

Cytokine	Group vaccinateda	Amt of cytokine expressed (pg/ml)b in:
		Medium	Medium with stimulating antigen
			Cellc	ConA	LPS
IL-2	HKGB15	24 ± 5	174 ± 41*	6,245 ± 2,411	27 ± 6
	IRGB15	26 ± 6	166 ± 26*	8,657 ± 2,653	28 ± 7
	CapGB15	26 ± 6	215 ± 81*	9,833 ± 1,515	21 ± 3
	Adjuvant	19 ± 2	28 ± 5 (HK)	8,118 ± 2,343	19 ± 2
	Adjuvant	19 ± 2	34 ± 2 (IR)	8,118 ± 2,343	19 ± 2
	Adjuvant	19 ± 2	29 ± 5 (Cap)	8,118 ± 2,343	19 ± 2
IL-12	HKGB15	206 ± 18	330 ± 48	561 ± 56	295 ± 29
	IRGB15	239 ± 37	491 ± 61	443 ± 115	241 ± 13
	CapGB15	233 ± 26	623 ± 85	556 ± 131	311 ± 139
	Adjuvant	207 ± 13	322 ± 40 (HK)	419 ± 48	263 ± 13
	Adjuvant	207 ± 13	432 ± 26 (IR)	419 ± 48	263 ± 13
	Adjuvant	207 ± 13	763 ± 102 (Cap)	419 ± 48	263 ± 13
IFN-γ	HKGB15	3,576 ± 298	45,452 ± 6,035*	43,047 ± 11,400	42,993 ± 5,886
	IRGB15	3,874 ± 223	48,507 ± 16,541*	36,361 ± 13,700	56,778 ± 7,525
	CapGB15	3,474 ± 372	59,907 ± 11,176*	58,193 ± 13,114	41,503 ± 5,886
	Adjuvant	3,129 ± 372	18,702 ± 3,949 (HK)	32,263 ± 6,035	47,240 ± 9,612
	Adjuvant	3,129 ± 372	31,742 ± 3,353 (IR)	32,263 ± 6,035	47,240 ± 9,612
	Adjuvant	3,129 ± 372	32,412 ± 6,482 (Cap)	32,263 ± 6,035	47,240 ± 9,612

^aThere were four mice per group, and spleens were processed as described in Materials and Methods.

^bResults are means ± SDs. *, P < 0.01 for comparison with the homologous adjuvant control.

^c“Cell” refers to the homologous stimulating antigen. Abbreviations in parentheses represent stimulating antigens: HK, heat-killed GB15; IR, irradiation inactivated GB15; Cap, capsule-negative GB15.

We then examined the expression of IL-4, IL-5, and IL-10 by these spleen cells when incubated in the presence of the three different B. mallei cell preparations (Table 3). Generally, we detected small amounts of IL-4 and IL-5 and moderate amounts of IL-10 in the 20- to 72-h incubation period in the presence of the B. mallei cells. At the same time, there was a slight increase in expression of IL-4 in the presence of ConA over that of control cells. Similarly, no expression or low expression of IL-5 was detected in the presence of ConA compared to control cells. LPS failed to stimulate the expression of IL-4 or IL-5 by these splenocytes. The expression of IL-10 (Table 3) was upregulated, even at the earliest time points (20 h). There was a slight-to-moderate increase in expression of IL-10 by these same splenocytes in the presence of ConA or LPS compared to the control cells. In summary, it appeared that both Th1-like cytokines (IL-2 and IFN-γ) and Th2-like cytokines (IL-4, IL-5, and IL-10) were expressed in the presence of the B. mallei cell preparations.

TABLE 3.

Expression of Th2-like cytokines from stimulated splenocytes obtained from B. mallei-vaccinated BALB/c mice

Cytokine	Group vaccinateda	Amt of cytokine expressed (pg/ml)b in:
		Medium	Medium with stimulating antigen
			Cellc	ConA	LPS
IL-4	HKGB15	30 ± 2	58 ± 15**	586 ± 131	30 ± 7
	IRGB15	27 ± 2	49 ± 6**	696 ± 270	25 ± 2
	CapGB15	24 ± 4	47 ± 18*	618 ± 324	23 ± 3
	Adjuvant	20 ± 1	21 ± 2 (HK)	470 ± 148	25 ± 2
	Adjuvant	20 ± 1	34 ± 2 (IR)	470 ± 148	25 ± 2
	Adjuvant	20 ± 1	29 ± 5 (Cap)	470 ± 148	25 ± 2
IL-5	HKGB15	29 ± 2	44 ± 15**	120 ± 21	44 ± 24
	IRGB15	40 ± 20	105 ± 40	53 ± 31	29 ± 2
	CapGB15	29 ± 9	35 ± 7**	88 ± 38	26 ± 4
	Adjuvant	25 ± 2	27 ± 4 (HK)	89 ± 29	30 ± 5
	Adjuvant	25 ± 2	83 ± 66 (IR)	89 ± 29	30 ± 5
	Adjuvant	25 ± 2	24 ± 3 (Cap)	89 ± 29	30 ± 5
IL-10	HKGB15	76 ± 7	483 ± 94**	491 ± 111	556 ± 163
	IRGB15	73 ± 7	613 ± 122**	519 ± 99	609 ± 116
	CapGB15	88 ± 13	1,175 ± 859**	449 ± 169	510 ± 165
	Adjuvant	80 ± 21	210 ± 32 (HK)	342 ± 48	283 ± 79
	Adjuvant	80 ± 21	375 ± 43 (IR)	342 ± 48	283 ± 79
	Adjuvant	80 ± 21	300 ± 69 (Cap)	342 ± 48	283 ± 79

^aThere were four mice per group, and spleens were processed as described in Materials and Methods.

^bResults are means ± SDs. Statistical significance: * or **, P < 0.05 or P < 0.01, respectively, for comparison with the homologous adjuvant control.

^c“Cell” refers to the homologous stimulating antigen. Abbreviations in parentheses represent stimulating antigens: HK, heat-killed GB15; IR, irradiation inactivated GB15; Cap, capsule-negative GB15.

Ig class and subclass response to nonviable B. mallei.

In addition to examining the cellular immune response to B. mallei, we wanted to examine the Ig class and Ig subclass response to the glanders organism. The HKGB15-, IRGB15-, and CapGB15-vaccinated mice gave a good IgG response (titers of 280,000, 1,000,000, and 300,000, respectively). The IgM response was approximately the same (titer of 10,000) against all the nonviable B. mallei cell preparations. The IgA immune response to the same B. mallei cells was generally not measurable (titer < 50), as was the case with the Ig immune response to the adjuvant-only control group (titer < 50).

In addition to examining the class of antibodies produced in BALB/c mice in response to the B. mallei cell preparations, we evaluated the IgG1 and IgG2a subclass response to B. mallei in the presence and absence of the adjuvant (Alhydrogel). Of the two Ig subclasses we examined which were produced in the presence of the adjuvant, IgG1 was the predominant Ig subclass induced by the three different B. mallei cell preparations, as shown in Table 4. We consistently found this result in studies on the immune response to B. mallei cells in BALB/c mice. We did not always see as much of a difference between the levels of IgG1 in response to IRGB15 or HKGB15 cells. The level of Ig subclasses in mice vaccinated with the adjuvant only was typically less than 50 (data not shown), which we considered to be a negative response.

TABLE 4.

Effect of adjuvant on ratio of IgG2a to IgG1 in response to B. mallei immunization in BALB/c mice

Expt	Treatmenta	Immunogenb	Titer (102)c of:		Ratio of titers (IgG2a/IgG1)
			IgG1	IgG2
I	With adjuvant (100 μg)	HKGB15	2,400 ± 924	575 ± 695	0.24
		IRGB15	12,800 ± 9,050	1,800 ± 1,007	0.14
		CapGB15	3,800 ± 3,020	1,900 ± 9	0.50
II	Without adjuvant (50 μg)	HKGB15	160 ± 113	44 ± 24	0.28
		IRGB15	2,330 ± 2,080	960 ± 370	0.41
		CapGB15	2,880 ± 4,909	1,387 ± 1,124	0.48

^aThe amount of adjuvant used in the study is described in Materials and Methods. The number in parentheses represents the amount of each immunogen used per immunization.

^bThe mice (four per group) were immunized twice, and sera were collected 21 days after the second booster.

^cThe Ig titers for mice treated with adjuvant only (experiment I) or PBS only (experiment II) were both less than 50, as determined with IRGB15 cells. Results are reported as means ± SD.

In the absence of the adjuvant there was still a good IgG1 and IgG2a subclass response to the IRGB15 and CapGB15 cells, even though one-half as many cells were used for the vaccination (Table 4, experiment II). In the case with the HKGB15 cells, the Ig subclass-specific titer decreased between 13- and 15-fold under the same conditions. Nevertheless, the IgG1 subclass-specific titer was still greater than the IgG2a subclass-specific titer in the absence of the adjuvant for the B. mallei cell preparations, and the adjuvant did not affect the ratio of IgG2a to IgG1.

In addition to evaluating the effect of the adjuvant on the immune response to B. mallei, we examined the effect of the amount of immunogen on the IgG1 and IgG2a subclass response in BALB/c mice. Mice were vaccinated twice with B. mallei IRGB15 cells (with adjuvant), ranging from 25 to 100 μg, or B. mallei CapGB15 cells (with adjuvant), ranging from 10 to 100 μg. Table 5 shows that the ratio of IgG2a to IgG1 did not change appreciably with different amounts of B. mallei IRGB15 cells (0.04 to 0.1) or B. mallei CapGB15 cells (0.05 to 0.62), although at the same time the titers of these two Ig subclasses generally decreased with dosage. In summary, the predominate Ig subclass antibody response to nonviable B. mallei cells was IgG1, and as a result the ratio of IgG2a to IgG1 was consistently less than 1.0. These results suggest that a Th2-like Ig subclass antibody response was induced by nonviable B. mallei cells in BALB/c mice independent of the adjuvant or dosage used in these studies.

TABLE 5.

Effect of B. mallei cell concentration on IgG1 and IgG2a production in BALB/c mice

B. mallei	Amta (μg)	Titerb (103) of:		Ratio of titers (IgG2a/IgG1)
		IgG1	IgG2a
IRGB15	100	2,880 ± 1,610	224 ± 64	0.1
	50	1,792 ± 512	76 ± 60	0.04
	25	1,536 ± 591	112 ± 106	0.1
CapGB15	100	600 ± 495	370 ± 315	0.62
	50	480 ± 539	66 ± 70	0.14
	25	1,040 ± 480	145 ± 130	0.14
	10	380 ± 301	21 ± 29	0.05

^aTwo immunizations were given 3 weeks apart, and sera were obtained 3 weeks after the last booster.

^bThe isotype titer is the mean of those obtained from four mice per group ± SD. The titer of mice treated with adjuvant only in both cases was less than 50 as determined with IRGB15 cells.

Challenge of B. mallei-vaccinated BALB/c mice.

We next challenged B. mallei-vaccinated mice to determine if there was any protective efficacy in the B. mallei cell preparations. Table 6 shows the results of this study. In the first part of the study (Table 6, group I), HKGB15, IRGB15, and CapGB15 B. mallei cells were each used to vaccinate mice and as boosters (five mice per cell type). Four weeks after the second injection, the vaccinated mice were challenged i.p. with 2.3 × 10⁸ B. mallei (GB15.1-2) cells (329 50% lethal doses [LD₅₀s]). The LD₅₀ of B. mallei GB15.1-2 is 7 × 10⁵ CFU/ml for 6- to 8-week-old BALB/c mice (10). As shown in Table 6, group I, in the three sets of vaccinated mice challenged with approximately 329 LD₅₀s, all but one of the mice did not survive the challenge. The spleen of the surviving mouse was greatly oversized and heavily infected with B. mallei cells.

TABLE 6.

Effect of parenteral challenge of B. mallei-vaccinated BALB/c mice

Group	Immunogena	No. of mice	Challengeb		No. of survivorsc
			B. mallei strain	No. of cells
I	HKGB15	5	GB15.1-2	2.3 × 108	0
	IRGB15	4	GB15.1-2	2.3 × 108	1
	CapGB15	5	GB15.1-2	2.3 × 108	0
II	IRGB15	5	GB15.1-2	2.8 × 108	1
	IRGB15	5	CapGB15	2.8 × 108	5
	IRGB15	5	CapGB15	6.5 × 108	4
	Adjuvant	5	GB15.1-2	2.8 × 108	0

^aTwo vaccinations were given per mouse as stated in Materials and Methods.

^bMice were challenged i.p. 3 to 4 weeks after the second vaccination.

^cMice were observed for 21 days after the challenge.

In the second group of mice shown in Table 6, group II, we wanted to demonstrate that although the B. mallei CapGB15 mutant was similar to the wild-type B. mallei strain in its inability to provide any protective efficacy as shown above, the outcome of challenging nonviable B. mallei-vaccinated BALB/c mice with the B. mallei CapGB15 mutant versus the B. mallei GB15.1-2 cells was much different. Three sets of mice were vaccinated twice with B. mallei IRGB15, and a fourth set of mice received only the adjuvant. One group of vaccinated mice and the group of mice receiving only the adjuvant were challenged with 2.8 × 10⁸ B. mallei GB15.1-2 cells (approximately 400 LD₅₀s). In the first group of mice, there was one surviving mouse 3 weeks after challenge, while in the second group of mice (adjuvant only), there were no survivors after challenge. Like the one surviving mouse from the first group of mice described above, the lone surviving mouse had a greatly oversized and heavily infected spleen (approximately 6 × 10⁷ CFU/ml). As for the two remaining groups of B. mallei IRGB15-vaccinated mice in this study, one was challenged with 2.8 × 10⁸ B. mallei CapGB15 cells (equivalent to approximately 400 LD₅₀s for B. mallei GB15.1-2 cells), and the other group was challenged with 6.5 × 10⁸ B. mallei CapGB15 cells (equivalent to approximately 930 LD₅₀s for B. mallei GB15.1-2 cells). The only reported LD₅₀ for B. mallei CapGB15 cells (DD3008) in BALB/c mice was >10⁶ cells by aerosol challenge (9). All but one mouse survived the challenge with the B. mallei CapGB15 cells at the lower and higher cell counts. Unlike the surviving mouse from the wild-type B. mallei GB15.1-2 challenge, the spleens of the surviving mice challenged with the B. mallei CapGB15 mutant cell appeared more normal, and they did not harbor any live B. mallei cells. The results of these challenge studies demonstrate that nonviable B. mallei cell preparations used in our studies were unable to protect vaccinated mice from a live challenge of B. mallei GB15.1-2 cells.

Challenge of vaccinated mice with less than a lethal dose of B. mallei.

During the course of our studies, we challenged a group of HKGB15-, IRGB15-, and CapGB15-vaccinated mice with approximately 2.4 × 10⁷ CFU or 34 LD₅₀s of B. mallei GB15.1-2 cells (Table 7). Unlike in the previous challenge studies described above, most of the challenged mice survived. We measured the Ig class- and subclass-specific antibody levels in the surviving mice. We saw a good IgG and IgM response in all the surviving, vaccinated mice except those inoculated with the adjuvant only. The mice that had been vaccinated with the B. mallei CapGB15 mutant produced twice as much of both IgG and IgM classes of antibodies as the mice vaccinated with either B. mallei HKGB15 or IRGB15 cells. The Ig2a-to-IgG1 ratio for these mice ranged from 0.25 to 1.0, with the highest ratio belonging to the B. mallei IRGB15-inoculated mice and the lowest belonging to the B. mallei CapGB15-inoculated mice. It is noteworthy that there were two surviving mice in the group of challenged mice inoculated with the adjuvant only. The difference between the number of mice surviving in the B. mallei (IRGB15)-vaccinated group (five of five) and the number of mice surviving in the adjuvant-only group (two of five) was not significant (P = 0.17). In the adjuvant-only mice, the IgG and IgM cell-specific antibody levels were the same (Table 7, adjuvant), although they were much lower than those in the vaccinated mice. However, when the IgG1 and IgG2a (IRGB15-specific) subclasses were determined, the IgG2a-to-IgG1 ratio was 4.0. This ratio was higher than that observed for the surviving mice in the B. mallei-vaccinated group. As in the previous study with a higher challenge dose, the spleens in the surviving mice given the lower challenge dose in both the B. mallei GB15.1-2-vaccinated mice and control mice were greatly enlarged and heavily infected with B. mallei. Although the outcome of this study with respect to the number of mice surviving challenge was different from that in the previous section with the higher-LD₅₀ dose challenge with B. mallei GB15.1-2 cells, the spleens in the surviving mice in both studies were similarly enlarged and infected.

TABLE 7.

Ig class and IgG1 and IgG2a subclass response in surviving B. mallei-immunized BALB/c mice after parenteral challenge

Immunogena	No. of survivorsb	Titer (103)c of:				Ratio of titers (IgG2a/IgG1)
		IgG	IgM	IgG1	IgG2a
HKGB15	4	320 ± 3.2	40 ± 0.2	320 ± 4.5	160 ± 3.0	0.5
IRGB15	5	320 ± 3.2	40 ± 0.3	1,280 ± 23	1,280 ± 14	1.0
CapGB15	5	640 ± 3.2	80 ± 0.4	2,560 ± 51	640 ± 4.5	0.25
Adjuvant	2	32 ± 0.3	32 ± 0.2	16 ± 0.2	64 ± 0.9	4.0

^aTwo immunizations were given per mouse as stated in Materials and Methods. All mice (n = 5 per group) were challenged i.p. with 2.4 × 10⁷ cells of GB15.1-2 4 weeks after the second immunization.

^bMice were observed for 21 days after the challenge.

^cSera were collected from the survivors and pooled, and titers were determined in triplicate and reported as means ± SDs. The titer for the group treated with adjuvant only was determined with IRGB15 cells.

DISCUSSION

The results of our studies suggest that nonviable B. mallei cells may not be a desirable vaccine candidate, at least in the present unattenuated form. We obtained a mixed Th1- and Th2-like cytokine response and a Th2-like Ig subclass response to the B. mallei cell preparations (14, 15). IRGB15 cells induced the highest level of IgG, followed by the CapGB15 mutant cells and then the HKGB15 cells. More revealing, however, was the Ig subclass response to B. mallei cells. In all cases, IgG1 was the predominate subclass response to the B. mallei cells, even in the absence of the Alhydrogel adjuvant used in our studies. Although aluminum hydroxide adjuvants are considered Th2-like stimulating adjuvants (6, 11), in our case a Th2-like Ig subclass response was seen even without the adjuvant.

The inability of the nonviable B. mallei cells to protect BALB/c mice from a parenteral challenge may be in part another characteristic like the mixed cytokine response to an inadequate candidate vaccine. Two different cell challenge concentrations were used in one of our studies: low, at 2.4 × 10⁷ CFU (34 LD₅₀s), and high, at 2.3 × 10⁸ to 2.8 × 10⁸ CFU (329 to 400 LD₅₀s). In the study with the higher cell challenge, the B. mallei-vaccinated mice did not survive the B. mallei GB15.1-2 challenge. Only one mouse from the B. mallei IRGB15-vaccinated group survived. However, for the challenge at the lower concentration, all the mice except one survived the challenge. These results were unexpected because the LD₅₀ for B. mallei in BALB/c mice was reported to be 7 × 10⁵ CFU (10). This was very close to the results of a previous study with BALB/c mice, which reported the LD₅₀ for a parenteral challenge to be approximately 4.2 × 10⁵ CFU (16). The results of our challenge study might be explained by at least two factors. First, the mice used in this study were much older and larger when they were challenged: approximately 6 to 8 weeks old in the initial study (10) versus 17 weeks old in this study. The older mice may have been able to tolerate the challenge dose better than the younger mice because of a more-developed immune system (18). Second, the LD₅₀ of the lower-dose challenge in our study was only 34-fold higher than the reported LD₅₀, which may be below or close to the LD₅₀ for 17-week-old BALB/c mice challenged with B. mallei. On the other hand, the difference between the reported LD₅₀ and our higher-dose challenge study was approximately 329-fold. These two factors, age and challenge dose, may account for the number of survivors in the low-dose challenge group and the number of survivors in the adjuvant control group. Nevertheless, it was interesting to see a difference between the number of survivors in the low-dose challenge group and the adjuvant control group of mice. Whether the B. mallei cell preparations had some efficacy in this study was not clear because of the small number of animals used. Furthermore, the spleens of all survivors from all groups in this study were still heavily infected with B. mallei cells. The fourfold increase in the IgG2a/IgG1 ratio between the B. mallei IRGB15-vaccinated mice and the mice inoculated with adjuvant only may not have been enough to completely protect the control mice.

Although the murine immune response appeared to be much the same for the wild type (capsulated) and the uncapsulated mutant of B. mallei, we saw a much different result in our challenge studies with these two strains. We confirmed the observation reported by DeShazer et al. (9), i.e., that the capsular polysaccharide of B. mallei was a major virulence factor. The capsule component is closely related to that of B. pseudomallei (17), the cause of melioidosis in human and animals. In this study, it was shown that the LPS and capsule structure of B. pseudomallei are responsible for both resistance and virulence of the microorganism (8, 17). DeShazer et al. (9) showed in two different animal models of B. mallei infection (Syrian hamsters and BALB/c mice) that the capsule-negative mutant was attenuated. They also suggested from the results of a failed rechallenge experiment that the capsule-negative mutant may not be a good vaccine candidate. Our studies found that two vaccinations with the irradiation-inactivated, capsule-negative strain of B. mallei does not provide any protection from a challenge with a wild-type strain of B. mallei. Whether the capsular polysaccharide can be a protective immunogen in another context, such as when it is coupled to a protein carrier, remains to be demonstrated. We are continuing our studies on the development of candidate vaccines for B. mallei infections. Whether a modified whole-cell or subunit vaccine will eventually prove to be efficacious awaits further investigation.