Vol. 30, No. 9JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1992, p. 2256-22640095-1137/92/092256-09$02.00/0Copyright © 1992, American Society for Microbiology

Cell-Mediated and Humoral Immune Responses Induced byScarification Vaccination of Human Volunteers with a NewLot of the Live Vaccine Strain of Francisella tularensis

DAVID M. WAAG,l* ALAN GALLOWAY 2 GUNNAR SANDSTROM,3 CHRISTOPHER R. BOLT,'MARILYN J. ENGLAND,' GENE 0. NELSON,4 AND JIM C. WILLIAMS"5

Bacteriology Division, Department ofPathogenesis and Immunology, 1 Medical Division,2 and Biometrics andInformation Management Division,4 U.S. Army Medical Research Institute of Infectious Diseases,

Fort Detrick, Frederick, Maryland 21702; Centerfor Biologics Evaluation and Research,Food and Drug Administration, Bethesda, Maryland 20892'; and Department of

Microbiology, National Defence Research Establishment,S-901 82, Umea, Sweden3

Received 18 March 1992/Accepted 8 June 1992

Tularemia is a disease caused by the facultative intracellular bacterium FranciseUla tularensis. We evaluateda new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccinationinduced humoral and cell-mediated immune responses. Indications of a positive immune response aftervaccination included an increase in specific antibody levels, which were measured by enzyme-linkedimmunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to wholeF. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P < 0.05) in immunoglobulin A(IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P < 0.01) in vaccinees14 days after accination. These data verify that this new lot of live F. tularensis vaccine is immunogenic.

Tularemia is a zoonotic disease caused by the facultativeintracellular bacterium Francisella tularensis, which ishighly virulent for humans, with only 10 to 50 microorgan-isms required to cause disease (16, 17). The disease occurs inthe Northern Hemisphere, with sporadic human cases andepidemic outbreaks. Vaccination with a live vaccine strain(LVS) or natural infection with F. tularensis protects againsttularemia (3, 6, 22).

Viable microorganisms may be necessary to induce pro-tective immunity in humans, because nonviable whole cellsor subfractions of killed F. tularensis do not induce protec-tion against challenge with highly virulent bacteria (7, 23).However, there is a moderation of clinical symptoms oftularemia in individuals previously vaccinated with the killedvaccine (23). Other studies show that vaccination with the F.tularensis LVS is efficacious. Volunteers vaccinated intra-dermally with F. tularensis LVS were afforded limitedprotection against aerosol challenge, because challengedoses of >2,000 virulent microorganisms caused illness (11).In another study, intradermal vaccination of volunteers withLVS followed by aerosol challenge with 2.5 x 104 virulentmicroorganisms 2 months later completely protected volun-teers against clinically recognized disease (6). However,protection from tularemia waned after 14 months because53% of the vaccinated volunteers were not protected againstvirulent challenge.

Cell-mediated and humoral immune responses in humanscan be measured shortly after LVS vaccination or tularemia(8, 21). The host defense against tularemia seems to bemediated by stimulated T lymphocytes. Thoracic duct lym-phocytes from rats infected with LVS and adoptively trans-ferred to naive rats enhanced the recipient's ability to clearF. tularensis from their spleens and livers. In contrast,

* Corresponding author.

passively transferred immune serum afforded undetectableprotection (9). Although heat-killed LVS stimulated anti-body production in rats, it did not stimulate cell-mediatedimmunity, as measured by macrophage migration inhibition.During World War II, Soviet scientists developed a vac-

cine based on an attenuated F. tularensis strain that affordedgood protection in humans (13, 22). An ampoule containingthe Soviet F. tularensis strain was transferred to the UnitedStates in 1956. From that preparation, a strain suitable forvaccination was isolated and was designated LVS (4). Ei-gelsbach and Downs (4) subsequently discovered that cul-turing of LVS on glucose cysteine blood agar yielded twocolony variants, a gray colony variant and a blue colonyvariant. The structural differences between the two variantsremain unknown; however, differences in the virulences ofthe two variants in both humans and animals are apparent.The lethal dose of the gray colony variant inoculated intra-peritoneally into mice and guinea pigs was at least 10-foldhigher than the lethal dose of the blue colony variant.Furthermore, the blue colony variant was judged to be moreimmunogenic than the gray colony variant, because miceand guinea pigs immunized subcutaneously with 102 or 103 ofthe blue colony variants had positive antigen-specific agglu-tination titers. Animals inoculated with the gray colonyvariant did not acquire positive agglutination titers. Subcu-taneous challenge of mice and guinea pigs with virulent F.tularensis showed that the blue colony variant inducedprotection, whereas animals immunized with the gray colonyvariant were as susceptible as nonimmunized controls. Inaddition to the heterogenicity in the vaccinogenic potentialof colony variants exhibited by different lots of LVS, certainundefined in vitro growth conditions can result in a loss inthe ability of LVS to induce protection in animals (5).Therefore, all lots of vaccine must be evaluated for theirimmunogenicities because of the lack of consistency inproduction of LVS. The old vaccine lot of F. tularensis LVS

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(NDBR 101) contained 99% blue and 1% gray colonies.While the new vaccine lot tested in the study described here(TSI-GSD-213) was derived from the same parent strain asthe NDBR 101 vaccine lot was, it contained 80% blue and20% gray colonies and had a higher residual moisture con-tent than the NDBR 101 vaccine lot did. In addition, the newvaccine was produced in a fermentation apparatus, whereasthe old vaccine was grown in bottles. These differencesbetween the old and the new vaccine lots required that theimmunogenicity of the new vaccine lot of F. tularensis LVSbe assessed in volunteers.

MATERIALS AND METHODS

Vaccine. A new vaccine lot of F. tularensis LVS, desig-nated TSI-GSD-213 (lot 1R), was produced at The SalkInstitute, Swiftwater, Pa. The vaccine was distributed in alyophilized form, and each vial contained approximately 7.0x 108 viable bacteria per ml of reconstituted vaccine.Sodium chloride (0.9%) was the placebo.

Vaccinees. Volunteers were recruited from a residentpopulation of U.S. Army personnel stationed at the U.S.Army Medical Research Institute of Infectious Diseases,Fort Detrick, Frederick, Md. Individuals were examined bya physician before enrolling in the study. Each gave writteninformed consent before participating in the study. Beforevaccination, individuals were bled, and baseline values weredetermined for the enzyme-linked immunosorbent assay(ELISA) and the lymphocyte proliferation assay. Volunteerswere randomized before receiving a single dose of vaccine(7.0 x 107 bacteria per 0.1 ml) or placebo by scarification ina coded fashion so that neither vaccinees nor investigatorsknew the composition of the injection. Vaccine or placebo(0.1 ml) was applied to the ventral forearm and was pressedinto the dermis 15 times with a bifurcated fork. Ninevolunteers received vaccine and nine volunteers receivedplacebo. The coded identities of the vaccinees and controlswere not broken until all the data were obtained and ana-lyzed. Volunteers were bled by venipuncture at 7, 14, 28,and 56 days after vaccination.Humoral immunity. The development of humoral immu-

nity after injection of the tularemia vaccine was evaluated byELISA and immunoblot.

(i) ELISA. The levels of class-specific (immunoglobulin A[IgA], IgG, and IgM) antibody in serum to irradiation-killedLVS cells were determined by an ELISA similar to thatdescribed previously for evaluating humoral immunity toCoxiella bumnetii (25). Viable LVS was irradiated with 2.1 x106 rads (2.1 x 10 Gy) of gamma irradiation (cobalt 60source) while it was frozen on dry ice, thawed, and thensuspended in sodium carbonate-bicarbonate buffer (pH 9.6)at a concentration of 1 mg (dry weight) per ml. Thiswhole-cell suspension was diluted 1:40 in sodium carbonate-bicarbonate buffer, and 0.05 ml of the mixture was seededinto all wells of 96-well Immunolon II microtiter plates (FlowLaboratories, McLean, Va.). The plates were dried over-night in a 37°C dry incubator. Wells of the entire plate wereblocked with carbonate-bicarbonate buffer containing 0.25%bovine gelatin (60 bloom) for 1 h at 37°C. Plates were thenwashed five times with phosphate-buffered saline (PBS)-Tween buffer at pH 7.4. Sera were diluted twofold from 1:16to 1:32,768 in microtiter test wells and in control wells, andthe plates were incubated in a humidified incubator at 37°Cfor 1 h. Microtiter plates were again washed five times withPBS-Tween buffer. Alkaline phosphatase-anti-immunoglob-ulin (anti-alpha chain, anti-gamma chain, or anti-mu chain;

Kirkegaard and Perry, Gaithersburg, Md.) conjugate inPBS-Tween-0.5% gelatin was added to the appropriatewells, and the plates were incubated at 37°C for 1 h in ahumidified incubator. After the plates were washed, enzymesubstrate in diethanolamine buffer (pH 9.8) was added andthe plates were incubated for an additional 1 h at 37°C. Theoptical densities of all wells were determined spectroscopi-cally at a test wavelength of 405 nm and a referencewavelength of 630 nm by using a microplate reader. As-signed titers represent the highest dilution of serum that gavea minimum difference of 0.05 optical density units whencomparing the test and control wells.

(ii) Immunoblot. Immunoglobulin class-specific antibodyreactivity to F. tularensis antigens was evaluated by immu-noblotting (2). Briefly, LVS or F. tularensis phenol waterextract (PWE), which contained lipopolysaccharide as thepredominant constituent, was suspended in Laemmli samplebuffer at a concentration of 1 mg (dry weight) per ml and wasboiled for 5 min. Lysates (750 pg of the LVS per 10-cm slot)were loaded onto a polyacrylamide gel (5% stacking gel,12.5% separating gel), and the bacterial components wereseparated electrophoretically. Bacterial components weretransferred electrophoretically to nitrocellulose by using 500mA of constant current for 16 h in Tris (25 mM)-Tricine (192mM) buffer (pH 10.4). The nitrocellulose was then blockedwith 3% bovine serum albumin (immunoglobulin free) andcut into strips. Sera (pre- and postvaccination) from volun-teers were diluted 1:500, incubated with the nitrocellulosestrips containing LVS antigens for 1 h, and washed. Anti-class-specific immunoglobulin (goat anti-human IgA, IgG, orIgM), which had been iodinated with 1"I using Iodogen (10),was added to individual nitrocellulose strips and incubatedfor 1 h. The nitrocellulose strips were washed six times inTris-buffered saline (containing 0.02 M EDTA, 0.25% bovineserum albumin, and 0.05% Nonidet P-40) at pH 7.4 anddried, and the bands that bound class-specific antibody werevisualized after exposure to X-ray film (Kodak X-omatic).

F. tularensis PWE was prepared as described previously(12). Briefly, F. tularensis LVS whole cells were irradiated(2.1 x 106 rads [2.1 x 104 Gy]) and suspended at a concen-tration of 10 mg/ml in water. An equal amount of 90% phenolwas added, and the suspension was heated to 68°C for 15min. The suspension was centrifuged at 10,000 x g for 30min, and the aqueous phase was collected. An equal amountof water was added to the phenol phase and the cells werereextracted a second and a third time. The PWE wasdialyzed and lyophilized before use. Selected samples weretreated with proteinase K (Sigma Chemical Company, St.Louis, Mo.) prior to electrophoresis. F. tularensis LVS orPWE was suspended at a concentration of 5 mg/ml in 50 mMTris buffer (pH 7.4). Proteinase K was added to a concen-tration of 1 mg/ml, and the suspension was incubated over-night at 45°C.

Cell-mediated immunity. The ability of F. tularensis LVSto initiate a cell-mediated immune response after vaccinationwas evaluated by using a lymphocyte proliferation assay.This test measures the ability of presensitized peripheralblood leukocytes to proliferate in response to irradiation-killed (2.1 x 10' rads [2.1 x 104 Gy]) LVS antigen in vitro.Peripheral blood cells from 40 ml of human blood wereseparated into two 50-ml conical centrifuge tubes. Each tubereceived 20 ml of Hanks' balanced salt solution. Each cellsuspension was layered onto 15 ml of Histopaque (Sigma),and the cells were separated by centrifugation at 500 x g for30 min. Leukocytes were collected from the central band,washed twice with Hanks' balanced salt solution containing

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0.2% bovine serum albumin (immunoglobulin free), andsuspended in RPMI 1640 medium containing 5% autologousor pooled human type AB (PHAB) serum (M.A. Bioprod-ucts, Walkersville, Md.). Viable peripheral blood lympho-cytes (PBL) (360,000) in a volume of 0.180 ml were seededinto individual wells of a 96-well tissue culture plate. Anoptimal amount of a mitogen-concanavalin A (ConA), phy-tohemagglutinin, or pokeweed mitogen-was added to theappropriate wells. Irradiation-killed LVS was also added toappropriate wells to give final concentrations of 550, 55, 5.5,0.55, or 0.055 ,ug/ml. C. bumetii chloroform-methanol resi-due vaccine (24) (5.5 ,ug/ml) was added to appropriate wellsas the control antigen. Ninety-six hours after initiating theculture, we added 1 ,Ci of [3H]thymidine to each well.Radiolabeled PBL were collected on glass fiber filters 20 hlater. Radiolabel uptake was determined by standard scintil-lation techniques. Stimulation indices (SIs) representing thecounts per minute of mitogen- or antigen-stimulated culturesdivided by the counts per minute of unstimulated controlwells were calculated.

RESULTS

Evaluation of humoral immunity. (i) ELISA. Antibodytiters directed against F. tularensis LVS were determined byELISA. Mean ELISA titers (IgA, IgG, and IgM) for non-vaccinated and vaccinated individuals are depicted in Fig. 1.The background levels (day 0) of IgA antibodies were thelowest of all antibody classes tested. Although vaccinationinduced a rise in IgG, IgM, and IgA titers, the IgA responsewas the most remarkable.Upon vaccination, the antigen-specific IgA, IgG, and IgM

titers rose by 28 days after vaccination. Of the three anti-body classes, the IgA titer showed the largest overall in-crease. The mean prevaccination IgA titer was 32, whereasthe mean prevaccination IgG and IgM titers in controls andvaccinees were 927 and between 128 and 141, respectively(Table 1). By 28 days after vaccination, the mean IgA titersof vaccinees rose to 406, the mean IgG titer rose to 1,896,and the mean IgM titer rose to 348. Antibody titers invaccinees continued to rise through day 56. Differences inIgA, IgG, and IgM ELISA titers between vaccine andcontrol groups were significant at theP < 0.01, P < 0.05, andP < 0.05 levels, respectively, by day 56, as determined byStudent's t test. The ranges of antibody titers in immunizedand control volunteers (Table 2) show the relatively highbackground level of IgG antibody. The data also show therelatively high IgA and IgG antibody titers that were attainedafter vaccination.The immunogenicity of LVS in individual volunteers was

also evaluated by determining the ability of the vaccine tostimulate a fourfold rise in ELISA antibody titers (Table 3).The sera of seven of nine (78%) vaccinated volunteersattained fourfold or greater increases in IgA antibodies,while the sera of five of nine (56%) and four of nine (44%)volunteers had fourfold or greater increases in IgG or IgMantibody, respectively. The sera of only two volunteers(22%) given placebo demonstrated a fourfold or greater risein titer of any antibody class. The sera of vaccinees showedaverage rises in IgA, IgG, and IgM titers of 95-, 18-, and11-fold, respectively. The average increase in the corre-sponding titers in the sera of the placebo group was twofoldfor all antibody classes.

(ii) Immunoblot. We used the highly sensitive immunoblottechnique to detect class-specific antibodies reactive toantigenic epitopes of F. tularensis cell lysates. Immunoblots

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DAYS AFTER VACCINATIONFIG. 1. Class-specific (IgA, IgG, and IgM) antibody titers against

F. tularensis LVS in sera collected from volunteers before vaccina-tion (day 0) or 7, 14, 28, or 56 days after vaccination with F.tularensis LVS (-) or placebo (O). Error bars signify two standarderrors.

incubated with prevaccination sera showed antibody-reac-tive bands that differed in size, specificity, and intensity for8 of 18 volunteers. While prevaccination sera from threevolunteers had scattered antibody reactivities in the 15- to62-kDa region (Fig. 2, lanes 1), prevaccination sera from fourother volunteers had binding activities that were concen-trated in the low-molecular-mass region (15 to 25 kDa), anda prevaccination serum sample from one volunteer reactedwith high-molecular-mass bands (33 to 62 kDa). Prevaccina-tion sera from 10 volunteers showed no reactivity in theimmunoblot.

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TABLE 1. Anti-F. tularensis LVS antibody titers in vaccinatedand control volunteers

ELISA antibody titer'Vaccinea Daysb

IgA IgG IgM

P 0 32 (2.2) 927 (2.1) 128 (1.8)V 0 32 (1.5) 927 (2.0) 141 (1.3)

P 7 32 (2.5) 597 (2.3) 149 (1.8)V 7 44 (1.6) 753 (1.9) 138 (1.8)

P 14 44 (2.2) 753 (2.2) 149 (2.6)V 14 59 (2.0) 878 (2.0) 149 (2.0)

P 28 35 (3.2) 553 (4.4) 161 (3.0)V 28 406 (5.3) 1,896 (2.1) 348 (1.9)

P 56 44 (2.0) 597 (3.0) 174 (2.4)V 56 790 (4.5)d 4,467 (3.6)e 861 (3.3)e

a Serum samples were obtained from volunteers vaccinated with placebo(P) or F. tularensis LVS (V).

b Serum samples were obtained from volunteers before vaccination (day 0)or at 7, 14, 28, or 56 days after vaccination.

c IgA, IgG, and IgM antibodies directed against F. tularensis LVS. Valuesare geometric means and (geometric standard deviations [unbiased]) ofELISA antibody titers directed against F. tularensis LVS in vaccine andplacebo groups.

d Significant differences between the two groups at the P < 0.01 level(Student's t test).

I Significant differences between the placebo and vaccine groups at the P <0.05 level (Student's t test).

When sera obtained throughout the study from placebo-vaccinated volunteers were evaluated by immunoblotting, itwas found that sera from two nonvaccinated persons hadminor reactivities (Fig. 2). IgA, IgG, and IgM antibodies hadidentical specificities. Serum from one individual had strongreactivity in the immunoblot, with similar numbers of anti-genic bands and intensities of banding as those of sera fromsome vaccinees. Sera from the remaining six controls had noreactivity with the F. tularensis antigen. We noted no

consistent antibody class-specific epitopes in this study. Forexample, IgA from one vaccinated volunteer recognized a15-kDa antigen, while IgG and IgM, but not IgA, fromanother vaccinee bound an antigen with an identical molec-ular mass.

TABLE 2. Range of anti-F. tularensis LVS antibody titers invaccinated and control volunteers

LVS antibody titer range on the following day afterVolunteer and inoculatione:

antibodya0 7 14 28 56

NonvaccineesIgA 8-64 8-128 16-128 8-128 8-64IgG 256-2,048 128-2,048 128-2,048 16-2,048 128-4,096IgM 64-256 64-256 32-512 32-1,024 32-512

VaccineesIgA 16-32 16-64 16-128 32-16,384 32-4,096IgG 256-2,048 256-2,048 256-4,096 512-8,192 512-32,768IgM 128-256 64-512 32-256 128-1,024 128-4,096

a Sera from immunized and control volunteers were assayed for F. tularen-sis-specific IgA, IgG, and IgM antibodies.

b Sera were collected from the volunteers before vaccination (day 0) and at7, 14, 28, and 56 days after vaccination. The ranges of serological titers of allimmunized or control volunteers are given.

TABLE 3. Increase in specific antibody titers to F. tularensisLVS in vaccinated and control volunteers

Volunteer and No. (%) of volunteersbfold increasea IgA IgG IgM

Vaccinees1 2 (22) 1 (11) 2 (22)2 0 (0) 3 (33) 3 (33)4 1 (11) 1 (11) 0 (0)8 0 (0) 3 (33) 1 (11)16 1 (11) 0(0) 1 (11)32 0 (0) 0 (0) 2 (22)64 3 (33) 0(0) 0(0)128 1 (11) 1 (11) 0 (0)512 1 (11) 0(0) 0(0)Meanc 95 18 11

Controls1 3 (33) 6 (67) 5 (56)2 6 (67) 1 (11) 3 (33)4 0(0) 1 (11) 1 (11)8 0 (0) 1 (11) 0 (0)Mean 2 2 2

a Maximum fold increase in anti-F. tularensis LVS ELISA titers in serumobtained before inoculation and at 7, 14, 28, or 56 days after inoculation.

b The number (percentage) of volunteers with an x-fold increase in IgA,IgG, and IgM antibody titers when pre- and postinoculation maximum titerswere compared.

c The mean fold increase in IgA, IgG, and IgM anti-F. tularensis LVSantibody titers in vaccinees and controls.

Sera from eight of nine vaccinees showed antibody-reac-tive bands in immunoblots (Fig. 3), although sera from threevaccinees exhibited low reactivities. The most notable cor-relate to vaccination was the presence of antigen bandingpatterns (resembling the rungs of a ladder) in immunoblots ofsera from five of nine vaccinees. This antigen bandingpattern was observed only for sera collected 28 and 56 days(Fig. 3, lanes 4 and 5, respectively) after vaccination. Wenoted approximately 18 discrete antigenic bands with aperiodicity of 2.5 kDa; the molecular masses of the bandsranged from 73 to 23 kDa. Although they were not distinctbands, antigenic components were detected between 73 kDaand the top of the blot (about 200 kDa). An antigenic bandingpattern exhibiting only IgM-reactive bands was seen inimmunoblots of sera from two vaccinees. Sera from threevaccinees displayed identical antigenic banding patternsexhibiting IgA-, IgG-, and IgM-reactive bands. This anti-genic profile was not detected in immunoblots of sera fromplacebo-injected volunteers.

Electrophoretically separated PWE of F. tularensis LVSwas immunoblotted and probed with serum collected from asingle volunteer 28 days after LVS vaccination (Fig. 4B).The antigen banding profile was identical to that in animmunoblot in which LVS was used as the antigen (Fig. 3and 4A). Treatment of LVS and PWE with proteinase Kbefore electrophoresis and immunoblotting did not alter theantibody specificity (Fig. 4, lanes 2). The antigen bandingpatterns seen in LVS and PWE immunoblots probed withimmune serum was identical to those seen in F. tularensisLVS, PWE, and lipopolysaccharide immunoblots probedwith monoclonal antibody to F. tularensis lipopolysaccha-ride (12). Therefore, in immunized volunteers, the majorantibody reactivity detected by the immunoblot assay wasdirected against a cellular constituent with an antigen band-ing pattern similar to that of lipopolysaccharide.

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separated by sodium dodecyl sulfate-polyacrylamide gel electropho-resis. The blots were probed with serum (1:500 dilution) from acontrol volunteer, and bound antibodies were detected with radio-labeled class-specific antibodies (anti-IgA, anti-IgG, and anti-IgM).Serum was collected before vaccination (lanes 1) and at 7 (lanes 2),14 (lanes 3), 28 (lanes 4), and 56 (lanes 5) days after vaccination.Molecular mass markers (in kilodaltons) are shown on the left.

Evaluation of cell-mediated immunity. The ability of PBLfrom vaccinated and nonvaccinated individuals to respond toirradiation-killed F. tularensis was measured in a lympho-cyte proliferation assay. Variables in the PBL cultures werethe serum source (autologous or PHAB) and the concentra-tion of killed LVS used as the recall antigen (0.055, 0.55, 5.5,55, or 550 ,ug/ml). The LVS recall antigen at 5.5 ,ug/ml wasthe most mitogenic dose of antigen, giving a mean baselineSI for PBL of prevaccinees of 5.0 (vaccinees) and 6.7(controls) when PBL were incubated in the presence ofautologous sera. The mean baseline SI for PBL of vaccineeswas 4.7 and 8.4 for controls when PBL were incubated in thepresence of PHAB (Table 4). However, the mitogenicity ofthe in vitro LVS cellular antigen (5.5 ,ug/ml) did not mask theability to stimulate selectively PBL from volunteers immu-nized with LVS, as described below.A comparison by Student's t test of vaccine immunoge-

nicity in populations of vaccinees and control volunteersshowed significant differences (Table 4). Highly significant(P < 0.01) differences in SIs between PBL of vaccinees andplacebo controls on day 14 indicated enhanced cell-mediatedimmunity. Ranges of SIs for all time points for PBL culturesincubated with 0.055, 0.55, 5.5, 55, and 550 ,ug of LVS per mlwere 1.07 to 7.50, 1.05 to 19.64, 1.02 to 42.53, 0.20 to 19.95,and 0.03 to 0.20, respectively. The serum source seemed tohave an impact on when significant differences between PBLof vaccinees and control groups occurred. Statistically sig-nificant differences in SIs were obtained earlier (day 14)when vaccinee PBL were incubated in PHAB than whenPBL were incubated in autologous serum. Vaccination with

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rated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The blots were probed with serum (1:500 dilution) from an F.tularensis LVS vaccinee, and bound antibodies were detected withradiolabeled class-specific antibodies (anti-IgA, anti-IgG, and anti-IgM). Serum was collected before vaccination (lanes 1) and at 7(lanes 2), 14 (lanes 3), 28 (lanes 4), and 56 (lanes 5) days aftervaccination. Molecular mass markers (in kilodaltons) are shown onthe left.

LVS did not nonspecifically enhance or suppress lympho-cyte responses, because the mitogenic responses for PBL ofvaccinees to ConA, phytohemagglutinin, and pokeweedmitogen did not differ significantly (P < 0.05) from those ofcontrols (data not shown). In addition, responses for PBL ofvaccinees to a nonrelated negative control antigen, the C.burnetii chloroform-methanol residue vaccine, were neversignificantly different (P < 0.05) from those of nonvaccinatedcontrols (data not shown). However, the specific stimulationof the immune system by F. tularensis LVS is notable. Byday 14 after vaccination, the mean lymphoproliferative re-sponses for PBL of vaccinees were greater than the mito-genic responses to ConA (positive control) (Fig. 5).The data were also evaluated for the ability of LVS

vaccination to induce a rise in SIs of cultured PBL. PBL ofsix of nine (67%) vaccinees attained at least a fourfoldincrease in SI throughout the course of the study (Table 5).In contrast, PBL of only one control attained an SI increaseof fourfold or greater. PBL of vaccinees displayed an aver-age 3.6- and 4.2-fold rise in SI when PBL were cultured withautologous or PHAB serum, respectively. In contrast, SIsfor PBL of placebo-injected volunteers exhibited 0.8-foldchanges during the evaluation period.

DISCUSSIONIn the present study, we showed that a new vaccine lot of

F. tularensis LVS induces antigen-specific humoral andcell-mediated immune responses in humans. The currenttularemia vaccine is made up of an attenuated, live F.

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FIG. 4. Immunoblot analysis of F. tularensis LVS (A) and PWE(B) separated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. LVS and PWE were either untreated (lanes 1) or treatedwith proteinase K (lanes 2). The blots were probed with serum

(1:500 dilution) that was collected 28 days after vaccination ofvolunteers with F. tularensis LVS vaccine. Bound antibodies weredetected with radiolabeled anti-IgG antibodies. Molecular massmarkers (in kilodaltons) are shown on the left.

tularensis strain that occurs in two colony phenotypes, blueand gray. One of these phenotypes (the blue variant) isimmunogenic and has been suggested to be of major impor-tance for the induction of protective immunity (4). Thereason for the switch of LVS from an immunogenic to a

nonimmunogenic variant is unknown. The nonimmunogenicvariant may have an altered cell surface (14) and wouldprobably be rapidly killed by nonspecific immune responsesin humans. Therefore, each lot of F. tularensis vaccineproduced is evaluated for its immunogenicity in humans.

Induction of humoral and cell-mediated immunity by othervaccine lots of LVS has been reported (8, 21). However, no

correlation between the titers of antigen-specific antibodiesin serum and the magnitude of in vitro lymphocyte prolifer-ative responses to recall antigens has been found (21). Wedetermined that immunization causes a general increase inspecific immune responsiveness, but the magnitude of thehumoral and cell-mediated immune responses did not corre-late between individuals. Individuals and time points thatshowed the highest antigen-specific antibody responses werenot necessarily those that exhibited the highest cell-mediatedimmune responses. This might be explained as heteroge-neous genetic predispositions to respond to antigenicepitopes. The correlation of Ia haplotypes with specificepitope responsiveness has not been studied with the LVSantigen.Measurement of F. tularensis antigen-specific serum anti-

bodies is commonly used to evaluate immunogenicity aftervaccination (21) and for the diagnosis of tularemia (19, 21).In previous reports, the choice of the immunoglobulin classmeasured was not critical because, in most cases, IgA, IgM,and IgG antibodies appeared simultaneously (1, 20). Al-though our results confirm that observation, our studiesshowed that antigen-specific increases in IgA titers werestatistically significant when compared to the rise in titer ofIgG or IgM. No early antigen-specific IgM titer rise wasdetected after vaccination. Although vaccination induced arise in antigen-specific IgA, IgG, and IgM titers, the IgAresponse was the best serological parameter of vaccination.IgA titers were highly significant (P < 0.01) within 56 days ofvaccination (Table 1). This may be due to low levels ofbackground absorbance with IgA compared with those withIgG, which appeared to bind nonspecifically to LVS cells(Fig. 1; Table 2). Although sera from some placebo-treatedcontrols contained IgA, IgG, and IgM antibodies that recog-nized LVS proteins in immunoblots, only IgG displayed ahigh level of nonspecific binding in ELISA. This differencemay be due to conformational or accessibility differencesbetween native LVS protein antigens in ELISA and dena-tured protein in immunoblots. The existence of IgG-bindingproteins, LVS antigenic epitopes that cross-react with otherbacterial antigens, or previous exposure to F. tularensis arepossible explanations for this apparent reactivity. The induc-tion of antigen-specific IgA antibody after immunization maybe explained by (i) stimulation of mucosal immunity by theintracutaneous route of vaccination, (ii) colonization of theliver by F. tularensis LVS, and/or (iii) the presence ofspecific IgA-inducing epitopes on the microorganism.The humoral immune response, as measured by immuno-

blotting, was heterogeneous. Sera from six of nine controlsand one of nine vaccinees did not show any reaction with F.tularensis antigens. Of the volunteers whose sera exhibitedantibody reactivities by immunoblotting, the numbers andintensities of antibody-reactive antigen bands were not reli-able predictors of vaccination effectiveness because theimmune responses of vaccinees and activities of some con-trols were somewhat heterogeneous. The heterogeneity ofantigen-specific antibody responses of vaccinees in thisstudy might be due to a T-cell-dependent antibody response,and thus may reflect the class II restriction of each volun-teer.The only distinguishing characteristic of the majority (five

of nine) of sera from vaccinated volunteers was the presenceof an antigen banding pattern in immunoblots (Fig. 3). Thetarget epitopes of the antigen banding profile are unknown,but they may involve lipopolysaccharide. The immunoblotof one vaccinee's day 28 serum against purified PWE andproteinase K-treated PWE (Fig. 4) appeared to be identical

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TABLE 4. Effect of F. tularensis LVS vaccination on in vitro lymphoproliferation to vaccine antigen

SI after receiving LVS recall antigen at a concn (pLg/ml) of d:Vaccine" Serumb Day-

55 5.5 0.55

P Auto 0 2.4 ± 2.2 6.7 ± 3.9 1.8 ± 2.6V Auto 0 1.4 ± 0.8 5.0 ± 1.6 0.9 ± 1.2

P Auto 7 1.3 ± 0.9 5.0 ± 3.2 0.9 ± 1.4V Auto 7 1.8 ± 1.4 6.1 ± 3.2 1.4 ± 2.1

P Auto 14 1.8 + 1.8 4.8 ± 2.5 2.1 ± 2.7V Auto 14 3.4 ± 3.5 11.5 ± 10.0 7.4 ± 8.8

P Auto 28 3.5 + 2.6 8.5 ± 6.2 3.7 ± 6.3V Auto 28 6.1 ± 3.7 18.0 ± 10.3e 9.7 ± 10.7

P Auto 56 2.2 ± 1.9 4.8 ± 3.6 1.3 ± 1.4V Auto 56 8.6 ± 5.9f 12.2 ± 4.7f 8.2 ± 7.9f

P PHAB 0 2.3 ± 2.3 8.4 ± 6.4 2.1 ± 3.9V PHAB 0 1.2 ± 1.0 4.7 ± 2.4 0.9 ± 1.4

P PHAB 7 0.9 ± 0.7 4.5 ± 3.3 0.6 ± 0.9V PHAB 7 1.7 ± 2.2 5.6 ± 3.4 0.9 ± 1.4

P PHAB 14 1.7 ± 1.6 3.5 ± 1.8 1.3 ± 1.8V PHAB 14 3.8 ± 3.3 14.9 ± 11.3f 8.7 ± 9g.e

P PHAB 28 1.7 ± 1.4 5.9 ± 2.9 1.5 ± 1.7V PHAB 28 4.2 ± 2.7e 13.1 ± 7.2f 7.0 ± 8.1l

P PHAB 56 1.3 ± 1.1 4.1 ± 3.7 1.8 ± 2.0V PHAB 56 3.9 ± 2.5f 11.3 ± 9,4e 7.7 ± 9.0

aPeripheral blood cells were obtained from volunteers vaccinated with placebo (P) or F. tularensis LVS vaccine (V).b Peripheral blood cells were cultured in the presence of 5% autologous serum (Auto) or 5% pooled human type AB serum (PHAB).c Peripheral blood cells were obtained from volunteers before vaccination (day 0) or at 7, 14, 28, or 56 days after vaccination. Values are mean ± standard

deviation (unbiased) SIs of vaccine and placebo groups.d Peripheral blood cells were cultured in the presence of irradiation-killed F. tularensis LVS at a concentration of 55, 5.5, or 0.55 pg/ml.e Significant differences between the placebo and vaccine groups at the P < 0.05 level (Student's t test).f Significant differences between the two groups at the P < 0.01 level (Student's t test).

to the antigen banding pattern evident in immunoblots withF. tularensis LVS. The immunoblot with serum from avaccinee obtained on day 28 also appeared to be identical toimmunoblots of anti-F. tularensis lipopolysaccharide mono-clonal antibody directed against PWE, purified lipopolysac-charide, and LVS (12). Therefore, lipopolysaccharide is alikely target of a humoral immune response seen in volun-teers vaccinated with LVS.

Sera collected during an outbreak of tularemia showedheterogeneous binding in immunoblots, and only one major,common band of 43 kDa was found (1). Only IgG antibodieswere identified in that study. One vaccinee in the presentstudy had IgG antibodies directed against a 46-kDa antigen.Another study (18) showed that sera from individuals whohad tularemia 8 to 24 months previously contained antibod-ies which recognized a dominant 17-kDa lipoprotein inimmunoblots (18). However, only a minority of serum sam-ples from individuals vaccinated with LVS 5 to 18 monthspreviously recognized the 17-kDa antigen in immunoblots.In our study, sera from two controls and four vaccineesrecognized an antigen of approximately 17 kDa. However, inthose individuals, antibody to the 17-kDa antigen was seen inthe preimmunization sera and was not enhanced by vacci-nation. Perhaps the different serological specificities seen inour study reflect differences between LVS vaccinees andpersons naturally infected with F. tularensis. The time

course after vaccination or infection may dictate the pre-dominance of carbohydrate reactivity in immune responses.

Intradermal vaccination with the F. tularensis LVS stim-ulated cell-mediated immunity, as measured by in vitrolymphocyte proliferation. Although the native microorgan-ism was sufficiently mitogenic to potentially mask low levelsof specific reactivity, the SIs of peripheral blood cells fromvaccinees rose significantly by day 14 postvaccination whencells were exposed to LVS in an assay with PHAB in theculture medium (Table 4). SIs of vaccinees remained signif-icantly (P < 0.05) greater than those of control volunteersthrough day 56 after vaccination. The stimulation of cellularimmune responses by LVS vaccination was also shown bymean in vitro lymphoproliferative responses of vaccinees toLVS that exceeded the response to the T-cell mitogen ConA(Fig. 5). Immunization with F. tularensis LVS did notsuppress or enhance cellular immune responsiveness tomitogens or C. bumnetii, an unrelated, gram-negative micro-organism. Protective immunity against F. tularensis isthought to be due to cell-mediated immunity (9). Cell-mediated immunity was reported to appear 1 to 4 weeks afterthe onset of disease (19) or after vaccination with the LVS ofF. tularensis (8, 21). The results of our study are consistentwith those results. Utilization of purified F. tularensis pro-teins (15) as recall antigens will be the subject of a futurereport.

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VACCINATION WITH FRANCISELLA TULARENSIS LVS

7

Positive Control

Negative Control

1 4 28 56

Days After VaccinationFIG. 5. Mean SIs of peripheral blood cells from volunteers vaccinated with F. tularensis LVS (-) or saline (LI). Cells were obtained before

vaccination and at 7, 14, 28, and 56 days after vaccination and were cultured in the presence of PHAB and LVS (5.5 ,ug/ml). The mean SIsof LVS-stimulated cells are shown relative to the mean ConA mitogenic response (positive control) and the mean response to C. bumetiichloroform-methanol residue antigen (negative control).

TABLE 5. Increase in specific lymphoproliferative responses toF. tularensis LVS in vaccinated and control volunteers

Fold No. (%) of volunteersbincreasea LVS autoc LVS PHAB

Vaccinees1 1 (11) 1 (11)2 1 (11) 2 (22)3 1 (1) 2(22)4 4 (44) 0 (0)5 1 (11) 2 (22)6 1 (11) 1 (22)7 0 (0) 0(0)11 0 (0) 1 (11)Meand 3.6 4.2

Controls1 7 (78) 7 (78)2 0(0) 2 (22)3 1 (11) 0(0)4 0 (0) 0 (0)5 1 (11) 0(0)Mean 0.8 1.0

a Maximum fold increase in peripheral blood lymphoproliferative SIs bycomparing individual preinoculation and maximum postinoculation SIs whenF. tularensis LVS (5.5 ,ug/ml) was used as the recall antigen.

b The number (percentage) of volunteers with an x-fold increase in periph-eral blood cell SIs when the maximum values obtained preinoculation andpostinoculation were compared.

c Peripheral blood cells were cultured in the presence of 5% autologousserum (auto) or PHAB.

d The mean fold increase in vaccinee and control SIs when peripheral bloodcells were incubated in the presence of F. tularensis LVS.

In conclusion, we found that a recently prepared vaccinelot of F. tularensis LVS induced cell-mediated and humoralimmune responses in volunteers. The humoral immuneresponse involved stimulation of IgA, IgG, and IgM antigen-specific antibodies. Although evaluation of cell-mediatedimmunity may still be needed to predict vaccine efficacy, arise in specific IgA antibody titers may be an excellentpredictor of vaccination effectiveness and exposure to thenative microorganism. ELISA or lymphocyte proliferationassays appear to be more predictive of vaccination effective-ness than immunoblot analysis. The immunoblot antigenicbanding patterns of sera from vaccinated and nonvaccinatedvolunteers appeared to be similar and difficult to interpret.However, at 28 days postinjection, antigenic banding pat-terns appeared in immunoblots of sera from a majority (fiveof nine) of vaccinees but were not present in immunoblots ofsera from placebo-injected volunteers. The identities of theantigen bands that make up the banding pattern are un-known, but preliminary results suggest that lipopolysaccha-ride may be involved.

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