Comparison of two immunization schedules for a Pseudomonas aeruginosa outer membrane proteins vaccine in burn patients

Vaccine 19 (2001) 1274 – 1283 www.elsevier.com/locate/vaccine

Comparison of two immunization schedules for a Pseudomonas aeruginosa outer membrane proteins vaccine in burn patients Dong Kun Kim a,*, Jeong Jin Kim a, Jong Hyun Kim a, Young Min Woo a, Sung Kim a, Dae Won Yoon a, Chang Sig Choi a, Ik-Sang Kim b, Wan Je Park c, Na-Gyong Lee c, Sang Bo Jung c, Bo Young Ahn c, Sung Woo Nam c, Suk Min Yoon c, Won Jung Choi c a

Hangang Sacred Heart Hospital, School of Medicine, Hallym Uni6ersity, Youngdungpo-ku, Seoul 150 -020, South Korea b Clinical Pharmacology Unit, College of Medicine, Seoul National Uni6ersity, SNUH, Seoul 110 -799, South Korea c R&D Center of Bioscience, Institute of Science and Technology, CheilJedang Corporation, Ichon, Kyonggi 467 -810, South Korea Received 5 April 2000; received in revised form 5 June 2000; accepted 6 July 2000

Abstract The aim of the present study was to compare two immunization schedules for a Pseudomonas aeruginosa outer membrane proteins (OMPs) vaccine in burn patients. In a double-blind, randomized and placebo-controlled clinical trial, 95 adult patients with burn injuries in 10% or greater of total body surface area were randomly allocated to either placebo or immunization groups. Three doses of the vaccine (0.5 or 1.0 mg) were administered intramuscularly at either 3- or 7-day intervals. The vaccine was well tolerated, and no severe adverse reactions were observed in any of the vaccinees. After three immunizations, 88 patients were available for evaluation of serum antibody titers. Elevation of OMPs-specific antibody titers in the immunization groups was significantly higher as compared with the placebo group, and the highest antibody response was obtained by immunization with 1.0-mg doses at 3-day intervals. Conventional blood culture, tissue culture of wound biopsy specimens and a nested polymerase chain reaction (PCR) assay of blood specimens were performed to determine the protective efficacy. The results of the nested PCR indicated that the overall detection rate of P. aeruginosa in blood was significantly lower among immunized patients than placebo patients (6.1 vs. 40.0%, PB0.001). Based on these results, we concluded that the P. aeruginosa OMPs vaccine is safe and highly immunogenic in burn patients, especially with 1.0-mg doses at 3-day intervals, and may be effective in conferring protection against P. aeruginosa bacteremia in burn patients. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Pseudomonas aeruginosa vaccine; Burn patient; Clinical trial; Immunization schedule

1. Introduction Burn patients are highly susceptible to infection with Pseudomonas aeruginosa which is one of the most predominant isolates recovered from burn wound infection [1 – 3]. The high rate of bacterial infection in burn patients is not only due to disruption of the mechanical barrier of the skin but also due to major alterations in the general immune system, and regional colonization with P. aeruginosa often leads to systemic infection, causing septic shock. P. aeruginosa is naturally resistant to most antibiotics and quickly develops resistance to * Corresponding author. Tel.: +82-2-26395442; fax: 82-2-6784386. E-mail address: [email protected] (D.K. Kim).

those commonly used [4,5]. For these reasons, development of prophylactic and therapeutic means to cope with this problem has been a subject of extensive research. Several attempts have been made to develop safe and effective vaccines against P. aeruginosa. Lipopolysaccharide (LPS), one of the major cell surface components, has been developed as vaccines in the form of conjugate with carriers. Although shown to be highly immunogenic and protective in human trials as well as in animal studies, they have not been routinely used because of the toxicity caused by the lipid A portion of LPS [6–11]. In addition, serotype specificity of LPS makes it difficult to develop a vaccine with a wide protective range. Outer membrane proteins (OMPs)

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D.K. Kim et al. / Vaccine 19 (2001) 1274–1283

also have been used as the target for vaccine development. A recombinant OprI protein and an OprF-OprI fusion protein of P. aeruginosa expressed in Escherichia coli were developed and tested in healthy humans [12,13]. They elicited high titers of specific serum antibodies. Meanwhile, a peptide vaccine derived from OprF was developed and tested for immunogenicity and protectivity in a mouse model [14,15]. We have been developing a P. aeruginosa vaccine that is composed of OMPs with molecular weight 10– 100 kDa from four P. aeruginosa strains and contains minimal contaminating LPS [16]. The safety and prophylactic efficacy of the vaccine have been demonstrated in animal model systems. It induced a high titer of anti-P. aeruginosa OMPs antibodies and protected mice against experimental infection with P. aeruginosa under the conditions of active and passive immunizations [17,18]. This OMPs vaccine has an advantage over LPS-based vaccines in that OMPs share homology among various immunotypes [19] and, thus, can afford cross-protection against heterologous P. aeruginosa strains [20]. In healthy humans, when given three times at 7-day intervals, the vaccine was well tolerated and highly immunogenic at 0.5- and 1.0-mg doses [21]. The immune sera from the vaccinees showed a significantly higher C1q-binding capacity and opsonophagocytic killing activity. When passively transferred to mice, it conferred protection against lethal infection with P. aeruginosa in a burn mouse model as well as in a normal mouse model [21]. The protective capacity of the OMPs-specific human antibodies was also confirmed using IgG purified from human normal plasma by affinity chromatography [22]. There have been several reports demonstrating that the immune system of burn patients is somewhat altered during thermal injury and that cellular and humoral immune response of burn patients would differ from those of healthy humans [23 – 27]. Therefore, immune responses to a vaccine observed in healthy people may not be obtained in burn patients. In the present study, we performed a clinical study of the P. aeruginosa OMPs vaccine in burn patients and compared two immunization schedules for the safety, immunogenicity and protective efficacy of the vaccine in order to determine an optimal dose and immunization schedule for burn patients.

2. Materials and methods

2.1. Vaccine The P. aeruginosa vaccine used in the clinical study was manufactured by CheilJedang (Ichon, Korea) as previously described [16]. The vaccine was composed of OMPs with a molecular weight range of 10 – 100 kDa,

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which was isolated from four attenuated P. aeruginosa strains belonging to Fisher-Devlin immunotypes 1, 2, 3 and 6. LPS content in the P. aeruginosa OMP vaccine was less than 20 ng per mg protein as determined by the chromogenic Limulus Amebocyte Lysate test using the QCL-1000 kit (Biowhittaker, USA). The vaccine was manufactured and tested according to the general guidelines of the Korean Pharmacopoeia for parenterally administered substances. Each vial of the vaccine contained 0.5 or 1.0 mg OMPs, 0.6 mg Na2HPO4, 0.1 mg KH2PO4, 4.4 mg NaCl, 0.1 mg KCl, and 20 mg D-mannitol. Vials for a placebo group contained the vaccine vehicle lacking OMPs. The vaccine content was reconstituted in sterile pyrogen-free distilled water immediately before use.

2.2. Study subjects Patients, aged 18 years or older, who had been admitted to the Hangang Sacred Heart Hospital (Seoul, South Korea) with burn injuries of 10% or greater of total body surface area were considered eligible for the study. Later, the degree of burn size for the eligibility was changed to the range of 10–50% of total body surface (Section 3). At admission, patients were asked about their medical history and underwent general physical examinations including electrocardiography, and clinical laboratory tests for blood and urine analysis. Patients who received inhalation injury or severe electrical burn injury were excluded from the study. Other exclusion criteria were impaired immune functions, underlying diseases, pregnancy or previous experiences of hypersensitivity to any proteins or vaccines. All the participating patients received the same clinical management during the study period. Antibiotic treatment was given to patients with burned surface greater than 20% of total body surface or when burn wound infection was suspected. Initially given were cefmetazol and amikacin, which were known to be the most effective chemotherapeutic agents against prevalent microorganisms at our burn unit according to the data of our infection-control committee. At the same time, infected burn wounds were debrided and tissue specimens were subjected to tissue culture and histologic examination. Based on the microbiological results, the antibiotic therapy was discontinued or switched to other antibiotic regimens. Taking into consideration antibiotic treatment and its regimens, 80–90% patients of each group were given the treatment and there were no significant differences between placebo and immunization groups. The study protocol was approved by the Institutional Review Board of the Hangang Sacred Heart Hospital and the Korea Food and Drug Administration. The clinical trial was performed according to the Korean Good Clinical Practice (KGCP).

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2.3. Immunization protocol

that of the highest titer among all patient sera. The dilution factor was 1600.

After providing informed written consent, study participants were randomly allocated in the ratio of 2:2:1, using a computer-generated randomization program, to receive three intramuscular injections into deltoid of either 0.5 or 1.0 mg vaccine or placebo control, respectively. Immunization schedule was either 3- or 7-day intervals (on days 0, 3 and 6 or on days 0, 7 and 14, respectively). The first injection was given as soon as possible after entry into the study, at the latest within 72-h post-burn injury. All vaccine recipients underwent a brief physical examination after each immunization and were under surveillance for immediate reactions at least for 30 min. Signs of systemic and local reactions or any changes of vital signs were monitored and recorded on a control sheet for each patient. Then 1 week after the third immunization, blood and urine samples were collected and analyzed for laboratory tests. Blood samples were taken from patients by venipuncture on days 0, 3, 7, 14 and 21 for the 3-day interval groups and on days 0, 7, 14, 21 and 28 for the 7-day interval groups. Sera were prepared from an aliquot of each blood sample and stored at −70°C until assayed. The rest of the sample was used for conventional blood culture in order to detect bacteremia or frozen immediately for nested polymerase chain reaction (PCR) analysis. Central line catheter tip culture or tissue culture and histologic examination of skin biopsy specimens were performed when invasive burn wound infection was suspected. Results obtained from the nested PCR were compared with those of conventional blood culture, tissue culture and histologic examination.

2.4. ELISA Serum antibody levels were determined by an enzyme-linked immunosorbent assay (ELISA) as described previously [21]. Goat anti-human immunoglobulin antibody conjugated with horseradish peroxidase was used as a secondary antibody and ophenylenediamine dihydrochloride as a chromogenic substrate. All samples were assayed in duplicate. Antibody levels were expressed as ELISA unit (EU) which was calculated as below:

EU= [(Asample /(Apositive

sera

−Anegative

control

control

−Anegative

)

control

)] ×dilution factor

where Asample sera is the absorbance of patient sera collected before or after immunization; Anegative control is the absorbance of the lowest titer and Apositive control is

2.5. Nested PCR An aliquot (400 ml) of a patient blood specimen was mixed with 600 ml of resuspension buffer (83 mM Tris–HCl, pH 8.5, 1.6 mM EDTA, 0.83% Tween 20) and boiled for 10 min. After centrifugation, the supernatant was recovered, and proteinase K was added to a final concentration of 600 mg ml − 1, incubated at 55°C for 2 h and inactivated by boiling for 10 min. DNA was then extracted with an equal volume of a mixture of phenol:chloroform (1:1, v/v), mixed with 1/10 vol. of 3 M sodium acetate (pH 5.2), followed by precipitation in isopropanol at 4°C for 30 min. After centrifugation, the recovered pellet was washed with ice-cold 70% ethanol, resuspended in 100 ml of sterile distilled water and used as a template for PCR. Two sets of PCR primers were purchased from Bioneer (Cheongwon, Korea). The first round PCR primers PL1 (5%-ATGGAAATGCTGAAATTCGGC3%) and PL2 (5%-CTTCTTCAGCTCGACGCGACG-3%) corresponded to the beginning and the end of the open reading frame of the P. aeruginosa oprL gene, respectively, and amplified a 504-base pair (bp) fragment [28]. The second set of primers, PL3 (5%-CGACCCGAACGCAGGCTATG-3%) and PL4 (5%-CGACCGGACGCTCTTTACCA-3%), were designed to amplify a 335-bp fragment of sequences within the first round PCR product. For the first PCR, 10 ml of template DNA, 10 ml of a primer mix (25 pmol each of PL1 and PL2), and 30 ml of sterile distilled water were added to the AccuPower™ PCR tube (Bioneer) containing 2.5 U Taq DNA polymerase and a deoxynucleotide mixture (dATP, dGTP, dCTP and dTTP; final concentration 250 mM each), and vortexed briefly. The reaction was carried out for 25 cycles using Perkin Elmer DNA Thermal Cycler 480 (Perkin Elmer, USA), and the reaction condition was as follows: initial template denaturation, 94°C for 2 min; denaturation, 94°C for 40 s; annealing, 55°C for 40 s; extension, 72°C for 40 s; final extension, 72°C for 7 min. A total of 5 ml of the first reaction product was taken and used as a template for the second PCR, which was performed in the same way as the first PCR except that PL3 and PL4 were used. After the second amplification, 15 ml of the reaction mixture was electrophoresed on a 1.5% agarose gel. The gel was stained with ethidium bromide and visualized on a UV transilluminator. Each set of experiments included a positive control reaction containing P. aeruginosa GN11189 DNA to confirm the success of amplification and a negative control lacking DNA template to check for adventitious contamination.

D.K. Kim et al. / Vaccine 19 (2001) 1274–1283

2.6. Statistical analysis For antibody levels elicited by immunization with the vaccine, geometric mean titers and standard deviations were calculated, and the differences in antibody titer among study groups at different time points were compared by the two-tailed Student’s t-test. The detection rates of P. aeruginosa determined by the nested PCR among study groups were compared by the chi-square analysis. The influence of age, sex, extent of burn, and time interval between immunization and the onset of burn injury on vaccine immunogenicity and efficacy as the outcome variables was analyzed with multiple regression analysis, or when appropriate, logistic regression analysis. The difference was considered to be significant at PB 0.05.

3. Results

3.1. Characteristics of the study subjects Among 100 burn patients screened for a period of 14 months, a total of 95 patients were enrolled in the study (Fig. 1). A total of 16 patients in the placebo group and 72 in the immunization groups (92.6% of the total patients enrolled) completed the study. Among the seven drop-outs, two were excluded from the study for medical reasons. One patient in the immunization groups developed an acute pulmonary complication 3 days after the first injection, and the other patient died of staphylococcal sepsis on day 11. The basic characteristics of the patients who completed the study are shown in Table 1. The mean age of the patients was

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36.7 years, and the oldest patient was 68 years old. The mean degree of burn size was 32.2%, and the mean time interval between the first immunization and burn injury was 41.6 h. There were no significant differences among study groups with respect to age, sex of patients, and the first immunization time after burn injury. But, significant differences were observed in mean degree of burn size between 3-day interval groups and the other three groups. Under the current guidelines of KGCP, death of patients enrolled for a clinical study greatly hampers the study: after the occurrence of the patient death described above, the burn size for the study enrollment was restricted to 10–50% of total body surface to avoid further death of study subjects. This resulted in a significant reduction in the mean degree of burn size of 3-day interval groups.

3.2. Safety of the 6accine No systemic or local reactions were observed in the placebo group. In the immunization groups, no serious systemic responses or changes in vital signs attributable to immunization were noted with two exceptions. One patient given 0.5-mg doses at 7-day intervals ran a fever of 39.0°C, 24 h after the second immunization and 18 h after the third immunization. Another patient given 0.5-mg doses at 3-day intervals developed a fever of 39.5°C after the second immunization. In both cases, the fever was transient and dissolved without treatment. As for local reactions, two patients receiving 1.0-mg doses complained of tenderness and developed erythema and induration at the injection sites, which were mild and self-limited. During the study, a patient who had received 0.5-mg doses at 7-day intervals died on

Fig. 1. Schematic diagram of the clinical study of P. aeruginosa OMPs vaccine burn patients. * The patient died of staphylococcal sepsis on day 11. ** The patient developed pulmonary complication on day 3.

16 19 20 88

1.0 0.5 1.0

3

13/3 17/2 17/3 71/17

13/3 11/6

Sex (male/female)

Burn size (%), mean9 S.D. (range)

35.5921.0 (15–95) 38.7 913.9 (15–65) 34.2912.4 (12–50) 25.3 911.0** (13–45) 27.4911.2* (10–50) 32.2914.9 (10–95)

Age (years), mean 9S.D. (range)

33.8 9 8.2 (19–47) 40.8 9 11.5 (25–68) 40.8 99.6 (24–56) 34.1 910.0 (19–55) 34.899.2 (19–51) 36.7 9 10.1 (19–68)

Only includes patients who completed the study. * PB0.05 versus the 0.5-mg doses, 7-day interval group. ** PB0.01 versus the 0.5-mg doses, 7-day interval group and PB0.05 versus the 1.0-mg doses, 7-day interval group.

a

Total

16 17

– 0.5

– 7

No. of subjects

Placebo group Immunization group

Dose (mg)

Interval (days)

Group

Table 1 Basic characteristics of burn patients enrolled in the clinical study of the P. aeruginosa vaccinea

44.1911.7 46.6 915.1 38.2 918.7 41.6915.9

(21–72) (19–71) (5–66) (5–72)

43.29 20.7 (8–70) 35.99 8.7 (12–46)

Time of first immunization after burn injury (h), mean9 S.D. (range)

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D.K. Kim et al. / Vaccine 19 (2001) 1274–1283

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Table 2 Antibody responses of burn patients to the P. aeruginosa OMPs vaccine Group

Placebo group Vaccination group

Interval Dose (days) (mg)

No. of subjects

GMTa 9 S.D. (ELISA unitsb) 0 days

3 days

7 days

14 days

21 days

28 days

141.59 72.9

354.3 9167.3*

368.1 9139.4*

508.1 9 281.8*

420.0 9223.4*

316.2 9134.1*

551.6 9 230.9*,**

550.8 9227.4*

622.7 9193.9*,**

499.0 9 300.7* 337.9 9 126.4* 437.8 9222.8*

755.0 9 271.8*,** 649.3 9 277.6*,** 949.0 9370.0*,**

783.1 9229.7*,** 779.6 9273.8*,** 661.2 9249.2* NT 917.3 9339.2*,** NT

–

–

16

162.8 9 126.7

7

0.5

17

111.4 9 68.8

3

1.0 0.5 1.0

16 19 20

118.3 9 56.9 152.2 995.0 193.9 9 99.1

NTc NT 146.1 995.0 157.4 9 86.3

a

GMT, geometric mean titer. ELISA unit = [(Asample sera−Anegative control)/(Apositive control−Anegative control)]×dilution factor. Asample sera is the absorbance of patient sera collected before or after immunization; Anegative control is the absorbance of the lowest titer and Apositive control is that of the highest titer among all patient sera. The dilution factor was 1600. c NT, not tested. * PB0.05 versus the corresponding pre-immune sera at the same day. ** PB0.05 versus the corresponding sera of the placebo group at the same day. b

day 11, which was confirmed to be not related to the immunizations but was due to staphylococcal sepsis.

3.3. Immunogenicity Burn patients received three doses of the vaccine or placebo at 7- or 3-day intervals. Sera collected from the patients were analyzed by ELISA for antibody levels against the P. aeruginosa OMPs, which were converted to EU. For all five groups including the placebo group, a significantly high rise in mean total antibody level was observed as early as day 7 when compared with the corresponding pre-immune sera (Table 2). But, when compared with the placebo group of the same day, only the 1.0-mg groups showed higher antibody titers. For the immunization groups, maximum antibody levels were generally reached at day 14 or 21 and were significantly higher than that of the placebo group. Immunization with 1.0-mg doses tended to induce a faster and higher antibody response than 0.5-mg doses regardless of immunization interval, indicating a dose-dependent response to the vaccine. Between two immunization intervals, 3-day intervals gave a faster and higher antibody response at both doses than 7-day intervals. There were no significant differences in antibody induction with respect to age, sex, extent of burn, and time interval between the first immunization and burn injury, as determined by multiple regression analysis (data not shown).

Fig. 2 illustrates the antibody responses of individual patients to the vaccine. The antibody levels of pre-immune sera from all burn patients were substantially low, and with a few exceptions, were below 400 EU. In the 7-day interval groups, three patients receiving 1.0mg doses showed a marked increase in antibody level even after the first immunization (on day 7) (Fig. 2a). At day 14, most immunized patients began to show a rise in antibody titer. After three immunizations, all immunized patients showed antibody levels greater than the corresponding pre-immune sera, and the ratios of the post- to pre-immune sera antibody levels were greater than 2, which is considered sero-converted according to previous criteria [21,29]. In the meantime, among the placebo patients, the levels of two patients rose sharply at day 21, which suggested bacterial infections. In fact, one patient who had an antibody level of 1340 EU at day 21, was found infected with P. aeruginosa as determined by tip culture from day 7 through the study period. The other patient with an antibody level of 850 EU had an Acinetobacter infection. The patients in the 3-day interval groups generally showed a much higher responses as compared with the 7-day interval groups (Fig. 2b). All but one vaccinated patient responded to the vaccine after three immunizations. These results indicate that the P. aeruginosa OMPs vaccine is highly immunogenic in burn patients, especially with 1.0-mg doses given at 3-day intervals.

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D.K. Kim et al. / Vaccine 19 (2001) 1274–1283

3.4. Protecti6e efficacy In order to detect P. aeruginosa and other bacterial infection in the blood of patients, conventional blood culture was routinely performed. Central line catheter tip culture and tissue biopsy for culture and histologic examination were carried out only when invasive burn wound infection was suspected. Among 13 placebo patients tested by blood culture technique, only one was found positive for P. aeruginosa at day 21, while none of 68 immunized patients were positive (Table 3). The small number of patients tested and low detection rates did not allow us to obtain meaningful data. Likewise, neither tissue biopsy nor tip culture revealed any significant differences between immunization and placebo groups at any time point during this study (data not shown). Since the detection rate of P. aeruginosa determined by conventional culture techniques was too low to evaluate the protective efficacy of the vaccine, we designed a nested PCR analysis for blood specimens. The nested PCR was based on the P. aeruginosa oprL gene

using two sets of primers, one of which has been demonstrated to be highly sensitive and specific for P. aeruginosa DNA [28]. Blood samples taken from the burn patients on days 0, 7, 14 and 21 were used for the nested PCR analysis. At day 0, the overall P. aeruginosa detection rate for the immunization groups was rather higher than the placebo group (PB 0.001) (Table 4). But, the rate began to decline at day 7 and, after three immunizations, became significantly lower than the placebo group. These results suggested that immunization with the vaccine provided protection against P. aeruginosa infection in these patients. Furthermore, a higher clearance rate of P. aeruginosa from immunized patients who had infection during the study (100 vs. 44.4% of placebo patients) suggested a therapeutic effect of the vaccine. There was no significant difference in the rate of detection for P. aeruginosa with respect to age, sex and interval between burn injury and the first immunization as determined by logistic regression analysis. With respect to the extent of burn size, however, a significantly greater detection rate was obtained with a higher degree of burning, especially in patients with

Fig. 2. Serum antibody responses of burn patients to immunization with the P. aeruginosa OMPs vaccine at 7-day (a) or 3-day (b) intervals. Burn patients were immunized three times with the vaccine at either 0.5-mg ( ) or 1.0-mg () doses. Patients in the placebo group were given vaccine vehicle lacking the OMPs (). The x-axis is the antibody titer of pre-immune sera, and y-axis is that of sera collected at the day indicated on the graph.

D.K. Kim et al. / Vaccine 19 (2001) 1274–1283

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Table 3 Results of the blood culture of burn patients Group

Placebo group Vaccination group

Detection rate (%) (no. of positive patients/no. of tested patients)

P. aeruginosa Other bacteria P. aeruginosa Other bacteria

7 days

14 days

21 days

0 (0/16) 6.3 (1/16) 0 (0/70) 2.9 (2/70)

0 (0/16) 12.5 (2/16) 0 (0/71) 5.6 (4/71)

7.7 (1/13) 7.7 (1/13) 0 (0/68) 4.4 (3/68)

burn size of 30% or greater of total body surface area (data not shown).

4. Discussion In this study, we investigated the immune response of burn patients to the P. aeruginosa OMPs vaccine given by two different schedules. The results of the study revealed that immunization with three does of 0.5- or 1.0-mg vaccine at 7- or 3-day intervals induced significantly higher antibody responses in burn patients when compared to the placebo group, suggesting the applicability of the OMPs vaccine to burn patients. Since the severity and extent of burn size may affect the immune response of patients to vaccines, it may not be possible to directly compare the four immunization groups for immunogenicity because of the differences in the degree of burn size. The serum antibody response of vaccinees, however, was dose-dependent regardless of immunization interval, and between two 1.0-mg dose groups with a similar mean burn size, 3-day interval immunization yielded the better results. Therefore, we concluded that immunization with 1.0-mg doses at 3-day intervals was optimal for burn patients. Meanwhile, the antibody titer against P. aeruginosa OMPs also increased with time in the placebo group, indicating an antibody induction by natural infection in burn patients. There are several lines of evidence suggesting that the immune system of burn patients is somewhat altered during thermal injury and that cellular and humoral immune responses of burn patients would differ from those of healthy humans [23 – 27]. It is also believed that the functional activity of antibodies raised by natural infection in burn patients may be different from those induced by active immunization and that the presence of specific antibodies in the patients may not correlate with protection against infection. Our study showed that antibodies elicited by natural infection in burn patients were not as effective in providing protection against P. aeruginosa in a mouse model as those induced by active immunization with the P. aeruginosa OMPs vaccine [30]. In addition, the sera from burn patients were not efficient in promoting opsonophagocytic-killing of P. aeruginosa, but

immunization of burn patients with the vaccine markedly enhanced the opsonic activity of the sera [30]. This difference may be related to the observation that antibodies raised in healthy volunteers by active immunization with the vaccine were mainly directed to native conformation-sensitive epitopes, while antibodies elicited in burn patients by natural exposure were directed to either linear or conformational determinants [31]. Anti-P. aeruginosa OMPs IgG antibodies purified from unimmunized normal human sera through affinity chromatography using the OMPs as ligands were highly protective in a mouse model as well as in in vitro assays [20,22]. Another line of evidence implying the importance of active immunization for protection against P. aeruginosa infection comes from a study on cystic fibrosis (CF) patients. Immunization of CF patients with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine elicited antibody responses to O-polysaccharide with high affinity which correlated with a reduced rate of lung infection with P. aeruginosa in vaccinated patients [32,33]. Such high affinity antibodies, however, were rarely acquired by natural infection with P. aeruginosa in the lungs of CF patients who developed chronic infection despite the high serum anti-P. aeruginosa LPS antibody levels. Determination of the survival rate of study subjects after immunization is critical in evaluating the protective efficacy of the vaccine. But, under the current regulations of KGCP, it was not possible to perform such a study. Thus, we attempted to assess the efficacy by determining P. aeruginosa bacteremia because the emergence of bacteremia is of substantial prognostic and therapeutic importance in burn patients. Conventional blood culture techniques have been routinely used to diagnose bacteremia in burn patients. Detection rates of microorganisms in blood specimens, however, were often lower than what would be actually present, probably due to decrease in the number of viable microorganisms resulting from antibiotic therapy and host defense mechanisms. Recently, Heininger et al. have demonstrated that an antibiotic treatment reduced the detection rate of E. coli in the blood of experimentally infected rats and that PCR significantly improved the detection rate even in the presence of the antibiotic [34]. PCR-based detection of infecting pathogens in

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Table 4 Detection of P. aeruginosa by nested PCR in the blood specimens of burn patients Group

Placebo group Vaccination group

Interval (days)

– 7 3

Dose (mg)

– 0.5 1.0 0.5 1.0

Subtotal

Detection rate of P. aeruginosa (%) (no. of positive patients/no. of tested patients) 0 days

7 days

14 days

21 days

15.4 18.2 0.0 21.1 25.0 18.6

42.9 27.3 30.0 5.3 15.0 16.7

33.3 8.3 25.0 5.3 5.0 9.5

40.0 0.0 7.1 5.3 10.0 6.1

(2/13) (2/11) (0/9) (4/19)* (5/20)* (11/59)**

(6/14) (3/11) (3/10) (1/19)* (3/20)* (10/60)**

(5/15) (1/12) (3/12) (1/19)* (1/20)* (6/63)**

(6/15) (0/13)* (1/14)* (1/19)* (2/20)* (4/66)**

* PB0.05 versus the placebo group at the same day. ** PB0.001 versus the placebo group at the same day.

clinical specimens has been employed to increase the sensitivity of diagnosis and evaluate the efficacy of vaccines in human trials [35 – 37]. Indeed, the results of the present study showed that blood culture technique is much less efficient than the nested PCR in detecting P. aeruginosa in blood specimens during antimicrobial treatment. The P. aeruginosa detection rates determined by the nested PCR in the immunized patient groups were significantly lower than that of the unimmunized patient group at day 14, when Pseudomonas infection is usually clinically overt in burn patients [38]. Furthermore, the clearance rates of P. aeruginosa in the immunization groups confirmed by negative conversion were significantly higher than that of the placebo group, suggesting a therapeutic effect of the vaccine in these patients. Since the rates of antibiotic treatment were similar among patient groups and PCR detection of P. aeruginosa is not affected by the presence of antibiotics, it is unlikely that antibiotic treatment influenced the detection rate of P. aeruginosa in patient blood specimens. A critical finding made in this study is that an immunization schedule as short as 3-day intervals induced a good antibody response, even higher than that induced by 7-day intervals. This fast antibody response in humans may be due to the fact that P. aeruginosa is an opportunistic pathogen and most people are exposed to or infected with P. aeruginosa during their lifetime. The antibody induction by immunization, therefore, is thought to be a memory response. Most human vaccines are designed for immunization at a few week- or several month-intervals, and there is no single vaccine currently available or being developed with this short immunization schedule. A fast antibody induction is essential for protecting burn patients in whom bacterial sepsis often occurs as early as 14 days after they receive thermal injury [38], and from this point of view, our finding implies a major benefit of using the OMPs vaccine to burn patients. Based on these results, we believe that further evaluation of this promising P.

aeruginosa vaccine in major burn is warranted and that immunization with three doses of 1.0 mg vaccine at 3-day intervals is optimal for immunoprophylaxis against Pseudomonas infection in burn patients.

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