Protective effects of affinity-purified antibody and ... - Wiley Online Library

Microbiol Immunol 2009; 53: 587–594 doi:10.1111/j.1348-0421.2009.00165.x

ORIGINAL ARTICLE

Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice Kiyoshi Moriyama1,4 , Jeanine P. Wiener-Kronish1,2,3,5 and Teiji Sawa1,6 1 Departments of Anesthesia and Perioperative Care and 2 Medicine, 3 Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94143, 5 Department of Anesthesia and Critical Care, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA; and 4 Department of Anesthesiology, Kyorin University School of Medicine, Tokyo 181-8611, 6 Department of Anesthesiology, Kyoto First Red Cross Hospital, Kyoto 605-0981, Japan

ABSTRACT Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide-induced immunosuppression by intraabdominal challenge of P. aeruginosa. Active immunization with either full-length PcrV1-294 or PcrV139-294 significantly improved the survival of mice infected with P. aeruginosa, while PcrV139-258, PcrV139-234, PcrV197294, and PcrV261-294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144-257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity-purified anti-PcrV IgG against either PcrV1294 or PcrV139-258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti-PcrV IgG and Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies that more potent anti-PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti-PcrV antibody against P. aeruginosa infection in humans. Key words affinity-purified antibody, component vaccine, Pseudomonas aeruginosa PcrV.

P. aeruginosa is one of the most common opportunistic pathogens causing lethal systemic infection in immunocompromised patients (1,2). Because P. aeruginosa is frequently resistant to a wide variety of antibiotics, infection with this organism may not be controlled by conventional antibiotic therapy (3). Therefore, there is a need for non-antibiotic based adjuvant therapies for P.

aeruginosa infection (4–6). Both active and passive immunization in high-risk patients are potential adjuvant strategies for controlling antibiotic-resistant P. aeruginosa infections (6). We and others have reported the protective effects of active and passive immunization against PcrV in lethal P. aeruginosa infections in animal models (7–11).

Correspondence Kiyoshi Moriyama, Department of Anesthesiology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Tel: +81 422 47 5511; fax: +81 422 43 1504; email: [email protected] Received 3 February 2009; revised 26 March 2009; accepted 3 July 2009. List of Abbreviations: aa, amino acid; anti-PcrV Rab, rabbit polyclonal anti-PcrV antibodies; cfu, colony forming units; control Rab, rabbit polyclonal control IgG; CP, cyclophosphamide; E. coli, Escherichia coli; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S transferase; i.p., intraperitoneally; LcrV, Yersinia V-antigen; Mab166, murine monoclonal anti-PcrV IgG; one-way ANOVA, one factorial analysis of variance; P. aeruginosa, Pseudomonas aeruginosa; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PcrV, P. aeruginosa V-antigen; Y. enterocolitica, Yersinia enterocolitica.

c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

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PcrV is a component of the P. aeruginosa type III secretion system and homologous to LcrV (7,12). More than 40 years ago protective antigenic characteristics of V-antigen were reported in Yersinia pestis (13,14). Vaccination against LcrV was found to be highly effective against both bubonic and pneumonic plague when tested in animal models (15–18). The monoclonal antibody against LcrV was protective against Yersinia pestis infection in mice (19). LcrV has been shown to be an integral component of translocation of the type III secretion system which mediates the delivery of type III secretory toxins into targeted eukaryotic cells (20). In P. aeruginosa, virulent strains expressing PcrV disable macrophage phagocytosis and induce acute lung injury by translocation of type III secretory toxins (7). In a clinical trial, ventilator-associated pneumonia due to P. aeruginosa type III secretory phenotypes was associated with increased neutrophilic apoptosis and persistent alveolar infection (6). Therefore, P. aeruginosa V-antigen PcrV appears to have a critical role in type III secretion-associated virulence. In our previous studies, we have demonstrated that the administration of anti-PcrV Rab prevents septic shock and acute lung injury in animal models of P. aeruginosa pneumonia, and that the effects of anti-PcrV Rab are independent of the Fc-fragments of IgG (7,8). In a murine model of chronic P. aeruginosa respiratory infection, anti-PcrV immunoglobulin G reduced the pulmonary inflammatory reaction (21). We recently generated a murine monoclonal anti-PcrV antibody, Mab166, that was comparable to antiPcrV Rab in decreasing mortality and acute lung injury in animals infected with P. aeruginosa (10,11). The characterization of Mab166 provided the evidence that, by blocking an epitope on PcrV, type III secretion-associated virulence could be inhibited (10). In this study, the PcrV regions that contribute to protective immunity against P. aeruginosa infection were investigated. This information could lead to a more optimal PcrV vaccine and antibody therapy against P. aeruginosa. By using a series of genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against these PcrV proteins, the efficacy of active and passive immunization was tested in mice with CP-induced immunosuppression by intraabdominal challenge with P. aeruginosa.

MATERIALS AND METHODS Reagents

CP was obtained from Bristol-Myers Squibb (Lyophilized Cytoxan, New York, NY, USA). Control Rab, anti-PcrV Rab, and Mab166 were prepared as described previously 588

(8,10). Control Mab was obtained from R&D systems (clone #133303, Minneapolis, MN, USA). P. aeruginosa strain

P. aeruginosa strain PA103 wild type was used in this study. Bacterial suspensions were prepared as previously described (22). Briefly, bacteria from a frozen stock were subcultured onto Vogel-Bonner minimal medium and inoculated into deferrated dialysate of trypticase soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma Chemical, St. Louis, MO, USA), 1% glycerol, and 100 mM monosodium glutamate. Cultures were grown at 33◦ C for 13 hr in a shaking incubator, then centrifuged at 8500 × g for 5 min. The bacterial pellets were washed three times in lactated Ringer’s solution and diluted into the appropriate number of cfu per milliliter as determined by spectrophotometry. The number of bacteria was confirmed by determining the cfu of diluted aliquots on sheep blood agar plates. PcrV vaccines

In a previous experiment, we constructed an E. coli plasmid library for the expression of GST-tagged truncated PcrV (10). Briefly, the coding sequences for PcrV were amplified from the chromosome of P. aeruginosa PA103 by PCR. The PCR fragments were then ligated in frame into the E. coli expression vector pGEX-2TK (Amersham Pharmacia Biotech, Piscataway, NJ, USA) to create GST-fusion protein constructs. Recombinant proteins were induced by isopropylthio-beta-galactoside and purified from the E. coli milieu using GST purification modules (Amersham Pharmacia Biotech). The bound recombinant proteins in the GST column were digested overnight with thrombin to cleave off the GST tag. The proteins were then eluted with reduced glutathione, dialyzed overnight against PBS, and applied to a detoxification column (Detoxi-Gel, Pierce, Rockford, IL, USA) to remove endotoxin. The amount of endotoxin in the final product was less than 2 endotoxin units/ml as measured by the limulus amebocyte lysate assay (Pyrochrome, Associates of Cape Cod, Falmouth, MA, USA). Affinity–purified polyclonal anti-PcrV IgG

Either full-length PcrV1-294 or PcrV139-258 was immobilized to the column using AminoLink kit (Pierce Biotechnology, Rockford, IL, USA). IgG fractions derived from rabbit anti-PcrV serum after protein A column chromatography were applied to the PcrV immobilized columns. The bound proteins were eluted by IgG elution buffer (Pierce Biotechnology) and dialyzed against PBS for 24 hr. The aliquots were stored at −80◦ C. c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

Affinity purified antibody against PcrV

Animals

The protocols for all animal experiments were approved by the animal research committee of the University of California, San Francisco, USA. Male Balb/c mice (Charles River Laboratories, Wilmington, MA, USA), 6–8 weeks old for active immunization studies or 8–12 weeks old for other studies, were housed in pathogen-free conditions, using cages with filter tops. Active immunization

A time course of the protocols for active and passive immunization, the induction of leukocytopenia, and survival studies after P. aeruginosa infection is summarized in Figure 1. Mice were immunized with one of six different PcrV vaccines including one full-length PcrV (PcrV1-294) and five truncated PcrV proteins (Fig. 2). Control mice were immunized with Freund’s adjuvant alone. On day 0, mice were injected with 30 μg of proteins emulsified in Freund’s complete adjuvant (Sigma Chemicals). The mice were boosted with an equal amount of the protein emulsified in Freund’s incomplete adjuvant (Sigma Chemicals) on day 30, and housed for another 30 days before survival study. For passive immunization, a designated dose of antibodies was injected i.p. into the mice 1 hr before bacterial challenge. Induction of leukocytopenia

Leukocytopenia was induced by the injection of CP (5 mg/mouse) i.p. (23,24). To evaluate leukocytopenia in these mice, blood samples were collected from tail veins 3 days after CP or saline injection (n = 3 in each group). Total leukocyte and differential cell counts were determined on a hemacytometer and by Wright-Giemsa staining. Body weights of the mice were also measured and compared for 3 days after CP or saline injection (n = 5 in each group). Survival studies

P. aeruginosa (5 × 106 cfu) suspended in 150 μl of a sterile saline solution was injected i.p. into mice. After the injection, the mice were returned to their cages, monitored regularly, and allowed access to food and water. Survival of the mice was monitored for 48 hr. Measurement of anti-PcrV titers

Sixty days after the first vaccination, serum samples from each mouse were collected from tail veins and titers against PcrV were measured by ELISA. Multi-well plates were coated with 100 ng of purified recombinant full-length PcrV, blocked with a solution of 1% bovine serum albumin, and aliquoted with dilutions of antisera. The plates c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

Fig. 1. The experimental protocol of our survival studies. For active immunization, mice were immunized with one of the PcrV vaccines with complete Freund’s adjuvant on day 0 and with incomplete Freund’s adjuvant on day 30. To induce leukocytopenia, mice received 5mg of CP on day 60. Mice were infected with 5 × 106 colony forming unit of P. aeruginosa on day 63, and survival was monitored until day 65. For passive immunization, mice were immunized with either anti-PcrV IgG or control IgG 1 hr before bacterial infection.

were washed and peroxidase-labeled antibodies against murine IgG (Sigma Chemicals) were added at 1:20 000. To evaluate the anti-PcrV IgG isotypes, goat anti-mouse IgG1, IgG2a, IgG2b or IgG3 (Sigma Chemicals) were added at 1:2000 as primary antibodies. The reaction was developed by using the peroxidase substrate 2,2 -azino-bis (3ethylbenzthiazoline-6-sulfonic acid). Titers were calculated as the maximum dilution of serum to give a 450-nm absorbance reading 0.1. Statistical analysis

The Mantel-Cox log rank test was used for assessment of mouse survival. One-way ANOVA followed by the Bonferroni multiple comparison test was used for comparison of other data except when otherwise indicated. Data are expressed as means ± SD. A P value < 0.05 was considered statistically significant.

RESULTS Induction of immunosuppression in mice

To induce immunosuppression in mice, 5 mg of CP or saline was injected i.p. Significant decreases in the numbers of total leucocytes, neutrophils and lymphocytes were observed in the CP-treated mice compared with those in the control mice (Fig. 3a). A significant decrease in body weight was observed in the CP-treated mice (Fig. 3b). Next, in another set of mice, P. aeruginosa (5 × 106 cfu) was injected i.p. 3 days after either CP or saline injection. It 589

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Fig. 2. Full-length PcrV and truncated PcrV vaccines used in this study. Scheme shows the minimal region to which murine monoclonal anti-PcrV IgG, Mab166, can bind. MW, predicted molecular weight.

was found that 80% of mice treated with saline and none of the CP-treated mice survived for 48 hr (Table 1).

Next, in the CP-treated mice, we tested the protective effect of active immunization with PcrV proteins against lethal P. aeruginosa infection. First, mice were actively immunized with one of six different PcrV (Fig. 2) or Freund’s adjuvant alone. Sixty days after the first shot of vaccine, anti-PcrV IgG titers were measured in the serum of the vaccinated mice (Fig. 4a). PcrV1-294, PcrV139-294, PcrV139-258 and PcrV139-234, but not PcrV197-294 and PcrV261-294, induced a titer increase against PcrV1-294. The titer induced by PcrV139-258 was higher than the

titer induced by full-length PcrV1-294 (P < 0.05). IgG isotypes in increased anti-PcrV titers were measured in three groups (PcrV1-294, PcrV139-294 and PcrV139-258) (Fig. 4b). In all three groups, an increase in IgG1 titer was predominant, followed by an increase in IgG2b titer as the second among four isotypes. The vaccinated mice were treated with 5 mg of CP on day 60, infected with a lethal dose of PA103 on day 63, and their survival monitored for 48 hr after infection (Table 2). Compared with the control group, active vaccination with a full-length PcrV1-294 significantly improved survival (P < 0.0001). Compared with the control group, PcrV139-294, which contains the complete Mab166 epitope and the carboxyl terminal tail, resulted in the same level of protection as full-length PcrV1294 (P < 0.0001). In contrast, PcrV139-258, which also

Fig. 3. (a) The effect of CP treatment on leukocyte profiles in peripheral blood. In the CP-treated group, mice received 5 mg of CP i.p. on day 0, and blood samples were collected on day 3. Control mice received saline. Data are expressed as means ± SD. ∗ , P < 0.01 compared

with the control group by unpaired t-test. (b) Body weight of mice after either CP or saline treatment. Data are expressed as means ± SD. ∗ , P < 0.05 compared with the saline-treated control group by unpaired t-test.

Active immunization against PcrV in immunocompromised mice

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Table 1. The effect of pretreatment with cyclophosphamide on the survival of mice infected with Pseudomonas aeruginosa

Group

Total, n

Survivors for 48 hr, n (%)

Control (saline-treated) Cyclophosphamide-treated

10 10

8 (80) 0 (0)∗

∗

, P < 0.0001 compared with mice treated with saline by the Mantel-Cox log rank test.

contains the complete Mab166 epitope but is without the carboxyl terminal tail, failed to demonstrate significant protection in comparison with the control group. Affinity-purified anti-PcrV antibody in immunocompromised mice

Mice treated with CP were passively immunized with either anti-PcrV IgG or control IgG i.p. 1 hr before P. aeruginosa infection, and their survival monitored for 48 hr (Table 3). Two different doses (100 μg and 10 μg) of either Mab166 or anti-PcrV Rab were tested in comparison with control Mab (100 μg) or control Rab (100 μg), respectively. Immunization of 100 μg of either MAb166 or anti-PcrV Rab significantly increased survival. However, neither 10 μg of MAb166 nor anti-PcrV Rab improved survival.

Fig. 4. (a) Anti-PcrV IgG titers in the serum of mice vaccinated with one of the PcrV vaccines. Data are expressed as means ± SD. ∗ , P < 0.05 compared with the PcrV1-294, PcrV139-294, and PcrV139234 groups, and analyzed by one-way ANOVA followed by the Bonferroni multiple comparison test. (b) Anti-PcrV IgG isotype titers in the


Next, CP-treated mice were passively immunized with 10 μg of affinity-purified anti-PcrV1-294 IgG (antiPcrV1-294 Rab) or anti-PcrV139-258 IgG (anti-PcrV139258 Rab) 1 hr before P. aeruginosa infection, and their survival at 48 hr was compared with the survival of mice immunized with 10 μg of the original anti-PcrV Rab or Mab166. The survival of the mice passively immunized with affinity-purified anti-PcrV Rab was significantly improved. Ten μg of affinity-purified anti- PcrV139-258 Rab demonstrated an efficacy comparable to 100 μg of the original anti-PcrV Rab and Mab166.

DISCUSSION P. aeruginosa is an opportunistic pathogen causing lethal infections in immunocompromised individuals, such as neutropenic and burns patients. In this study, we induced immunosuppression in mice by CP treatment to test the effects of active and passive immunization against lethal P. aeruginosa infection. The CP treatment caused panleukocytopenia and loss of body weight in mice within 3 days. CP-treated mice died from a significantly smaller dose of P. aeruginosa than the lethal dose in normal mice (data not shown). These results suggest that CP treatment induces immunosuppression in mice, decreases immunity against P. aeruginosa, and decreases the lethal dose of P. aeruginosa. By using this immunocompromised mouse

serum of mice vaccinated with one of the PcrV vaccines. Data are expressed as means ± SD. ∗ , P < 0.01 compared with IgG2a, IgG2b and IgG3, analyzed by one-way ANOVA followed by the Bonferroni multiple comparison test.

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Table 2. The effect of active immunization against PcrV on the survival of immunocompromised mice infected with Pseudomonas aeruginosa

Group

Total, n


Control (Freund’s adjuvant alone) PcrV1-294 (Full-length PcrV) PcrV139-294 PcrV139-258 PcrV197-294 PcrV139-234 PcrV261-294

20 20 20 15 10 10 10

3 (15) 13 (65)∗ 13 (65)∗ 6 (40) 4 (40) 2 (20) 2 (20)

∗ , P < 0.0001 compared with the control group by the Mantel-Cox log rank test.

Table 3. The effect of passive immunization against PcrV on the survival of immunocompromised mice infected with Pseudomonas aeruginosa

Group

Dose, μg

Total, n


Control Mab Mab166 (10μg) Mab166 (100μg) Control Rab Anti-PcrV Rab (10 μg) Anti-PcrV Rab (100 μg) Anti-PcrV1-294 Rab Anti- PcrV139-258 Rab

100 10 100 100 10 100 10 10

10 10 10 20 10 20 10 10

2 (20) 3 (30) 8 (80)∗ 4 (20) 3 (30) 19 (95) ∗∗ 7 (70) 8 (80) ∗∗∗

∗ , P < 0.01 compared with the control Mab group, ∗∗ , P < 0.0001 compared with the control Rab group, ∗∗∗ , P < 0.05 compared with the anti-PcrV Rab (10 μg) group, by the Mantel-Cox log rank test.

model, we tested whether active and passive immunization against PcrV protected the mice from lethal P. aeruginosa infection. PcrV, the V-antigen of P. aeruginosa, is one of the translocational components of the type III secretion system encoded by the pcrGVHpopBD operon of the genome with four other proteins, PcrG, PcrH, PopB and PopD (12,25,26). Through the translocational process, virulent P. aeruginosa delivers exoenzyme S and its associated toxins into the cytosol of targeted eukaryotic cells (25). PcrV consists of 294 amino acids and has a 57% amino acid sequence similarity with Yersinia LcrV (10). Vaccination of mice with recombinant PcrV was protective, similarly to what has been reported for LcrV in Yersinia (7). The murine monoclonal anti-PcrV IgG, Mab166, binds to the PcrV region of 144–257 aa (10). This PcrV region overlaps in their aligned sequences with the blocking epitope of Yersinia LcrV (10,20). In the case of Yersinia, the aminoterminal of LcrV includes the region that induces IL-10 592

production in macrophages through the toll-like receptor 2 (27). However, it has been reported that P. aeruginosa PcrV does not induce IL-10 production in infected hosts and the PcrV region corresponding to the IL-10-inducing region of LcrV fails to show any functional association with toll-like receptor 2 (27,28). Therefore, although the amino-terminal of Yersinia LcrV must be important for inducing an anti-inflammatory response (29), no functional importance has been reported for the amino-terminal of PcrV. In fact, in this study, active immunization with a carboxyl terminal half of PcrV showed the same efficacy as immunization with full-length PcrV against lethal P. aeruginosa infection. The blocking epitope region of LcrV includes the hypervariable region found in Y. enterocolitica LcrV (30). The hypervariable region of LcrV appears to account for the inability of anti-LcrV antibodies against three different Yersiniae species to cross block (30). This implies that the blocking mechanism involves the hypervariable region, which is located in the center of the blocking epitope. The Mab166 epitope of PcrV contains ahelical regions, which have high sequence similarity to the corresponding regions of LcrV blocking epitope, in its amino- and carboxyl-terminals (109–156 aa and 227–294 aa) (20,30). Therefore, although there are no reports concerning high sequence variation of PcrV among P. aeruginosa strains, the Mab166 epitope and carboxyl terminal tail regions may be important for preserving a three dimensional structure in inducing a protective immune response against P. aeruginosa infection. In this study, we tested six different PcrV vaccines including full-length PcrV (PcrV1-294) and five truncated PcrV proteins. We initially thought that vaccination with PcrV proteins containing the Mab166 epitope might induce protective immunity against lethal P. aeruginosa infection. The truncated PcrV proteins were constructed based on the fact that the shortest PcrV region bound to Mab166 was a PcrV region 144–257 aa (Fig. 2) (10). The outcome of this experiment enabled us to determine the region of PcrV that contributes to antigenicity and protection against lethal P. aeruginosa infection. Active vaccination with either full-length PcrV1-294, PcrV139-294, PcrV139-258 or PcrV139-234 led to significant increases in anti-PcrV titers in the serum of vaccinated mice, while PcrV197-294 and PcrV261-294 failed to induce increases in anti-PcrV titers. These results suggest that the internal region containing the Mab166 epitope and downstream carboxyl-terminal is the most antigenic region of PcrV. Among four IgG isotypes, increases in IgG1 titers were predominant. Increases in IgG2b titers were also detected, consistent with the fact that the Mab166 was IgG2b (10). Active immunization with a full-length PcrV1-294 significantly improved the survival of immunocompromised c 2009 The Societies and Blackwell Publishing Asia Pty Ltd


mice infected with a lethal dose of P. aeruginosa, as reported previously in normal Balb/c mice (7). The fact that PcrV139-294 shows the same protective effect as fulllength PcrV1-294 implies that both antigenicity of, and protection by, PcrV vaccines are derived from the carboxyl terminal half of PcrV. However PcrV139-258, which contains the complete Mab166 epitope but not the carboxyl terminal tail of PcrV, was not as protective as full-length PcrV1-294 and PcrV139-294, although vaccination with PcrV139-258 induced the greatest anti-PcrV titer increase in the serum of vaccinated mice. In addition, PcrV197294 and PcrV139-234, both containing the partial Mab166 epitope, failed to produce a significant increase in protection. Therefore, although to induce an anti-PcrV immune response a vaccine needs to contain the complete Mab166 epitope, the PcrV region 144–258 aa, , the carboxyl terminal tail region (261–294 aa) of PcrV downstream of the Mab166 epitope was found to induce the best protective immunity against lethal P. aeruginosa infection. Passive immunization with either anti-PcrV Rab or Mab166 protected immunocompromised mice from lethal P. aeruginosa infection in a dose-dependent manner, as reported previously in normal Balb/c mice (7,10). Affinity purification of anti-PcrV Rab against PcrV1-294 significantly enhanced the efficacy of protection by passive immunization against lethal P. aeruginosa infection. Passive immunization with only 10 μg of the affinity-purified anti-PcrV Rab IgG produced effects comparable to immunization with 100 μg of the original anti-PcrV Rab and Mab166. These results suggest that the highly protective effects of anti-PcrV Rab against P. aeruginosa are not due to synergistic effects of the background fraction of polyclonal immunoglobulins which do not bind to PcrV. Passive immunization with affinity-purified anti-PcrV Rab against PcrV139-258 also demonstrated a protective effect as great as that found with immunization with affinity-purified anti-PcrV Rab against PcrV1-294. This result was contradictory in view of the fact that, in active immunization, PcrV139-258, which lacks the carboxyl terminal tail of PcrV, failed to demonstrate protection. This mismatch between the results of active and passive immunization implied to us that, although protective immunity does indeed come from blocking the Mab166 epitope region of PcrV, as we initially thought, the carboxyl terminal tail of PcrV helps to provoke effective protective immunity in vaccinated mice. In our previous experiments, although murine monoclonal Mab166 demonstrated blocking effects comparable to those of anti-PcrV Rab, this result occurred in the setting of equal dose administration (10). The increased efficacy of affinity-purified anti-PcrV Rab suggests that more potent antibodies than Mab166 can be created against lethal P. aeruginosa infection. Moreover, this increased effect must be important for creating an c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

effective component vaccine for PcrV, although its mechanisms for inducing enhanced immunity were unclear in this study. In this study, active immunization with PcrV protected immunocompromised mice from lethal P. aeruginosa infection. Among various truncated PcrV vaccines, a PcrV protein containing both the Mab166 epitope region and the carboxyl terminal tail was the most effective. Passive immunization with affinity purified anti-PcrV Rab against PcrV proteins containing the Mab166 epitope produced higher protection than immunization with the original anti-PcrV Rab or Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies the possibility of creating more potent anti-PcrV antibodies. These outcomes will be crucial in developing the most efficacious PcrV vaccine for active immunization and in generating the most effective blocking anti-PcrV antibody against P. aeruginosa infection in humans.

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