Protection against Exotoxin A (ETA) and Pseudomonas aeruginosa Infection in Mice with ETA-Specific Antipeptide Antibodies

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170–2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [³H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of P. aeruginosa in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of P. aeruginosa. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from P. aeruginosa infection.

Pseudomonas aeruginosa is the leader among gram-negative organisms in causing burn wound infections (8), and exotoxin A (ETA) is one of the major virulence factors produced by this organism. P. aeruginosa ETA was first discovered and purified by Liu et al. (13). Since then, ETA has proven to be toxic for a wide variety of mammalian cells in vitro (19, 21) and lethal for many animal species (2, 20). In mice, ETA is approximately 10,000 times more lethal than lipopolysaccharide from P. aeruginosa (22). In vitro, ETA is produced by 95% of P. aeruginosa clinical isolates (3). ETA is an ADP-ribosylating toxin that catalyzes the transfer of ADP-ribose from NAD to eukaryotic elongation factor 2, resulting in the inhibition of protein synthesis and ultimately cell death (10, 11).

ETA is a heat-labile, 613-amino-acid (aa) single polypeptide chain with a molecular weight of 66,583 (7). X-ray crystallography studies and deletion mutation analysis of ETA revealed three structural domains (1, 9). Domain I of ETA includes aa 1 to 252 (Ia) and 365 to 395 (Ib) (9) and is associated with binding to the receptor of target cells. Domain II, aa 253 to 364, is believed to be involved in translocation of a 37-kDa enzymatically active fragment of ETA across the membrane of the endocytic vesicle to the cytoplasm of the target cell (9). Domain III, aa 396 to 613, constitutes the enzymatic portion of ETA (9, 11). To date, several studies have been conducted in order to understand the immunochemistry of ETA and to identify the immunodominant neutralizing epitopes of this molecule (4, 15, 16, 17, 18, 24, 25). Such studies are essential for the development of immunotherapeutic approaches for treating infections caused by toxin-producing strains of P. aeruginosa and for elucidating the structure-function relationship of ETA. They are also of great value to investigators interested in developing ETA-derived immunotoxins (6). Previously, we reported successful induction of neutralizing antipeptide antibodies to a short amino acid sequence representing a portion of the enzymatic domain of ETA (aa 596 to 625, designated peptide 11) (5). These antibodies provided in vitro protection to monolayers of 3T3 fibroblasts against ETA-induced inhibition of protein synthesis by specifically blocking ADP-ribosyltransferase activity (5). Antibodies to the 13 aa within the carboxyl half of peptide 11 were more efficient than antibodies to peptide 11 itself in neutralizing the cytotoxic and enzymatic activities of ETA. In the same study, we identified another synthetic peptide encompassing a region within the translocation domain of ETA (aa 289 to 333), which induced antibodies with moderate ability to neutralize the cytotoxic activity of ETA in vitro (5). Four synthetic peptides encompassing regions within the binding domain of ETA failed to induce ETA-neutralizing antibodies (5). In the present study, we examined the potential of neutralizing antipeptide antibodies to confer protection against ETA or infection with an ETA-producing strain of P. aeruginosa in mice. The ability of these synthetic peptides to induce a state of active immunity against ETA in mice was also examined.

Effect of antipeptide antibodies in providing protection against ETA in mice.

Affinity-purified antibodies to selected synthetic peptides (3, 6, 9, and 11) encompassing regions within the translocation and enzymatic domains of ETA (Fig. 1) were used in these studies (5). The 50% lethal dose (LD₅₀) (23) of ETA in Swiss Webster outbred mice was determined to be approximately 300 ng when it was injected intraperitoneally (i.p.). Two LD₅₀s of ETA were preincubated with antibodies (400 μg) for 1 h at 37°C. The mixture was then injected i.p. into mice, which were observed daily for mortality for a period of 6 days or longer (12, 14). Antibodies to ETA, peptides 6 and 11 (enzymatic domain), or peptide 9 (translocation domain) completely protected mice against the lethal effects of ETA (Table 1). Antibodies to peptide 3, which significantly cross-reacted with ETA but failed to neutralize its cytotoxicity in vitro (5), did not provide protection to mice challenged with ETA and therefore served as a negative control in this study (Table 1).

FIG. 1.

The synthetic peptides correspond to different regions within domains II (translocation) and III (enzymatic) of ETA. ETA and the synthetic peptides are not drawn to scale.

TABLE 1.

Effects of ETA cross-reacting antipeptide antibodies on survival of mice challenged with ETA^a

Antibody specificity	ETA domainb	No. of surviving animals/no. challenged (%)c
None		0/5 (0)
ETA	Holotoxin	5/5 (100)∗
Peptide 6	III (Enz)	5/5 (100)∗
Peptide 11	III (Enz)	5/5 (100)∗
Peptide 9	II (Trans)	5/5 (100)∗
Peptide 3	II (Trans)	0/5 (0)

^aTwo LD₅₀s (600 ng/mouse) of ETA (List Biological Laboratories, Campbell, Calif.) were mixed with affinity-purified antibodies (400 μg/mouse) (5). The mixture was incubated for 1 h at 37°C and then injected into the peritoneal cavities of mice (five mice per group). Animals were observed daily for up to 6 days, and the number of dead animals per group was recorded. No change in the mortality rate was observed after day 6. (Female Swiss Webster mice [18 to 20 g] were purchased from Jackson Laboratory, Bar Harbor, Maine.) 

^bEnz, enzymatic domain; Trans, translocation domain. 

^c∗, Statistically significant difference (P < 0.05) from the group of mice not receiving any treatment (according to Fisher’s exact test). 

Active immune protection against ETA in mice.

BALB/c mice were assigned to different groups, as described in Table 2. Mice were immunized with 20 ng of ETA or 50 μg of synthetic peptides along with complete Freund’s adjuvant. Likewise, keyhole limpet hemocyanin (KLH) was given with complete Freund’s adjuvant. All booster immunizations were delivered along with incomplete Freund’s adjuvant. Serum samples taken after seven immunizations from the different groups of ETA- and peptide-immunized mice (groups I to IV) contained high levels of ETA cross-reacting antibodies (data not shown). No ETA cross-reacting antibodies were detected in serum from group V mice, which were immunized with KLH (Table 2). Only mouse sera from groups I (immunized with ETA) and III (immunized with conjugated peptide 11) were capable of conferring significant protection against the cytotoxic activity of ETA (P < 0.05 [one-way analysis of variance]), as measured by incorporation of [³H]leucine into 3T3 fibroblasts (data not shown). Mouse serum from group I (immunized with ETA), group II (immunized with peptide 11, enzymatic domain), or group III (immunized with conjugated peptide 11, enzymatic domain) significantly interfered with the enzymatic activity of ETA in vitro, as measured by transfer of ¹⁴C-labeled ADP-ribose from NAD to eukaryotic elongation factor 2 (data not shown). Mouse serum from group IV (immunized with peptide 9, translocation domain) or group V (immunized with KLH) had no effect on ETA enzymatic activity in vitro. All of the different groups of BALB/c mice were challenged with a lethal dose of ETA (1 μg). The survival rate among mice immunized with ETA, peptide 11, or conjugated peptide 11 was 100% (Table 2). Eighty percent of the mice immunized with peptide 9 survived ETA challenge (Table 2). Immunization with KLH did not provide mice with active immunity to ETA and served as a negative control (Table 2).

TABLE 2.

Active immune protection against ETA in mice^a

Mouse group	Immunogen	No. of surviving animals/no. challenged (%)b
I	ETA	5/5 (100)∗
II	Peptide 11	5/5 (100)∗
III	Conjugated Peptide 11c	5/5 (100)∗
IV	Peptide 9	4/5 (80)∗
V	KLH	0/5 (0)

^aFour-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, Maine) (five per group) were repeatedly immunized for 7 months with immunogen (20 ng of ETA, 50 μg of synthetic peptide, or 50 μg of KLH along with Freund’s adjuvant), according to the protocol described previously (5). After the antibody immune response reached a plateau level, as determined by enzyme-linked immunosorbent assay (5), all mice were challenged with a lethal dose of ETA (1 μg/mouse) i.p. Mice were observed daily for 6 days, and the number of dead animals was recorded accordingly. No change in the mortality rate was observed after day 6. 

^b∗, Statistically significant difference from the control group (immunized with KLH), according to Fisher’s exact test (P < 0.05). 

^cPeptide 11 conjugated to KLH (Sigma, St. Louis, Mo.), as previously described (5). 

Protection of mice against infection with a toxin-producing strain of P. aeruginosa by antibodies to ETA or ETA synthetic peptides.

P. aeruginosa PA103, which produces large amounts of ETA and small amounts of alkaline protease, but no elastase, was used in this study. This particular strain is routinely used to study pathogenesis due primarily to ETA. Once they have been characterized with this infection model, antipeptide antibodies will be tested in mice infected with other, more virulent strains of P. aeruginosa. Mice were injected i.p. with cyclophosphamide at 250 mg/kg of body weight to suppress their immune systems (12, 14). Subcutaneous (s.c.) injection of 5 × 10⁵ CFU of PA103 into immunosuppressed mice caused 80 to 100% death between day 1 and day 4 (n = 10) (data not shown). As shown in Fig. 2, a combination of the antibiotic amikacin and anti-ETA antibodies was more efficient than amikacin alone in providing protection to mice infected with P. aeruginosa (100% versus 30%, respectively). Anti-ETA alone provided 50% protection to infected mice (Fig. 2). Polyclonal anti-KLH antibodies alone or in combination with amikacin did not provide significant protection (10 and 30% respectively [data not shown]). When mice were infected with a toxin-deficient strain of P. aeruginosa (PA103dtox), amikacin alone provided a significant level of protection (90%) compared to mice infected with the organism without any treatment (30%) (P < 0.05) (data not shown). When peptide-specific antibodies were tested in a similar experiment, it was determined that a combination of anti-peptide 11 or anti-peptide 6 antibodies and amikacin provided mice infected with PA103 with 90 and 100% protection, respectively (Fig. 3). Survival among infected animals treated with anti-peptide 6 (50% survival) or anti-peptide 11 (50% survival) alone was not statistically different at day 6 from survival among infected animals which did not receive any treatment (20% survival). Administration of amikacin alone increased the survival rate from 20 to 30% only (Fig. 2 and 3). These data indicated that neither antibodies (anti-ETA or antipeptide antibodies) nor antibiotic treatment alone was sufficient to provide significant protection to Pseudomonas-infected animals. However, a combination of antibodies and antibiotic therapy provided significant protection to infected mice. A combination of antibodies to peptide 9 or peptide 3 and amikacin had no significant effect on the survival rate among infected mice (30% and 20%, respectively [data not shown]). This was a surprising observation, since anti-peptide 9 antibodies provided mice with passive and active protection against ETA itself (Tables 1 and 2). A higher concentration of antibodies to peptide 9 than antibodies to peptides 6 and 11 was needed to provide protection against ETA-induced inhibition of protein synthesis in 3T3 fibroblasts (5). Similarly, it is possible that a higher concentration of antibodies to peptide 9 is required to provide protection against infection with P. aeruginosa. Antibodies to peptide 3, with or without amikacin, had no positive effect on the survival of Pseudomonas-infected animals and were not expected to have any because these antibodies did not neutralize ETA in vitro (data not shown).

FIG. 2.

In vivo protection against infection with a toxin-producing strain of P. aeruginosa (PA103 wild type [wt]) by using a combination of amikacin and polyclonal anti-ETA antibodies. Mice (n = 10) were injected with cyclophosphamide (Sigma Chemical Co., St. Louis, Mo.) at 250 mg/kg 4 days prior to bacterial challenge. On day 0, all mice were infected s.c. on the back with 5 × 10⁵ CFU of P. aeruginosa PA103 wt. One group of mice (wt+Am) received amikacin (6 mg) i.p. at 16 h postinfection and s.c. at 22 h postinfection. A second group (wt+anti-ETA) was treated i.p. with antibodies to ETA (400 μg) at 30 min postinfection. Another group (wt+Am+anti-ETA) was treated with a combination of amikacin and anti-ETA. The asterisk indicates a statistically significant difference from the group which did not receive any treatment (wt) (Fisher’s exact test).

FIG. 3.

In vivo protection against infection with a toxin-producing strain of P. aeruginosa (PA103 wild type [wt]) by using antipeptide antibodies. Mice (n = 10) were injected with cyclophosphamide 4 days prior to bacterial challenge. On day 0, all mice were infected with 5 × 10⁵ CFU of PA103 wt s.c. on the back. One group of mice (wt+Am) received amikacin (6 mg) i.p. at 16 h and s.c. at 22 h postinfection. Other groups (wt+Am+anti-P6 and wt+Am+anti-P11) received, in addition to amikacin, antipeptide antibodies (400 μg) i.p. at 30 to 60 min postinfection. Asterisks indicate statistically significant differences from the group not receiving any treatment (wt only) (Fisher’s exact test).

The above data support the potential use of peptide 11 or antibodies to peptide 6 for active and passive immunization, respectively, against ETA or infection with ETA-producing strains of P. aeruginosa. Although ETA-neutralizing antibodies have been previously reported (4), our study is provocative, as it represents the first reported incidence of ETA-neutralizing antibodies being successfully generated by using synthetic peptides for animal immunization. We believe that a combination of antibiotic and antipeptide antibody therapy could be beneficial to immunosuppressed patients (e.g., burn patients) in combating P. aeruginosa infection. Human polyclonal or monoclonal antibodies generated against peptides representing neutralizing epitopes of ETA might have the advantage of minimal risk of antigenic cross-reactivity and autoimmunity.