Ability of synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model

Vaccine, Vol. 13, No. 16, pp. 1750-1753, 1995 Elsevier Science Ltd Printed in Great Britain 0264-41 OX/95 $1 O+O.OO

0264-410X(95)00166-2

Ability of synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model Eileen E. Hughes*

and H.E. Gilleland

Jr*‘f

Three synthetic peptides (Nos 9, 10 and 18) representing surface-exposed, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa were each conjugated to the carriers keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), with the conjugates being used to immunize mice intranasally. Mice were also immunized intranasally with a KLHIBSA carrier control or with a peptide No. 8 conjugate as a negative control. An immunoglobulin G response reactive wlith P. aeruginosa whole cells was demonstrated by enzyme-linked immunosorbent assay (ELISA) of sera from mice immunized with peptide 9, 10 or 18, whereas no whole-cell reactivity by ELISA was detected in sera from mice immunized with peptide 8. Upon pulmonary challenge of immunized mice with a Fisher-Devlin immunotype 4 strain of P. aeruginosa, only those mice immunized with peptide 9 or peptide IO had a significantly greater survival rate compared to control mice immunized with the carriers alone. Peptides 9 (TDA YNQKLSERRAN) and 10 (NATAEGRAINRRVE) have potential for further development as a protective vaccine against P. aeruginosa infections. Keywords: P.scuti~)n~o~~us uerugino.w:

peptide

vaccine;

outer membrane

(OM) protein

F

Pseudomonas aeruginosa is generally

considered to be an opportunistic pathogen of importance in the immunosuppressed, in burn patients, and in children with cystic fibrosis, in whom P. aeruginosa causes chronic pulmonary infections. For possible immunotherapy of P. aeruginosa infections, our laboratory group has explored the vaccine potential of outer membrane (OM) protein F (OprF in the proposed nomenclature of Hancock et al. ‘). Purified protein F has been shown to be a protective vaccine in several animal models, including a rat model of chronic pulmonary infection2m5, a murine acute infection model’, and a murine burn wound sepsis mode17.8. Since the protein F preparation used in the animal studies is unsuitable for use in humans, we have been examining the vaccine potential of synthetic peptides that represent surface-exposed, linear B-cell epitopes of

OM protein F as possible safe, immunogenic alternatives. In previous studies that examined nineteen synthetic peptides of OM protein F, we demonstrated that peptides 9 and lo9 and peptide 181° from the carboxyterminal portion of protein F had potential for use in a synthetic peptide vaccine, based on data from P. aeruginosa whole-cell ELISA, flow cytometry and opsonization studies with the various peptide-directed antisera. In addition, peptides 9 and 10 confer protection upon use as active immunogens in the rat model of chronic P. aeruginosa infection’ ‘. The purpose of the pulmonary present study was to determine whether peptides 9, 10 and 18 could afford protection against acute pulmonary P. aeruginosa infection in a murine model upon use as an active vaccine. This represents the first time peptide 18 has been tested as a protective vaccine.

*Department of Microbiology and Immunology, Louisiana State University Medical Center, School of Medicine in Shreveport, 1501 Kings Highway, Shreveport, LA 711303932, USA. ~To whom all correspondence should be addressed. (Received 21 March 1995; revised 9 August 1995; accepted IO August 1995)

MATERIALS

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METHODS

Bacterial strains and culture conditions The strains of P. aeruginosa used were ATCC 27313 that corresponds to a Fisher-Devlin immunotype 2

Protein F synthetic peptides confer protection: E. M. Hughes and H. E. Gilleland Jr

(Difco O-l 1) and ATCC 27315 (Difco O-l; FisherDevlin immunotype 4). All ATCC strains were obtained from the American Type Culture Collection, Rockville, MD. All strains were grown at 30°C with shaking in BBL nutrient broth (Becton-Dickinson Microbiology Systems, Cockeysville, MD), or on nutrient agar (Difco, Detroit, MI) plates. Synthetic

peptides and conjugation

procedure

Peptide 8 (MKQYPSTSTTVEGHT), residues 242256 of the mature protein F molecule, peptide 9 (TDAYNQKLSERRAN), residues 261-274, peptide 10 (NATAEGRAINRRVE), residues 305-318, and peptide 18 (NEYGVEGGRVNAVG), residues 282-295, were synthesized and provided as lyophilized powder by the Peptide Synthesis Unit of the Core Laboratories, Louisiana State University Auxiliary Enterprises (New Orleans, LA). Each of the synthetic peptides was conjugated to keyhole limpet hemocyanin (KLH) (Calbiochem, San Diego, CA) or bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO) by glutaraldehyde coupling at pH 8.3 as described previously’. For conjugation to BSA, 20 mg ml- ’ of peptide and 100mgml-l of filter-sterilized BSA solution were used in order to achieve the appropriate molar ratio of peptide to carrier12. Animals and immunization

protocol

Groups of 5-week-old, specific-pathogen-free ICR mice (Harlan Sprague Dawley, Indianapolis, IN) were immunized intranasally with 100 ~1 PBS (pH 7.5) containing either 150 ,ug of crosslinked KLH with 25 pug of cholera toxin B subunit (CT-B; List Biologicals, Campbell, CA), or 150 pugpeptide-KLH conjugate with 25 ,ug of CT-B on day 0. The second dose, administered intranasally on day 14, consisted of 50~1 PBS containing 100 yg crosslinked BSA or 100 pug peptide-BSA conjugate. BSA conjugates were used for the second dose in an effort to diminish any nonspecific protection mediated by multiple doses of KLH. All together, this study included the following five immunization groups: the carrier control group immunized with KLH, followed by BSA without any synthetic peptide, and the four groups each immunized with one synthetic peptide conjugated to KLH for the first immunizing dose and conjugated to BSA for the second immunizing dose, i.e. the peptide 8-immunized group, the peptide 9-immunized group, the peptide lo-immunized group, and the peptide 18immunized group. Six weeks after the second immunization, three mice from each of the five groups were killed by cervical dislocation, the thoracic cavity opened, the blood aspirated to obtain serum samples and the lungs lavaged with three successive washes of 0.75 ml chilled PBS (pH 7.2). Sera and lung lavage fluids were stored at - 20°C until assay by ELISA as described below. The remaining animals were challenged using the acute pneumonia model described below. Monitoring

the antibody response

The antisera were tested using separate ELISAs, performed as described previously’, to determine the immunoglobulin G (IgG) and immunoglobulin A (IgA)

titers to various antigens. For one assay, each of the synthetic peptides was used as the ELISA antigen. The coating of u.v.-irradiated flat-bottom 96-well Immulon 1 microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) with syntheJ,c peptide solution at a was performed by the concentration of 25 p method of Boudet et al. $Trnl as previously described’. Each of the peptide-directed antisera was tested in comparison with the KLH control antiserum against peptides 8, 9, 10 and 18 and protein F purified from cell envelopes of the PA01 strain of P. aeruginosa6. In a second ELISA, whole bacterial cells were used as the ELISA antigen. Wells of Immulon 1 microtiter plates were coated with a suspension of P. aeruginosa cells prepared in a fashion similar to that of Abdillahi and Poolman’ as described previously’. Plates coated with cell suspensions of the challenge strain and one heterologous immunotype (FD4 and FD2, respectively) were prepared and probed with sera and lung lavage fluids from the carrier controlimmunized group and each of the peptide-immunized groups.

Acute pneumonia model P. aeruginosa acute pneumonia was induced in immunized mice by a procedure similar to that used by Eckhardt et al. I5 Briefly, mice were anesthetized with sodium pentobarbital i.p., held upright, and 50 ~1 of cell suspension prepared as previously described’ from midlogarithmic phase nutrient broth cultures was instilled into the nose. The fluid was aspirated during a period of anesthesia-induced, deep hyperventilation. The concentration of bacterial cells corresponded to approximately a 2 LD,, (ranging from 3.0 x lo6 to 6.0 x lo6 c.f.u.) as experimentally derived for that challenge experiment. The animals were monitored for death over 96 h. Survival of mice within each of the peptide-immunized groups was compared to that of the carrier-immunized group by Fisher’s Exact test using the EpiStat program (Tracy L. Gattis, Round Rock, TX) on an IBM personal computer; P values co.05 were considered significant. All animals used in this study were handled in accordance with the guidelines of the Louisiana State University Medical Center-Shreveport Animal Care and Use Committee.

RESULTS AND DISCUSSION The synthetic peptide immunization protocol was successful in eliciting immunoglobulin G (IgG) serum antibodies reactive both with the immunizing peptide and with protein F (Table I). Note that antisera to peptides 8, 9, 10 and 18 reacted at high titers to the immunizing peptide (1493-2347); however, little to no crossreactivity to the heterologous peptides was observed among these peptide-directed antisera (Table 1). In addition, the peptide-directed antisera reacted at intermediate to high titers to purified protein F (240-8533) (Table I). Consistent with our previous findings’.“, antisera to peptides 9, 10 and 18 reacted strongly with whole cells of P. aeruginosa FD4, the challenge strain, at titers ranging from 1009 to 5897 and with whole cells of the FD2 strain at titers ranging from 228 to 1920 (Table I). Antisera from mice immunized with either the

Vaccine 1995 Volume 13 Number 18 1751

Protein F synthetic peptides confer protection: E.M. Hughes and H.E. Gil/eland Jr Table 1

Mean titers of IgG antibodies

in antisera from mice from each vaccine group as determined

Mean titera determined

by ELISA with various antigens

by ELISA with each of the following as antigen:

Vaccine group

Peptide 8

Peptide 9

Peptide 10

Peptide 18

Protein F

FD2’ cells

FD4 cells

Carriers Peptide Peptide Peptide Peptide

1.7k2.9 1493r978 5+5 o+o 1.7+2.9

lOk8.7 16.7e20.2 1920+1109 3.3k2.9 5+5

Ok0 1.7k2.9 1.7k2.9 2347~2423 1.7k2.9

O&O 1.7*2.9 1.7k2.9 1.7k2.9 1707k-739

6.7k11.5 240?346 25601t2217 8533*2956 5973+3910

3.3+2.9 6.7211.5 1067+1293 1920+905 2282357

1.2e2.3 7+7.6 1009+1081 5897k4293 2103+3610

8 9 10 18

“Mean*S.D.

of three determinations.

‘FD, Fisher-Devlin

immunotype

Table 2 Protection afforded by mucosal immunization with synthetic peptides in a murine acute pneumonia model upon challenge with FD4 immunotype /? aeruginosa Vaccine group

No. survived/total

Carriers Peptide Peptide Peptide Peptide

23150 15132 32149 39150 13133

8 9 10 18

No.

% survival

P”

46 47

0.56 0.04’
;z 39

aP value determined by Fisher’s Exact test, compared to carrier control group. Asterisk denotes significant values at BO.05

carriers alone or with peptide 8 did not react with whole cells of either serotype (Table I). Our immunization protocol involved a 6-week interval between the final immunization and challenge; little, if any, whole-cell reactive IgA was detectable in the sera and neither IgG nor IgA was detectable in lung lavage fluids from the peptide-immunized mice at the time of challenge (data not shown). Immunized mice were challenged intranasally in a fashion similar to that of Eckhardt et al. I5 Consistent with their results”, all deaths of unprotected animals occurred within 4 days; in our hands, death usually occurred within 18-72 h. Table 2 shows the protection afforded by synthetic peptide immunization upon challenge with the FD4 strain. Immunization with carriers alone resulted in 46% (23 of 50) of these mice surviving challenge (Table 2); immunization with peptide 8 yielded similar results with 47% (15 of 32) survival. The results obtained with peptide 8 are consistent with our belief that this region is not exposed on the surface of the bacterial cell”. Immunization with peptides 9 and 10, two of our proposed surface-exposed epitopes’.‘” mediated significant protection, with 65% (32 of 49) and 78% (39 of 50) survival, respectively, as compared to immunization with the carriers alone. However, immunization with peptide 18, an epitope that elicits whole-cell reactive antibodies” (Table I), failed to confer protection, with 39% (13 of 33) survival of mice immunized with this peptide (Table 2). Interestingly, the protection observed appears to correlate with results obtained in earlier studies’,” where antisera to peptides 9 and 10, but not to peptide 18, mediated opsonic phagocytosis of P. aeruginosa by human polymorphonuclear leukocytes at a level significantly greater than that of antisera to crosslinked KLH. Peptides 9 and 10, but not peptides 18 or 8, had earlier been tested for possible vaccine efficacy using the rat model of chronic pulmonary infection”. In comparison to rats immunized with crosslinked KLH,

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in which 67% presented with severe pulmonary lesions upon death seven days after pulmonary challenge with agar-encased P. aeruginosa, immunization with peptide 10 was more successful at protecting rats from severe lesions than was immunization with peptide 9; 17.4% of peptide lo-immunized rats presented with severe pulmonary lesions vs 30.4% of rats immunized with with those peptide 9’ ’ . These results are consistent obtained in the present study, in that peptide 10 appears to be both a more potent immunogen than peptide 9 in terms of the ability to elicit whole-cell reactive antibodies (Table I) and a more protective immunogen than peptide 9 in terms of the ability of P. aeruginosa immunized mice to survive pulmonary challenge (Table 2). Data from two other laboratory groups support the identification of the peptide 10 epitope as being surfaceexposed and protective. Rawling et al. I6 identified the TAEGRAIN portion of peptide 10 as being a surfaceexposed loop in their proposed model for the topology of protein F within the outer membrane. Furthermore, TAEGRAIN was bound by their oRsonic monoclonal antibody MA5-8. von Specht et al. found that their peptide D5 (residues 308-326 in mature protein F) elicited antibodies that reacted with intact whole cells of P. aeruginosa, confirming our results with peptide 10 (residues 305-318). Additionally, antibodies induced in rabbits against peptide D5 were protective against P. aeruginosa infection in severe combined immunodeficient (SCID) mice”. Thus, there is complete agreement among the three laboratory groups that an epitope in the region of our peptide 10 is surface-exposed, with vaccine potential. The situation with peptide 9 is less clear-cut. Rawling et al. I6 place peptide 9 in the periplasm in their topological model, and von Specht et al. d’d not find evidence of surface reactivity for antibodies elicited in rabbits by their larger peptide D4 (residues 260-292 of mature protein F). These findings conflict with our data showing surface reactivity by whole-cell ELISA, flow cytometry and two opsonophagocytic assays’,” for murine antibodies elicited by peptide 9 (residues 261274). Furthermore, we now have evidence of vaccine efficacy for peptide 9 in two different animal models. Further studies are required to resolve these conflicting data. In conclusion, we have tested the vaccine efficacy of three proposed surface-exposed epitopes of OM protein F of P. aeruginosa. Mucosal immunization with synthetic peptides 9 and 10, but not peptide 18, confers protection from subsequent P. aeruginosa acute pneumonia. This indicates that the linear epitopes represented by peptides 9 (TDAYNQKLSERRAN) and 10

Protein F synthetic peptides confer protection: E. M. Hughes and H. E. Gilleland Jr

(NATAEGRAINRRVE) have potential development as protective immunogens.

for

further

ACKNOWLEDGEMENTS This work was supported by a grant from the Louisiana Affiliate, American Heart Association and by funds from the Center for Excellence in Cancer Research, Treatment, and Education, Louisiana State University Medical Center-Shreveport.

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REFERENCES Hancock, R.E.W., Siehnel, R. and Martin, N. Outer membrane proteins of Pseudomonas. Mol. Microbial. 1990, 4, 1069-1075 Gilleland, H.E. Jr, Gilleland, LB. and Matthews-Greer, J.M. Outer membrane protein F preparation of Pseudomonas aeruginosa as a vaccine against chronic pulmonary infection with heterologous immunotype strains in a rat model. Infect. Immun. 1988,56, 1017-1022 Gilleland, H.E., Jr, Gilleland, L.B., Hughes, E.E. and MatthewsGreer, J.M. Recombinant outer membrane protein F of Pseudomonas aeruginosa elicits antibodies that mediate opsonophagocytic killing, but not complement-mediated bacteriolysis, of various strains of /? aeruginosa. Curr. Microbial. 1992, 24, l-7 Gilleland, H.E., Gilleland, L.B. and Fowler, M.R. Vaccine efficacies of elastase, exotoxin A, and outer-membrane protein F in preventing chronic pulmonary infection by Pseudomonas aeruginosa in a rat model. J. Med. Microbial. 1993, 38, 79-86 Fox, C.W., Campbell, G.D. Jr, Anderson, W.M., Zavecz, J.A., Gilleland, L.B. and Gilleland, H.E. Jr. Preservation of pulmonary function by an outer membrane protein F vaccine: a study in rats with chronic pulmonary infection caused by Pseudomonas aeruginosa. Chest 1994, 105, 1545-l 550 Gilleland, H.E. Jr, Parker, M.G., Matthews, J.M. and Berg, R.D. Use of a purified outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine in mice. Infect. Immun. 1984, 44, 49-54

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Matthews-Greer, J.M. and Gilleland, H.E. Jr. Outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine against heterologous immunotype strains in a burned mouse model. J. Infect. Dis. 1987, 155, 1282-1291 Matthews-Greer, J.M., Robertson, D.E., Gilleland, L.B. and Gilleland, H.E. Jr. Pseudomonas aeruginosa outer membrane protein F produced in Escherichia co/i retains vaccine efficacy. Curr Microbial. 1990, 20, 171-175 Hughes, E.E., Gilleland, L.B., and Gilleland, H.E. Jr. Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of /? aeruginosa. Infect. Immun. 1992, 60, 3497-3503 Gilleland, H.E. Jr, Hughes, E.E., Gilleland, L.B., MatthewsGreer, J.M. and Staczek, J. Use of synthetic peptides to identify surface-exposed, linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa. Curr. Microbial. 1995, 31, 279-286 Gilleland, L.B. and Gilleland, H.E. Jr. Synthetic peptides representing two protective, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa elicit whole-cell-reactive antibodies that are functionally pseudomonad specific, Infect. Immun. 1995, 63, 2347-2351 Harlow, E. and Lane, D. Antibodies-a Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1988 Boudet, F., Theze, J. and Zouali, M. UV-treated polystyrene microtiter plates for use in an ELISA to measure antibodies against synthetic peptides. J. Immunol. Meth. 1991, 142, 73-82 Abdillahi, H. and Poolman, J.T. Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. EMS Microbial. Lett. 1987, 48, 367-371 Eckhardt, A., Heiss, M.M., Ehret, W. et a/. Evaluation of protective mAbs against Pseudomonas aeruginosa outer membrane protein I by Clq binding assay. Zbl. Bakt. 1991, 275, 100-111 Rawling, E.G., Martin, N.L. and Hancock, R.E.W. Epitope mapping of the Pseudomonas aeruginosa major outer -membrane oorin orotein OorF. infect. lmmun. 1995, 63, 38-42 von Specht,’ B.-U., Knapp, B., Muth, G. et a/. Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P aeruginosa outer membrane protein F and outer membrane protein I fusion proteins. Infect. Immun. 1995, 63, 1855-l 862

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