The e (P4) Outer Membrane Protein of Haemophilus influenzae

Vol. 59, No. 9

INFECTION AND IMMUNITY, Sept. 1991, p. 3191-3198 0019-9567/91/093191-08$02.00/0 Copyright C 1991, American Society for Microbiology

The e (P4) Outer Membrane Protein of Haemophilus influenzae: Biologic Activity of Anti-e Serum and Cloning and Sequencing of the Structural Gene BRUCE A. GREEN,* JOHN E. FARLEY, TAMMY QUINN-DEY, ROBERT A. DEICH, AND GARY W. ZLOTNICK Praxis Biologics, Inc., 300 E. River Road, Rochester, New York 14623 Received 7 May 1991/Accepted 22 June 1991

Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti-e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae.

tective against Hib meningitis. To further investigate this protein and to determine the degree of antigenic conservation among NT H. influenzae and the biologic activity of anti-e serum against NT H. influenzae, we purified the e protein and produced an anti-e polyclonal antiserum. This article reports that the purified protein elicited biologically active antibodies against NT H. influenzae and that anti-e OMP serum showed synergistic BC activity when mixed with antiserum against the recombinant PCP protein. We also report the cloning and sequencing of the gene encoding this protein and its expression in Escherichia coli as a lipoprotein. These results show that the e protein is a vaccine candidate against NT H. influenzae.

Nontypeable (NT) Haemophilus influenzae isolates are causative agents of a wide range of human diseases including tracheobronchitis, pneumonia, epiglottitis, and otitis media (2, 28, 36). In in vivo studies, Barenkamp (1) has shown that experimental otitis media with effusion in chinchillas caused by NT H. influenzae can be prevented by passive administration of antiserum reactive with non-lipooligosaccharide (LOS) NT H. influenzae surface components. Brinton et al. (4) have reported that active immunization with LKP pili or passive immunization with anti-LKP sera could protect

chinchillas against otitis media caused by NT H. influenzae organisms with homologous pili. While most of the work on outer membrane proteins (OMPs) of H. influenzae has focused on type b strains (Hib), some OMPs of Hib have been shown to be targets for bactericidal (BC) antibody in both Hib and NT H. influenzae strains (12, 33). OMPs which have been investigated in vitro for use as potential vaccine candidates against NT H. influenzae include the Hi-PAL, or P6 protein (11, 12, 27), the PCP protein (6), and a 105-kDa OMP (33). Extensive work with the Hi-PAL protein in this and other laboratories has demonstrated that it is a target for BC antibody in Hib and NT H. influenzae and for protective antibody in Hib (12, 25, 26). Our laboratory has also recently shown that antibodies against two different OMPs, recombinant PAL and PCP, can have synergistic BC effects when mixed together (6). This effect may make it desirable for

MATERIALS AND METHODS Bacteria. Clinical isolates of NT H. influenzae P860295, N10, N90, 0127E, N47, Hst-36, 0135E, N0246E, N0133E, N1955, Hst-34, P861454, P880859, Hst-34, G23, and 0204E were kindly provided by Porter Anderson, Rochester, N.Y., Eric Hansen, Dallas, Tex., Charles Bluestone, Pittsburgh, Pa., and Robert Daum, New Orleans, La. H. influenzae isolates were subcultured once by overnight incubation at 37°C on brain heart infusion (BHI) agar (Difco Laboratories, Detroit, Mich.) supplemented with hemin (Sigma Chemical Co., St. Louis, Mo.) at 10 ,ug/ml and NAD (Sigma) at 2 ,ug/ml. Isolates were stored without further passage at -70°C in BHI agar containing 20% glycerol. E. coli strains used in this study were HB101 (3), KH802 (19), and JM103 (22). Purification of e protein from Hib Eagan. e protein was purified from outer membrane fractions of Hib Eagan prepared as described by Zlotnick et al. (39). After some contaminating proteins were removed by extraction with 50 mM Tris HCI-5 mM EDTA (pH 8) (buffer A) containing 1% sarcosyl followed by extraction with 1% Zwittergent 3-12 (Calbiochem Corp., San Diego, Calif.) in buffer A, e protein was solubilized by extraction of the insoluble fraction with

vaccines against NT H. influenzae to be composed of multiple purified OMPs, thus possibly making them more effective and broader in scope. The P4 or e protein has been reported to be constant in size in all Hib isolates examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). However, Granoff and Munson (10) reported that antiserum directed against P4, when administered to infant rats, was nonpro-

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Corresponding author. 3191

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GREEN ET AL.

1% Zwittergent 3-14 in buffer A. Solubilized e protein was partially purified by passage over a DEAE Biogel-A (BioRad Laboratories, Richmond, Calif.) column equilibrated with buffer A containing 1% Zwittergent 3-14 (Calbiochem). Under these conditions, the e protein did not bind to the column and was in the fall-through peak. The soluble e protein was then absorbed onto a hydroxylapatite (Bio-Rad) column equilibrated in the above buffer and eluted with 0.3 M dibasic sodium phosphate (pH 8) containing 1% Zwittergent 3-14. The eluted e protein was precipitated with ethyl alcohol, washed with 1% octylglucoside-50 mM Tris HCl (pH 8), and solubilized in buffer A containing 1% Zwittergent 3-14. This purified e protein was used for further experiments. Isolation of DNAs. Plasmid DNA was isolated by SDSalkaline extraction (19). Chromosomal DNA was isolated by the procedure of Marmur (20). DNA restriction fragments were isolated from agarose gels (FMC Corp., Marine Colloids Div., Portland, Me.) by electrophoresis onto NA45 nylon (Schleicher & Schuell, Inc., Keene, N.H.) and elution in 1 M NaCl-10 mM Tris hydrochloride (pH 8.0)-i mM EDTA. Restriction endonucleases were purchased from New England Biolabs, Inc. (Beverly, Mass.), and used according to directions of the manufacturer. Cloning of the gene encoding the e protein. The e protein gene was isolated from the H. influenzae Rd strain KW20b-A Charon 4 chromosomal gene bank described previously (8) and plaqued on E. coli KH802. Plaques expressing the e protein were identified by a plaque lift assay (8) with anti-e monoclonal antibodies (MAbs) (see below). Bacteriophage from positive plaques was isolated and replaqued on a fresh lawn of E. coli KH802 and rescreened by using anti-e MAbs. Phage from positive plaques was isolated in CsCl gradients, and the DNA was isolated by phenol extraction (18). DNA from phage expressing the e protein all contained a single 15-kb EcoRI fragment. The e protein gene was subcloned on a -1.6-kb EcoRI-SspI fragment into pUC19 and saved as plasmid pPX513. DNA sequencing. DNA sequencing of the Haemophilus e lipoprotein (he!) gene was performed by dideoxynucleotide sequencing (32) directly from plasmid clones or from M13mpl8 and M13mpl9 (22) clones of the EcoRI-SspI and EcoRI-HindIII fragments. Sequencing primers were made on a 380B DNA synthesizer (Applied Biosystems, Foster City, Calif.). DNA and protein sequence analyses were done on an IBM-AT computer with Microgenie software (Beckman Instruments, Inc., Palo Alto, Calif.). DNA hybridization analysis. The EcoRI-SspI fragment of plasmid pPX513 was labeled with digoxigenin-dUTP (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) by following the directions of the manufacturer and used as a probe for hybridization analysis. Hybridization and detection of probes were carried out by following the directions supplied with the Genius labeling and detection kit (Boehringer Mannheim). Production of antiserum directed against e protein. Antiserum to e protein was produced in New Zealand White rabbits by intramuscular injection of 10 ,ug of purified protein emulsified in Freund incomplete adjuvant (Difco). Blood was taken before immunization and was the source for normal rabbit serum. Animals received booster injections on days 30 and 60 after the primary immunization. Animals were bled 10 days after the last injection, and sera were tested for anti-e protein antibodies by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) analysis. Production of MAbs. MAbs against the e protein were

INFECT. IMMUN.

produced by immunizing BALB/c mice with e proteinenriched OMP fractions (i.e., before column chromatography) emulsified in incomplete Freund adjuvant (Difco). Mice were boosted intravenously 2 weeks later with the OMP fractions in saline. Spleen cells were isolated and fused as previously described (8). Hybridoma cell culture supernatants were screened by ELISA against purified e protein and NT H. influenzae S2 (a derivative of Hib Eagan) LOS (8). Cell culture supernatants were also screened for reactivity to e protein and other NT H. influenzae OMPs by Western blot. After screening, three hybridomas, which did not react with either strain S2 LOS or any other NT H. influenzae OMP, were chosen for further study, namely, EPR-35-24, EPR-52.1, and EPR-17.1. Surface reactivity of MAbs with NT H. influenzae was determined by using an indirect immunofluoresence assay which was previously shown to be specific for surface-exposed epitopes (12). BC assays. BC assays were performed against NT H. influenzae as previously described (11) by using precolostral calf serum as the complement source. Endpoint titers are reported as the reciprocal of the highest dilution of antiserum capable of killing >50% of the NT H. influenzae cells as compared with control wells containing no antiserum. Assays to determine possible synergistic effects of anti-e and anti-rPCP sera (6) were performed as described by Deich et al. (6). Each antiserum was diluted to the BC endpoint titer of the more active antiserum, and the two antisera were mixed 1:1 in the first well of a microtiter plate (increasing the dilution of each antiserum in the first well by twofold). Standard BC assays were then performed against NT H. influenzae cells. The endpoint titer was reported as the highest dilution of antiserum capable of killing >50% of NT H. influenzae versus control wells. Thus, a titer of 1:160 means that each antiserum was diluted to 1:160 in that well. Protein determination. Protein was determined by using the method of Lowry et al. (17) as modified by Peterson (31). SDS-PAGE. SDS-PAGE was performed in a mini-gel system (70 by 100 mm) (Bio-Rad) by using the method of Laemmli (15). Samples were reduced with P-mercaptoethanol in sample preparation buffer and boiled for 5 min. Gels were run at 150 V (constant voltage). Separated proteins were detected by staining with Coomassie brilliant blue G-250 (Sigma). LOS was detected in SDS-polyacrylamide gels by using the silver staining method of Tsai and Frasch (34). Proteinase K was obtained from Sigma. Globomycin treatment of E. coli expressing e protein. Logarithmic-phase E. coli cells expressing e protein were treated with globomycin (generously provided by Henry Wu, Bethesda, Md.) to inhibit signal peptidase II, as previously described (8). Treated and untreated cells were run on SDS-polyacrylamide gels. e protein was detected by Western blot analysis by using anti-e MAbs as probes. Western blot analysis. Western blot analysis of proteins separated by SDS-PAGE was done as described previously (11). Nucleotide sequence accession number. The hel gene DNA sequence reported in this article has been submitted to GenBank and assigned accession number M68502.

RESULTS Purification of e protein. Hib Eagan was chosen as the source of the e protein since our preliminary studies indicated that there was a high degree of antigenic crossreactivity between strain Eagan e protein and NT H. influ-

VOL. 59, 1991

e

1

B.

A. 1

2 3

MW

1

2 3

-43 KD-25 KD-18 KD-

3

4

5

6

47-

24-

-

....:..:.

FIG. 1. SDS-PAGE analysis of e protein samples. A 10-,ug amount of purified e protein isolated from Hib Eagan, either with or without proteinase K treatment, was loaded and run on 15% SDSpolyacrylamide gels. Gels were stained with either Coomassie brilliant blue (A) or the silver stain (B) of Tsai and Frasch (34). Positions of molecular size standards are shown between the two gels. Lanes: 1, untreated e protein; 2, e protein treated with proteinase K; 3, 10 ng of Haemophilus LOS treated with proteinase K.

proteins. e protein was purified by using a combination of differential detergent extraction to quickly remove some contaminating proteins and then column chromatography to produce purified protein. Nondenaturing detergents were used throughout the procedure to minimize the possibility of destroying epitopes on the protein. An SDSpolyacrylamide gel of the purified protein is shown in Fig. 1. The gel is shown stained with Coomassie brilliant blue in Fig. 1A and silver stained to look for LOS contamination in Fig. 1B. These results show the purified e protein migrating as a single band at the expected molecular size of approximately 28 kDa. The silver stain in Fig. 1B shows the purified protein to contain