Immunization of mice with P6 of nontypeable Haemophilus influenzae: kinetics of the antibody response and IgG subclasses

Vaccine 18 (2000) 29±37

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Immunization of mice with P6 of nontypeable Haemophilus in¯uenzae: kinetics of the antibody response and IgG subclasses Wesam H. Badr a, Donna Loghmanee a, Richard J. Karalus b, Timothy F. Murphy b,c,d, Yasmin Thanavala a,* a

Department of Molecular Immunology, Roswell Park Cancer Institute, Elm & Carlton Streets, Bu€alo, NY 14263, USA b Department of Microbiology, State University of New York at Bu€alo, USA c Division of Infectious Diseases of the Department of Medicine State, University of New York at Bu€alo, USA d Western New York Veterans A€airs Healthcare System, Bu€alo, NY 14215, USA Received 16 December 1998; received in revised form 3 March 1999; accepted 30 March 1999

Abstract The kinetics of the anti-P6 antibody response was characterized in three strains of mice of di€erent haplotypes (Balb/c; H-2d, C3H/H; H-2k, SJL/J; H-2s). Anti-P6 antibodies were measured on a weekly basis by enzyme-linked immunosorbent assay (ELISA). The primary response peaked 2 or 3 weeks after the initial injection with 40 mg of puri®ed P6. The response remained at a plateau for 8±10 weeks. A maximum titer of 1:1,638,400 was attained and then steadily declined. To study the ability of P6 to generate a recall response, we opted to boost the vaccinated mice with a known subimmunogenic dose of live nontypeable Haemophilus in¯uenzae (NTHI) bacteria. After the anti-P6 antibody titers in the primed animals had stayed at baseline levels for 2 weeks, the mice were injected intraperitonealy with 108 cfu of NTHI in sterile saline. This challenge with live NTHI bacteria induced a very rapid and strong secondary antibody response in all mice. Finally, we demonstrated that these murine anti-P6 sera were 100% bactericidal against three strains of NTHI when tested in a complement dependant bactericidal assay. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Nontypeable Haemophilus in¯uenzae; Antibody responses; Bactericidal

1. Introduction Nontypeable Haemophilus in¯uenzae (NTHI) is an important human respiratory tract pathogen of children and adults. The bacterium is a common cause of otitis media in children. In addition, NTHI causes lower respiratory infections in adults with chronic lung disease [1]. Both infections are associated with substantial morbidity and account for enormous health care costs. Therefore, a great need exists for a vaccine to

* Corresponding author. Tel.: +1-716-845-8536; fax: +1-716-8458906. E-mail address: [email protected]€alo.edu (Y. Thanavala)

prevent infections caused by NTHI in these two populations. P6 is a 16 kDa peptidoglycan-associated lipoprotein which is present in the outer membrane of all strains of NTHI [2,3] and shows a high degree of sequence conservation among strains [4,5]. This latter observation is signi®cant in view of the antigenic heterogeneity of other surface proteins and lipooligosaccharide [6±11]. The following lines of evidence suggest that antibodies to P6 are protective from infection. (1) In an infant rat model of H. in¯uenzae type b infections antibodies to P6 are protective [12]; (2) In a rat model, respiratory clearance of NTHI is enhanced following mucosal immunization with P6 [13]; (3) Immunization with P6 a€ords protection in the chinchilla model of otitis media [14]; (4) Immunization of animals with P6 induces antibodies that are bactericidal to most strains

0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 1 6 6 - 8

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of NTHI [15,16]; (5) human antibody to P6 is bactericidal [17]; (6) otitis prone children have a lower level of antibodies to P6 in their serum compared to nonotitis prone children [18], and (7) antibodies to P6 in nasopharyngeal secretions and breast milk are associated with a decreased incidence of colonization with NTHI [19,20]. The observations regarding bactericidal antibody are especially relevant because protection from otitis media due to NTHI is associated with serum bactericidal antibody [21,22]. Therefore, since P6 is capable of generating bactericidal antibodies, vaccination with P6 may induce a protective immune response in the immunized host and human trials to study the immune response to P6 should be undertaken. However, the kinetics of the immune response to P6 and the isotype and subclass responses have not been well studied previously. The present study represents an extensive characterization of the immune response to P6 in mice. The response to immunization with P6 in three strains of mice which di€er in MHC haplotype was studied; the nature of the primary and secondary antibody response, the isotype and subclass responses to P6 and the bactericidal property against three strains of NTHI were characterized.

2. Material and methods 2.1. Animals Balb/c mice of MHC haplotype H-2d were obtained from Springville Laboratories (Springville, N.Y.) and Taconic Laboratories (Germantown, N.Y.). C3H and SJL/J mice of haplotypes H-2k and H-2s respectively were obtained from Jackson Laboratories. All mice were female and 8±10 weeks old at the time of use. 2.2. Induction of anti-P6 antibodies Mice (®ve animals/group) were immunized intraperitonealy with 40 mg P6 emulsi®ed in Complete Freund's Adjuvant (CFA). The mice were bled retro-orbitally on a weekly basis. Titers of anti-P6 speci®c antibodies were measured by an enzyme-linked immunosorbent assay (ELISA) assay. 2.3. Induction of anti-NTHI antibodies Three groups of mice (®ve animals/group) were immunized intraperitonealy with a 100 ml saline suspension of 106, 107 and 108 colony forming units (cfu) of live NTHI respectively. The mice were bled retroorbitally on a weekly basis. The sera obtained were analyzed for antibodies to P6 and NTHI by ELISA.

2.4. Bacterial strains NTHI strains 1479, 5657 and 2019 recovered from the sputum of adults with chronic bronchitis in Bu€alo, New York, were used in the bactericidal assay. Puri®ed P6 was obtained from strain 1479 only. 2.5. Puri®cation of OMP P6 A modi®cation of a previously described method was used to purify P6 from NTHI strain 1479 [23,24]. Bacteria were grown overnight in a brain±heart infusion (Difco, Bridgeport, NJ) supplemented with hemin and nicotinamide adenine dinucleotide both at 10 mg/ ml. Bacteria were harvested by centrifugation at 9,000 g for 20 min at 48C. Cells from 8 l of culture were suspended in 250 ml of phosphate bu€ered saline (PBS) and centrifuged again. Cells were suspended in 250 ml of bu€er B (1% sodium dodecyl sulfate, 0.1 M Tris, 0.5 M NaCl, 0.1% b-mercaptoethanol, pH 8) and sonicated six times for 10 s with a Branson Soni®er (Danbury, CT) using a large tip with a setting of six. The suspension was gently shaken at 378C for 30 min and centrifuged at 21,000 g for 30 min at room temperature. The supernatant was discarded. The above sequence of suspending, sonicating, shaking, and centrifuging was repeated three times with bu€er B plus RNAse A (10 mg/ml) and two additional times using bu€er B alone. The resulting pellet was resuspended in 48 ml bu€er A (0.01 M Tris, 0.15 M NaCl, pH 7.4) and incubated at 658C for 30 min to release P6 from peptidoglycan. The suspension was centrifuged for 1 h at 308C and the supernatant, which contained P6, was saved. The supernatant was subjected to di®ltration and then concentration by approximately 10 fold using an Amicon Ultra®ltration Cell (Danvers, MA) with a PM10 membrane. P6 was quantitated using the Sigma Protein Assay Kit (Sigma, St. Louis, MO) which is based on the method of Lowry. The purity of each preparation was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS±PAGE). The preparations consistently revealed a single band in the 16 kDa size range. 2.6. Enzyme-linked immunosorbent assay (ELISA) Ninety-six well ¯at bottom plates (Becton± Dickenson) were coated with 100 ml of a solution containing 3 mg/ml P6 in coating bu€er (0.1 M sodium carbonate, 0.1 M sodium bicarbonate, pH 9.6) to detect anti-P6 antibodies. Alternatively, 100 ml of 5  108 cfu/ml of NTHI suspension in coating bu€er was used to coat plates for detection of anti-NTHI antibodies. Plates were incubated at 48C for 36 h and then washed with PBS. Wells were blocked with 200 ml

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Fig. 1. Two groups of 5 mice (BALB/c, H-2d) each received a single intraperitoneal injection of 40 mg of P6 protein emulsi®ed with CFA. Secondary antibody responses were elicited upon challenge with an injection of 108 cfu of whole NTHI bacteria in sterile saline. The anti-P6 antibody detected in naõÈ ve mice against the booster dose of NTHI is depicted in the inset of Fig. 1. Anti-P6 antibody levels were measured weekly and for selected weeks the titers are indicated on the graph by solid arrows. Results are expressed as end point log10 titers.

3% bovine serum albumin (BSA) overnight, then washed with PBS. Test sera (100 ml) serially diluted from 1:100 to 1:1,638,400 in assay diluent (0.05% Tween-20 in PBS) was added to wells and incubated at 378C for 2 h. All samples were tested in duplicate wells. A negative control of assay diluent with no serum was included in each assay. Wells were washed ®ve times with 0.5% Tween-20 PBS. Alkaline phosphatase conjugated goat anti-mouse IgG, IgA and IgM (Sigma, St. Louis, MO; 100 ml of a 1:350 dilution in 0.05% Tween-20 in PBS) was added to all wells and incubated for 60 min at room temperature. Wells were washed eight times with 0.5% Tween-20 PBS and once with PBS. Color was developed by adding 100 ml 1 mg/ ml p-nitrophenyl phosphate in 10 mM diethanolamine and incubating for 30 min in the dark. The plates were read after 30 min at a single wavelength of 405 nm using an automated microtiter plate reader (Bio-tek Instruments, Winooski, VT). The reactive titer of an antiserum was determined as the reciprocal of the dilution uniformly yielding a 0.100 absorbance value over the pre-immune serum sample.

2.7. Isotype and IgG subclass distribution of anti-P6 antibody response The isotype and IgG subclass distribution was determined using the Mouse Typer Sub-isotyper Kit (Bio Rad Laboratories, Richmond, CA). Wells were coated with P6 as described above. Sera were serially diluted in 0.05% Tween-20 PBS and incubated on the P6coated wells for 2 h at 378C. Wells were washed and 100 ml rabbit anti-mouse subclass speci®c antiserum (1:2 dilution) was added to the wells and incubated for 1 h at room temperature. The wells were washed and 100 ml of 1:3000 dilution of goat anti-rabbit horseradish peroxidase conjugate was added to the wells and incubated for 1 h at room temperature. Color was developed by adding peroxidase substrate solution (2,2 '-azino-di [3-ethyl-benzthiazoline sulfonate] and H2O2) and incubating for 30 min at room temperature. The reaction was stopped with 100 ml 2% oxalic acid. Plates were read on an automated microtiter plate reader at OD 405 nm (Bio-tek Instruments, Winooski, VT).

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2.8. Bactericidal assays A volume of 10 ml of pre-warmed chocolate broth (protease peptone No. 3, corn starch, potassium phosphate dibasic, NaCl, 10 mg/ml hemin, 10 mg/ml nicotinamide adenine dinucleotide) was inoculated with approximately 50 colonies of NTHI from a plate grown overnight. The OD600 of this fresh inoculum was 0.05. The culture was grown with shaking at 378C in 5% CO2 to an OD600 of 0.2 [05  108 cfu/ml]. The culture was diluted 10ÿ4 in Gey's balanced salt solution (GBSS) containing 10% BSA. A complement source was prepared by adsorption of normal human serum over a Protein G column. Aliquots were stored at ÿ708C until used. A quantitative ELISA was done to measure IgG in the protein G-adsorbed serum. Adsorption with protein G removed >96% of the IgG from normal human serum. Controls were included in each assay to con®rm that the complement source alone did not have bactericidal activity [25]. A second control, containing complement source, bacteria and heat inactivated normal human serum was included to con®rm that the complement source supported complement-mediated killing. The reaction mixture contained: 25 ml bacterial dilution, 22 ml complement source, 12.5 ml of serum dilution and 190.5 ml GBSS with 2.5% fetal bovine serum. Negative control reactions contained all components except complement. An additional negative control included complement in the absence of serum to ensure that the complement source was not bactericidal in the absence of mouse serum. Positive control reaction mixtures contained known rabbit hyperimmune serum in place of the mouse serum samples. The reaction mixtures were incubated in polypropylene tubes with caps in a 378C water bath with shaking. A 25 ml aliquot of each reaction mixture was plated onto chocolate agar plates in duplicate at 0, 30 and 60 min. The plates were incubated overnight at 378C in 5% CO2 and colonies were counted.

3. Results 3.1. Kinetics of the anti-P6 antibody response Initially, Balb/c mice were immunized intraperitonealy with puri®ed P6 using three di€erent schedules. Two groups of mice were immunized either with a single injection of 25 mg P6 with CFA or two injections of 25 mg P6 one week apart (CFA, IFA respectively). The anti-P6 antibody responses elicited in mice by these two schedules (data not shown) were less than that elicited by the third and last schedule of a single dose of 40 mg P6 administered with CFA. This ®nal

immunization schedule was used for all subsequent experiments. Two groups of mice (®ve animals per group) were immunized with a single dose of 40 mg of P6 with CFA and showed a brisk IgG response which was detectable 1 week following immunization and reached a peak titer of log10 6.2 (i.e. 1:1,638,400 titer, Fig. 1) by the second week. This antibody level remained at a plateau for the next 10 weeks, after which it slowly declined and at week 24 a titer of log10 4.41 could still be detected (1:25,600). The antibody response gradually declined until it was undetectable 60 weeks after the initial immunization. To study the ability of P6 to generate a recall response, two options presented themselves (a) to boost with a known subimmunogenic dose of P6, or (b) with a known subimmunogenic dose of live NTHI bacteria. The second approach was chosen because if P6 is to be used as a vaccine, the human host will need to generate a recall response to whole bacteria. A review of the literature for experiments that utilized live or attenuated bacteria for secondary booster studies did not detail such a dose. The method of administration of the organism varied but was usually noninvasive and the doses utilized ranged from 105 to 108 cfu. To determine the subimmunogenic dose of the NTHI that would be appropriate to elicit a secondary antibody response in primed animals, the following experiment was carried out. Three groups of Balb/c mice (®ve animals per group) were injected intraperitonealy with 106, 107 or 108 cfu of NTHI. This invasive method of administration was chosen since it was the ®rst time live NTHI bacteria were being used to elicit a recall antibody response. The anti-P6 antibody response was followed on a weekly basis for all three groups. The anti-P6 antibody titer did not di€er in any of the groups irrespective of the dose of NTHI used for the immunization. All three doses of bacteria stimulated the same very weak antiP6 antibody response; a maximum titer of 1:100 was measured (see inset Fig. 1). The highest dose of 108 cfu was chosen to boost the P6 immunized mice. After the anti-P6 antibody titer had stayed at baseline levels for 2 weeks, the two groups of Balb/c mice were injected intraperitonealy at week 62 with 108 cfu of NTHI in sterile saline. An immediate and strong secondary antiP6 antibody response (1:409,600) was elicited (Fig. 1) in all animals. Recall that this dose when given to unprimed naõÈ ve mice only elicited a very weak anti-P6 response (1:100) at all time points measured (Fig. 1 inset). A similar series of experiments was performed in two additional strains of mice of di€erent MHC haplotypes (H-2k,s). Thus in C3H/H mice following a single injection of 40 mg P6 in CFA, a peak antibody response of end-point log10 5.6 (titer 1:144,160) was detected at week 4 (Fig 2a). The response gradually

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Fig. 2. Two groups of 5 mice [(a) C3H/H, H-2k and (b) SJL/J, H-2s] each received an intraperitoneal injection of 40 mg of P6 protein emulsi®ed with CFA. In C3H/H mice, secondary antibody responses were elicited upon a booster injection of 108 cfu of whole NTHI bacteria in sterile saline. Anti-P6 antibody levels were measured weekly and for selected weeks the titers are indicated on the graph by solid arrows. Results are expressed as end point log10 titers. SJL/J mice were sacri®ced at 24 weeks due to development of spontaneous tumors.

decreased and by week 24 an end point titer of 1:16,000 could be measured which was sustained for an additional 12 weeks. The response was no longer detectable at week 58. These animals were monitored for an additional 8 weeks when the anti-P6 antibodies remained undetectable. At week 66 these animals received an intraperitoneal injection of 108 cfu of live NTHI in sterile saline and an immediate and strong secondary anti-P6 response was elicited (Fig 2a). In SJL/J mice immunized with 40 mg P6, a peak anti-P6 response was measured at week 3 (end point titer 1:104,320, Fig. 2b). The response declined steadily

and by week 24 an antibody titer of 1:400 could be detected. All animals in this group were sacri®ced shortly thereafter due to development of spontaneous tumors. This strain is known to be genetically predisposed to lymphoma development. 3.2. Immunoglobulin isotype and subclass responses to P6 We have measured anti-P6 speci®c subclass responses in all three strains of mice. As shown in Table

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1 the overall responses are somewhat similar but there are some di€erences that warrant discussion. It is worth recalling that overall the highest P6 antibody titers were detected in Balb/c mice (Fig. 1) compared to C3H and SJL strains (Fig. 2 a, b). This is further re¯ected in the serum dilutions at which optimal isotype responses could be determined (refer to legend of Table 1). In Balb/c mice 1 week following immunization antiP6 antibodies of IgM, IgG1, IgG2a and IgG2b could all be readily detected. Antibodies of the IgG3 subclass were detected but at low levels and these did not increase signi®cantly with time. By week 2 the levels of IgG1, 2a and 2b increased substantially and these levels were sustained until week 9. IgM levels decreased after the second week but could still be detected at week 9. No serum P6 speci®c IgA responses were ever detected in any of the strains studied. All P6 speci®c antibodies always had the kappa light chain. In C3H/H strain P6 speci®c IgM, IgG1, IgG2a, IgG2b and IgG3 antibodies could all be detected. By week 2 there was a large increase in antibodies of IgG1, IgG2b and IgG3 subclass and by week 4 further increases were detected in IgG1 and IgG3. Antibodies in IgG2a subclass increased slowly and also declined more rapidly. By 9 weeks after the antigen injection, IgM and IgG2a levels had dropped substantially and modest decreases were also observed in both IgG1 and IgG3. The levels of P6 IgG2b antibodies were sustained.

Finally, in SJL/J mice very high levels of IgM, IgG1 and IgG2b subclasses could be detected by week 1. IgG3 levels were observed but at lower levels and did not increase signi®cantly with time. IgG2a antibodies were not detected. This was not surprising as this strain does not make IgG2a subclass but instead makes the rarer IgG2c subclass. All commercially available subclass speci®c sera are against determinants on IgG2a with marginal cross-reactivity with IgG2c and this is evident in our results (Table 1c). At week four the levels of all subclasses were maintained with the exception that IgM levels decreased. 3.3. Bactericidal antibody response to P6 To determine whether antibodies to P6 were bactericidal, assays were performed with three clinical isolates of NTHI. Fig. 3 shows that anti-P6 serum tested at a concentration of 5% was highly bactericidal for all three strains. Negative controls included serum in the absence of a complement source and the complement source in the absence of anti-P6 serum. Both of these controls showed no killing. In addition, pre-immune serum from the mice was tested in the same assay as the immune serum. The pre-immune sera showed no killing.

4. Discussion Previous studies have evaluated the antibody re-

Table 1 Anti-P6 immunoglobulin isotype and subclass responses in three strains of micea,b IgMc

IgG1

(a) Serum is diluted 1:6,400; cSerum is diluted 1:400 Week 0 Preimmune 0.080 0.106 Week 1 0.665 0.465 Week 2 0.614 1.247 Week 4 0.346 1.087 Week 9 0.248 1.013 (b) Serum is diluted 1:400; cSerum is diluted 1:100 Week 0 Preimmune 0.062 0.068 Week 1 1.262 0.378 Week 2 1.235 1.523 Week 4 1.284 2.060 Week 9 0.278 1.195 (c) Serum is diluted 1:400; cSerum is diluted 1:100 Week 0 Preimmune 0.077 0.071 Week 1 1.186 1.179 Week 2 0.612 1.247 Week 4 0.660 1.169 Week 9 0.307 0.854 a

IgG2a

IgG2b

IgG3

IgA

Kappa

Lambda

0.082 0.311 1.270 1.448 1.347

0.048 0.326 1.202 1.301 0.913

0.024 0.143 0.334 0.255 0.277

0.038 0.068 0.003 0.000 0.038

0.033 0.346 1.087 1.151 1.200

ÿ0.055 0.069 0.002 0.005 0.029

0.063 0.364 0.622 1.270 0.370

0.066 0.809 2.106 1.980 1.895

0.068 0.226 1.129 1.419 0.816

0.060 0.067 0.065 0.053 0.097

0.072 0.830 1.211 1.830 1.554

0.065 0.063 0.065 0.098 0.111

0.072 0.101 0.100 0.185 0.281

0.077 2.039 2.043 2.056 0.965

0.070 0.561 0.582 0.756 0.657

0.069 0.079 0.097 0.089 0.079

0.078 1.207 1.789 1.842 0.960

0.099 0.085 0.083 0.060 0.066

Values are expressed as OD at 405 nm. In two groups of ®ve mice each. a. BALB/c. b. C3H/H and c. SJL/J mice. The mice received a single intraperitoneal immunization of 40 mg of P6 with CFA adjuvant. Sera were collected retroorbitally on a weekly basis but only samples of selected weeks were chosen for measurement in this subclass assay. b

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Fig. 3. Determination of the bactericidal activity of anti-P6 antibodies in sera from mice immunized with a single dose of 40 mg in CFA. Killing of 3 strains of NTHI (1479 W, 2019 Q, and 5657 R was achieved with sera from immunized mice. Percent killing by preimmune sera is shown by open symbols.

sponse to P6 following immunization by various routes in various animal species. Green et al. raised antiserum in rabbits by immunizing with two or three doses of native [15] and recombinant [16] P6. Serum contained IgG antibodies and was bactericidal for most strains of NTHI. P6 has been tested as a vaccine antigen in the chinchilla model of otitis media [14,26]. Green et al. [26] immunized chinchillas with three doses of P6, administered together with two other outer membrane proteins. Antibodies to P6 were detected in serum but no bactericidal activity was present. By contrast, DeMaria et al. [14] immunized chinchillas with four doses of P6 and detected serum bactericidal antibody. Numerous di€erences in the methods used in the two studies likely account for the di€erences in antibody response observed. These di€erences include use of combinations of antigens in the Green study, di€erent methods of purifying P6, di€erent immunization schedules, di€erent adjuvants, and di€erent methods for determination of bactericidal antibodies. Of interest, DeMaria et al. [14] used the same method of purifying P6 as was used in the present study. Kyd et al. [13] immunized rats with P6 by direct injection into Peyer's patches followed by intratracheal boost. Animals developed both systemic and mucosal antibody responses and serum bactericidal antibodies to NTHI were detected. Yang et al. [27] have conducted a very interesting study on the importance of the lipid moiety on the immunoprotective properties of P6 and other physiochemical characteristics of this molecule. They conclude that removal of the palmitoyl moiety from the protein causes a slight diminution on the levels of anti-P6 antibodies produced. Thus, the lipid moiety may have adjuvant-like properties in that it facilitates

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micelle formation. However, they suggest that the protective property of anti-P6 antibodies may be due to opsonic activity of complement rather than bactericidal. Finally, Hotomi et al. [28] have studied the antiP6 antibody response elicited by intranasal immunization of OMP P6 in BALB/c mice administered with cholera toxin B subunit as a mucosal adjuvant. Speci®c enhancement of the response with IL-5 cytokine treatment was also observed. Anti-P6 serum IgG and nasopharyngeal IgA levels were the most enhanced by simultaneous intranasal treatment with IL-5. The underlying mechanism of speci®c cytokine modulation of the murine anti-P6 response remains unclear. Following intranasal administration, OMP P6 is thought to be absorbed in the nasal mucosa and to stimulate lymphocytes in the nasal associated lymphoid tissue. Long term responses were not reported in this study. In order to compare the immune responses to P6 with that seen against other bacterial proteins, a search of the literature revealed that Peterson et al. [29] were the ®rst to describe a murine neutralizing MAb antibody of the IgG2b subclass functioning both in vitro and in vivo against the Lipopolysacchride (LPS) of Chlamydia pneumoniae. The limitation however was that this neutralizing activity of MAbCP-33 was found to be bacterial strain-speci®c. In our study, a strong anti-P6 antibody of the IgG2b subclass was obtained and among the longest sustained in the three strains of mice used in our study. Furthermore, the anti-P6 antibody obtained from Balb/c mice proved to be 100% bactericidal against all three strains of NTHI used in our study. Little is known about the kinetics of the antibody response to P6. In addition, nothing is known about the IgG subclass response following immunization with P6. We have characterized the kinetics and duration of the antibody response to P6 in mice and also analyzed the isotype and subclass of the antibodies elicited. In addition, we have studied the antibody response to P6 in three strains of mice of di€erent MHC haplotypes (H-2d,k, and s) in order to determine the potential for immunization in individuals with diverse haplotypes. Immunization with a single dose of puri®ed P6 elicited strong and sustained antibody responses in all three strains of mice. In Balb/c mice, serum antibody levels persisted for at least 55 weeks. Of particular note, was the immediate, strong secondary response that was elicited after a single booster dose of 108 cfu of live bacteria. This has the most signi®cant clinical implication. The observation that whole NTHI bacteria can elicit a strong secondary response in mice that were primed with a puri®ed outer membrane protein, in this case P6, is unprecedented and lends signi®cant support for the choice of this protein as a vaccine candidate.

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Various IgG subclasses mediate di€erent e€ector functions on the antigenic target. Immunization with P6 elicited antibodies dominated by IgG2b and IgG1 subclasses (Table 1). Immunization of mice with a peptide derived from the major outer membrane protein of Chlamydia trachomatis induced a strong IgG response dominated by IgG1 subclass [30]. Interestingly, of the murine subclasses, IgG2b has the greatest capacity to activate complement [31]. Human antibody responses to other bacterial proteins have been studied extensively. Musher et al. [32] has studied the genetic regulation of the capacity to make IgG to pneumococcal capsular polysaccharides (PPS) in humans. In an extensive study they found that those who failed to make IgG to an individual PPS also failed to make IgM or IgA to that antigen. The responder status of the o€spring was highly related to that of the parents. The IgG level in some cases was associated with Gm (23)+ allotype. The Human Leucocyte Antigen (HLA) type was not associated with the antibody response. Thus, the response to PPS in humans was shown to be highly variable and heritable in a mixed, co-dominant fashion. In their attempts to develop a vaccine against streptococcal infections in the newborn, Feldman et al. [33] promote type III capsular carbohydrate as a vaccine candidate. They demonstrate that the natural human IgG antibody response is opsonically active in a complementdependent manner. Hetland et al. [34] studied classical complement activation of anti-lipoarabinomannan (anti-LAM) antibodies induced by whole bacilli of Mycobacterium bovis BCG and Mycobacterium tuberculosis products. Sera from Indian tuberculosis patients contained more anti-LAM antibodies than sera from healthy Indians. Levels of anti-LAM IgG2, but not anti-LAM IgM directly correlated with classical complement activation induced by BCG in the sera. They conclude that levels of anti-LAM IgG may be important for the extent of phagocytosis of M. tuberculosis by mononuclear phagocytes. The development of bactericidal antibodies to NTHI is of particular interest because the presence of bactericidal antibodies is associated with protection from infection in the case of otitis media in children. Shurin et al. [22] demonstrated that the absence of bactericidal antibody to NTHI was associated with susceptibility to otitis media caused by the bacterium. Faden et al. [21] studied the bactericidal antibody response to homologous strains of NTHI in children with otitis media. Their studies showed that otitis media elicits strainspeci®c bactericidal antibodies and that these antibodies are associated with protection from reinfection with the same strain. In view of the strong association of the presence of bactericidal antibodies with protection from infection, an antigen, which is capable of eliciting a bactericidal antibody response, may serve as

an e€ective vaccine antigen. Using three strains of mice of di€erent MHC haplotypes, we have demonstrated a strong sustained primary antibody response following immunization with a single dose of P6, the ability to recall a secondary immune response (following booster injection with a subimmunogenic dose of NTHI whole bacteria) and the bactericidal nature of these antibodies against three strains of NTHI. These observations along with earlier work in chinchillas [14] and rats [13] indicate that P6 is an important candidate for human vaccine development. Our future studies will focus on characterizing the immune response to P6 in humans.

Acknowledgements This work was supported by NIH grant AI19641 from the National Institutes of Allergy and Infectious Diseases, and by the Department of Veterans A€airs.

References [1] Murphy TF, Sethi S. Bacterial infection in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992;146:1067±83. [2] Murphy TF, Nelson MB, Dudas KC, Mylotte JM, Apicella MA. Identi®cation of a speci®c epitope of Haemophilus in¯uenzae on a 16,600-dalton outer membrane protein. J Infect Dis 1985;152:1300±7. [3] Deich RA, Metcalf BJ, Finn CW, Farley JE, Green BA. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus in¯uenzae. J Bacteriol 1988;170:489±98. [4] Murphy TF, Bartos LC, Campagnari AA, Nelson MB, Apicella MA. Antigenic characterization of the P6 protein of nontypable Haemophilus in¯uenzae. Infect Immun 1986;54:774±9. [5] Nelson MB, Munson Jr RS, Apicella MA, Sikkema DJ, Molleston JP, Murphy TF. Molecular conservation of the P6 outer membrane protein among strains of Haemophilus in¯uenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms. Infect Immun 1991;59:2658±63. [6] Troelstra A, Vogel L, van Alphen L, Eijk P, Jansen H, Dankert J. Opsonic antibodies to outer membrane protein P2 of nonencapsulated Haemophilus in¯uenzae are strain speci®c. Infect Immun 1994;62:779±84. [7] Duim B, Dankert J, Jansen HM, van Alphen L. Genetic analysis of the diversity in outer membrane protein P2 of non-encapsulated Haemophilus in¯uenzae. Microbial Pathogen 1993;14: 451±62. [8] Bell J, Grass S, Jeanteur D, Munson Jr RS. Diversity of the P2 protein among nontypeable Haemophilus in¯uenzae isolates. Infect Immun 1994;62:2639±43. [9] Sikkema DJ, Murphy TF. Molecular analysis of the P2 porin protein of nontypeable Haemophilus in¯uenzae. Infect Immun 1992;60:5204±11. [10] Weiser JN. The oligosaccharide of Haemophilus in¯uenzae. Microbial Pathogen 1992;13:335±42. [11] Campagnari AA, Gupta MR, Dudas KC, Murphy TF, Apicella

W.H. Badr et al. / Vaccine 18 (2000) 29±37

[12] [13]

[14]

[15]

[16]

[17]

[18] [19]

[20]

[21]

[22] [23]

MA. Antigenic diversity of lipooligosaccharides of nontypeable Haemophilus in¯uenzae. Infect Immun 1987;55:882±7. Munson Jr RS, Grano€ DM. Puri®cation and partial characterization of outer membrane proteins P5 and P6 from Haemophilus in¯uenzae type b. Infect Immun 1985;49:544±9. Kyd JM, Dunkley ML, Cripps AW. Enhanced respiratory clearance of nontypeable Haemophilus in¯uenzae following mucosal immunization with P6 in a rat model. Infect Immun 1995;63:2931±40. DeMaria TF, Murwin DM, Leake ER. Immunization with outer membrane protein P6 from nontypeable Haemophilus in¯uenzae induces bactericidal antibody and a€ords protection in the chinchilla model of otitis media. Infect Immun 1996;64:5187±92. Green BA, Quinn-Dey T, Zlotnick GW. Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus in¯uenzae against multiple clinical isolates of H. in¯uenzae type b. Infect Immun 1987;55:2878±83. Green BA, Metcalf BJ, Quinn-Dey T, Kirkley DH, Quataert SA, Deich RA. A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus in¯uenzae elicits biologically active antibody against both nontypeable and type b H. in¯uenzae. Infect Immun 1990;58:3272±8. Murphy TF, Bartos LC, Rice PA, Nelson MB, Dudas KC, Apicella MA. Identi®cation of a 16,600-dalton outer membrane protein on nontypable Haemophilus in¯uenzae as a target for human serum bactericidal antibody. J Clin Invest 1986;78:1020±7. Yamanaka N, Faden H. Antibody response to outer membrane protein of nontypeable Haemophilus in¯uenzae in otitis-prone children. J Pediatr 1993;122:212±8. Harabuchi Y, Faden H, Yamanaka N, Du€y L, Wolf J, Krysto®k D, Tonawanda/Williamsville Pediatrics. Nasopharyngeal colonization with nontypeable Haemophilus in¯uenzae and recurrent otitis media. J Infect Dis 1994;170:862±6. Harabuchi Y, Faden H, Yamanaka N, Du€y L, Wolf J, Krysto®k D. Human milk secretory IgA antibody to nontypeable Haemophilus in¯uenzae: possible protective e€ects against nasopharyngeal colonization. J Pediatr 1994;124:193±8. Faden H, Bernstein J, Brodsky L, Stanievich J, Krysto®k D, Shu€ C, Hong JJ, Ogra PL. Otitis media in children. I. The systemic immune response to nontypable Haemophilus in¯uenzae. J Infect Dis 1989;160:999±1004. Shurin PA, Pelton SI, Tazer IB, Kasper DL. Bactericidal antibody and susceptibility to otitis media caused by nontypeable strains of Haemophilus in¯uenzae. J Pediatr 1980;97:364±9. Munson RS, Grano€ DM. Puri®cation and partial characterization of outer membrane proteins P5 and P6 from Haemophilus in¯uenzae type b. Infect Immun 1985;49:544±9.

37

[24] Murphy TF, Bartos LC, Campagnari AA, Nelson MB, Apicella MA. Antigenic characterization of the P6 protein of nontypeable Haemophilus in¯uenzae. Infect Immun 1986;54:774±9. [25] Yi K, Sethi S, Murphy TF. Human immune response to nontypeable Haemophilus in¯uenzae in chronic bronchitis. J Infect Dis 1997;176:1247±55. [26] Green BA, Vazquez ME, Zlotnick GW, Quigley-Reape G, Swarts JD, Green I, Cowell JL, Bluestone CD, Doyle WJ. Evaluation of mixtures of puri®ed Haemophilus in¯uenzae outer membrane proteins in protection against challenge with nontypeable H. in¯uenzae in the chinchilla otitis media model. Infect Immun 1993;61:1950±7. [27] Yang YP, Munson Jr RS, Grass S, Chong P, Harkness RE, Gisonni L, James O, Kwok Y, Klein MH. E€ect of lipid modi®cation on the physicochemical, structural, antigenic and immunoprotective properties of Haemophilus in¯uenzae outer membrane protein P6. Vaccine 1997;15(9):976±87. [28] Hotomi M, Yokoyama M, Kuki K, Togawa A, Yamanaka N. Study on speci®c mucosal immunity by intranasal immunization of outer membrane protein P6 of Haemophilus in¯uenzae with cholera toxin B subunit. Acta Oto-LaryngologicaÐSupplement 1996;523:150±2 [Clinical Trial. Journal Article. Randomized Controlled Trial]. [29] Peterson EM, de la Maza LM, Brade L, Brade H. Characterization of a neutralizing monoclonal antibody directed at the lipopolysaccharide of Chlamydia pneumoniae. Infection and Immunity 1998;66(8):3848±55. [30] Su H, Parnell M, Caldwell HD. Protective ecacy of a parenterally administered MOMP-derived synthetic oligopeptide vaccine in a murine model of Chlamydia trachomatis genital tract infection: serum neutralizing IgG antibodies do not protect against chlamydial genital tract infection. Vaccine 1995;13:1023± 32. [31] Oi VT, Vuong TM, Hardy R, Reidler J, Dang J, Herzenberg LA, Stryer L. Correlation between segmental ¯exibility and e€ector function of antibodies. Nature 1984;307:136±9. [32] Musher DM, Groover JE, Watson DA, Pandey JP, RodriguezBarradas MC, Baughn RE, Pollack MS, Graviss EA, de Andrade M, Amos CI. Genetic regulation of the capacity to make immunoglobulin G to pneumococcal capsular polysaccharides. Journal of Investigative Medicine 1997;45(2):57±68. [33] Feldman RG, Breukels MA, David S, Rijkers GT. Properties of human anti-group B streptococcal type III capsular IgG antibody. Clin Immunol and Immunopath 1998;86(2):161±9. [34] Hetland G, Wiker HG, Hogasen K, Hamasur B, Svenson SB, Harboe M. Involvement of antilipoarabinomannan antibodies in classical complement activation in tuberculosis. Clin Diag Lab Immunol 1998;5(2):211±8.