INTRODUCTION

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It has long been known that normal human sera and sera from infant or adult rats are often bactericidal for unencapsulated Haemophilus influenzae and for type b H. influenzae. Further, any given isolate of H. influenzae undergoes high-frequency switching between a serum-resistant and a serum-sensitive state (17, 18). The molecular basis of this phenotypic variation has been the subject of extensive study over the past several years. It has been shown to be the result, at least in part, of phase variation in the expression of surface-exposed lipopolysaccharide (LPS) antigenic structures. These include phosphorylcholine (ChoP) and gal1,4gal, both of which mimic structures found on the surface of host cells (18, 19). Substitution of LPS by ChoP is mediated by the lic1 locus of H. influenzae (17). Expression of gal1,4gal requires the lic2 and lgtC loci (8, 13). Expression of ChoP increases the sensitivity of H. influenzae isolates to normal human sera, since human serum contains C-reactive protein, which binds to the ChoP, activating complement and killing the bacteria (19). In contrast, sera from normal rats contain very low levels of C-reactive protein, and expression of ChoP increases the resistance of H. influenzae isolates to the bactericidal activity of rat sera (18). Expression of gal1,4gal increases sensitivity to rat sera, but not to human sera (18). This suggests that rats, but not humans, often have naturally occuring antibodies to this epitope, which is also found on human glycolipids.

Like humans and rats, laboratory rabbits often have naturally occuring serum bactericidal activity for H. influenzae. In this study, we report that this activity is mediated by immunoglobulin M (IgM) antibodies directed against phase-variable LPS structures. We were able to determine, in part, the target of the bactericidal antibodies. The epitopes targeted include gal1,4gal, the structure which confers serum resistance in human serum, but not ChoP, which appears to mask bactericidal epitopes in some strains.

	MATERIALS AND METHODS

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Bactericidal assay. H. influenzae strains used in this study are listed in Table 1. The procedure for the bactericidal assay was modified from that described by Shurin et al. (16). Except as indicated (Table 2), bactericidal assays were carried out by using strain KW20 from the strain collection at MedImmune (KW20-MI) (see Table 1), grown to mid-log phase in brain heart infusion medium (Difco) supplemented with 10 µg of nicotinamide adenine dinucleotide per ml and either Levinthal's supplement (added at 10%, vol/vol [1]) or 10 µg of hemin per ml. Reactions were carried out in 96-well polystyrene plates with round-bottomed wells. Each reaction (0.1-ml final volume) contained 10 µl of complement (3- to 4-week-old rabbit complement; Pel-Freez, Brown Deer, Wis.), approximately 200 bacteria, and varying dilutions of test serum. The diluent was Gey's balanced salt solution (Sigma Chemical Co., St. Louis, Mo.). The reactions were incubated for 30 min at 37°C on a rocking platform. The plate was then placed on ice, and 20 µl from each fraction was spotted onto a GC-hemin plate, (prepared from GC II agar [BBL Microbiology Systems] supplemented with Isovitalex [1%, vol/vol, BBL] and 10 µg of hemin per ml). The agar plates were allowed to dry and were then incubated overnight at 37°C in 5% CO₂. The endogenous complement in test sera was inactivated by heating for 30 min at 56°C, and the sera were diluted to final concentrations ranging from 1:20 to 1:1,280. All reactions were carried out in duplicate, and the colony counts were averaged. The bactericidal titer of a serum was defined as the highest dilution resulting in 50%-or-greater reduction in viable bacteria, compared to control wells in which bacteria were incubated with complement but no serum. Each lot of complement purchased was prescreened to ensure that it would support the killing of H. influenzae KW20 by antiserum to outer membrane and that killing in the absence of added serum was minimal.

Flow cytometric detection of antibody bound to bacterial cells. Bacteria were grown to mid-log phase as described above, then washed in Hanks' balanced salt solution (Life Technologies, Grand Island, N.Y.) containing 5% fetal bovine serum and were suspended in the same buffer to approximately 5 × 10⁸ CFU/ml. A 25-µl volume of bacterial suspension was added to 25 µl of diluted serum (final serum dilution, 1:20 or 1:50). Bacteria were incubated with serum for 1 h at 4°C and were then washed and suspended in 50 µl of phycoerythrin-conjugated goat anti-rabbit Ig (-Ig) or anti-rabbit IgM (-IgM) (Southern Biotechnology Associates, Inc., Birmingham, Ala.) (final antibody concentration of 5 µg/ml). After 1 h of incubation at 4°C in the dark, the bacteria were washed and resuspended in 1 ml of Hanks' balanced salt solution-5% fetal bovine serum for analysis by flow cytometry. Samples were acquired and analyzed on a FacStar Plus flow cytometer by using CellQuest software from Becton Dickinson Immunocytometry Systems (San Jose, Calif.). Logarithmic amplification of all parameters was used with a side scatter threshold. An electronic gate was used to limit acquisition and analysis to the singlet bacteria population. Ten thousand events per sample were collected and analyzed for the detection of bound antibodies as indicated by a shift in the fluorescent signal above background.

Enzyme-linked immunosorbent assay (ELISA). Anti-LPS antibodies were measured as follows: a suspension of LPS (purified from strain KW20 by hot phenol-water extraction as described [4]) was diluted to 10 µg per ml in Dulbecco's phosphate-buffered saline (PBS; Hyclone, Logan, Utah) containing 10 mM MgCl₂ and was used to coat Immulon 2HB plates (Dynex Technologies, Inc., Chantilly, Va.) (100 µl per well). Plates were incubated overnight at 4°C and then wells were filled with blocking solution consisting of 5% bovine serum albumin fraction V (Sigma) in PBS containing 0.2% Tween 20, and the plates were incubated for 1 h. The wells were washed once with PBS-0.2% Tween 20 using an automatic plate washer (model 96PW; TECAN U.S., Hillsborough, N.C.). A 100-µl aliquot of serum (serially diluted in blocking buffer to a final concentration of from 1:10 to 1:1,280) was added to each well and was incubated for 3 h at room temperature. The plate was washed four times, and 100 µl of secondary antibody (horseradish-peroxidase-conjugated goat anti-rabbit IgG or anti-rabbit IgM, diluted in blocking buffer to 1:5,000; Southern Biotechnology Associates) was added and incubated for 2 h. After washing, bound antibody was detected by using the 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). The optical densities at 405 nm were determined 30 min after addition of substrate by using a Vmax Kinetic Microplate Reader (Molecular Devices Corporation, Menlo Park, Calif.). Each reaction was carried out in duplicate, and the results were averaged. The anti-LPS titer of a serum was defined as the last dilution giving an absorbance greater than twice the background value.

	RESULTS AND DISCUSSION

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Phase-variable LPS structures affect killing by bactericidal rabbit sera. In preliminary experiments (data not shown) we tested the bactericidal activity of rabbit antisera to a number of different recombinant H. influenzae proteins that had been cloned from strain KW20 as hexahistidine-tagged proteins, denatured with guanidine, purified by nickel-affinity chromatography, and renatured by dialysis as described (7). Control sera were from rabbits that had been immunized with an outer membrane (OM) preparation of KW20 prepared by Triton X-100 extraction as described by Loeb & Smith (10). Additional control sera were from unimmunized rabbits or rabbits immunized with antigens cloned from Streptococcus pneumoniae or Staphylococcus aureus. We found that over half of the antisera against H. influenzae antigens were bactericidal for H. influenzae KW20, with bactericidal titers as high as 2,560. However, equally high bactericidal titers were seen in many of the antisera to gram-positive antigens, as well as some of the sera from unimmunized rabbits. This suggests that bactericidal activity for unencapsulated H. influenzae may occur naturally in rabbits (as it does in humans and in rats) and is not necessarily the effect of immunization with a given protein antigen. We are therefore unable to draw conclusions about the ability of any of the H. influenzae proteins to elicit bactericidal antibodies.

Bactericidal activity is the result of IgM antibodies directed against LPS. The results of the experiments described above suggest that the bactericidal activity we saw in certain rabbit sera was the result of antibody to LPS. However, this is not necessarily the case. As noted above, the bactericidal activity of normal human serum for H. influenzae isolates expressing ChoP is not antibody mediated, but results from C-reactive protein binding to the ChoP on the bacterial surface and activating complement. Further, there is precedent for LPS structure affecting the susceptibility to killing mediated by protein antigens: gonococci with sialylated LPS are resistant not only to killing by normal human sera, but also to killing by antisera to the major OM protein, protein 1 (20).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Preincubation of sera with LPS reduces both bactericidal activity and binding of IgM to the bacterial cell surface. Rabbit sera ME46 () and ME109 () were incubated for 1 h in varying concentrations of LPS before use in the bactericidal assay (panel A) and flow cytometric determination of binding to bacteria (panel B). The LPS concentrations indicated in each panel are the final concentrations during incubations of bacteria with sera. The final serum concentrations were 1:100 for the bactericidal assay and 1:20 for flow cytometry. Serum ME109 was raised against a surface antigen of S. pneumoniae (our unpublished data).

Implications for evaluation of vaccine candidates. While the attempt to demonstrate bactericidal activity of immune sera is a customary part of the evaluation of H. influenzae vaccine candidates (2, 3), obtaining convincing, reproducible evidence of bactericidal activity is often difficult (11) due to the existence in many laboratory animals of naturally occurring bactericidal activity towards H. influenzae. The level of bactericidal activity in individual animals varies and may increase during the life of the animal, independent of the immunogen. Researchers have tried to minimize the effect of these antibodies. For example, Loeb reported that the sera of rabbits immunized with H. influenzae OM protein a (also known as P1) were bactericidal even after adsorption with capsular polysaccharide and LPS and concluded that antibodies to protein a were bactericidal (9). Our data suggest that the problem of naturally occuring antibodies is compounded by the fact that they are directed against phase-variable epitopes. In practice, adsorption of sera with purified LPS may not remove all the anti-LPS antibodies, because the bacterial culture used for any individual bactericidal assay may express LPS with a different structure from that of the purified LPS.