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Bactericidal Antibody Responses Elicited by a Meningococcal Outer Membrane Vesicle Vaccine with Overexpressed Factor H–Binding Protein and Genetically Attenuated Endotoxin

  1. Oliver Koeberling1,
  2. Anja Seubert2 and
  3. Dan M. Granoff1
  1. 1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California
  2. 2Novartis Vaccines, Siena, Italy
  1. Reprints or correspondence: Dan M. Granoff, 5700 Martin Luther King Jr. Way, Oakland, CA 94609 (dgranoff{at}chori.org).
 
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Abstract

Background. Outer membrane vesicle (OMV) vaccines from mutant Neisseria meningitidis strains engineered to overexpress factor H-binding protein (fHbp) have elicited broadly protective serum antibody responses in mice. The vaccines investigated were not treated with detergents to avoid extracting fHbp, which is a lipoprotein. Because of their high endotoxin content, the vaccines would not be safe to administer to humans.

Methods. We prepared a native OMV vaccine from a strain engineered to overexpress fHbp and in which the gene encoding LpxL1 was inactivated, which reportedly decreases endotoxin activity.

Results. The OMV vaccine from the mutant had a similar or lower ability to induce the expression of proinflammatory cytokines by human peripheral blood mononuclear cells, compared with a detergent-extracted wild-type OMV, and 1000–10,000-fold lower activity than a native wild-type OMV. In mice, the OMV vaccine from the mutant elicited higher serum bactericidal antibody responses to a panel of heterologous N. meningitidis strains than did a control multicomponent recombinant protein vaccine or a detergent-extracted OMV vaccine that has been demonstrated to confer protection against meningococcal disease in humans.

Conclusions. The data illustrate the potential to develop a broadly immunogenic native OMV vaccine that has decreased endotoxin activity and is potentially suitable for testing in humans.

No broadly effective vaccine is available for Neisseria meningitidis group B strains, which account for half of meningococcal cases in the United States [1, 2] and >80% of cases in Europe [3, 4]. The group B capsule is structurally similar to antigens expressed by neural tissues and is therefore a poor immunogen that also has the potential to elicit autoantibodies. Thus, a polysaccharide-protein conjugate vaccine is unlikely to be feasible for the prevention of group B disease [5].

Novel antigens discovered by “genome mining” are currently under investigation as group B vaccines. One highly promising candidate is factor H-binding protein (fHbp), which has also been known as genome-derived neisserial antigen 1870 [6] and LP2086 [7, 8]. fHbp is a surface-exposed lipoprotein present in all N. meningitidis strains [6]. This protein can be subclassified into 3 variants on the basis of sequence similarity and antigenic cross-reactivity. In general, antibodies prepared against fHbp variant (v.) 1 have been shown to be bactericidal against strains expressing fHbp from the v.1 group but not against strains expressing v.2 or v.3 proteins (and vice versa) [6, 9]. The v.1 antigen is part of a promising investigational meningococcal vaccine consisting of 3 recombinant proteins, 2 of which are fusion proteins expressing 2 antigens each (i.e., a total of 5 antigens) [10]. In humans, this vaccine has elicited serum bactericidal antibody responses to genetically diverse N. meningitidis strains [11].

Outer membrane vesicle (OMV) vaccines are safe [12, 13] and efficacious against meningococcal disease [14, 15]. An OMV vaccine was licensed in New Zealand and controlled a long-standing group B epidemic [16–19]. However, serum bactericidal antibodies elicited by OMV vaccines are directed primarily at a major porin protein, PorA [20], which is immunodominant [21] and antigenically variable [22, 23]. OMV vaccines are treated with detergents to extract lipooligosaccharide (LOS) and decrease endotoxin activity. This procedure also removes detergent-soluble antigens, such as fHbp or GNA2132, which in mice have elicited broadly protective serum antibody responses [6, 24, 25].

To increase protective activity, we previously prepared “native” OMV vaccines from N. meningitidis strains engineered to overexpress fHbp v.1 [26, 27]. The serum samples from immunized mice conferred broader bactericidal activity against genetically diverse N. meningitidis strains than did serum samples from control mice immunized with recombinant fHbp (rfHbp) v.1 or a native OMV vaccine prepared from the corresponding wild-type strain [26, 27]. The native OMV vaccines were prepared without the use of detergents, to avoid extracting fHbp. Thus, the endotoxin activity was too high for the vaccine to be administered safely to humans.

In the present study, we prepared a native OMV vaccine from a N. meningitidis mutant strain engineered to overexpress fHbp and in which the lpxL1 gene encoding a late-functioning acyltransferase also was inactivated. The deletion resulted in penta- instead of hexaacylated lipid A, which in previous studies decreased endotoxin activity while retaining adjuvant activity [28–30]. Our hypothesis was that this OMV vaccine would be less toxic than a native OMV prepared from a wild-type strain while retaining the ability of the mutant OMV to elicit serum anti-fHbp antibodies with broad bactericidal activity.

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Methods

Meningococcal strains. Meningococcal strains used in this study are described in table 1. Strain H44/76 and mutants derived from this strain were used to prepare the OMV vaccines. This strain expresses an fHbp v.1 protein with an amino acid sequence identical to that of strain MC58 [6], which provided the gene to overexpress fHbp v.1 (referred to in table 1 as subvariant 1.1). The other 6 strains expressed PorA proteins heterologous to that of the H44/76 vaccine strain and also expressed different subvariants of fHbp v.1 (table 1).

Growth conditions. N. meningitidis strains were grown at 37°C on GC agar plates in an atmosphere containing 5% CO2 or in Mueller-Hinton broth containing 0.25% glucose and 5 µg/mL chloramphenicol or 80 µg/mL kanamycin as required.

Electrophoretic studies. SDS-PAGE was performed with 4%–20% gradient gels (Invitrogen). For Western blot analyses, proteins were transferred onto nitrocellulose membranes, and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG/A/M (Invitrogen). For resolution of LOS, the samples were separated by SDS-PAGE at a constant of 20 mA for ∼1.5 h. The gel was agitated for 1 h in a solution of 40% ethanol and 5% acetic acid and treated for 5 min with 0.7% (wt/vol) periodic acid dissolved in the same solution. After being washed with H2O, the gels were stained with 0.67% (wt/vol) silver nitrate in 0.019 mol/L NaOH and 0.4% NH4OH for 10 min, washed with H2O, and developed in a solution containing 50 mg/L citric acid and 0.015% (vol/vol) formaldehyde. The reaction was stopped with 50% methanol.

Recombinant DNA methods. Transformation of N. meningitidis and overexpression of the fHbp v.1 encoded on plasmid pFP12-fHbp was performed as described elsewhere [26, 27]. To generate the LpxL1 knockout (KO) mutant, the lpxL1 gene from strain MC58 was amplified by polymerase chain reaction (PCR) using primers LpxL1_forHindIII (5′-CCCAAGCTTATCCTTCGGGGATGCAGGTC-3′) and LpxL1_revXbaI (5′-GCTCTAGAGCCGTCTGAACGTAGTCAGTAAAAATCGGGGC-3′). The lpxL1 fragment was cloned into HindIII- and XbaI-digested plasmid pUC18, resulting in plasmid pUCLpxL1. An internal 204-bp fragment of the lpxL1 gene was deleted by inverse PCR with plasmid pUCLpxL1 as template using primers LpxL1_del1 (5′-AACTGCAGCGGTGAAGTGCGGATACAGG-3′) and LpxL1_del2 (5′-ACGCGTCGACAGGATTTCGGACGCAACG-3′). A kanamycin-resistant cassette was ligated with the product from the inverse PCR, resulting in plasmid pUCLpxL1kan.

OMVs. The OMV vaccine consisted of membrane blebs released by the bacteria into the supernatant and isolated as described elsewhere [21]. Relative amounts of fHbp in the different OMV preparations were visualized by Western blot using a murine anti-fHbp monoclonal antibody (MAb), JAR 3 (figure 1) [25]. As shown in figure 1A, 0.013 µg of native OMV protein prepared from the LpxL1 KO mutant with overexpressed fHbp contained slightly more fHbp than 0.05 µg of OMV protein from the wild-type strain. Thus, the mutant OMV contained ∼4–5-fold more fHbp than did the wild-type OMV. Silver staining (figure 1B) showed a slightly lower molecular mass of the LOS from the LpxL1 mutant compared with that of the wild type. This observation was consistent with a structural change in the LOS from hexa- to pentaacylated lipid A caused by deletion of the gene encoding LpxL1.

Figure 1
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Figure 1

A, Factor H-binding protein (fHbp) variant (v.) 1 in outer membrane vesicle (OMV) preparations, as detected by Western blot analysis (WT, native OMV prepared from the H44/76 wild-type strain; fHbp KO, OMV from a mutant of H44/76 in which the gene encoding fHbp was inactivated; LpxL1 KO OE fHbp, OMV from a mutant of H44/76 with inactivated LpxL1 and overexpressed fHbp; rfHbp, recombinant v.1 protein). The primary antibody was the anti-rfHbp monoclonal antibody JAR 3. B, Silver-stained SDS-PAGE of lipooligosaccharide in OMV preparations from strain H44/76. Amounts of vesicles loaded in each lane were standardized on the basis of total protein content.

Extraction of LOS from OMV was performed on the basis of the published deoxycholate procedure for production of the OMV vaccine by the Norwegian Institute of Public Health, Oslo [32]. Silver staining showed that 0.2 µg of detergent-extracted OMV contained less LOS than 0.02 µg of native OMV (figure 1B, right). These data were consistent with removal of the majority of the LOS by the detergent treatment. As expected, the detergent treatment also removed most of the fHbp (figure 1A, right).

Serological analysis. Total antibody responses to fHbp, NadA, GNA2132, and LOS were measured by ELISA, as described elsewhere [25]. The recombinant protein antigens on the plate consisted of purified His-tagged fHbp, NadA [33], or GNA2132 [24] (encoded by genes from strains MC58, 2996, and NZ98/254, respectively). LOS was purified from the wild-type and LpxL1 mutant strains of H44/76, using the procedure described by Westphal et al. [34]. The secondary antibody was goat anti-mouse IgG/A/M conjugated to alkaline phosphatase.

Complement-mediated bactericidal antibody responses were measured as described elsewhere using washed, log-phase bacteria grown in Mueller-Hinton broth with 0.25% glucose and 0.02 mmol/L CMP-NANA [35]. The buffer was Dulbecco's PBS (Sigma-Aldrich) containing 0.9 mmol/L CaCl2 × 2 H2O, 0.5 mmol/L MgCl2 × 6 H2O, and 1% (wt/vol) bovine serum albumin. The complement was human serum from a healthy adult with no detectable intrinsic bactericidal activity. Bactericidal titers were defined as the serum dilution resulting in a 50% decrease in the number of colony-forming units after 60 min of incubation at 37°C compared with the number at time 0 in negative control reactions.

Absorption of anti-fHbp antibodies. The absorption was performed as described elsewhere [27], using a column containing His-tagged rfHbp or, as a negative control, recombinant His-tagged NadA bound to Sepharose (GE Healthcare). Nonspecific binding sites were blocked with normal mouse serum, and the column was washed to remove unbound rfHbp. To compensate for dilution and incomplete recovery of antibodies during the absorption, the anti-LOS titers of the absorbed serum pools were adjusted to match the respective titers before absorption.

Cytokine assay. After obtaining informed consent, blood was collected from healthy donors, and the buffy coats were fractionated by Ficoll density centrifugation. The peripheral blood mononuclear cell (PBMC) layer was recovered, washed 3 times with RPMI 1640 medium, and resuspended in complete medium (RPMI 1640, l-glutamine, and 25 mmol/L HEPES containing 10% fetal calf serum [HyClone] and 1% penicillin/streptomycin/glutamine). PBMCs were cultured in 96-well flat-bottom plates at a density of 4×105 cells per well. Serial 10-fold dilutions (from 1 to 0.00001 µg/mL final concentration) of native OMV were added, prepared from the H44/76 wild type, the LpxL1 KO mutant with overexpressed fHbp, or detergent-treated wild-type OMV. The samples were incubated for 4 h at 37°C. Cytokine secretion was measured by Bio-Plex analysis (Bio-Rad) according to the manufacturer's instructions using the human 27-plex panel. The following soluble proteins were assayed: interleukin (IL)-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, eotaxin, basic fibroblast growth factor, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, interferon (IFN)-γ, inducible protein 10, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, platelet-derived growth factor BB, RANTES, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor. The concentrations of cytokines were analyzed using the Bio-Plex System and Bio-Plex Manager software (version 4.1).

Immunization. Mice were immunized with native OMV vaccine prepared from the LpxL1 KO strain of H44/76 with increased expression of fHbp. Control vaccines consisted of a clinical lot (FMEN0405) of detergent-extracted OMV vaccine prepared from strain H44/76 and a multicomponent recombinant protein vaccine containing GNA2091 fused with fHbp v.1, GNA2132 fused with GNA1030, and NadA [10]. The Norwegian Institute of Public Health provided the detergent-extracted OMV vaccine preadsorbed with aluminum hydroxide [15]. Novartis Vaccines provided the recombinant proteins. The recombinant antigens (300 µg/mL) were formulated in our laboratory with 3 mg of aluminum hydroxide per milliliter of water containing 10 mmol/L histidine and 9 mg/mL NaCl.

Groups of 4–6-week-old female CD-1 mice (Charles River Breeding Laboratories) were immunized intraperitoneally (10–15 mice per group). For each injection, the mice received a dose of 2 µg of protein from native or detergent-extracted OMV vaccine, which was absorbed with 600 µg of aluminum hydroxide. The total dose of the recombinant protein vaccine was 60 µg (20 µg of each protein), which was absorbed with 600 µg of aluminum hydroxide. Three injections of vaccine were given, separated by 3 weeks. Blood was collected 3 weeks after the third injection. Serum samples were separated by centrifugation and stored frozen. The animal experiments complied with the relevant guidelines of Italy and the institutional policies of Novartis Vaccines.

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Results

Cytokine release from human PBMCs. Figure 2 shows the dose responses for 4 proinflammatory cytokines, TNF-α, IL-1β, IL-6, and IL-8, as measured in experiments with PBMCs from 2 donors. Small doses of native OMV from the wild-type strain elicited high levels of each of the cytokines, whereas much higher doses of native OMV prepared from the LpxL1 KO mutant with overexpressed fHbp or of the detergent-treated OMV from the wild-type strain were required to stimulate cytokines. For example, for donor 2 (figure 2A, right), a dose of 4×10−6µg/mL of native OMV from the wild-type strain elicited the same concentration of TNF-α as did 3×10−2 µg/mL of native mutant OMV (ratio, 1:7500) or 2.5×10−3 µg/mL of detergent-extracted wild-type OMV (ratio, 1:625). For each of the 4 cytokines and both donor's PBMCs, the native OMV from the mutant elicited cytokine responses that were lower than or similar to those elicited by the detergent-extracted OMV from the wild-type strain.

Figure 2
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Figure 2

Release of the proinflammatory cytokines interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α after incubation of peripheral blood mononuclear cells (PBMCs) with different concentrations of outer membrane vesicle (OMV) for 4 h. Left, PBMCs from donor 1, experiment 1; right, PBMCs from donor 2, experiment 2. OMV vaccines tested were native OMV prepared from either the H44/76 wild-type (WT, native) (white squares, solid lines) or an H44/76 mutant with inactivated LpxL1 and overexpressed factor H-binding protein (mutant, native) (black squares, solid lines) and a detergent-extracted OMV from the H44/76 wild-type strain (WT, extracted) (white circles, dashed lines).

Of the other 23 cytokines measured, 6 (IL-1ra, G-CSF, IFN-γ, MCP-1, MIP-1α, and MIP-1β) were above background levels after incubation of PBMCs with OMV from the wild-type strain. As shown in figure 3, for each of these cytokines the native OMV from the mutant had much lower stimulating activity than did native OMV from the wild-type strain (>500- to 10,000-fold) and similar or lower stimulating activity compared with the detergent-extracted OMV from the wild-type strain.

Figure 3
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Figure 3

Release of cytokines in response to incubation of human peripheral blood mononuclear cells (PBMCs) for 4 h with different concentrations of outer membrane vesicle (OMV).

Serum antibody responses by ELISA. Figure 4 summarizes the serum antibody responses to rfHbp, NadA, and GNA2132, the 3 antigens in the recombinant protein vaccine responsible for eliciting bactericidal antibodies [10]. We also measured titers of antibody to LOS from the wild-type and LpxL1 KO mutant N. meningitidis strains, which had been used to make the OMV vaccines; the respective anti-LOS titers were nearly identical. Therefore, data are shown only for the mutant LpxL1 KO LOS.

Figure 4
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Figure 4

Serum antibody responses to factor H-binding protein (fHbp) (variant 1), NadA, GNA2132, and lipooligosaccharide (LOS) (LpxL1 knockout mutant), as measured by ELISA. Shown are results for (left to right) mice immunized with aluminum hydroxide (black bars), Norwegian outer membrane vesicle (OMV) vaccine (OMV, Norway) (checkered bars), recombinant protein vaccine (striped bars), and native OMV prepared from H44/76 with inactivated LpxL1 and increased expression of fHbp (OMV, mutant) (gray bars). Error bars show the 95% confidence intervals (CIs) for the geometric mean titers (GMTs).

Mice immunized with the recombinant proteins had high titers of serum antibody to each of the respective antigens in the vaccine and no detectable serum antibody responses to LOS. Mice immunized with the Norwegian OMV vaccine had low anti-fHbp titers (geometric mean titer [GMT], 1:80) and undetectable titers of antibody to the other antigens tested. In contrast, mice immunized with the native OMV vaccine prepared from the LpxL1 KO mutant with overexpressed fHbp had high antibody responses to fHbp and LOS. The anti-fHbp GMT was 1:100,000, similar to that in the mice given the recombinant proteins (GMT, 1:70,000).

The titers shown in figure 4 were measured in serum samples that had been incubated with the antigen at 4°C for 18 h. The respective titers to rfHbp were similar when the incubation was performed at 37°C for 2 h (figure 5). However, the anti-LOS GMT decreased by >2 log10 when the incubation was performed at 37°C for 2 h (figure 5).

Figure 5
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Figure 5

Effect of temperature on measurement of titers of antibody to lipooligosaccharide (LOS) and factor H-binding protein (fHbp) (variant 1) by ELISA in serum samples from mice immunized with mutant outer membrane vesicle. Serum samples were incubated for 2 h at 37°C or for 18 h at 4°C. After the plates were washed, bound immunoglobulin was measured by use of goat anti-mouse IgG/A/M conjugated to alkaline phosphatase. Error bars show 95% confidence intervals (CIs) for the geometric mean titers (GMTs).

Serum bactericidal antibody responses. The serum bactericidal antibody responses of each of the vaccine groups were high when measured against strain H44/76 (GMT, >1:1000) (figure 6). This strain was used to prepare the OMV vaccines, and it expressed fHbp with an amino acid sequence identical to that of fHbp in the recombinant protein vaccine. Interestingly, mice given the mutant native OMV with increased fHbp had a 10-fold higher GMT against H44/76 than did mice given the recombinant protein or detergent-extracted OMV vaccines. The higher bactericidal antibody responses to the mutant OMV may have reflected the combined bactericidal activity of homologous anti-PorA and anti-fHbp antibodies.

Figure 6
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Figure 6

Serum bactericidal activity (shown as geometric mean titers [GMTs]) in immunized mice, as measured against Neisseria meningitidis strain H44/76 (used to prepare the outer membrane vesicle [OMV] vaccines; see text) and 6 additional strains with PorA molecules heterologous to that of the vaccine strain and expressing subvariants of factor H-binding protein (fHbp) variant 1 (see table 1). Bars represent the GMTs from assays of 3 serum pools from each vaccine group (serum samples from 5 mice per pool), as described for figure 4; error bars show 95% confidence intervals (CIs). There were 2 serum pools for the Norwegian OMV vaccine group, and the bars represent the GMTs and ranges of the values. titers (GMTs).

The other 6 test strains had PorA that was heterologous to that of the vaccine strain, and they expressed subvariants of fHbp v.1 (table 1). The serum bactericidal antibody titers in mice given the Norwegian detergent-extracted OMV vaccine were <1:10 for all of these strains. The titers in mice immunized with the recombinant protein vaccine were high for some of these strains (e.g., CA0408 or 4243) but were low (e.g., NZ98/254) or undetectable (e.g., Z1092) for others. In contrast, in mice immunized with the native OMV vaccine prepared from the mutant, high bactericidal antibody titers developed for all 6 heterologous strains. The GMTs for the 6 heterologous strains were <1:10 in mice given the detergent-extracted Norwegian OMV vaccine, 1:82 in mice given the recombinant protein vaccine, and 1:797 in mice given the native OMV from the LpxL1 KO mutant with overexpressed fHbp (P<.001).

Table 1
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Table 1

Neisseria meningitidis strains.

To investigate the contribution of anti-fHbp antibodies to bactericidal activity in the serum of mice immunized with the mutant OMV, we absorbed the serum pools using a column that contained rfHbp-His bound to nickel-Sepharose. After absorption, the serum samples were no longer bactericidal against the 2 heterologous stains tested, but the control serum samples absorbed on a nickel-Sepharose column containing recombinant His-tagged NadA retained bactericidal activity (table 2).

Table 2
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Table 2

Effect of absorption of antibodies to factor H-binding protein (fHbp) on the bactericidal activity of serum samples from mice immunized with outer membrane vesicle (OMV) vaccine from a mutant LpxL1 knockout strain of H44/76 with overexpressed fHbp.

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Discussion

Detergent-extracted OMV vaccines are safe [13] and effective (reviewed in [36]). Their major limitation is strain-specific serum bactericidal antibody responses. To broaden bactericidal antibody responses, OMV vaccines have been prepared from >1 strain [37] or from multiple strains engineered to express >1 PorA molecule [38–41]. However, it is difficult to provide broad coverage against endemic disease in large geographic areas with a vaccine that elicits bactericidal antibodies directed against PorA, because the strains are genetically diverse and express many different PorA molecules. Moreover, certain PorA serosubtypes have been reported to be poorly immunogenic [42].

Recently, Weynants et al. [43] prepared detergent-treated OMV vaccines from mutant N. meningitidis strains engineered to overproduce several surface-exposed outer membrane proteins that normally are present in low copy number (TbpA, Hsf, NspA, and Omp85). They found that overproduction of >1 minor protein was required to elicit serum bactericidal antibodies to heterologous strains. They theorized that a critical density of antibodies on the surface of the organism was required to engage C1q and activate classic complement pathway bactericidal activity. We have postulated a similar mechanism to explain why 2 nonbactericidal anti-fHbp MAbs that recognized nonoverlapping epitopes became bactericidal when tested in combination [25, 44]. However, mice immunized with native OMV vaccines prepared from mutant N. meningitidis strains with overproduction of fHbp had high serum bactericidal antibody responses against heterologous strains [26, 27]. Therefore, with a native OMV vaccine and overproduction of fHbp, a single antigen was sufficient to elicit polyclonal bactericidal antibodies.

In the present study, we constructed a mutant N. meningitidis strain that both overexpressed fHbp and contained the LpxL1 KO mutation. Although inactivation of the lpxL1 gene of N. meningitidis has been reported to decrease endotoxin activity [28–30], other published data have indicated that non-LOS components of the bacterium can be potent inducers of cytokines [45]. Therefore, it was not clear how much attenuation we would achieve with a native OMV vaccine prepared from an LOS mutant with overexpressed fHbp. It was also unclear whether the OMV vaccine from the LOS mutant would retain sufficient immunogenicity, because some of the adjuvant properties of LOS may depend on the presence of hexaacylated LOS [46].

Our data showed that a native OMV vaccine from the mutant strain had 1000–10,000-fold lower activity in stimulating human PBMCs to release proinflammatory cytokines than did a control native OMV from the wild-type strain. Furthermore, the native OMV vaccine from the mutant had similar or lower activity in stimulating proinflammatory cytokines compared with a detergent-treated OMV in which most of the LOS had been extracted. Although additional studies are needed, these data suggest that native OMV vaccines prepared from LpxL1 KO strains may be safe to administer to humans.

The native OMV from the LpxL1 KO mutant with overexpressed fHbp elicited serum bactericidal antibody responses to strain H44/76 (from which the vaccine was prepared) that were 10-fold higher than those elicited by a clinical lot of the Norwegian detergent-extracted OMV vaccine, which was also prepared from strain H44/76 and which protected humans from meningococcal disease [15]. Mice immunized with the native OMV prepared from the mutant strain also had higher and broader bactericidal antibodies than did control mice immunized with a multicomponent recombinant protein vaccine containing fHbp as one of its antigens.

Neither the detergent-treated nor the native OMV vaccine elicited serum antibodies to NadA or GNA2132 (figure 4). NadA was found to be immunogenic during infection in humans [47] and was also immunogenic in mice immunized with an OMV vaccine prepared from an N. meningitidis strain other than H44/76 [48]. The H44/76 vaccine strain does not have the gene encoding NadA (authors' unpublished data), and natural expression of GNA2132 may have been too low for the OMV vaccines to elicit anti-GNA2132 antibody responses.

Mice immunized with the mutant OMV vaccine had high titers of both anti-LOS and anti-fHbp antibodies (figure 4). However, the serum bactericidal antibodies appeared to be directed against fHbp, because depletion of anti-fHbp antibodies resulted in the loss of serum bactericidal activity (table 2). Antibodies to fHbp both activate complement-mediated bactericidal activity and inhibit binding of factor H to the bacterium [44]. The latter enhances the susceptibility of N. meningitidis to complement-mediated bactericidal activity, and it is difficult to separate the 2 functions of the anti-fHbp antibodies. Therefore, we cannot exclude the possibility that the anti-LOS antibodies elicited by the native OMV vaccine contributed to bactericidal activity in the presence of anti-fHbp inhibition of factor H binding to the bacterium. This possibility, however, seems unlikely, because the anti-LOS antibodies bound poorly at 37°C (figure 5), the temperature used to measure bactericidal activity. Furthermore, in our previous study, mice immunized with a native OMV vaccine prepared from an fHbp KO mutant that was given with rfHbp had high titers of anti-fHbp antibodies, but these antibodies were not bactericidal and did not activate C3b deposition on the bacterial surface of heterologous strains [26]. Thus, the combination of anti-LOS antibodies elicited by the native OMV vaccine and anti-fHbp antibodies elicited by rfHbp was not sufficient for bactericidal activity. In contrast, serum samples from mice immunized with a native OMV with overexpressed fHbp were bactericidal and activated C3b deposition, and these activities were eliminated by absorption of the anti-fHbp antibodies. Conceivably, certain conformational fHbp epitopes important for eliciting antibodies with broad bactericidal activity are better expressed by fHbp in the OMV than by rfHbp. Although the actual mechanism remains to be defined, the data illustrate the potential to develop a broadly immunogenic native OMV vaccine that has decreased endotoxin activity and that is potentially suitable for testing in humans.

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Acknowledgments

We thank Brunella Brunelli, Serena Giuntini, and Esther Mungai for technical assistance; Mariagrazia Pizza (Novartis Vaccines, Siena, Italy) for providing the recombinant protein antigens; and Johan Holst (Norwegian Institute of Public Health, Oslo) for providing the detergent-extracted OMV vaccine. The anti-LOS immunotype monoclonal antibodies were a gift from W. Zollinger (Walter Reed Army Institute of Research, Washington, DC).

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Footnotes

  • Potential conflicts of interest: D.M.G. is the principal investigator of laboratory research conducted on behalf of Children's Hospital Oakland Research Institute, which is funded by grants from Novartis Vaccines and Diagnostics and Sanofi Pasteur; he also holds a paid consultancy from Novartis and is an inventor on patents or patent applications in the area of meningococcal B vaccines. A.S. is an employee of Novartis Vaccines, Siena, Italy.

  • Financial support: National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH; Public Health Service grant R01 AI46464); Novartis Vaccines. The laboratory work was performed, in part, in a facility funded by the National Center for Research Resources, NIH (Research Facilities Improvement Program grant CO6 RR-16226). Work was also performed at Novartis Vaccines, Siena, Italy, during a sabbatical year by 2 of the investigators (O.K. and D.M.G.).

  • Received November 13, 2007.
  • Accepted January 24, 2008.
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