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Children's Hospital Oakland Research Institute, Oakland, California
Background.
Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants.
Methods.
We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine.
Results.
Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies.
Conclusions.
The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine.
Outer membrane vesicle (OMV) vaccines elicit protective immunity against Neisseria meningitidis group B disease (reviewed in [1]). Recently, an OMV vaccine received a provisional license in New Zealand and was introduced for widespread immunization in response to a group B epidemic that has been ongoing there for more than a decade [24]. One important limitation of OMV vaccines is that they elicit bactericidal antibody responses that are largely directed against surface-exposed loops of PorA [5], a major porin protein, and there is considerable PorA antigenic diversity in strains causing endemic meningococcal disease [6]. Thus, OMV vaccines are of greatest use for prevention of epidemic disease caused by a predominant (clonal) meningococcal strain, such as in New Zealand [4].
Recent efforts to develop group B meningococcal vaccines have focused on antigenically conserved antigens, such as neisserial surface protein A (NspA) [7, 8], or a number of other novel proteins (referred to as "genome-derived neisserial antigens" ) discovered during the N. meningitidis MC58 genome sequencing project [9]. Among the latter is GNA1870, a lipoprotein of unknown function that is presently being evaluated for use in a recombinant protein vaccine [10, 11]. GNA1870 can be subdivided into 3 variant groups on the basis of amino-acid variability and antigenic cross-reactivity. Strains expressing GNA1870 in the variant 1 (v.1) group account for 60% of the disease-producing group B isolates [11]. In a previous study, mice immunized with a recombinant GNA1870 (rGNA1870) v.1 protein vaccine developed serum bactericidal antibody responses against most, but not all, strains expressing subvariants of the GNA1870 v.1 protein [10]. Thus, GNA1870 is a promising antigen for inclusion in a protective meningococcal vaccine, but it would be desirable to improve the breadth of the protective antibody responses elicited by the recombinant protein.
In the present study, we investigated serum antibody responses elicited in mice after immunization with an OMV vaccine prepared from a N. meningitidis strain genetically engineered to overexpress GNA1870 v.1 protein. Our hypothesis was that the functional activity of antibodies elicited by the overexpressed native GNA1870 v.1 protein anchored in the OMV might be greater than that elicited by a rGNA1870 protein vaccine or by a conventional OMV vaccine.
MATERIALS AND METHODS
Bacterial strains. The 7 N. meningitidis strains used in this study are listed in table 1. Strain RM1090 naturally expresses low levels of a GNA1870 variant 2 (v.2) protein. The other 6 strains express subvariants of GNA1870 v.1 proteins [10, 11] and are genetically diverse on the basis of their genetic lineages as defined by electrophoretic cluster analysis [12, 13] and/or sequencing typing [14].
Overexpression of GNA1870 v.1 protein by N. meningitidis.
We prepared a mutant strain of RM1090 in which the GNA1870 v.2 gene was inactivated (RM1090GNA1870) by use of homologous recombination with plasmid pBSUDGNA1870ERM [11] (gift from J. Adu-Bobie, Chiron Vaccines) and erythromycin selection (5 g/mL). RM1090GNA1870 was transformed with pFP12-GNA1870, as described elsewhere [15], to generate a second-generation mutant that overexpressed GNA1870 v.1 protein. All strains containing the introduced pFP12-GNA1870 shuttle vector were grown in the presence of 5 g/mL chloramphenicol.
Vaccines.
The wild-type (wt) and mutant RM1090 strains were inoculated into Mueller-Hinton broth containing 0.25% glucose and were incubated at 37°C with rocking until the optical density measured at 620 nm reached 0.81.0. Phenol was added (0.5% wt/vol), and the broth was left to incubate overnight at 4°C, to kill the bacteria. The bacterial cells were pelleted by centrifugation (at 10,000 g) for 30 min at 4°C and were frozen and stored at -20°C until they were used for preparation of the OMV vaccines. The OMV vaccines were prepared as described elsewhere without the use of detergents, to avoid extraction of the GNA1870 lipoprotein [16]. The rGNA1870 protein vaccine was purified and expressed in E. coli, as described elsewhere [10], by use of a GNA1870 DNA sequence encoding 6 carboxy-terminal histidines and devoid of the amino terminal sequence coding for the putative leader peptide.
Immunization.
N. meningitidis OMV preparations and rGNA1870 protein were adsorbed with an equal volume of aluminum phosphate adjuvant (1% Alhydrogel [wt/vol; Superfos Biosector] that had been incubated with PBS to convert aluminum hydroxide to aluminum phosphate). Groups of female CD1 mice, 46 weeks old (10 mice/group; Charles River Breeding Laboratories), were immunized intraperitoneally (ip). Each mouse received a dose containing 5 g of total protein (5 g of OMV preparations or rGNA1870 protein or 2.5 g each of OMV preparations and rGNA1870 protein). A total of 3 injections were given, each separated by 3 weeks. Two weeks after the third dose, anesthetized mice were killed by cardiac puncture. Serum samples were separated and were stored frozen at -20°C.
Anti-GNA1870 antibody response.
Serum antibody titers against GNA1870 were measured by ELISA, which was performed as described elsewhere [10]. The secondary detecting antibodies were alkaline phosphataseconjugated goat anti-mouse antiserum specific for IgM, IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotech).
Complement-mediated bactericidal antibody activity.
The bactericidal assay was performed as described elsewhere [17], and log phase bacteria grown in Mueller Hinton broth supplemented with 0.25% glucose were used. The final reaction mixture contained 20% (vol/vol) complement and serial 2-fold dilutions of test serum diluted in Gey's buffer. Bactericidal titers were defined as the serum dilution resulting in a 50% decrease in colony-forming units per milliliter after a 60-min incubation of bacteria in the reaction mixture, compared with the value in controls at time 0. The complement source was human serum from a healthy adult that had no detectable intrinsic bactericidal activity and was qualified as described elsewhere [18].
Binding of antibodies to the surface of live encapsulated N. meningitidis.
The ability of anti-GNA1870 antibodies to bind to the surface of live N. meningitidis was determined by flow-cytometric detection of indirect fluorescence, which was performed as described elsewhere [19].
Control antibodies and antiserum.
Controls in the different experiments included mouse monoclonal antibodies (MAbs) specific for groups B and C polysaccharide capsules [20, 21]), PorA P1.2 [19], and GNA1870 v.1 protein (JAR3) [10]. In addition, hyperimmune antiserum from mice immunized with rGNA1870 proteins (expressed in E. coli from the genes of N. meningitidis strains MC58, 2996, and M1239 [v.1, v.2, and variant 3 {v.3} proteins, respectively]) and purified as HisTag proteins, as described elsewhere [10, 11], was used.
Activation of human complement deposition on the surface of live encapsulated meningococci.
Anti-GNA1870 antibodydependent deposition of C3b or iC3b on the bacterial surface of live N. meningitidis bacteria was determined by flow cytometry [22].
Passive protection in infant rats.
The ability of antiserum to confer passive protection against N. meningitidis group B bacteremia was tested in infant rats challenged ip with group B strain NZ98/254, and this experiment was performed as described elsewhere [17, 22, 23].
RESULTS
Surface accessibility of GNA1870 in N. meningitidis.
To determine whether the GNA1870 protein expressed by strain RM1090 that was transformed with pFP12-GNA1870 is an integral part of the OMV and is surface exposed, we measured binding of anti-GNA1870 antibody to live encapsulated bacterial cells by flow cytometry (figure 2). Positive control MAbs specific for group C capsular polysaccharide (figure 2, column 2) or PorA (anti-P1.2, column 3) showed strong binding to the wt (row B) and 2 mutant RM1090 strains: RM1090GNA1870 (row A) and the RM1090 strain overexpressing GNA1870 v.1 protein (row C). Compared with cells incubated with the negative control antiserum (column 1), the RM1090 strain overexpressing GNA1870 v.1 protein showed strong binding with both a MAb specific for GNA1870 v.1 protein (column 4) and polyclonal anti-GNA1870 antiserum prepared in mice immunized with v.1, v.2, and v.3 proteins (columns 5 and 6). In contrast, there was no significant binding of these antibodies with the mutant RM1090GNA1870 strain or with the wt RM1090 strain, which naturally expresses low levels of a GNA1870 v.2 protein. Thus, in the RM1090 strain overexpressing GNA1870 v.1 protein, GNA1870 is anchored in the OMV and is surface exposed.
There was no evidence of complement deposition when the bacterial cells of either test strain were incubated with the human complement source alone or with complement and a 1 : 40 dilution of negative control serum from mice immunized with aluminum phosphate (black areas of panels in column 1). Similarly, there was no detectable C3 deposition with heat-inactivated complement plus 5 g/mL of a positive control mouse MAb to GNA1870 v.1 protein (JAR 3) (black areas of panels in column 2). In contrast, the addition of active complement to 1 g/mL anti-GNA1870 MAb (white area under the curve of panels in column 2) or to 25 g/mL of a positive control group B anticapsular MAb (white area under the curve of panels in column 1) elicited deposition of C3 on the bacterial surface of both test strains, as evidenced by increases in the percentages of bacteria showing strong immunofluorescence.
The panels in columns 36 show the effect of adding complement to dilutions of serum pools from groups of mice immunized with different vaccines. The addition of complement to a 1 : 100 dilution of serum from mice immunized with the rGNA1870 protein vaccine (column 3) or the OMV vaccine prepared from the wt RM1090 strain (column 4) or the OMV vaccine prepared from strain RM1090GNA1870 mixed with rGNA1870 protein (column 5) did not activate C3 deposition. In contrast, dilutions of 1 : 100 or 1 : 400 of serum samples from mice immunized with the OMV vaccine prepared from the RM1090 strain overexpressing GNA1870 v.1 protein activated strong C3 deposition against both test strains (column 6).
Role of anti-GNA1870 antibody in functional activity.
The higher functional activity of the antiserum from mice immunized with the OMV vaccine prepared from the RM1090 strain overexpressing GNA1870 v.1 protein could have resulted from antibodies elicited by antigens other than GNA1870. To investigate this possibility, we used an anti-GNA1870 affinity column to absorb a serum pool from mice immunized with the OMV vaccine prepared from the RM1090 strain overexpressing GNA1870 v.1 protein. By ELISA, 99% of the anti-GNA1870 antibodies were removed, and the absorbed antiserum lost all ability to activate human C3 deposition on N. meningitidis strain NZ98/254 (table 3). In contrast, there was no effect on C3 deposition by absorbing the serum pool on an anti-NadA affinity column, which served as a negative control. Table 3 also summarizes the bactericidal titers of the absorbed serum pools as measured against strains Cu385 and M6190. Absorption of the anti-GNA1870 antibodies completely removed the bactericidal activity against strain Cu385 but had no significant effect on the titers against strain M6190. This latter result was expected, because, as noted above, strain M6190 expresses a PorA with a homologous serosubtype to that of the RM1090 vaccine strain, and the bactericidal anti-PorA antibodies would not be removed by absorption with the GNA1870 or NadA affinity columns. In other experiments, we tested bactericidal activity against a PorA-deficient mutant of strain M6190 that was prepared by selection with complement and anti-PorA P1.2 antibody. The results are summarized in table 4. In contrast to the results with the wt M6190 parent strain, the PorA-deficient mutant was susceptible to complement-mediated bactericidal activity of antibodies elicited by the rGNA1870 protein vaccine (geometric mean titer, 1 : 1000) or the vaccine prepared from the RM1090 strain overexpressing GNA1870 v.1 protein (geometric mean titer, 1 : 775) but not by antibodies elicited by the OMV vaccine prepared from the wt RM1090 strain (geometric mean titer, <1 : 10; see Discussion).
DISCUSSION
In previous studies, mice immunized with a rGNA1870 protein vaccine developed high serum bactericidal antibody responses against most strains expressing GNA1870 proteins of the homologous variant group [10, 11]. However, a number of strains that expressed subvariants of the respective GNA1870 protein were resistant to anti-GNA1870 complement-mediated bactericidal activity.
In an effort to enhance protective activity, we investigated the immunogenicity of an OMV vaccine prepared from a N. meningitidis strain that was genetically engineered to overexpress GNA1870 v.1 protein. The serum anti-GNA1870 antibodies elicited by this modified GNA1870-OMV vaccine had a greater bactericidal response against N. meningitidis strains expressing subvariants of the GNA1870 v.1 protein than those elicited by the rGNA1870 protein vaccine or the OMV vaccine prepared from the wt RM1090 strain or a mixture of rGNA1870 protein and the OMV vaccine prepared from the wt RM1090 strain. The antibodies elicited by the OMV vaccine prepared from the RM1090 strain overexpressing GNA1870 v.1 protein also gave greater C3 deposition on the surface of strains NZ98/254 and M1390 (figure 6, column 6) and greater passive protection against bacteremia in infant rats challenged with strain NZ98/254 (figure 7). The ability of antibodies to confer passive protection in the absence of bactericidal activity in an animal model has been reported elsewhere [10, 22], and it likely results from opsonization [24, 25].
O'Dwyer et al. [26] recently described the results of immunizing mice with an OMV vaccine prepared from a commensal N. flavescens strain engineered to express another conserved meningococcal vaccine candidate, NspA. N. flavescens does not naturally express PorA or NspA. The immunized mice developed NspA-specific serum opsonophagocytic activity. After absorption of antibodies against wt N. flavescens OMV vaccine, the residual anti-NspA antibodies conferred passive protection to mice given a lethal challenge of an encapsulated N. meningitidis strain. However, in contrast to the results with the modified GNA1870-OMV vaccine investigated in the present study, the recombinant NspAN. flavescens OMV vaccine did not elicit serum bactericidal antibody responses.
Defining the mechanisms by which the modified GNA1870-OMV vaccine elicits serum antibodies that have broader functional activity than those elicited by the rGNA1870 protein vaccine will require further study. OMV vaccines are complex mixtures of antigens and would be expected to elicit antibodies directed against a number of antigenic targets. Our serum absorption studies demonstrated that the ability of the OMV vaccine prepared from RM1090 overexpressing GNA1870 v.1 protein to elicit serum bactericidal antibodies to strain Cu385 and to activate C3 deposition on strain NZ98/254 was a result of anti-GNA1870 antibodies (table 3). Recently, the region of GNA1870 comprising aa 100254 has been shown to be important in eliciting bactericidal responses [27]. Conceivably, expression of the native GNA1870 protein anchored within the OMV permitted better expression of certain conformational epitopes in this region than in the corresponding portion of the nonlipidated recombinant protein vaccine.
An important second property of the modified GNA1870-OMV vaccine is its ability to elicit serosubtype-specific bactericidal activity against PorA. This result was expected on the basis of results from previous studies that identified PorA as an important antigenic target of antibodies elicited by OMV vaccines [5, 28] and was confirmed in the present study by the lack of bactericidal activity in serum samples from mice immunized with a conventional OMV vaccine when they were tested against a mutant strain of M6190 deficient in PorA expression (table 4). Unexpectedly, the PorA-deficient mutant strain was susceptible to bactericidal activity by antibodies elicited by the rGNA1870 protein, whereas the parent PorA-sufficient wt strain was resistant (table 4). By flow cytometry and Western blot, the encapsulated PorA-deficient mutant strain had more surface-accessible GNA1870 than the PorA-sufficient wt strain (authors' unpublished data), which likely accounted for the susceptibility of the PorA-deficient mutant strain to bactericidal activity by anti-GNA1870 antibodies. Thus, another unanticipated advantage of the modified GNA1870-OMV vaccine over a conventional OMV vaccine is the ability of the former to decrease the likelihood of immune selection of PorA-deficient escape mutants [2933], which can decrease the susceptibility of strains to the bactericidal activity of anti-PorA antibodies [34].
Acknowledgments
We thank Zyde Raad, Patricia Zuno-Mitchell, and Maggie Ching, for excellent technical assistance; Gregory Moe and Alexander Lucas (Children's Hospital Oakland Research Institute), for invaluable critical comments on the manuscript; and Jo-Anne Dillon (University of Saskatchewan, Saskatoon, Saskatchewan, Canada), for the pFP12 shuttle vector used for the expression of GNA1870.
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