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*
Chiron Vaccines, Emeryville, CA 94608;
Chiron Vaccines, Siena, Italy; and
Children’s Hospital Oakland Research Institute, Oakland, CA 94609
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(2
8) sialic
acid, has been attributed to immunologic tolerance induced by prenatal
exposure to host polysialyated glycoproteins. Substitution of
N-propionyl (N-Pr) for
N-acetyl groups on the meningococcal B polysaccharide, and
conjugation of the resulting polysaccharide to a protein carrier, have
been reported to yield a conjugate vaccine that elicits protective Abs
with minimal autoantibody activity. To characterize the protective
epitopes on the derivatized polysaccharide, we isolated 30
anti-N-Pr meningococcal B polysaccharide mAbs. These
Abs were heterogeneous with respect to complement-mediated bactericidal
activity, fine antigenic specificity, and autoantibody activity as
defined by binding to the neuroblastoma cell line, CHP-134, which
expresses long-chain (28)-linked polysialic acid. Eighteen of the
Abs could activate complement-mediated bacteriolysis. Seven of these 18
Abs cross-reacted with N-acetyl meningococcal B
polysaccharide by ELISA and had strong autoantibody activity. Thus,
N-Pr meningococcal B polysaccharide conjugate vaccine has
the potential to elicit autoantibodies. However, 7 of the 18
bactericidal mAbs had no detectable autoantibody activity. These Abs
may be useful for the identification of molecular mimetics capable of
eliciting protective Abs specific to the bacteria, without the risk of
evoking autoimmune disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polysaccharide vaccines are available for prevention of disease caused by serogroup A, C, Y, and W135 strains. These vaccines are used infrequently because plain polysaccharide vaccines tend to be poor immunogens in infants and young children, the age groups with the highest incidences of meningococcal disease. Efforts are underway to develop more effective meningococcal A and C vaccines by conjugating the polysaccharides to protein carriers using approaches that proved highly effective for Haemophilus type b conjugate vaccines (4, 5, 6). However, no vaccine is currently available for prevention of disease caused by serogroup B strains, which account for 50% or more of invasive isolates in Europe and North America (2, 3). Further, as described below, the conventional conjugate vaccine approach has not proven promising for prevention of disease caused by meningococcal serogroup B strains.
The group B polysaccharide capsule is composed of a homolinear polymer
of (28) N-acetyl
(N-Ac)3
neuraminic acid (polysialic acid). This capsule is an important
virulence determinant: for example, mutants deficient in capsular
expression are serum sensitive and nonpathogenic. Evidence also
indicates that serum Ab to the group B polysaccharide confers
protection against disease by activating complement-mediated
bacteriolysis and/or opsonization (4, 7). Efforts to employ the
meningococcal B capsular polysaccharide as a vaccine component have
been hampered by its very poor immunogenicity, even when conjugated to
a carrier protein (8). The poor immunogenicity is attributed to
immunologic tolerance induced by fetal exposure to cross-reactive
polysialylated glycoproteins, such as the neural cell adhesion
molecule, expressed in a variety of host tissues (9, 10). Jennings,
Roy, and Gamian (11); Jennings, Gamian, and Ashton (12); and Jennings
(13) have pursued an innovative strategy for overcoming immunologic
tolerance to this polysaccharide by substitution of
N-propionyl (N-Pr) for N-Ac
groups, and conjugation of the resulting N-Pr meningococcal
B polysaccharide to a protein carrier. The resulting conjugate vaccine
is reported to be highly immunogenic in experimental animals, including
nonhuman primates (14), eliciting IgG Abs that activate
complement-mediated bacteriolysis and passively protect experimental
animals challenged with N. meningitidis group B. These Abs
also appear to have less autoantibody activity than Abs raised to the
native meningococcal B polysaccharide.
One potential concern about the use of the Jennings N-Pr meningococcal B conjugate vaccine is that a subset of the Abs elicited by this vaccine have anti-host Ab activity (see, for example, Fusco et al. (14) and Häyrinen et al. (15)). Although there is no direct evidence that such autoantibodies are deleterious to the host, it is difficult to eliminate this possibility. For example, Abs capable of binding to host polysialic acid could evoke autoimmune inflammatory disease, or could cross the placenta and adversely affect neurologic development in the fetus (16, 17).
The ideal meningococcal B vaccine should elicit protective Abs but pose
a minimal risk of evoking autoantibodies. Given that it is both
unlikely and difficult to prove that meningococcal B
polysaccharide-based vaccines do not elicit autoantibodies,
alternatives must be considered. One approach is to use noncapsular Ags
(reviewed in 4 . However, because of strain heterogeneity, and
temporal and geographic shifts in the predominant strains causing
disease, this approach requires the use of Ags from multiple
meningococcal B strains. Another approach would be to use molecular
mimetics, such as peptides, of meningococcal B polysaccharide epitopes
that are unique to the pathogen. This approach has been used
successfully by Westerink et al. to induce Abs to meningococcal C
polysaccharide ((29) N-Ac neuraminic acid (18)).
The purpose of the present study was to prepare a panel of murine mAbs to N-Pr meningococcal B polysaccharide, with the goal of identifying Abs that react with epitopes that are specific for meningococcal B organisms and that show no cross-reactivity or minimal cross-reactivity with host polysialic acid. Such Abs could be useful for identifying molecular mimetics for inclusion in a meningococcal B vaccine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N-Ac meningococcal B polysaccharide was purified from the supernatant of broth cultures of Neisseria meningitidis group B (strain B11), as described by Bartoloni et al. (19). For preparation of the derivatized polysaccharide, the N-Ac groups of N-Ac meningococcal B polysaccharide were removed by heating a solution of the polysaccharide (13.3–16.6 mg/ml) in 2 M NaOH to 110°C for 6 h in the presence of sodium borohydride (1 mg/ml), as described by Jennings, Roy, and Michon (20). The deacetylated polysaccharide was exhaustively dialyzed in saturated sodium bicarbonate buffer, then stirred with an excess of propionic anhydride for 12 h at ambient temperature. The solution was exhaustively dialyzed in water and the N-propionylated meningococcal B polysaccharide was recovered by lyophilization.
For preparation of the conjugate vaccine, the N-Pr meningococcal B polysaccharide was partially hydrolyzed in 10 mM sodium acetate, pH 5.5, at 50°C for 2 h. The resulting mixture of oligosaccharides was fractionated on a Q-Sepharose FF column (Pharmacia, Uppsala, Sweden) (approximately 10–15 mg of sialic acid per ml of gel) by using a stepwise gradient elution consisting of low-salt (100 mM sodium chloride) and high-salt (500 mM sodium chloride) solutions in 5 mM sodium acetate buffer, pH 6.5. As determined by analytical chromatography on a MonoQ column (Pharmacia) small oligosaccharides with a degree of polymerization (Dp) ranging from 2 to 6 were eluted with low-salt buffer, and intermediate-sized oligosaccharides (Dp range 7 to 20, average Dp = 13) were eluted with high-salt buffer.
A terminal aldehyde group was generated at the nonreducing end of the intermediate-sized oligosaccharides by an oxidation reaction using 100 mM sodium periodate for 15 to 30 min at ambient temperature in the dark. Excess ethylene glycol was used to quench the reaction and the product was desalted on a Sephadex G-25 column (Pharmacia) in water and lyophilized.
The oligosaccharide-protein conjugate was prepared by stirring a mixture of terminal aldehyde-containing N-Pr meningococcal B oligosaccharide with tetanus toxoid (molar ratio of 200:1, respectively) in 0.75 M potassium phosphate buffer, pH 9.0, with sodium cyanoborohydride (40 mg/ml) for 1 day at 40°C, then 2 days at ambient temperature. The resultant N-Pr meningococcal B oligosaccharide-tetanus toxoid conjugate was purified by gel permeation chromatography on Sephadex G-100 (Pharmacia) using PBS as the eluting buffer. Sialic acid and protein content of the conjugate vaccine were measured by the Svennerholm resorcinol reaction (21) and Lowry et al. (22) assays, respectively. On a weight basis, the final saccharide to protein ratio of the conjugate was determined to be 0.15. A Western blot employing both anti-tetanus toxoid-specific and anti-N-Pr meningococcal B-specific antisera was used to confirm that the protein and saccharide contained in the final product were covalently linked (data not shown).
Preparation of mAbs
Four- to six-week-old female CD1 mice, obtained from Charles River Italia S.P.A. (Calco, Italy), were vaccinated with an N-Pr meningococcal B-tetanus toxoid conjugate vaccine prepared as described above. The 0.1-ml dose contained 2.5 µg of sialic acid and 16 µg of tetanus toxoid protein, and was administered i.p. together with CFA. In individual experiments, two or three injections were given, each separated by 1 mo, with the final dose given without adjuvant. Three days later, animals were sacrificed and their spleen cells were fused with myeloma cells P3X63-Ag8.653 at a ratio of 5 spleen cells to 1 myeloma cell. After 2 wk of incubation in HAT-selective medium, hybridoma supernatants were screened for Ab-binding activity to N-Pr meningococcal B polysaccharide by an ELISA described below. Specificity of Ab binding was demonstrated by inhibition with high m.w. soluble N-Pr meningococcal B polysaccharide (25 µg/ml). Hybridomas secreting reactive Ab were cloned by limiting dilution, and then expanded and frozen for subsequent use in tissue culture, or for ascites production in BALB/c mice.
A total of 46 cell lines were prepared from three fusions. Of the 46
cell lines, the Abs from 30 were characterized. The isotypes of the
mAbs were determined using a solid-phase Ab capture ELISA, performed as
described below, and alkaline phosphatase-conjugated polyclonal Ab
specific for each of the mouse IgG subclasses, IgM, IgA, and 
and
light chains (Zymed, South San Francisco, CA). Among the 30 Abs,
one was an IgM and the remaining 29 were IgG (3 IgG1, 3 IgG2a, 15
IgG2b, and 8 IgG3). All of the Abs had light chains. The isotypes
and other selected characteristics of these Abs are summarized in Table I
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mAbs were partially purified from tissue culture fluid or mouse ascites by ammonium sulfate fractionation and exhaustive dialysis. The Abs were quantified by an ELISA capture assay performed as previously described (7). In the present study, the "capture" Ab consisted of affinity-purified rabbit anti-mouse IgG, IgM, and IgA (Zymed) diluted to 1 µg/ml in PBS (pH 7.4) and adsorbed to microtiter plates (Immulon 2; Dynatech Laboratories, Chantilly, VA) by incubation overnight at 4°C. Bound mouse mAb was detected using alkaline phosphatase-conjugated rabbit anti-murine IgG, IgM, and IgA polyclonal Abs (Zymed). The Ig concentration of the partially purified mAb preparations were calculated from standard curves prepared from titrations of commercial murine IgG1, IgG2b, IgG3, and IgM standards (Southern Biotechnology Associates, Birmingham, AL) at concentrations ranging from 4 ng/ml to 500 ng/ml. The concentration of selected mAbs also was measured by a commercial radial immunodiffusion assay performed according to the manufacturer’s instruction (The Binding Site Limited, Birmingham, U.K.).
Binding to N-Pr meningococcal B polysaccharide
A solid-phase ELISA was used to assess the binding of the mAbs to biotinylated N-Pr meningococcal B polysaccharide bound to avidin-coated microtiter wells, using a procedure previously described (23). Specificity of antigenic binding was confirmed by demonstration of inhibition of binding in the presence of an excess of soluble N-Pr meningococcal B polysaccharide (25 µg/ml). The ability of N-Pr meningococcal B oligomers (Dp range of 3 to 6; average = 3.8) to inhibit binding also was determined by a competitive solid-phase ELISA performed as described above, except that the mAbs were prediluted to concentrations to yield an OD of 0.5 to 1.0 after approximately 30 min of incubation with substrate. The mAbs were added to wells of replica plates, each containing a final inhibitor concentration of 25 µg/ml of high m.w. N-Pr meningococcal B polysaccharide, or oligomers of N-Pr meningococcal B polysaccharide, or buffer alone. The plates were covered and incubated overnight at 4°C. The following day, the contents of the wells were aspirated, and the plates washed five times with washing buffer. Bound Ab was detected by alkaline phosphatase-conjugated rabbit anti-murine IgG, IgM, and IgA polyclonal Ab. Percent inhibition was calculated by comparing absorbance values at 405 nm after 30 min of incubation with substrate in wells that contained the inhibitor and the corresponding wells without inhibitor.
Cross-reactivity with N-Ac meningococcal B polysaccharide
The mAbs were evaluated for their ability to cross-react with the N-Ac meningococcal B polysaccharide as demonstrated by direct binding in a solid-phase ELISA. The method used was similar to that described above for the N-Pr meningococcal B polysaccharide ELISA, except that N-Ac meningococcal B polysaccharide coupled to adipic acid dihydrazide was used as the solid-phase Ag instead of biotinylated N-Pr meningococcal B polysaccharide.
Autoreactivity
The 30 mAbs were evaluated for autoreactivity to host polysialic acid expressed by the human neuroblastoma cell line CHP-134 (24) using flow cytometric detection of indirect immunofluorescence. Cells from nearly confluent cultures were collected in 50-ml centrifuge tubes and centrifuged (1000 x g). After the supernatant was decanted, 5 ml of blocking buffer (PBS containing 1% (w/v) BSA and 0.1% (w/v) sodium azide) was added to resuspend the cells. The cells were then counted in a hemacytometer and divided into two equal aliquots. One aliquot was incubated for 2 h at ambient temperature with exoneuraminidase (10 U/108 cells; Sigma, St. Louis, MO); the other aliquot was treated identically but without enzyme. The exoneuraminidase treatment cleaves the surface polysialic acid and provides a control in the assay for specificity of Ab binding to polysialic acid. After incubation, the cells from each aliquot were distributed among individual reaction tubes so that each tube contained 106 cells. To wash the cells, 2 ml of blocking buffer were added to each reaction tube, the tubes were centrifuged (208 x g) for 6 min at 20°C, and the supernatant was removed by aspiration. The washed cells were incubated for 2 h on ice in a total volume of 200 µl with either no Ab, or the indicated concentration (usually 10 or 100 µg/ml) of the test or control Ab.
At the end of the 2 h, blocking buffer (2 ml) was added to each reaction tube, and the tubes were centrifuged as before. Following centrifugation, the supernatant was aspirated and the cells incubated for 1 h at ambient temperature with 150 µl of FITC-conjugated F(ab')2 fragment goat anti-mouse IgG (4 µg/ml; Jackson ImmunoResearch, West Grove, PA). After washing with blocking buffer, 400 µl of 0.25% formaldehyde in PBS buffer was added to the cells, and the cells were analyzed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA).
Control Abs in the assay included: 1) an IgG mAb of irrelevant specificity (VIG10, as a negative control); 2) an IgM anti-polysialic acid mAb (2-1B (25), as a positive control); and 3) an anti-CD56 mAb specific for the protein backbone of neuronal cell adhesion molecule (NCAM; Immunotech, Marseille, France).
Binding of mAbs to the bacterial surface
The ability of the anti-N-Pr meningococcal B
polysaccharide mAbs to bind to the surface of pathogenic strains of
N. meningitidis group B was determined using a flow
cytometric detection of indirect immunofluorescence assay. Two fully
encapsulated meningococcal B test organisms were used, strain 8047 (the
strain used to measure bactericidal activity, see below) and strain
NMB. A third, unencapsulated strain, M7, which is a
transposon-containing mutant of NMB (26), was used as a negative
control for specificity of Ab binding to the encapsulated bacteria.
Bacterial cells grown to mid-log phase in Mueller-Hinton broth and
0.25% glucose were harvested and resuspended in blocking buffer at a
density of 
108 cells per ml. The mAbs (concentration of
10 or 100 µg/ml) were then added and the mixture was incubated for
2 h on ice. Following two washes with blocking buffer, the cells
were incubated with FITC-conjugated F(ab')2 fragment goat
anti-mouse IgG (H+L) (Jackson ImmunoResearch), fixed with 0.25%
formaldehyde in PBS buffer, and analyzed by flow cytometry.
Positive control Abs included meningococcal-specific serotyping and subtyping mAbs (MN2C3B and MN16C13F4; Rijksinstituut Voor Volksgezondheid en Mileu, Bilthoven, The Netherlands). The negative control consisted of a mouse IgG mAb (VIG10) of irrelevant specificity.
Complement-dependent bactericidal Ab activity
The bactericidal assay was adapted from the method previously described (7), with the following modifications. The test organism, strain 8047, was grown for approximately 2 h in Mueller-Hinton broth containing 0.25% glucose, which rendered the organism more resistant to complement-mediated bacteriolysis by endogenous "natural" Abs, compared with organisms grown in Mueller-Hinton without supplemental glucose (D. M. Granoff, unpublished observations). In the serum bactericidal assay, several complement sources were tested (e.g., human serum, infant rabbit serum; see Results). The complement source was used at 20% (v/v) in the final reaction mixture, along with serial twofold dilutions of the mAbs in Gey’s buffer containing 1% BSA (instead of barbital buffer as previously described by Mandrell, Azmi, and Granoff (7)). Serum bactericidal titers were defined as the Ab concentration resulting in a 50% decrease in CFU per ml after 60 min of incubation of bacteria in the reaction mixture, compared with control bacteria at the initiation of the incubation period.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A solid-phase ELISA was used to assess the concentration-dependent
binding of the mAbs to N-Pr meningococcal B polysaccharide
in the presence of dilution buffer alone, or dilution buffer containing
25 µg/ml of soluble N-Pr meningococcal B polysaccharide
inhibitor. Figure 1 shows data from
testing four Abs (SEAM 2, 3, 16, and 18). The concentrations of each of
the 30 mAbs sufficient to yield an OD of 0.5 after 30 min of incubation
with substrate ranged from less than 0.01 µg/ml to 25 µg/ml
(Table I
).
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Figure 2 depicts the inhibition of
binding of four anti-N-Pr meningococcal B polysaccharide
mAbs to solid-phase N-Pr meningococcal B polysaccharide by
either 25 µg/ml of soluble high m.w. N-Pr meningococcal B
polysaccharide, or 25 µg/ml of soluble N-Pr meningococcal
B oligosaccharides (Dp < 6; mean = 3.8). There was 75 to
95% inhibition of Ab binding in the presence of the high m.w.
polysaccharide of all the mAbs tested. However, as shown in Figure 2, differences in fine antigenic specificity of the different Abs are
evident from the results of inhibition studies performed with lower
m.w. oligomers. For example, binding of mAbs SEAM 3 and 18 to
solid-phase N-Pr meningococcal B polysaccharide is
completely inhibited by the soluble oligosaccharide, while there is no
significant inhibition (less than 20%) of the binding activity of mAb
SEAM 2 or 16.
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). Some of the
Abs showed strong cross-reactivity, such as mAb SEAM 18, which gave a
positive signal when tested at 0.5 µg/ml. Other Abs, such as mAb SEAM
12, were positive only when tested at 10-fold or higher concentrations.
In contrast, other Abs, such as mAbs SEAM 2 and 3, were negative when
tested at up to 50-fold higher Ab concentrations.
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). Autoreactivity
The ability of the anti-N-Pr meningococcal B
polysaccharide mAbs to cross-react with host polysialic acid was
assessed by indirect fluorescence flow cytometry. In this assay, the
human neuroblastoma cell line CHP-134, which expresses on its surface
long-chain polysialic acid associated with NCAM, serves as a cellular
marker for human polysialic acid Ags. In a typical experiment (Fig. 4), cells incubated without a primary
mAb, or with a control murine mAb having an irrelevant antigenic
specificity, show very little fluorescence (approximately 98% of the
cells have <10 U of fluorescence, panel
A). In contrast, virtually all cells treated with the
anti-N-Ac meningococcal B polysaccharide murine mAb,
2-1B, fluoresce strongly (panel B,
left). This fluorescence is decreased to control
levels when the Ab is incubated with cells that had been pretreated
with neuraminidase (panel B,
right). Similarly, cells treated with anti-CD56
fluoresce strongly (panel C). With
this Ab, the fluorescence is unaffected by pretreatment of the cells
with neuraminidase, since the CD56 determinant is located in the
protein backbone of NCAM and is unaffected by the removal of polysialic
acid with neuraminidase.
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D), and is considered as negative in this assay. In
contrast, mAb SEAM 35 shows strong polysialic acid-specific binding
when tested at 10 or 100 µg/ml (panels
E and F), and is considered positive. A
few anti-N-Pr meningococcal B polysaccharide mAbs show
binding when tested at 100 µg/ml, but appear to be negative when
tested at 10 µg/ml (see, for example, mAb SEAM 12 in Fig. 4, panels G (10 µg/ml) and H (100
µg/ml)). A rare mAb appeared to have weak reactivity that was
unaffected by the pretreatment of the neuroblastoma cell line cells
with neuraminidase (see monoclonal SEAM 7, panel
I). The autoreactivity of such Abs with polysialic
acid was, therefore, considered indeterminate in this assay. The
results of autoantibody activity for each of the 30
anti-N-Pr meningococcal B polysaccharide mAbs are
summarized in Table I.
As shown in Table I, none of the 15 Abs that were negative for binding
to N-Ac meningococcal B polysaccharide by ELISA (i.e., fine
antigenic specificity groups III or IV) was found to bind to polysialic
acid in the CHP-134 assay. In contrast, all 11 Abs showing autoantibody
activity in the CHP-134 assay also showed cross-reactivity with
N-Ac meningococcal B polysaccharide in the ELISA (fine
antigenic specificity groups I and II). There were four Abs in groups I
and II that cross-reacted with N-Ac meningococcal B
polysaccharide by ELISA that were either negative for autoantibody
activity (mAbs SEAM 10, 20, and 21) or were indeterminate (monoclonal
SEAM 16). Whether these results reflect insufficient sensitivity of the
autoantibody assay, or whether these N-Ac meningococcal B
cross-reacting Abs truly lack autoantibody activity to host polysialic
acid, requires additional study.
Bacterial binding assay
The ability of the anti-N-Pr meningococcal B polysaccharide mAbs to bind to the surface of pathogenic strains of N. meningitidis group B also was determined using a flow cytometric detection of indirect immunofluorescence assay. As described in Materials and Methods, two fully encapsulated meningococcal B test organisms were used, strain 8047 (the strain used to measure bactericidal activity, see below) and strain NMB. A third unencapsulated strain, M7, which is a transposon-containing capsular-deficient mutant of NMB, was used as a negative control for specificity of Ab binding to the encapsulated bacteria.
Figure 5 A–G shows
the results from a representative bacterial Ab-binding experiment.
Anti-N-Pr meningococcal B polysaccharide mAbs SEAM 3 and 18
show strong capsular-specific binding to both encapsulated test strains
(panels D and E,
respectively). In contrast, mAbs SEAM 9 and 10 were negative in this
assay (panels F and G,
respectively). As summarized in Table I, 19 of the 30
anti-N-Pr meningococcal B polysaccharide mAbs tested
showed evidence of bacterial binding when tested at 100 µg/ml. For
each of these, no Ab binding was detected with the unencapsulated
mutant. Two additional Abs showed evidence of weak binding to both
encapsulated and nonencapsulated mutant strains (see, for example, mAb
SEAM 7, as depicted in Fig. 5H). Therefore, although
these Abs are polysaccharide specific, based on their inhibition in an
ELISA (Table I and Fig. 1), their specificity of binding with respect
to the bacteria was considered indeterminate. The remaining 9 Abs were
negative in the bacterial binding assay.
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The percent survival of N. meningitidis strain 8047
when incubated with different concentrations of Ab and 20% complement
is shown for four mAbs (Fig. 6). Each Ab
was tested with three different complement sources: infant rabbit serum
pool I, infant rabbit serum pool II, and human agammaglobulinemia
serum. For anti-N-Pr meningococcal B polysaccharide Abs
SEAM 5 (IgG3) and 12 (IgG2a), a similar dose response was observed with
each of the three complement sources. In contrast, mAb SEAM 18 (IgG2b)
required higher Ab concentrations to elicit bacterial killing in the
presence of human complement than were required with either source of
rabbit complement. mAb SEAM 3 (IgG2b) showed effective killing when
tested with one of the two rabbit complement sources, less effective
activity with the second rabbit complement source, and no activity with
the human complement source (despite excellent bacterial binding as
determined by the flow cytometric immunofluorescence assay; Fig. 5).
The ability of each of the mAbs to activate bacteriolysis in the
presence of rabbit complement pool 1 and/or human complement is
reported in Table I. There were examples of mAbs from each of the four
fine antigenic specificity groups that could activate
complement-mediated bacteriolysis.
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), but were able to
activate bacteriolysis despite their apparent low avidity. However,
both of these Abs required relatively high concentrations to elicit
bacteriolysis (Ab concentrations for 50% lysis in the presence of
rabbit complement of 18 and 35 µg/ml vs 1 to 3 µg/ml for more
active Abs such as SEAM 28 or 3). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, there was
extensive heterogeneity in both the isotype distribution as well as
fine antigenic specificities. Differences in fine antigenic specificity
were defined by recognition of long or short epitopes, the latter
defined by the ability of small N-Pr meningococcal B
oligosaccharides (Dp < 6) to inhibit Ab binding to
N-Pr meningococcal B polysaccharide. The mAbs also could be
subdivided by their ability to recognize polysialic acid epitopes, as
indicated by cross-reactivity with N-Ac meningococcal B
polysaccharide in an ELISA (Table I). In addition, the Abs differed
with respect to their relative avidities for binding to N-Pr
meningococcal B polysaccharide. For example, approximately 30%
required high concentrations to yield positive binding in an ELISA (see
for example SEAM 2, Fig. 1), whereas other Abs gave strong positive
signals when tested at concentrations as low as 0.01 µg/ml (SEAM 18,
Fig. 1). The Abs also differed with respect to their ability to
activate complement-mediated bactericidal activity against strains of
N. meningitidis group B, and autoantibody activity to
polysialic acid as assessed by the CHP-134 neuroblastoma cell-binding
assay.
The diverse characteristics of the Abs in this panel (Table I) are
strikingly different from those described recently by Pon et al. (27).
They reported that an N-Pr meningococcal B polysaccharide
conjugate vaccine evoked a narrow immune response dominated by Abs
recognizing a long polysaccharide epitope that did not cross-react with
N-Ac meningococcal B polysaccharide. Further, they found
that only Abs recognizing a long epitope were bactericidal in the
presence of complement. In contrast, most (20 of 30) of the mAbs in our
panel recognized a short epitope. Further, of the 18 mAbs that were
able to mediate bacteriolysis in the presence of complement, 11 were
specific for a short epitope. The basis for the different results in
our study and the previous study by Pon et al. (27) is not clear. They
could relate to the smaller average chain length of saccharide in the
conjugate vaccine used to elicit the Abs in our study (average Dp of 13
vs an estimated 35 to 40 in the conjugate vaccine used in the earlier
study). The different antigenic specificities also could be a result of
the use of different adjuvants in the two studies (CFA vs RIBI
adjuvant), or different mouse strains (CD1 vs BALB/c).
Based on their analysis of anti-N-Pr meningococcal B mAbs, and on earlier studies of polyclonal sera by the same group (28), Pon et al. suggested that N-Pr meningococcal B polysaccharide mimics a unique meningococcal B polysaccharide epitope that is characterized by being "extended" (or long) and possibly involving associated nonpolysaccharide molecules (27, 28). However, in the panel of anti-N-Pr meningococcal B mAbs described here, there was no correlation between any particular fine antigenic specificity (short or long epitopes, N-Ac meningococcal B polysaccharide cross-reactive or noncross-reactive) and the ability of the Ab to activate complement-mediated bactericidal activity. Further, in other studies not presented, some of the bactericidal mAbs recognizing short segments, such as mAbs SEAM 3 and 18, also have been shown to have opsonic activity for N. meningitidis group B organisms in vitro (A. F. M. Verheul, H. Snippe, and B. Bernaissa-Trouw, Utrecht University, The Netherlands, personal communication). The SEAM 18 mAb also has been shown to confer passive protection against meningococcal B bacteremia and meningitis in infant rats (H. Käyhty et al., National Public Health Institute, Helsinki, Finland, personal communication). Taken together, the present data provide proof that there is more than one epitope on N-Pr meningococcal B polysaccharide that can mimic protective epitopes on the bacterial surface of encapsulated N. meningitidis group B, and that these N-Pr meningococcal B polysaccharide epitopes can exist in short or long saccharide segments.
A second important finding of the present study was that 11 of the 18
mAbs that mediated bactericidal activity had autoantibody activity
defined by their cross-reactivity with human polysialic acid. This
group of autoantibodies included representatives from fine antigenic
specificity groups I and II that recognized short or long epitopes,
respectively. Virtually all Abs positive for autoantibody activity
cross-reacted with N-Ac meningococcal B polysaccharide in an
ELISA. This correlation was expected, since the structure of the
meningococcal B polysaccharide and host polysialic acid expressed by
this cell line are reported to be the same (i.e., (28)
N-Ac neuraminic acid (9)). Although the Ab repertoire in
response to N-Pr meningococcal B polysaccharide conjugate
vaccines may be variable in different animal strains given different
vaccine compositions, the present data underscore the potential of
protective epitopes on this derivatized polysaccharide to elicit
autoantibodies. Despite the apparent safety of a prototype
N-Pr meningococcal B conjugate when tested in subhuman
primates (14), the present autoantibody results raise concerns about
the safety of such vaccines for human use.
Finally, for the purpose of developing anti-meningococcal B vaccines based on molecular mimetics, Abs that are bactericidal in the presence of complement, but that do not cross-react with human polysialic acid Ags, are of particular interest for Ag selection. Of the 30 anti-N-Pr meningococcal B polysaccharide mAbs studied, eight Abs (SEAM 1, 2, 3, 5, 7, 8, 16, and 48) exhibited these desired characteristics. A second group of three mAbs (SEAM 12, 18, and 28) were weakly cross-reactive with polysialic acid and may also be useful for identifying molecular mimetics of unique meningococcal B polysaccharide epitopes. This collection of 11 mAbs includes representatives from each of the 4 fine antigenic specificity groups and of 3 isotypes (IgG2a, IgG2b, and IgG3).
Vaccines containing molecular mimetics of N-Pr meningococcal B polysaccharide may be capable of eliciting protective Abs against meningococcal B disease with minimal risk of autoantibody activity. The underlying principle that a mimetic can elicit a protective anti-polysaccharide Ab response was demonstrated by Westerink et al. (18) and Agadjanyan et al. (29). In the studies of Westerink et al., they prepared a vaccine from a small peptide having an amino acid sequence corresponding to a loop segment of a murine anti-idiotypic Ab. The anti-idiotypic Ab had been prepared against an anti-meningococcal C polysaccharide mAb and had been shown to be a mimetic of group C meningococcal polysaccharide (30). The isolated peptide vaccine also was able to elicit an Ab response against meningococcal C polysaccharide, and protected mice against a lethal challenge by serogroup C meningococci. The bactericidal, nonautoreactive N-Pr meningococcal B polysaccharide mAbs described here might be useful for preparing anti-idiotypic Abs and applying this approach to the development of a safe meningococcal B vaccine. Alternatively, the mAbs could be useful in screening combinatorial libraries such as phage display peptide libraries (31) or combinatorial molecule pools to identify mimetics of unique meningococcal B epitopes. These possibilities are currently being investigated.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dan M. Granoff, Chiron Vaccines, 4560 Horton Street, R-311, Emeryville, CA 94608-2916. E-mail address:
3 Abbreviations used in this paper: N-Ac, N-acetyl; N-Pr, N-propionyl; Dp, degree of polymerization; NCAM, neuronal cell adhesion molecule.
Received for publication November 10, 1997. Accepted for publication January 23, 1998.
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28 N-acetyl neuraminic acid, the capsular polysaccharide of Neisseria meningitidis serogroup B. J. Infect. Dis. 172:1279.[Medline]
28 linked polysialosyl units in a neural cell adhesion molecule. Biochem. Biophys. Res. Commun. 112:482.[Medline]
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). Different ranges
for the x-axis in the data are used, wherein mAbs SEAM 3,
SEAM 16, and SEAM 18 are shown at 0.0001 to 1 µg/ml, and mAb SEAM 2
is shown at 0.1 to 100 µg/ml.
); infant rabbit complement II (•); and human complement
(