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Infection and Immunity, April 2000, p. 1871-1878, Vol. 68, No. 4
Department of Laboratory Medicine, University
of California,1 and Center for
Immunochemistry, VA Medical Center,2 San
Francisco, California; Max Planck Institut für Molekulare
Genetik, D-14195 Berlin, Germany3;
Department of Vaccinology, National Institute of Public
Health,4 and Department of
Pharmacognosy, Institute of Pharmacy, University of
Oslo,5 Oslo N-0403, Norway
Received 8 November 1999/Accepted 21 December 1999
The meningococcal PorA protein is considered a promising vaccine
candidate. Although much is understood regarding the structure of PorA
proteins, little is known about the structure-function relationships of
PorA antibodies. The aim of this study was to compare the functional
and molecular characteristics of a human monoclonal antibody (MAb) and
three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs
207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both
bactericidal and opsonophagocytic for P1.7-expressing meningococci,
whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated
neither effector function. Epitope mapping with synthetic peptides
revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ,
which is the same specificity motif that a previous study had
established for SS269 and MN14C11.6. Nucleotide and amino acid sequence
analyses of the variable regions of the four MAbs showed that the SS269
VH region belonged to the VH3 family and was approximately
70% homologous to those of the murine MAbs which were all from the
7183 family, whereas the SS269 VL region belonged to the
V Disseminated meningococcal infection
is a fulminant disease with high morbidity and mortality
(3). Immune defense against this illness depends on
recognition of the bacterial surface by antibody and activation of
complement (32). The current meningococcal vaccine is
composed of the meningococcal capsular polysaccharides of serogroups A,
C, Y, and W135 (7). Although the efficacy of the vaccine in
adults and older children is quite high, the response is age dependent
in younger children, and children younger than 2 years of age respond
poorly (13, 25, 41). In addition, the vaccine does not
include the serogroup B capsular polysaccharide, as it is poorly
immunogenic (68), and therefore vaccination does not protect
against disease due to serogroup B strains that are responsible for the
majority of meningococcal infections in many countries (58).
Thus, the development of a vaccine for protection against serogroup B
infection has focused on outer membrane proteins as alternative targets
(50).
The PorA class 1 outer membrane protein is a major outer membrane porin
protein expressed by almost all meningococcal strains (23, 64,
65). Sequence comparisons of PorA proteins have shown strong
homology among the proteins with the major variation confined to two
discrete regions termed variable region 1 (VR1) and VR2 (42,
62). Based on a two-dimensional topology model, PorA has eight
predicted surface-exposed loops, and VR1 and VR2 are located at the
apices of the two longest loops: loops 1 and 4, respectively
(62). Epitope mapping with murine monoclonal antibodies
(MAbs) and synthetic peptides confirmed the surface exposure of loops 1 and 4 (62) and showed that most epitopes recognized by
serosubtyping MAbs are localized to VR1 and VR2 (42, 43).
PorA is highly immunogenic in humans following infection or
immunization (16, 26, 63), and the specific antibodies
induced predominantly recognize epitopes within VR1 and VR2 as well as exhibit both bactericidal and opsonic functions (1, 39, 45, 52,
53). PorA-specific murine MAbs that bind to epitopes within VR1
and VR2 are bactericidal in vitro and protective in infant rats when
administered passively (51, 55, 56). Thus, PorA protein is
considered to be an important vaccine candidate either alone
(54) or when administered as an outer membrane vesicle conjugated with the Haemophilus influenzae type b capsular
polysaccharide (44).
Molecular epidemiological investigations have revealed that the P1.7
epitope within the VR1 of PorA is one of the most common serosubtype
epitopes expressed by bacteria isolated from cases of meningococcal
disease. The P1.7 epitope is expressed by epidemic strains of serogroup
A meningococci isolated in West Africa and China (4, 64) and
by serogroup B meningococci of the ET-5 complex, which have been
commonly isolated in Europe, North America, and South America for
decades (12). Recently, a hexavalent PorA meningococcal
outer membrane vesicle vaccine was developed which covered more than
80% of the meningococcal PorA subtypes isolated in many countries
(16). Vaccination of adult volunteers with the PorA vaccine
induced bactericidal antibodies that were predominantly directed
against P1.7 VR1 epitopes and to a lesser degree against P1.16 VR2
epitopes (52-54).
In a study of the human immune response to the P1.7 PorA protein,
Delvig et al. reported the development of a human MAb, SS269, which was
derived from the peripheral blood B lymphocytes of a volunteer
immunized with an outer membrane vesicle meningococcal group B vaccine
(19). A peptide analysis of the binding specificity of SS269
revealed that it was specific for an epitope present at the apex of the
P1.7 VR1 loop which overlapped with the epitope recognized by murine
P1.7-specific MAbs A'dam and MN14C11.6 (19). But despite
the recognition of similar linear epitope sequences by the three MAbs
and the fact that SS269 was an immunoglobulin G3 (IgG3) isotype known
to fix complement (18), SS269 did not function in
complement-dependent opsonophagocytosis or bactericidal effector assays
as efficiently as the murine MAbs (19, 53). These findings
suggest that the effector functions of P1.7-specific murine antibodies
may not necessarily emulate those of human antibodies despite the
recognition of similar epitopes.
In this study, we compared the effector function activities, peptide
epitope specificities, and immunoglobulin variable region gene
sequences of human MAb SS269 and three P1.7-specific murine MAbs to
better understand the structure-function relationship of PorA
antibodies. The data show that distinct differences exist in the
functional activities and variable region gene sequences of human and
murine P1.7-specific MAbs despite their recognition of similar
epitopes. In addition, we describe the expression of the Fab fragment
of MAb SS269 with binding function for a meningococcal strain
expressing the P1.7 epitope.
Neisseria meningitidis strains.
Strains 44/76
and 44/76-SL (B:15:P1.7,16:L3,7,9 ET-5), 188/87 (B:15:P1.7,16d ET-5),
29019 (A:4/21:P1.7,10, subgroup V), and 7973 (C:2:P1.2:L3,7) have been
described previously (21, 27, 40, 64). Stock cultures were
maintained in 10% skim milk at Hybridoma cell lines.
The human hybridoma SS269 (IgG3, Bactericidal activity.
The bactericidal activities of the
four MAbs were tested against strain 44/76-SL by using human serum as a
complement source as previously described (1). Briefly, the
assays were performed in microtiter plates with a bacterial inoculum of
approximately 100 CFU per well. MAbs were tested against the test
strain either as purified antibodies or as ascites fluid. When used as
ascites fluid, the concentration of antibody was determined by using a capture enzyme-linked immunosorbent assay (ELISA) assay
(45). Increasing concentrations of MAb were incubated with
the bacteria prior to initiation of the bactericidal reaction with
human serum at a final concentration of 25%. After 30 min at 37°C,
brain heart infusion agar was added, and the plates were incubated
overnight at 37°C in 5% CO2. Colonies were counted, and
the percent kill was calculated by comparing test samples with controls
containing MAb and heat-inactivated human serum. The results are
presented as the lowest concentration of MAb resulting in greater than
50% killing of the strain.
Opsonophagocytic activity.
The opsonophagocytic activities
of the different MAbs were tested against viable strain 44/76-SL by
using human serum as a complement source and human peripheral blood
polymorphonuclear leukocytes (PMN) as effector cells as previously
described (1). The test was performed by using flow
cytometry to measure respiratory burst (RB) by the PMN effector cells
following phagocytosis of the test strain (1). Briefly,
increasing concentrations of MAb determined as described above were
incubated with log-phase-growth meningococci for 30 min at 37°C.
Human serum at a final concentration of 10% was added as a complement
source, and the mixture was further incubated for 10 min at 37°C.
Finally, PMN from a donor heterozygous for the Fc Epitope mapping.
The synthesis of N-terminally
acetylated peptides on pins was performed as described previously
(19). Briefly, the solid-phase synthesis of peptides on
polyethylene rods was carried out using a commercial kit (Cambridge
Research Biochemicals, Cambridge, United Kingdom). Synthetic peptides
on pins composed of 4 to 10 amino acids from the PorA P1.7 loop of
meningococcal strain H44/76 were synthesized to contain all possible
sequences which spanned the NGGAS epitope of MAb SS269 and the ASGQ
epitope of MAb MN14C11.6. The pins were screened by ELISA for
reactivity to MAbs 207,B-4 and 208,D-5, and the assay was repeated
twice for each MAb. Cell culture supernatants of each MAb were diluted
1:1,000 in phosphate-buffered saline (PBS) containing 1.0% bovine
serum albumin (BSA) and incubated with the pins for 90 min. MAb binding
to peptide sequences was detected by incubation of the pins for 90 min
in a 1:1,500 dilution of affinity-purified horseradish
peroxidase-conjugated goat anti-murine IgG (H+L; Bio-Rad, Richmond,
Calif.) followed by azino-3-ethylbenzthiazodinsulfonate (Sigma, St.
Louis, Mo.) substrate development. Control peptides reacting with a MAb
supplied by the manufacturer were included to verify peptide coupling
during synthesis.
PCR primers.
PCR primers for amplifying the Fab fragments of
human immunoglobulin genes and mouse immunoglobulin genes were modified
from those used by Chazenbalk et al. (14) and by
Kettleborough et al. (34), respectively. Recognition
sequences of restriction enzymes XhoI, SpeI,
XbaI, or SacI were integrated into different primers to facilitate the cloning procedure outlined below. All primers
that resulted in PCR products are listed in Table
1.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Functional Activities and Immunoglobulin Variable
Regions of Human and Murine Monoclonal Antibodies Specific for the P1.7
PorA Protein Loop of Neisseria meningitidis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1-b family and was less than 40% homologous to those of the murine
MAbs which were all members of the V
1 family. The Fab fragment of
SS269 was cloned and expressed in Escherichia coli and was
shown by enzyme-linked immunosorbent assay analyses to bind as well as
intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We
conclude that distinct differences exist in the effector function
activities and variable region gene sequences of human and murine
P1.7-specific MAbs despite their recognition of similar epitopes.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C. Organisms were cultivated on
gonococcal agar base (Difco, Detroit, Mich.) containing 2% IsoVitaleX
(Becton Dickinson, Mountain View, Calif.) at 37°C in 5%
CO2.
light chain) (19) and three BALB/c murine hybridomas,
207,B-4 (IgG2a, light chain), 208,D-5 (IgA, light chain), and
MN14C11.6 (IgG2a, light chain) (2), were selected for
this study. The four hybridomas produce MAbs that recognize epitopes
within the P1.7 loop of the PorA protein. Hybridomas 207,B-4 and
208,D-5 were prepared by immunizing mice with outer membrane vesicles
from strain 188/87. The cell line MN14C11.6 was obtained from M. Maiden
and I. Feavers of the National Biological Standards Board, Potters Bar,
United Kingdom.
RIILRHR
allotype (66) which had been primed with dihydrorhodamine
123 (Molecular Probes, Eugene, Oreg.) were incubated with the opsonized bacteria at an effector/target ratio of 1:25 for 10 min at 37°C, after which RB was analyzed by flow cytometry. The results are presented as the lowest concentration of MAb resulting in RB in greater
than 50% of the PMNs.
TABLE 1.
Oligonucleotide sequences of
PCR primers
Immunoglobulin gene cloning and in vitro expression. Total RNA was prepared from approximately 106 hybridoma cells of SS269, 207,B-4, 208,D-5, and MN14C11.6 by using an acid guanidinium thiocyanate-phenol-chloroform method as described by the manufacturer (TriPure Reagent; Boehringer Mannheim, Indianapolis, Ind.). Five micrograms of total RNA was used for each first-strand cDNA synthesis reaction using the Superscript Preamplification System (Gibco BRL, Gaithersburg, Md.). The cDNAs coding for the heavy and light chains were then amplified by using the sets of primers listed in Table 1. Each PCR reaction was performed in a 50-µl volume containing 2.5 U of Extend Long Template DNA polymerase (Boehringer Mannheim) and AmpliWax PCR Gem 50 according to the Hot-Start protocol of the manufacturer (Perkin-Elmer, Norwalk, Conn.). PCR amplifications were done according to the following protocol: denaturation for 3 min at 94°C; 40 cycles of 40 s at 92°C, 40 s at 55°C, and 1.5 min at 72°C; and a final extension time of 5 min at 72°C.
Two independent PCR products from each heavy and light chain derived from the four MAbs were selected for the subsequent cloning and sequencing procedures. Except for the heavy chain of MN14C11.6, the heavy- and light-chain PCR products were purified with QiaQuick PCR Purification Kit (Qiagen, Valencia, Calif.), double digested with either XhoI-SpeI or XbaI-SacI (Boehringer Mannheim), respectively, separated by agarose gel electrophoresis, and extracted from the gel with the QiaQuick Gel Extraction Kit (Qiagen). The products were then ligated into the corresponding site of the phagemid vector pComb3 (kindly provided by C. F. Barbas III, Scripps Research Institute, La Jolla, Calif.). Each ligation reaction was incubated overnight at 16°C in a 20-µl volume containing 1 U of T4 ligase (Boehringer Mannheim), and approximately 0.25 µg of PCR product and 0.25 µg of pComb3 vector. The ligated DNA molecules (2 µl each) were then transformed into E. coli XL-1 Blue MRF' using an Electroporator II (Invitrogen, Carlsbad, Calif.). After an overnight incubation of the bacteria on Luria-Bertani agar plates supplemented with carbenicillin (20 µg/ml) and tetracycline (10 µl/ml), plasmids were prepared from single colonies with a Qiaprep Spin Miniprep Kit (Qiagen) and examined by agarose gel electrophoresis. The cloned inserts were then sequenced with primers pComb3-1 (GGTGGCGGCCGCAAATTCTATTTC) for the variable region of the heavy chain, pComb3-1013rev (GGCGACTAGCTAGTTTAGAATTCG) for the CH1 region of the heavy chain, pComb3-1013 (CGAATTCTAAACTAGCTAGTCGCC) for the variable region of the light chain, and pComb3-1166rev (TCACTATAGGGCGAATTGGGTACC) for the CL region of the light chain at the Biomolecular Research Center, University of California at San Francisco. The heavy-chain PCR product of MN14C11.6 was ligated into plasmid pCRII with a TA Cloning Kit (Invitrogen) and sequenced with universal primers of the Sp6 promoter and the T7 promoter. Heavy-chain and light-chain gene fragments of the human hybridoma cell line SS269 were first cloned into separate pComb3 vectors and, after insertion of both gene fragments was confirmed by DNA sequencing, the heavy-chain fragment was excised and religated into the vector containing the light chain. This vector was then converted from a phage-display form to a soluble Fab producing form by removing the nucleotide sequence encoding the phage protein PIII from the vector followed by self-ligation. Soluble Fab was prepared from the cell pellet of a 10-ml overnight growth of E. coli XL-1 Blue MRF' by repeatedly freezing (in a dry ice-ethanol bath) and thawing (in a 37°C water bath) the bacteria four times as previously described (8). Following centrifugation, the supernatant containing soluble Fab molecules was stored at70°C prior to further characterization.
Whole-cell ELISA. Bacteria grown overnight on plates were suspended to an optical density at 650 nm of 0.1 in PBS as described previously (31). Aliquots of 75 µl of the suspension were dispensed into wells of microtiter plates, and the plates were dried overnight. After a washing with PBS, nonspecific binding sites were blocked with PBS containing 0.1% BSA for 1 h. Wells were washed and incubated for 1 h with either a 1:10 dilution of the cell culture supernatant of MAb SS269 or a 1:10 dilution of the supernatant of an E. coli lysate containing the Fab fragment of MAb SS269. The binding of the MAb or its Fab fragment was detected with affinity-purified alkaline phosphatase-conjugated goat anti-human lambda chain antibody (Sigma) followed by p-nitrophenyl phosphate (Sigma) substrate development. Nonspecific absorbance was determined by reacting control wells with blocking solution in the absence of bacteria.
DNA and amino acid sequence analyses. The DNA and protein sequence analysis software Lasergene (DNAStar, Madison, Wis.) was used for sequence editing and basic sequence analyses. The specific sequence domains of the immunoglobulin genes were defined and the domains of the different immunoglobulin genes were compared by using the computer program SAW (University of Alabama at Birmingham). Human immunoglobulin germ line gene comparisons were performed by using the V BASE database (http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) and comparisons of murine germ line genes were performed by using DNAPLOT (http://www.genetik.uni-koeln.de/dnaplot). Similarities between germ line genes and the immunoglobulin gene sequences were calculated with MegAlign of Lasergene. Hydrophilicity structure predictions of MAb heavy chains were determined by using GPMAW protein analysis software (Lighthouse data, Odense, Denmark) based on the values of Kyte and Doolittle (37).
Nucleotide sequence accession numbers. The nucleotide sequences of the MAb variable region genes have been deposited in GenBank under accession numbers AF191789 (207,B-4 VH), AF191790 (207,B-4 VL), AF191791 (208,D-5 VH), AF191792 (208,D-5 VL), AF191793 (MN14C11.6 VH), AF191794 (MN14C11.6 VL), AF191092 (SS269 VH), and AF191795 (SS269 VL).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bactericidal activity of the P1.7 MAbs.
The bactericidal
activities of human MAb SS269 and murine MAbs 207,B-4, 208,D-5, and
MN14C11.6 were tested against strain 44/76-SL by using human serum as
the complement source. As shown in Table
2, no bactericidal killing of strain
44/76-SL was detected for either human IgG3 SS269 or mouse IgA 208,D-5
at concentrations of the MAbs that exceeded 400 µg/ml. In contrast,
both mouse IgG2a 207,B-4 and IgG2a MN14C11.6 showed high bactericidal
activity. The lowest concentrations of the two murine MAbs resulting in greater than 50% killing of the test strain were found to be in the
range of 0.5 to 1.0 µg/ml (Table 2).
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Opsonophagocytic activity of the P1.7 MAbs.
The opsonic
function of the MAbs was measured with PMN as the effector cells and
human serum as the complement source. The lowest concentrations of the
four MAbs showing opsonic activity against strain 44/76-SL were
determined as RB in more than 50% of PMN based on flow cytometric
analyses. As shown in Table 2, the results paralleled those of the
bactericidal assays in that SS269 and 208,D-5 showed little or no
detectable opsonic activity, whereas 207,B-4 and MN14C11.6 were opsonic
for the test strain at concentrations of 1.1 and 0.2 µg/ml,
respectively. The dose-response curves of the opsonic activities showed
that the curve of SS269 function was considerably less steep than those
for 207,B-4 and MN14C11.6 and that SS269 did not induce RB in 50% of
the PMNs at a concentration of 400 µg/ml (Fig.
1). In contrast, 207,B-4 and MN14C11.6
demonstrated high opsonophagocytic activity with >80% of PMNs
exhibiting measurable RB at a concentration of the MAbs of <10
µg/ml. In addition, MAb 208,D-5, the IgA isotype, was negative for
opsonic potential at a concentration of 100 µg/ml.
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Linear epitope mapping.
We next investigated the epitope
specificities of the MAbs for the P1.7 VR1 region using synthetic
peptide analyses. The epitope specificities of two of the MAbs, SS269
and MN14C11.6, had been determined previously (19).
Synthetic peptides on pins composed of 4 to 10 amino acids from the
P1.7 loop of meningococcal strain H44/76 were synthesized to contain
all possible sequences which spanned the NGGAS epitope of MAb SS269 and
the ASGQ epitope of MAb MN14C11.6 and were used to map the epitope
specificities of murine MAbs 207,B-4 and 208,D-5. As shown in Fig.
2, MAb 207,B-4 bound most strongly to
peptides containing the sequence ASGQV and bound less efficiently to
peptides which contained only the sequence ASGQ (summarized as ASGQv).
In contrast, MAb 208,D-5 bound strongly to ASGQ, and further addition
of amino acids found in the P1.7 sequence did not influence its binding
efficiency. Neither MAb bound to peptide sequences which lacked the
glutamine (Q) residue. These results have been summarized in Table
3.
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Immunoglobulin variable region gene analyses.
We next cloned
the variable region genes for the VH and VL
regions of the four MAbs and determined their nucleotide sequences. The
deduced amino acid sequences were aligned and are shown in Fig.
3. All sequences were unique based on a
search of the GenBank database. The results of the gene family
assignments of the variable region gene sequences are summarized in
Table 3.
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gene was assigned to the V1-b family and was most
similar to germ line gene VL-47.1 (67), with a similarity of
97.9% (without including the first 12 nucleotides which contained an
artificial restriction enzyme insertion). All three murine V gene
fragments belonged to the V1 gene family but showed the highest
sequence homology to different germ line genes (17, 46, 59).
Both 207,B-4 and 208,D-5 shared high homology (98.6 and 97.6%,
respectively) to K1A5, whereas the V gene of MN14C11.6 showed 99%
homology to the germ line gene K5.1. In addition, all three murine MAbs
used the same J segment genes for both the heavy and light chains,
whereas D gene utilization differed (Table 3).
A similarity comparison of the VH and VL amino
acid sequences of the four MAbs revealed that the heavy chain of SS269
was approximately 70% homologous to those of the murine MAbs and that its light chain was less than 40% homologous. In contrast, high homology was observed among the heavy and light chains of the three
murine MAbs: 95.2% between the VH of 207,B-4 and 208,D-5, 89.5% between the VH of 207,B-4 and MN14C11.6, 89%
between the VH of 208,D-5 and MN14C11.6, 96.8% between the
VL of 207,B-4 and 208,D-5, 94.4% between the
VL of 207,B-4 and MN14C11.6, and 94.4% between the
VL of 208,D-5 and MN14C11.6. A hydrophilicity structural prediction comparison of the human and murine MAb VH
regions showed the greatest difference in the CDR3 region where the
human MAb was considerably more hydrophilic than the three murine MAbs
(results not shown).
Expression of the SS269 Fab fragment in E. coli.
In the early stages of this study, we repeatedly obtained two PCR
products from SS269 VH gene cDNA, one that was 348 bp in length and a second that was 360 bp. The DNA sequences of each product
indicated that both sequences were in frame and belonged to the human
VH3 family but that only the 348-bp product had been spliced to the CH1
3 domain.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The meningococcal PorA outer membrane protein is considered a promising vaccine candidate because it is expressed by nearly all meningococcal strains (23), it is highly immunogenic in humans (16, 26), and bactericidal antibodies directed against PorA protect against meningococcal infection in an animal model (56). Although much is understood regarding the structure of PorA proteins (42, 62), little is known about the structure-function relationships of PorA antibodies. To this end, we took advantage of the fact that the PorA protein is one of only a few meningococcal proteins against which human MAbs have been reported (19, 22). We compared the functional and molecular characteristics of a human MAb directed against the P1.7 epitope of the PorA protein to those of three murine P1.7-specific MAbs and found that distinct differences exist in their opsonophagocytic and bactericidal effector function activities and variable region gene sequences despite their recognition of similar epitopes.
Murine MAbs 207,B-4 and MN14C11.6 were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 and murine MAb 208,D-5 initiated neither effector function. The ability of 207,B-4 and MN14C11.6 to initiate complement-dependent effector mechanisms is consistent with their IgG2a isotype which is known to activate complement efficiently (38), and the observed bactericidal activity of MN14C11.6 is in agreement with previous reports (53, 62). In contrast, SS269, an IgG3 isotype known to fix complement (18) did not mediate bactericidal or opsonic functions. This was not due to low affinity of SS269 for P1.7 since the affinity of SS269 is similar to that of murine MAb A'dam which is bactericidal and opsonic for P1.7 meningococci (19), suggesting that a structural characteristic of SS269 other than isotype or affinity influences its functional activity. Interestingly, Pollard et al. concluded that the bactericidal responses to a P1.7 strain in children following meningococcal infection derived from qualitative rather than quantitative differences in the complement-dependent functional activity of human anti-meningococcal IgG antibody (49). The finding that 208,D-5 did not initiate complement-dependent effector functions is presumably due to its IgA isotype constant region, which is a poor activator of complement compared with those of IgG and IgM (33). This result is consistent with the reported lack of opsonophagocytic activity exhibited by human PorA-specific IgA antibodies induced during meningococcal disease (39).
The fact that SS269 was not bactericidal is in accordance with the study by Delvig et al. which reported no bactericidal killing of a P1.7 strain by SS269 (19). However, in contrast to the findings presented here, they also found that SS269 was efficient in the opsonophagocytosis of strain 44/76. This difference is most likely due to the fact that their opsonophagocytosis experiments were performed with ethanol-fixed bacteria (19), whereas viable bacteria in the log phase of growth were used as targets for opsonophagocytosis in the present report. Our data showed only low levels of opsonophagocytic activity by SS269 against viable bacteria which did not reach 50% RB of the neutrophils for the maximum concentration tested. This contrasted with MAbs 207,B-4 and MN14C11.6, which showed strong opsonic activity. Apparently, the binding of SS269 to its epitope renders the MAb nonfunctional for bactericidal and opsonic activities despite it being an IgG3 subclass antibody which normally is highly efficient for both effector functions (11). Further investigation is required to determine the degree to which SS269 is representative of the human immune response to P1.7 epitopes.
Peptide epitope mapping revealed that MAbs 207,B-4 and 208,D-5 recognized amino acid sequences in the presumed apex of the P1.7 loop (62), which is the same site of binding established previously for MAbs SS269 and MN14C11.6 (19). The four MAbs shared recognition of the ASGQ motif in which alanine and serine were found to be necessary for epitope binding by all of the MAbs. It is likely that these two amino acids form key hydrogen bonds with contact residues of the Fab antigen combining sites as proposed for the recognition of the PorA P1.16 epitope by a murine MAb (61). For the three murine MAbs, glycine was also required for binding. For 207,B-4 and 208,D-5, all four amino acids in the ASGQ motif were required for binding although the binding of 207,B-4 was improved by the addition of a C-terminal valine residue (ASGQv). In contrast, glycine and glutamine were not necessary for SS269 epitope recognition but rather for increased binding efficiency and, also unlike the murine MAbs, SS269 required NGG for its minimal epitope (19). It may be that SS269 recognizes a conformational epitope only partially mimicked by short peptides as postulated previously (19). A possible explanation for the poor functional activities of SS269 is that it may not recognize its epitope through a peptide-induced tight conformational fit as described for the interaction of a bactericidal murine MAb directed against the P1.16 epitope in loop 4 of PorA (60). This in turn would increase the off-rate of the SS269-P1.7 complex resulting in poor induction of complement-mediated bactericidal and phagocytic activities. This suggestion is supported by the reported low avidity of SS269 for a P1.7 strain (19).
Nucleotide sequence analyses revealed that the VH region of
SS269 belonged to the VH3 gene family and that all three murine VH regions were encoded by genes belonging to the VH7183
family. Based on a phylogenetic association of progenitor
VH gene segments, these two families are classified as
members of VH subgroup III (57). It is
interesting to note that both of these families are more frequently
utilized by self-reactive antibodies than other families (5,
20). We determined that the amino acid sequence of the
VH region of SS269 was approximately 70% similar to those
of the murine MAbs, which is in agreement with the finding that nearly
all human VH3 family members are at least 70% homologous to mouse
subgroup III gene families, including the 7183 family (28).
Molecular modeling studies have demonstrated that such VH
subgroup and family identity predicts similarity in the solvent-exposed 
loop subdomains of FR1 and FR3 both within and across species (35). This is especially important for FR3, since residues
within FR3 can influence the conformation of CDR2 (15),
suggesting that conservation of FR3 residues across species may provide
initial family-associated constraints to antibody affinity for specific antigen epitopes. Thus, our data indicate that the immunoglobulin response of humans and mice to P1.7 epitopes may be biased toward the
expression of related subgroup III family VH genes, perhaps as the result of evolutionary pressure to generate related antigen binding site conformations reactive toward P1.7 epitopes.
Alternatively, the subgroup III relatedness of the four MAbs may simply
reflect the reported overexpression of VH3 and VH7183 gene families in humans and mice, respectively (36, 69).
Since neither the isotype nor the affinity for P1.7 accounted for the poor bactericidal and phagocytic effector functions of SS269, it may be that structural differences between the VH regions of SS269 and those of 207,B-4 and MN14C11.6 gave rise to effector function differences. Although the VH regions of the three MAbs shared structural characteristics, differences in key portions of these regions were apparent, which is consistent with the report that differences exist between humans and mice in the structural repertoire of the VH germ line gene segments (6). Certainly, the species restriction of natural meningococcal infection to humans supports the concept of a species-specific antibody response to P1.7 resulting in divergent levels of immunological protection. This explanation for the observed effector function differences is supported by two studies which demonstrated that differences mainly in the CDR3 portion of the VH region between antibodies with identical VL and constant regions can affect complement activation by IgG (29, 30). Importantly, the CDR3 region of SS269 was three amino acids shorter in length with a higher degree of hydrophilicity than those of 207,B-4 and MN14C11.6.
We cloned and expressed the Fab fragment of SS269 and found that it bound as well as the intact SS269 MAb to strain 44/76 expressing a P1.7,16 PorA protein. When tested against strains expressing P1.7,10 and P1.2 PorA proteins, the Fab of SS269, unlike intact SS269, showed reactivity toward both strains, albeit well below that observed for strain 44/76. Since PorA proteins are known to express silent P1.7 epitopes which are physically masked in the native protein (9, 43), it may be that the SS269 Fab due to its smaller size reacts with P1.7 epitopes not otherwise accessible to the intact SS269 MAb. Considering that P1.7-expressing strains are frequently isolated as causative agents of serogroup B infections in the continuing absence of a group B vaccine (12, 24), the successful cloning and expression of a functional SS269 Fab provides the means by which anti-P1.7 human antibodies can be generated for the passive immunization of high-risk individuals (22). Such clinically relevant antibodies could be constructed with bactericidal and phagocytic effector functions by combining human constant regions with either human or murine variable regions specific for P1.7 epitopes as described by Norderhaug et al. (47).
In conclusion, the data presented here contribute to our understanding of the human immune response to the meningococcal PorA protein. Given the importance of the PorA protein as a vaccine candidate, further studies of the immunochemistry of PorA antibodies are warranted.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Institutes of Health grant AI32944 (G.A.J.) and the Department of Veterans Affairs (J.M.G.).
We gratefully acknowledge receipt of hybridoma cell lines and MAbs from Jan Kolberg, Department of Vaccinology, National Institute of Public Health, Oslo, Norway, and the measurement of bactericidal activity performed by Arne Høiby, Department of Bacteriology, National Institute of Public Health, Oslo, Norway. We also gratefully acknowledge the receipt of PCR primers for the MN14C11.6 VH gene from Martin Maiden and Ian Feavers, National Biological Standards Board, N.I.B.S.C., Potters Bar, United Kingdom.
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FOOTNOTES |
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* Corresponding author. Mailing address: VA Medical Center, Dept. 111W1, 4150 Clement St., San Francisco, CA 94121. Phone: (415) 221-4810, x2303. Fax: (415) 221-7542. E-mail: jarvis{at}itsa.ucsf.edu.
Report no. 92 from The Center for Immunochemistry.
Present address: BioGenex, San Ramon, CA 94583.
Editor: T. R. Kozel
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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