| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
3 Gene-Disrupted Mice Selectively Deficient in the Dominant IgG Subclass Made to Bacterial Polysaccharides. II. Increased Susceptibility to Fatal Pneumococcal Sepsis Due to Absence of Anti-Polysaccharide IgG3 Is Corrected by Induction of Anti-Polysaccharide IgG11
Departments of
*
Pediatrics,
Biochemistry, and
Pathology, Case Western Reserve University School of Medicine, and Division of Infectious Diseases, Allergy, Immunology, and Rheumatology, Rainbow Babies and Children’s Hospital, Cleveland, OH 44106
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3 H chain constant region gene via targeted mutagenesis. Mutant
mice lacking IgG3 were backcrossed for 10 generations to wild-type (WT)
BALB/c mice to generate BALB/c mice that have complete absence of IgG3.
WT mice immunized with type 3 Streptococcus pneumoniae
capsular PS made anti-PS IgM, IgG3, and small quantities of IgG1,
which opsonized S. pneumoniae for killing by
polymorphonuclear leukocytes. These mice were protected against death
from lethal doses of type 3 S. pneumoniae. In contrast,
IgG3-/- mice made similar titers of anti-PS IgM and
IgG1 as WT mice but no IgG3, and had poorly opsonic sera with
significantly increased mortality after S. pneumoniae
challenge. Immunization of IgG3-/- mice with type 3
S. pneumoniae PS conjugated to carrier protein
CRM197-elicited IgM and high-titer IgG1 Abs, restored serum
opsonization, and gave protection from mortality after S.
pneumoniae, challenge comparable to WT mice. We conclude that
mice lacking the dominant IgG3 subclass made to bacterial PS are more
susceptible to fatal S. pneumoniae sepsis than WT mice,
but that IgG1 induced by a S. pneumoniae glycoconjugate
can adequately protect against S. pneumoniae sepsis.
This model suggests that IgG subclass of anti-PS Ab is an important
component of immunity to encapsulated bacteria. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is clear from studies in humans and animals that anti-PS immune responses differ from the immune responses generated by other Ags. The ability to respond to PS occurs late in mammalian development, while anti-protein Ab can be generated from a very young age (2, 5, 7). In addition, PS do not yield an anamnestic response with multiple exposures so that Ab titers do not boost beyond their original level after primary immunization. This lack of apparent memory and the ability of athymic mice to respond to PS has led to the concept that PS are so-called T cell-independent Ags (2, 7). Finally, and most relevant to function of anti-PS Abs, PS elicit Ab in humans and animals that are remarkably isotype restricted to IgM and only one or two IgG subclasses (IgG3 in mice, IgG2 and IgG1 in humans; Refs. 3, 4, 6 and 8). The IgG3 subclass restriction of murine anti-PS IgG Ab has been described for a variety of PS including group A streptococcus carbohydrate, dextran, LPS, capsular PS of Hemophilus influenzae type b, Streptococcus pneumoniae, and the group C meningococcus among others. Similarly, IgG subclass restriction to primarily IgG2 has been described after immunization in humans with these PS, while protein Ags elicit IgG1, IgG3, and IgG4, but not usually IgG2 (6, 7, 8). Thus, the ontogeny and character of the mouse IgG3 anti-PS Ab response is very similar to that seen with human IgG2, and they are considered immunologically comparable.
The clinical relevance of the human IgG subclass response to bacterial
PS is supported by the observation that children deficient in IgG2 are
more susceptible to sinus and pulmonary infections with encapsulated
bacteria such as pneumococcus. However, controversies exist as to
whether the dominant anti-PS subclass has improved functional
capabilities over other IgG subclasses, and whether the absence of this
isotype is clinically relevant (9, 10, 11, 12, 13). We previously
reported on the construction of an animal model of IgG subclass
deficiency, in which the 3 CH1 region of the Ig gene was disrupted
via gene targeting and homologous recombination rendering the mice
deficient in the dominant IgG subclass made to bacterial PS
(14). These mice were found to lack serum IgG3, and were
unable to produce anti-PS IgG3 after PS immunization. However, due
to the mixed genetic background of these mice, it was difficult to
compare their PS immune responses to the previously published
literature on host response to PS and infection with encapsulated
bacteria, since most previous studies primarily have used BALB/c mice.
Therefore, we backcrossed the IgG3-/- mice for
10 generations to BALB/c mice to develop an inbred BALB/c-background,
IgG3-deficient mouse. This mouse also has complete absence of serum and
membrane IgG3, and cannot make IgG3 after immunization with bacterial
PS. To elucidate whether the isolated absence of the dominant
anti-PS IgG subclass increases susceptibility to infection with
encapsulated bacteria, these mice were immunized with purified
pneumococcal capsular PS or capsular PS conjugated to carrier protein,
and then challenged with type 3 S. pneumoniae. We now
demonstrate that the absence of IgG3 anti-PS Abs after immunization
with pneumococcal PS greatly increases susceptibility to fatal
pneumococcal sepsis, while induction of isotype switching to IgG1
anti-PS Abs via immunization of IgG3-/-
mice with a pneumococcal glycoconjugate vaccine corrects this defect
and allows protection similar to that seen in wild-type (WT)
mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IgG3-deficient mice described previously (14) were backcrossed to BALB/cAnTAC mice for 10 generations (Taconic Farms, Germantown, NY). WT and IgG3-/- BALB/c mice were housed in microisolator cages in a pathogen-free satellite facility of the Animal Resource Center of Case Western Reserve University (Cleveland, OH). The Animal Care and Use Committee of Case Western Reserve University approved the animal protocols that were used. Animals were fed an ad libitum diet of autoclaved Teklad mouse chow (Harlan Teklad, Madison, WI).
PCR to demonstrate genotype of BALB/c-backcrossed mice
Genomic DNA was obtained from mouse tail clippings of the BALB/c
WT and the BALB/c-IgG3-/- (C
129(B6)-Igg3tm1N10) female mice. Tail
clippings were incubated at 55°C overnight with 0.36 mg proteinase K
per milliliter of 50 mM Tris/50 mM EDTA/100 mM NaCl/1% SDS solution
(pH 8.0), then phenol/chloroform isopropanol purified. Genomic DNA was
amplified in a 50-µl reaction mix of PCR buffer, 5 mM
MgCl2, 0.75 mM dNTPs, 2.5 U Taq DNA
polymerase (Life Technologies, Rockville, MD), and 25 pmol of each
primer used. These primers were specific for the BALB/c 3 H chain
genomic DNA (Integrated DNA Technologies, Coralville, IA). The WT
primers (CH-1 up, 5'-TCAAACCTAGCTGCTAATTC-3'; CH-1 down,
5'-TGGATATGATCATTGACAGG-3') amplify a 759-bp fragment of the CH1 region
and the knockout (KO) primers (Neo2,5'-CTTGGGTGGAGAGGCTATTC-3';
Neo3,5'-CAACGCTATGTCCTGATAGC-3') amplify a 628 bp fragment of the
neomycin cassette that was inserted via homologous recombination. The
cycle program was conducted on a GeneAmp PCR Thermacycler (Applied
Biosystems, Foster City, CA) and the PCR products were analyzed on a
2% Tris acetate EDTA agarose gel.
ELISA and Western blot to measure isotype of Abs
The quantity of serum Ig of various isotypes in WT and IgG3-/- mice was measured via isotype ELISA as we have previously described (14). Briefly, 96-well microtiter plates (Corning Plasticware, Corning, NY) were coated with 0.1 µg/well anti-murine Ig (Southern Biotechnology Associates (SBA), Birmingham, AL) and blocked with 1% BSA (Sigma-Aldrich, St. Louis, MO) in PBS (50 mM phosphate, 150 mM NaCl, pH 7.2). Blocked wells were incubated overnight with serial dilutions of sera from unimmunized WT and IgG3-/- mice. Bound Igs were detected by the addition of a 1/1500 dilution of isotype-specific alkaline phosphatase (AP)-conjugated goat polyclonal Abs (SBA), followed by development with 100 µl/well p-nitrophenyl phosphate, disodium chromogenic substrate (Sigma-Aldrich). Absorbance was determined at 415 nm using a Dynatech MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). Purified murine Igs (SBA) served as control proteins and were used to generate standard curves that allowed derivation of microgram per milliliter values.
Western blot was also used to confirm absence of IgG3 Ab in
IgG3-/- mice via discontinuous SDS-PAGE in a
gel consisting of 12% acrylamide. Sera from WT and mutant mice were
diluted in PBS and then 1/1 in sample buffer (SDS reducing buffer),
while purified mouse IgG3 (
FLOPC 21, Sigma-Aldrich) served as a
positive control. Each sample was loaded onto the acrylamide gel and
electrophoresed for 60 min at 150 V. Following electrophoresis,
proteins were transferred to a nitrocellulose membrane (Sigma-Aldrich)
in 25 mM Tris, 192 mM glycine, and 20% methanol transfer buffer (pH
8.3) for 80 min at 80 V. The membrane was then blocked overnight at
4°C in 1% BSA-PBS, and goat anti-mouse IgG3-AP (SBA) was then
added. The membrane was washed three times and stained using
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium
alkaline phosphatase substrate tablets (Sigma-Aldrich). Kaleidoscope
prestained standards (Bio-Rad, Richmond, CA) were used as markers.
Anti-S. pneumoniae PS ELISA
Abs against type 3 S. pneumoniae were measured in sera as previously described (14, 15, 16). Ninety-six-well microtiter plates (Corning Plasticware) were coated with 10 µg/ml PS from the type 3 capsule (Advanced Type Culture Collection (ATCC), Manassas, VA). Unbound sites were blocked with 1% BSA/PBS. Cross-reactive anti-cell wall PS Abs were removed by adsorbtion of all sera with 50 µg of S. pneumoniae cell-wall PS per milliliter of serum before use (University of Rochester, Rochester, NY; Ref. 17). Anti-PS Ab levels in serial dilutions of sera taken from weekly bleeds were then detected by the addition of subclass-specific AP-conjugated polyclonal Abs (SBA) followed by the addition of 100 µl p-nitrophenyl phosphate, disodium chromogenic substrate (Sigma-Aldrich). Absorbance was then determined at 410 nm, and Ab titer was determined via a linear fit for OD values of five dilutions, and calculation of reciprocal dilutions that gave 50% of the maximum absorbance.
ELISA spot analysis of surface IgG3
The presence or absence of mouse B cells secreting IgG3 was measured by computer-assisted, single-cell resolution ELISPOT analysis (18). Nitrocellulose-based microtiter plates (Millipore, Bedford, MA) were coated with 2 µg/ml of unlabeled goat anti-mouse IgG3 (SBA) and incubated overnight at 4°C. The plates were washed three times with PBS, and then blocked with 10% FBS in PBS (Life Technologies), and then washed again with PBS. Spleens aseptically removed from three naive BALB/c WT and three BALB/c IgG3-/- mice were placed into RPMI 1640 media (Life Technologies). Single-cell suspensions of 1.25 x 107 cells/ml were prepared by grating the spleens over a wire mesh. AP-labeled goat anti-mouse IgG3 Ab at a 1/1000 dilution (SBA) was added to the suspensions. A total of 100 µl/well of AP-labeled Ab solutions were aliquoted to the microtiter plate. Murine myeloma cells that produce IgG3 mAb to high m.w. PS of Pseudomonas aeruginosa LPS were used as IgG3-secreting control cells (19). These cells were prepared in serial dilutions from 1 x 104 cells/ml to 1 x 102 cells/ml, and then combined with secondary Ab as described above, and 100 µl of the cell suspensions were put into the appropriate wells of the microtiter plate. Nonsecreting Sp2/0 murine myeloma cells were used as a negative control. The plates were then incubated at 37° for 18 h, and washed with PBS-Tween 20 (0.05%) followed by PBS. Substrate solution was prepared by adding one tablet of Sigma-Fast 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium alkaline phosphatase substrate (Sigma-Aldrich) to 10 ml of deionized water, and 100 µl was added to each well. Plates were then washed again, blotted dry, and allowed to dry overnight. Spots representing Ab-secreting cells were detected using a computer-assisted ImmunoSpot series I Image Analyzer (Cellular Technology, Cleveland, OH; Ref. 18), and all samples were analyzed in quadruplicate.
Immunization of mice with PS, PS-protein conjugate vaccines, or passive antisera, and bacterial challenge with S. pneumoniae
WT and IgG3-/- mice were immunized i.p. with 0.5 µg of the type 3 PS (ATCC) or 0.5 µg of the type 3 PS covalently linked to the carrier protein CRM197, a nontoxic mutant of diphtheria toxin (0.93 mg/ml type 3 pneumococcal PS linked to 1.73 mg/ml CRM197, kindly supplied by Dr. R. Eby, Wyeth Lederle Vaccines, West Henrietta, NY; Ref. 20) on days 0 and 13 without adjuvant, after dose-response experiments indicated that this dose elicited an optimal Ab response in BALB/c mice (data not shown). Sera from naive and immunized mice were obtained from 200 µl whole blood acquired weekly via venipuncture of the tail vein and, after preincubation with cell wall PS, were assayed for Abs in ELISA or used in opsonophagocytosis experiments as described in the next section. In passive immunization experiments, WT or IgG3-/- mice were given 200 µl of sera i.p. obtained from wk 4–5 after PS immunization of the WT mice described previously before bacterial challenge.
Actively or passively immunized mice were challenged with varying concentrations of type 3 S. pneumoniae (strain A66.3, kindly supplied by Dr. D. Briles, University of Alabama, Birmingham, AL) given i.p. Frozen glycerol stocks of bacteria were streaked overnight on tryptic soy agar II plates (TSA II; BD Biosciences, Mountain View, CA) and then grown in Todd Hewitt Broth containing 0.5% yeast extract (Difco Laboratories, Detroit, MI) until 1 x 108 CFU/ml was achieved. Bacteria were then serially diluted in sterile, chilled PBS, and both WT and IgG3-/- mice were injected i.p. with various doses of pneumococci (5 x 102–1 x 105 CFUs) in a volume of 100 µl. Previous experiments (data not shown) had shown that 10 CFU was the minimal lethal dose in naive BALB/c mice, thus, challenge doses ranged from 50–10,000x minimum lethal dose. Challenge inoculum was confirmed by plating aliquots of the inoculum on TSA II plates. The outcome measured was death, and mice were observed for 8 days postchallenge.
Ab and complement-mediated opsonization and killing
The ability of sera from immunized WT and IgG3-/- mice to opsonize pneumococcus for uptake and killing by human polymorphonuclear leukocytes (PMN) was determined using a modification of an opsonophagocytosis assay as previously described (21, 22). Type 3 S. pneumoniae was grown in Todd Hewitt broth with 0.5% yeast extract to a concentration of 1 x 108 CFU/ml, resuspended in sterile PBS, and diluted to 1 x 105 CFU/ml in RPMI 1640 medium (Life Technologies) with 10% FBS. PMN were isolated from 30 ml heparinized blood by Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient separation. RBC were lysed with 0.2% saline followed by 1.6% saline to restore physiological salt concentration. Recovered PMNs were washed in HBSS without calcium chloride, magnesium chloride, magnesium sulfate, or phenol red (Life Technologies), and resuspended in RPMI 10% FBS to a concentration of 2 x 107 cells/ml.
Pooled sera from mice week five postimmunization with either pure PS or glycoconjugate were preabsorbed with pneumococcal C PS as described previously for ELISA, and then heat inactivated at 56°C for 20 min. ELISA showed equivalent titers of IgM, IgG2b, and IgG1 anti-PS Abs in pooled KO vs WT sera (data not shown). A total of 100 µl of bacterial preparation (1 x 104 CFU), 50, 25, 12.5, or 6.25 µl of sample sera, 100 µl of PMNs, and 40 µl of human sera obtained from an agammaglobulinemic patient as the complement source were combined. The final volume was brought to 400 µl with RPMI 10% FBS and incubated in a rotator for 90 min at 37°C. Aliquots (100 µl) from each tube were diluted in RPMI with 10% FBS, and 10 µl of each dilution was plated in duplicate on TSA II plates with 5% sheep blood (BD Biosciences) and incubated overnight at 37°C. CFU were counted and calculated for each original reaction tube. Percentage of killing was calculated using the following formula: percent killing = 100 - (100 x (CFU/CFU in the tube without Ab)).
Statistics
Differences in mortality after pneumococcal challenge were
determined via 
2 analysis. Serum Ig
concentration group means were compared using the two-sample Student’s
t test. Where variances between groups were unequal, the
Wilcoxon rank sum test was used as the nonparametric alternative to the
two-sample t test. Values of p 
0.05 were
considered significant. Statistical analyses were performed on the
StatView statistical software (Abacus Concepts, Berkeley, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IgG3-/- mice were backcrossed to BALB/c
mice for 10 generations. To confirm that the genotype was consistent
with deletion of the original 54 bp of the CH1 region of the 3 gene
and insertion of the neomycin resistance cassette (neo'), PCR was
performed with primers designed to amplify neo’ and the 54 bp that had
been deleted from the original gene. PCR demonstrated the correct
genotype in the homozygous IgG3-/- BALB/c mice
after the 10-generation backcross (Fig. 1
a).
|
3 allele had no serum IgG3 as
determined by Western blot (Fig. 1
b). To determine absence
of B cells secreting IgG3, computer-assisted ELISPOT assay that is
sensitive enough to detect one Ab-secreting B cell per million cells
was also performed on WT and mutant mouse splenocytes
(18). There were no detectable IgG3-secreting B cells in
the BALB/c IgG3-/- mice using this assay (Fig. 1c). Naive IgG3-/- mice have decreased IgA, IgG2b, and IgG2a compared with WT BALB/c mice
Resting Ig levels were measured in naive IgG3-/- and WT mice by ELISA using purified isotype-specific mouse Abs to generate standard curves. IgG3 was absent in mutant mice as expected. There were also decreased levels of serum IgA, IgG2b, and IgG2a in IgG3-/- mice compared with WT mice (0.077 vs 0.122 mg/ml IgA, p = 0.003; 0.175 vs 0.243 mg/ml IgG2b, p = 0.002; and 0.066 vs 0.032 mg/ml IgG2a, p = 0.005). However, quantities of serum IgM and IgG1 in naive BALB/c IgG3-/- mice did not significantly differ from those observed in naive WT mice.
Ab response to immunization with the purified capsular PS of S. pneumoniae
BALB/c mice immunized with the purified capsular PS of type 3
pneumococcus made high titers of IgM and IgG3 anti-PS Abs and low
titers of IgG1 and IgG2b anti-PS Abs (Fig. 2a).
IgG3-/- BALB/c mice also made IgM and low
titers of IgG1 and IgG2b anti-PS Abs that did not differ from peak
titers obtained from WT mice (p = 0.24, 0.56,
and 0.19, respectively; Fig. 2b). However, no detectable
IgG3 anti-PS Abs were found after PS immunization in the
IgG3-/- mouse. Neither group of mice made
detectable quantities of anti-PS IgA Abs after immunization.
|
To determine whether pneumococcal PS-CRM197
glycoconjugate vaccine induced anti-PS Ab isotype switching to IgG1
in the backcrossed BALB/c IgG3-/- mice, and to
assess the protective efficacy of IgG1 anti-PS Ab in isolation from
serum IgG3, mutant, and WT mice were immunized twice >2 wk with a
vaccine consisting of the type 3 pneumococcal capsule conjugated to
CRM197, a nontoxic single amino acid mutant of
diphtheria toxin. This carrier protein, when conjugated to bacterial
PS, has been previously shown to induce anti-PS Ab responses in a
manner resembling T-dependent Ags, and to induce isotype switching in
mice from IgG3 to IgG1 (16, 20, 23). WT BALB/c mice made
higher titers of anti-PS Abs than when immunized with pure PS,
particularly IgG1 anti-PS Abs in addition to IgM, IgG3, and small
amounts of IgG2b (Fig. 3a).
IgG3-/- BALB/c mice made similar peak titers of
IgM, IgG2b, and IgG1 anti-PS Abs as WT mice
(p = 0.87, 0.09, and 0.37,
respectively), and as expected, no detectable IgG3 (Fig. 3b).
|
WT mice immunized with type 3 pneumococcal PS and then challenged
with lethal doses of type 3 pneumococci were substantially protected
against mortality until high challenge doses were reached
(>10,000–100,000 CFU; Table I).
IgG3-/- mice, although protected from mortality
at the lowest challenge dose (500 CFU) and despite having similar
titers of IgM and IgG1 anti-PS Abs as WT mice, had significantly
higher mortality than WT mice at all other challenge doses (5,000,
10,000, and 100,000 CFU; p < 0.02, 0.05, and 0.02,
respectively; Table I).
|
To demonstrate that the observed increased susceptibility to
pneumococcal sepsis in IgG3-/- mice was due to
Ab deficiency and not to other non-Ab immunological dysfunction,
another group of PS-immunized IgG3-/- mice were
passively immunized with pooled anti-sera obtained from WT mice 5
wk after PS immunization, before bacterial challenge. PS-immunized
IgG3-/- mice pretreated with sera from
PS-immunized WT mice were protected from mortality except at the
highest challenge dose (Table I).
IgG3-/- mice that make IgG1 anti-PS Abs after immunization with pneumococcal-CRM197 glycoconjugate vaccine are protected from fatal pneumococcal sepsis
WT mice immunized with type 3 pneumococcal
PS-CRM197 glycoconjugate vaccine were better
protected from fatal pneumococcal sepsis even with high inoculum
bacterial challenge (100,000 CFU) than PS-immunized WT mice (90%
survival vs 40%; p < 0.02; Table I). Similarly,
IgG3-/- mice immunized with
PS-CRM197 were significantly better protected
than PS-immunized IgG3-/- mice after 5,000,
10,000, or 100,000 CFU bacterial challenge (p =
0.001, 0.0003, and 0.001, respectively; Table I). In addition, there
were no statistical differences between survival of
glycoconjugate-immunized WT or IgG3-/- mice,
and all were well-protected against fatal pneumococcal sepsis.
Sera from PS-immunized IgG3-/- mice are poorly opsonic for uptake and killing of pneumococcus by human PMN in a complement-dependent assay
Pooled sera obtained 5 wk after PS immunization from WT and KO
mice containing equivalent ELISA titers of IgG1, IgG2b, and IgM
anti-PS Ab were used in an assay designed to measure the
complement-dependent ability of PS-specific Abs to opsonize
pneumococcus for uptake and killing by human PMN. Sera from
PS-immunized IgG3-/- mice were poorly opsonic
for killing of pneumococcus by PMN compared with WT sera. Although neat
postimmunization IgG3-/- sera opsonized
bacteria in the presence of complement for some killing of the
bacterial inoculum, opsonic ability quickly declined with dilutions of
sera, so that no killing was obtained with the 1/8 dilution. In
contrast, sera from WT mice immunized with purified PS were highly
opsonic for PMN-mediated killing of pneumococcus at all the dilutions
tested (Fig. 4a).
|
Immunization of IgG3-/- mice with the type
3 pneumococcal-CRM197 glycoconjugate vaccine
yielded higher titers of anti-PS Ab than PS immunization, normal
isotype switching to anti-PS IgG1, and pooled sera that were highly
opsonic for uptake and killing of the bacteria by PMN. Pooled
IgG3-/- mouse sera after glycoconjugate
immunization was at least 4-fold more opsonic than sera from
PS-immunized IgG3-/- mice, since significant
bacterial killing was obtained at a serum dilution of 1/32, similar to
bacterial killing observed using sera obtained from WT mice immunized
with the glycoconjugate (Fig. 4b). WT mice immunized with
the glycoconjugate also achieved higher anti-PS Ab titers than WT
mice immunized with purified PS, and their sera were also more opsonic
than sera from PS-immunized WT mice (Fig. 4b).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although IgG3-/- mice did not make IgG3 anti-PS Abs in response to immunization with purified pneumococcal type 3 PS, they did make predominantly IgM and low titers of IgG1 and IgG2b anti-PS Abs that were very similar in titer to that produced by WT mice. These results differ from our previous observation that the mixed background agouti 129/Black 6 IgG3-/- mice made more IgM than WT mice after immunization with certain bacterial PS (14). However, increased IgM was not seen after immunization with all PS, and we did not use type 3 pneumococcal PS in these mixed background mutant mice. In addition, the previous mixed background mouse had significant individual variability in Ab responses that has been greatly reduced by the 10-generation backcross to the inbred BALB/c genetic background.
The absence of IgG3 after immunization with the T-independent
pneumococcal PS rendered the IgG3-/- mice more
susceptible to fatal pneumococcal sepsis than WT mice, similar to the
increased susceptibility of humans with IgG2 subclass deficiency to
infection with encapsulated bacteria (8, 9, 10, 11). In addition,
sera obtained from these mice were much less opsonic for uptake and
killing of the bacteria by human PMN, suggesting that IgG3 anti-PS
Abs are the major opsonic Ab isotype after PS immunization. Because the
ability of anticapsular pneumococcal Abs to promote opsonophagocytosis
correlates closely with protection from pneumococcal bacteremia, the
opsonic capability of IgG3 postimmunization with TI bacterial PS may be
an important component of subclass specific immunity to the
pneumococcus (25). However, IgM and small amounts of IgG1
anti-PS Abs produced were capable of protecting mice from fatality
after challenge with the lowest dose of pneumococci. Because
macrophages and neutrophils are not thought to have µFcRs
(26), it seems possible that the partial protection
observed in the IgG3-/- mice against low dose
challenge with pneumococci may be a result of the ability of the
anti-PS IgM to fix complement to the bacterial surface and
partially opsonize the bacteria for relatively inefficient phagocytic
clearance mediated primarily by phagocyte complement receptors.
Alternatively, the low titer of IgG1 anti-PS Abs in combination
with complement deposition mediated by IgM anti-PS Abs may have
been adequate to opsonize small numbers of pneumococci for uptake by
macrophages with FcR to which IgG1 was capable of binding (27, 28).
Conjugation of bacterial PS to proteins has been used to enhance immunogenicity of bacterial PS and yields vaccines that function as T-dependent Ags. Glycoconjugates induce booster responses after repeated immunization, induction of memory and carrier protein-specific CD4+ T cell help, increased avidity of anti-PS Ab, and isotype switching to IgG1 in both mice and humans (7, 16, 20, 23, 29). However, IgG3, the dominant mouse subclass made to bacterial PS, has been thought to have characteristics that enhance anti-PS Ab function. For example, mouse anti-PS IgG3 exhibits Fc-mediated cooperative binding that increases functional affinity for binding to multivalent PS epitopes such as those on bacterial capsules (30, 31), and mouse IgG3 has been found to provide more potent protection against pneumococcus compared with other isotypes (12). Thus, it has been hypothesized that the dominant anti-PS Ab IgG subclass functions in a superior manner against encapsulated bacteria than other IgG subclasses. Nevertheless, our data clearly show that induction of high-titer IgG1 anti-PS Abs against the type 3 pneumococcal capsular PS using the glycoconjugate T cell-dependent form of the PS in IgG3-/- mice yields efficient opsonization and killing of pneumococcus, as well as in vivo protection that is not distinguishable from protection afforded WT mice that produce IgG3. These data are similar to the observation that some humans with complete IgG2 H chain gene deletion may make other anti-PS IgG subclasses that appear to substitute adequately in function for IgG2 (32). However, because the overall titers of anti-PS Ab after glycoconjugate immunization were much higher than titers induced by the purified PS, it remains a possibility that mouse IgG3 may function in a superior manner in comparison to other IgG subclasses against encapsulated bacteria on a per microgram basis.
Although we found no differences in postimmunization anti-PS Ab
titers with PS or the T-dependent glycoconjugate vaccine between
IgG3-/- and WT mice (except for absence of IgG3
in mutant mice), the BALB/c IgG3-/- mice had
significantly lower resting serum levels of IgA, IgG2a, and IgG2b
compared with WT BALB/c mice. Because the mutation in the 3 gene
preserved the switch region, and because we induced normal isotype
switching of anti-PS Ab to IgG1, it seems unlikely that the
decreased serum levels of IgA, IgG2a, and IgG2b was due to inhibition
of downstream isotype switching. Other possible reasons for the
decreased levels of IgA, IgG2a, and IgG2b include reduced pool of IgA,
IgG2a, or IgG2b+ B cells, alterations in B cell
secretion rates of these isotypes, or increased catabolism of these
Abs. Since this model was a narrowly directed mutation model, it seems
more likely the complete absence of the IgG3 response to Ags, lack of
IgG3 immune complexes, lack of IgG3 B cell receptors, and interactions
with FcRs during development may exert indirect effects on B cells
destined to secrete these other isotypes. Further investigation will be
required to determine whether the altered resting levels of these Ab
isotypes is a result of decreased production or increased catabolism
or both.
We conclude that the new IgG3-/- BALB/c mouse seems to be an appropriate model of human IgG subclass deficiency in the sense that there is increased susceptibility to infection with the encapsulated pathogen, S. pneumoniae, similar to that seen in humans, that is isotype-specific. In addition, this model suggests that isotype switching that occurs with T-dependent glycoconjugate immunization yields IgG1 anti-PS Abs that are capable of opsonizing pneumococcus for uptake and killing by phagocytes, and that are highly protective in vivo. Further studies will be required to determine whether subclass-specific anti-PS Ab functional differences that occur with human anti-PS Abs are relevant to protection against infection with encapsulated bacteria in humans.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. John R. Schreiber, Departments of Pediatrics and Pathology, Case Western Reserve University, Division of Pediatric Infectious Diseases/Allergy/Immunology/Rheumatology, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address: jrs3{at}po.cwru.edu
3 Abbreviations used in this paper: PS, polysaccharide; WT, wild type; AP, alkaline phosphatase; PMN, polymorphonuclear leukocyte; KO, knockout; TSA II, tryptic soy agar.
Received for publication November 16, 2001. Accepted for publication January 18, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines. J. Immunol. 161:3393.This article has been cited by other articles:
|
|
 |
|
  K. Kaur, S. Chowdhury, N. S. Greenspan, and J. R. Schreiber Decreased expression of tumor necrosis factor family receptors involved in humoral immune responses in preterm neonates Blood, October 15, 2007; 110(8): 2948 - 2954. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tian, A. Groner, M. Boes, and L.-a. Pirofski Pneumococcal Capsular Polysaccharide Vaccine-Mediated Protection against Serotype 3 Streptococcus pneumoniae in Immunodeficient Mice Infect. Immun., April 1, 2007; 75(4): 1643 - 1650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Colino and C. M. Snapper Dendritic Cell-Derived Exosomes Express a Streptococcus pneumoniae Capsular Polysaccharide Type 14 Cross-Reactive Antigen That Induces Protective Immunoglobulin Responses against Pneumococcal Infection in Mice Infect. Immun., January 1, 2007; 75(1): 220 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Kirimanjeswara, P. B. Mann, M. Pilione, M. J. Kennett, and E. T. Harvill The Complex Mechanism of Antibody-Mediated Clearance of Bordetella from the Lungs Requires TLR4 J. Immunol., December 1, 2005; 175(11): 7504 - 7511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Loke, X. Zang, L. Hsuan, R. Waitz, R. M. Locksley, J. E. Allen, and J. P. Allison Inducible costimulator is required for type 2 antibody isotype switching but not T helper cell type 2 responses in chronic nematode infection PNAS, July 12, 2005; 102(28): 9872 - 9877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. O. Gor, X. Ding, D. E. Briles, M. R. Jacobs, and N. S. Greenspan Relationship between Surface Accessibility for PpmA, PsaA, and PspA and Antibody-Mediated Immunity to Systemic Infection by Streptococcus pneumoniae Infect. Immun., March 1, 2005; 73(3): 1304 - 1312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Kobrynski, A. O. Sousa, A. J. Nahmias, and F. K. Lee Cutting Edge: Antibody Production to Pneumococcal Polysaccharides Requires CD1 Molecules and CD8+ T Cells J. Immunol., February 15, 2005; 174(4): 1787 - 1790. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Q. Khan, Q. Chen, Z.-Q. Wu, J. C. Paton, and C. M. Snapper Both Innate Immunity and Type 1 Humoral Immunity to Streptococcus pneumoniae Are Mediated by MyD88 but Differ in Their Relative Levels of Dependence on Toll-Like Receptor 2 Infect. Immun., January 1, 2005; 73(1): 298 - 307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. K. Buchwald, A. Lees, M. Steinitz, and L.-a. Pirofski A Peptide Mimotope of Type 8 Pneumococcal Capsular Polysaccharide Induces a Protective Immune Response in Mice Infect. Immun., January 1, 2005; 73(1): 325 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Maaser, M. P. Housley, M. Iimura, J. R. Smith, B. A. Vallance, B. B. Finlay, J. R. Schreiber, N. M. Varki, M. F. Kagnoff, and L. Eckmann Clearance of Citrobacter rodentium Requires B Cells but Not Secretory Immunoglobulin A (IgA) or IgM Antibodies Infect. Immun., June 1, 2004; 72(6): 3315 - 3324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Lefeber, B. Benaissa-Trouw, J. F. G. Vliegenthart, J. P. Kamerling, W. T. M. Jansen, K. Kraaijeveld, and H. Snippe Th1-Directing Adjuvants Increase the Immunogenicity of Oligosaccharide-Protein Conjugate Vaccines Related to Streptococcus pneumoniae Type 3 Infect. Immun., December 1, 2003; 71(12): 6915 - 6920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. D. de Stahl, J. Dahlstrom, M. C. Carroll, and B. Heyman A Role for Complement in Feedback Enhancement of Antibody Responses by IgG3 J. Exp. Med., May 5, 2003; 197(9): 1183 - 1190. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
