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Department of Microbiology and Immunology, Medical College of Ohio, Toledo, OH 43614
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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knockout mice showed that
stimulation of TI-2 Ab responses by IL-12 was only partially dependent
on IFN-. The ability of IL-12 to dramatically enhance TI Ab
responses suggests that IL-12 will be useful as a powerful vaccine
adjuvant to induce protective immune responses against encapsulated
pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 has been shown to enhance cell-mediated immunity through
activation of T and NK cells, inducing these cells to secrete large
amounts of IFN- (5). It is now established that IL-12 also enhances
production of certain Ab isotypes (6, 7, 8, 9, 10, 11), indicating that the cytokine
will be useful as a vaccine adjuvant for stimulating protective humoral
immunity. The major influence on B cells is increased isotype switching
to IgG2a and IgG3, an effect that is mediated by IFN- (8, 9, 12, 13, 14, 15). Other influences of IL-12 appear to be IFN- independent
(13) and may be caused by different intermediary cytokines or by direct
stimulation of B cells. The latter mechanism is suggested by our recent
finding that B cells can directly bind IL-12 (16).
NK cells are a major target for the actions of IL-12 and have also been
thought to be a principal cell type involved in regulating TI-2 Ab
responses. Evidence that NK cells play a pivotal role in TI-2 Ab
responses includes the ability of NK cells or their products to enhance
in vitro IgG responses to anti-IgD-dextran (17) and inhibition of
these responses by anti-IFN- Ab (18). Furthermore, injection of
mice with poly(I):poly(C) can lead to an NK cell-dependent enhancement
of 2,4,6-trinitrophenyl (TNP)-specific IgG2a after immunization
with TNP-LPS (19). Thus, it is possible that IL-12 will be an effective
vaccine adjuvant for TI-2 Ags through activation of NK cells.
We now report that injection of mice with IL-12 at the time of vaccination enhances Ab responses to TI-2 Ags in a manner similar to its effects on TD Ab responses. Furthermore, examination of various immunodeficient and knockout (KO) mice suggests a novel mechanism for the influence of IL-12 on TI humoral immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 mice 6–8 wk old were obtained from the
National Cancer Institute (Bethesda, MD). C57BL/6 TCR
ß-
- double-KO mice, CD3
transgenic
mice, (C57BL/6J x CBA/J)F1 mice, and BALB/c IFN-
KO (GKO) mice were all obtained from The Jackson Laboratories (Bar
Harbor, ME). The mice were housed in the animal facility at the Medical
College of Ohio, and all experimental procedures performed on them
adhered to an approved Institutional Animal Care and Use
Committee protocol.
IL-12 treatment and immunization strategy
Recombinant murine IL-12 was provided by Genetics Institute (Cambridge, MA). Groups of three or four mice were injected i.p. for 3 consecutive days (days -1, 0, and +1) with 1 µg IL-12 diluted in PBS that contained 1% normal mouse serum. This amount of IL-12 did not result in any apparent toxicity. Control mice received PBS-1% normal mouse serum vehicle only.
Mice were immunized i.p. on day 0 with Ag precipitated in alum or emulsified in CFA (Life Technologies, Grand Island, NY) as specified in Results. Preparation of Ag in alum was performed by mixing 300 µl PBS containing 500 µg of Ag with 160 µl of 10% aluminum potassium sulfate (Fisher Scientific, Pittsburgh, PA), adjusting the pH to 6.5, and washing the precipitate three times with PBS. Ags included 50 µg/mouse of DNP-OVA and DNP-Ficoll (both from Biosearch Technologies, San Rafael, CA) as model TD and TI-2 Ags, respectively. In addition, the commercial polysaccharide vaccines that were used included 1) 115 µg/mouse of PNU-Imune 23 (Lederle Laboratories Division, American Cyanamid, Pearl River, NY), a polyvalent pneumococcal vaccine consisting of a mixture of purified capsular polysaccharides from 23 serotypes of S. pneumoniae, and 2) 20 µg/mouse of Menomune-A/C/Y/W-135 (Connaught Laboratories, Swiftwater, PA), a meningococcal vaccine consisting of purified capsular polysaccharides from four serogroups of N. meningitidis. In some experiments, mice were boosted i.p. on day 28 with Ag emulsified in IFA (Life Technologies). Sera were prepared by the induction of bleeding from the orbital plexus.
Detection of Ab levels by ELISA
Anti-DNP Ab levels were measured by isotype-specific ELISAs as previously described (9, 13), with some modifications. Briefly, microtiter plates (Nalge Nunc International, Naperville, IL) were coated overnight with 10 µg/ml DNP-BSA (Biosearch Technologies) in PBS. After washing with PBS containing 0.3% Brij-35 (Sigma Chemical, St. Louis, MO), the plates were blocked for 1 h at room temperature with PBS containing 5% FCS (HyClone Laboratories, Logan, UT) and 0.1% Brij-35 (Sigma). The plates were then incubated with serial dilutions of mouse sera for 2 h at room temperature, and bound Ab was detected with alkaline phosphatase, which was conjugated to goat anti-mouse Ig (Sigma) for detection of total Ab or to specific goat anti-isotype Ab (Southern Biotechnology Associates, Birmingham, AL) for detection of individual isotypes. After incubation at room temperature for 1 h, p-nitrophenyl substrate was added, and color development was read at 405 nm with an ELISA microplate reader (Bio-Tek Instruments, Winooski, VT). The isotype specificities and appropriate working dilutions of the Ab-enzyme conjugates were determined by titration against standard myeloma proteins of known isotypes (Sigma). Specificity of the assay for DNP was confirmed by lack of binding of the mouse sera to BSA-coated wells.
Abs specific for pneumococcal and meningococcal polysaccharides were measured by coating microtiter plates at 37°C for 2 h with 100 µg/ml poly-L-lysine (Sigma) in PBS. The plates were washed with PBS, and 10 µg/ml PNU-Imune 23 or Menomune A/C/Y/W-135 in PBS was added to each well overnight. The remainder of the assay was performed as described above for anti-DNP Ab measurement. No binding of antisera was observed using plates coated only with poly-L-lysine.
Total serum Ig was measured in a similar manner using plates coated with goat anti-mouse Ig (Southern Biotechnologies). Concentrations of bound Ig were determined by comparison to a standard curve generated using purified myeloma protein (Sigma).
Statistical analyses were performed using the Mann-Whitney U test. Titers were calculated by fitting the data to a generalized four-parameter logistics curve using Titercal software.
Cell cultures
Single-spleen cell suspensions obtained from BALB/c mice were cultured in 96-well microtiter plates (2 x 105 cells/well in a volume of 200 µl/well) in RPMI 1640 containing 10% (v/v) FCS (HyClone Laboratories), 1 mM sodium pyruvate (Life Technologies), 4 mM L-glutamine (Life Technologies), and 10 µg/ml gentamicin (Sigma). The cells were exposed to 20 µg/ml Salmonella typhimurium LPS (Sigma) and 5 ng/ml recombinant murine IL-12. Cells were harvested after 3 days for cytokine analysis.
Cytokine RT-PCR
Total RNA was isolated from spleens using Trizol reagent (Life
Technologies, Gaithersburg, MD). cDNA synthesis was performed using an
RT kit (Life Technologies) utilizing oligo(dT)16–18
primers. The cDNA was amplified using specific primers for IFN-
,
IFN-, and hypoxanthine phosphoribosyl transferase (HPRT). The sense
and antisense primers had the following sequences: IFN-,
5'-GACTCATCTGCTGCTTGGAATGCAACCCTCC-3' and
5'-GACTCACTCCTTCTCCTCACTCAGTCTTGCC-3'; IFN-,
5'-TGAACGCTACACACTGCATCTTGG-3' and 5'-CGACTCCTTTTCCGCTTCCTGAG-3';
and HPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and
5'-GATTCAACTTGCGCTCATCTTAGGC-3'. PCR amplification was performed by
mixing 2 µl of cDNA, 5 µl of 2.5 mM deoxynucleotide triphosphates
(Invitrogen, San Diego, CA), 0.5 µl of Taq DNA polymerase (2.5 U;
Life Technologies), and 2 µl of 20 µM primer in a total volume of
50 µl in 60 mM Tris-HCL buffer (pH 8.5) containing 75 mM
(NH4)2SO4 and 2 mM
MgCl2. The mixture was incubated at 95°C for 5 min and
then subjected to the following amplification profile: 1 min at 95°C,
1 min at 56°C, and 1 min at 72°C for a duration of 35 cycles. This
was followed by a final extension for 10 min at 72°C. The PCR
products were separated on a 2.5% agarose gel and stained with
ethidium bromide. The bands were visualized and photographed under UV
transillumination.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 has been shown to stimulate cell-mediated immunity through
increased IFN- secretion by T cells and NK cells (5, 20, 21). IL-12
has also been shown to play a pivotal role in regulating TD Ab
responses (6, 7, 8, 9, 10, 11). We have now investigated the influence of IL-12 on
IgG Ab responses to the TI-2 Ag, DNP-Ficoll, and compared the observed
effects with those seen with the TD form of DNP conjugated to OVA.
BALB/c mice were injected with 1 µg of IL-12 or PBS vehicle i.p. on
days -1, 0, and +1 and with DNP-OVA or DNP-Ficoll emulsified in CFA on
day 0. Analysis of individual Ab isotypes revealed increases in the
levels of DNP-specific IgG2a and IgG3 in IL-12-treated mice compared
with control mice (Fig. 1
). Significant
increases were observed in mice immunized with either the TD or TI-2
form of DNP, although the effects were more dramatic in the former
group. In both cases, the enhancement of IgG2a and IgG3 Abs reached a
maximum on day 21 and remained elevated for at least 35 days. There was
no significant difference in expression of total serum Ig between mice
given Ag only and mice given Ag plus IL-12 (8.5–23.9 mg/ml total Ig
for mice given Ag only and 5.45–27.5 mg/ml total Ig for mice given Ag
and IL-12). The results demonstrate that the influence of IL-12 is
similar in both TD and TI-2 humoral immune responses and suggest that
IL-12 would be an effective adjuvant for TI-2 polysaccharide vaccines.
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The influence of IL-12 on anti-DNP responses after
immunization with DNP-Ficoll was intriguing, but the magnitude of the
effects was less than that observed with a TD Ag. The next series of
experiments was thus performed to determine how IL-12 might stimulate
IgG Ab responses to other TI-2 Ags, especially polysaccharide Ags that
are of medical importance in humans. For this purpose, BALB/c mice were
immunized with a meningococcal polysaccharide vaccine (Menomune)
consisting of the A, C, Y, and W-135 capsular serogroups. Vaccine was
administered i.p. to adult mice together with three daily doses of
IL-12 or PBS vehicle. It was found that levels of IgG2a and IgG3
anti-polysaccharide Abs were dramatically enhanced by IL-12
administration compared with mice not exposed to IL-12 (Fig. 2). In fact, the mice mounted only very
weak or no IgG2a responses unless they had been inoculated with both
vaccine and IL-12. Levels of total and IgG1 Abs were somewhat increased
by IL-12 exposure, IgM was slightly suppressed, and there was no
detectable effect on IgG2b production.
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The findings with the meningococcal vaccine were confirmed using
another TI-2 vaccine, the pneumococcal vaccine (PNU-Imune 23), which
consists of purified capsular polysaccharides from 23 serotypes of
S. pneumoniae. Again, it was found that mice that were
treated with IL-12 had significantly enhanced IgG2a and IgG3 responses
(Fig. 3). There were no dramatic
differences in the Ab responses before and after boosting,
demonstrating that reimmunization is not necessary to obtain
enhancement of TI-2 Ab responses by IL-12. Total Ab and IgG1 levels
were also enhanced by IL-12 treatment on day 21 (Fig. 3A),
as well as 14 days after boosting (Fig. 3B). Significant
differences in IgM levels after exposure to IL-12 were only detectable
2 weeks after boosting (Fig. 3B). IgG2b levels were
undetectable in both IL-12-treated and control mice throughout the
course of the experiment. Thus, IL-12 enhances anti-polysaccharide
Ab responses in adult mice to vaccines that are currently used in
humans with limited success.
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To assess the involvement of T cells in mediating the effects of
IL-12 on TD and TI-2 Ab responses, mice specifically lacking T cells,
i.e., C57BL/6 ß-- TCR double-KO mice,
were analyzed. TCR KO mice were immunized with either DNP-OVA or
DNP-Ficoll and treated concurrently with IL-12 or PBS vehicle. As
expected, neither PBS-treated nor IL-12-treated TCR KO mice responded
to immunization with the TD Ag, DNP-OVA. TCR KO mice responded to
DNP-Ficoll, and the responses were essentially identical with those of
C57BL/6 wild-type (WT) mice (Fig. 4).
While IL-12 had little effect on total Ab production in either strain,
it was still able to enhance production of IgG2a DNP-specific Abs in KO
mice and to the same degree as seen in WT mice. These results confirm
the TI nature of the response and demonstrate that IL-12 can mediate
its effects in the absence of T cells.
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There is evidence that NK cells are responsible for regulating
TI-2 Ab responses (1, 17, 18, 19, 22). It is also known that IL-12
activates NK cells (5, 21). Therefore, to investigate the role of NK
cells in the enhancement of IgG2a anti-DNP responses by IL-12, mice
that lack both T and NK cells were inoculated with either DNP-OVA or
DNP-Ficoll and IL-12. The animals used for this experiment were
(C57BL/6 x CBA)F1 mice that are transgenic for human
CD3 (The Jackson Laboratories). Introduction of this transgene has
led to a complete blockage of both T lymphocyte and NK cell development
in the recipient mice, but B cell development is normal (23). The
CD3 mice failed to respond to immunization with DNP-OVA, regardless
of whether they received PBS or IL-12 treatment. Unexpectedly, it was
found that exposure of these animals to DNP-Ficoll in the presence of
IL-12 resulted in typical enhancement of IgG2a anti-DNP Ab
responses (Fig. 5A). The level
of enhancement was actually more striking in CD3 mice compared
with WT controls because of the nearly complete absence of IgG2a Ab
produced by CD3 mice not inoculated with IL-12. The IgG2a response
in CD3 mice reached a maximum at day 14 and began to decline by day
21, whereas IgG2a levels in control mice remained elevated, suggesting
that NK cells are needed to sustain IgG2a responses. Although in this
experiment IL-12 showed little enhancement of IgG3 anti-DNP levels
in WT mice, it clearly stimulated IgG3 production in CD3 mice (Fig. 5B). With regard to other isotypes, IL-12 treatment of WT
mice caused reduced production of IgG1 and IgG2b anti-DNP Ab and
had no effect on IgM Ab (data not shown). In CD3 mice, on the other
hand, IL-12 caused suppression of IgM but had no effect on IgG1 and
IgG2b levels. The complete absence of T and NK cells in CD3 mice
after immunization with DNP-Ficoll with or without IL-12 was confirmed
by flow cytometry (data not shown).
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mice were immunized with
meningococcal polysaccharide vaccine. It was found that CD3 mice
that were given the vaccine and IL-12 showed a significant increase in
IgG2a on day 14 in comparison with controls. On day 21, IgG2a still
appeared to be elevated, but the differences between control and
IL-12-treated mice were not statistically significant (Fig. 6). Taken together, our results provide
evidence that the mechanism of the influence of IL-12 on IgG2a and IgG3
TI-2 Ab responses does not involve NK or T cells, although these cells
might influence expression of other isotypes. Our laboratory has
previously shown that activated B cells express a receptor for IL-12
(16), suggesting that IL-12 directly activates B cells. Alternatively,
IL-12 could stimulate secretion of intermediary cytokines from cells
other than T or NK cells, and these cytokines may then mediate the
observed effects.
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IFN- induced by IL-12 plays a pivotal role in enhancement of
IgG2a and IgG3 during TD immune responses (8, 9, 13). To investigate
the role of IFN- in stimulating IgG2a and IgG3 Ab production during
TI-2 responses, BALB/c mice were immunized with DNP-Ficoll and injected
with either PBS vehicle or IL-12 as described above. Analysis of
splenic mRNA 12 h later revealed that IFN- levels were
substantially increased after exposure to IL-12 (Fig. 7). The results were identical regardless
of whether alum or CFA was used as an adjuvant. The ability of IL-12 to
induce large amounts of IFN- mRNA during a TI-2 response suggests
that IFN- may be important in the observed enhancement of Ab
production.
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, WT and GKO mice were
immunized with Menomune or DNP-Ficoll and simultaneously injected with
either PBS vehicle or IL-12. WT mice treated with Ag and IL-12 had a
three- to 10-fold enhancement of serum IgG2a levels in comparison with
mice that received only Ag and PBS vehicle (Table I). GKO mice immunized in the same manner
showed less enhancement but still tended to have increases in levels of
IgG2a. However, the differences were not statistically significant. In
the case of IgG3, two- to threefold enhancement by IL-12 was observed
in both WT and GKO mice, except that GKO mice immunized with DNP-Ficoll
produced large amounts of IgG3 Ab regardless of whether they were
treated with IL-12 or PBS vehicle. The absence of IFN- in GKO mice
after immunization with DNP-Ficoll with or without IL-12 was verified
by RT-PCR. These results suggest that enhancement of IgG2a by IL-12 is
dependent on IFN-, whereas the increase in levels of IgG3 is wholly
IFN--independent.
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Experiments involving injection of mice with exogenous IFN- or
poly(I):poly(C), which induces the secretion of the cytokine by
macrophages, have indicated that IFN- can enhance IgG2a production
(24). Expression of IFN- mRNA was therefore examined to determine
whether IFN- plays a role in IL-12 enhancement of IgG2a responses
against TI Ags. There were no differences in the levels of IFN- mRNA
in either BALB/c WT or GKO mice 12 h after immunization with Ag
and PBS or Ag and IL-12. In addition, both WT and CD3 spleen cells
stimulated in vitro with LPS demonstrated comparable levels of IFN-
mRNA whether exposed to PBS or IL-12 (Fig. 8). The constitutive expression of
IFN- in WT, GKO, and CD3 mice indicates that IFN- is not
involved in mediating the effects of IL-12 on TI Ab responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, whereas enhancement of IgG2a expression
appeared to be IFN- dependent. These results are the first to
demonstrate an adjuvant effect of IL-12 on TI Ab responses and suggest
that IL-12 will be useful for inducing protective responses against
bacterial pathogens.
IL-12 was found to have similar effects on TD and TI-2 responses to the
DNP hapten. In both cases, specific IgG2a and IgG3 anti-DNP serum
levels were significantly increased by simultaneous administration of
Ag and IL-12, while IgG1 expression did not appear to be affected at
the time points analyzed. IL-12 was most effective when given at the
time of initial priming and had little influence on TI-2 Ab responses
when administered only at boosting (R. M. Buchanan and D. W.
Metzger, unpublished observations). Use of TCR
ß-- double-KO mice confirmed the TI
nature of the response to DNP-Ficoll and the fact that the mechanism
for IL-12-mediated enhancement did not involve T cells. However, the
observed effects of IL-12 in the responses of both WT and TCR KO mice
to DNP-Ficoll was of a lesser magnitude than that observed in WT mice
against DNP-OVA. This likely reflects a property of the individual
DNP-Ficoll preparation rather than the fact that it is a TI-2 Ag, since
use of other TI-2 Ags such as bacterial capsular polysaccharides
yielded levels of IL-12 enhancement similar to those seen with TD Ags
(8, 9). We also recently demonstrated that SCID mice reconstituted with
human peripheral blood lymphocytes could mount primary Ab responses to
N. meningitidis serogroup C polysaccharide if the mice were
treated with human IL-12 at the time of cell transfer (25). However, in
those experiments, it was unclear whether IL-12 was actually
stimulating specific Ab-producing B cells or simply aiding in
engraftment of the transferred population. It was previously
established that IL-12 enhances in vivo TD production of IgG2a in
response to protein and hapten-carrier Ags (7, 8, 9, 10, 11, 13). Administration
of the cytokine suppresses IgG1 production, but this suppression is
only temporary and IgG1 production is eventually somewhat enhanced (8, 9). While TD Ags stimulate conventional B cells, TI-2 Ags are thought
to preferentially activate cells with the B-1 phenotype (26) and fail
to induce isotype switching. Since B-1 cells inhibit responses by
conventional B cells (27) and IL-12 inhibits B-1 cell function (28, 29), one influence of IL-12 may be to allow conventional B cells to
respond to TI-2 Ags, thus resulting in the observed enhancement of IgG
production.
Several groups have reported that NK cells play a major role in the
stimulation of IgG TI-2 responses through release of IFN-. It has
been shown that Ig secretion induced in vivo or in vitro in a TI manner
can be increased by NK cell activation (19) and inhibited by NK cell
depletion (17, 19). Ab neutralization of IFN- reverses the influence
of NK cells (18). Recently, a role for endogenous IL-12 in TI responses
was proposed by Koh and Yuan (22) based on the finding that Ab
responses induced by TNP-LPS and BCL1 tumor cells were
inhibited by neutralization of IL-12. Since IL-12 is a known activator
of NK cells, we investigated the role of these cells in IL-12-mediated
enhancement of TI-2 Ab responses. For this purpose, we used mice that
are transgenic for the human CD3 gene and that lack T and NK cells
(23). It was found that exposure of these animals to a TI-2 Ag in the
presence of IL-12 resulted in typical enhancement of IgG2a and IgG3 Ab
responses. Thus, in our system, NK cells are not required for
stimulation of TI IgG production by IL-12. However, NK cells do appear
to be important in maintaining IL-12-induced IgG expression over an
extended period of time. While WT and CD3 mice showed no differences
in responsiveness to IL-12 on day 14 after immunization, CD3 mice
did demonstrate lower IgG2a responses compared with WT mice on day 21
and thereafter. Therefore, although NK cells are not strictly required
for the influence of IL-12, they may appear to be critical depending on
the time of experimental observation.
IFN- is known to be a switch factor for both IgG2a and IgG3 (9, 12, 13, 14, 15), the major isotypes induced by IL-12, and high levels of
IFN- mRNA were detected in the spleens of mice injected with TI-2 Ag
and IL-12. Nevertheless, in the absence of the two cell types
responsible for IFN- production (T and NK cells), IL-12 still
significantly enhanced TI-2 Ab responses. This suggests that IFN-
either is not involved in IL-12-mediated enhancement of TI-2 Ab
responses or is being produced by another cell type. B cells have been
reported to produce IFN-, particularly after stimulation with IL-12
and IL-18 (24, 30, 31). Furthermore, we have detected IFN- mRNA in
CD3 spleen cells that have been activated in vitro with LPS and
IL-12 (R. M. Buchanan, B. P. Arulanandam, and D. W.
Metzger, unpublished observations). To directly assess the role of
IFN- in mediating IL-12 enhancement, we examined TI-2 responses in
GKO mice and found that enhancement of IgG2a by IL-12 was absent, while
IgG3 secretion appeared to be totally independent of IFN-. In our
earlier studies using TD Ags (13), we similarly found that IL-12 could
enhance the levels of some IgG isotypes in mice genetically deficient
in IFN- expression. Production of Ab in response to TD Ags was low
in GKO mice, but injection of IL-12 significantly enhanced IgG1 and
IgG2b levels. In fact, IgG1 levels in some cases were reconstituted by
IL-12 to the same levels seen in WT mice. However, the expression of
IgG2a and IgG3 with or without IL-12 treatment was completely dependent
on IFN-. The mechanisms involved in IL-12 enhancement in the absence
of IFN- are unknown but could involve other intermediary cytokines
or a direct stimulation of B cells. We recently showed that IL-12 binds
to the surface of activated human and murine B cells (16), which
suggests that postswitched cells can respond directly to IL-12, a
mechanism that would be consistent with results in both TD and TI Ag
systems. Finkelman et al. (24) previously reported that IFN- has the
ability to stimulate IgG2a secretion in mice. In our studies, however,
there were no differences in expression of IFN- mRNA in BALB/c,
BALB/c GKO, or CD3 mice after immunization with a TI Ag with or
without IL-12, indicating that IFN- did not play a role in the
observed enhancement of IgG2a by IL-12.
The findings reported here may be of immediate clinical significance, since S. pneumoniae and N. meningititis are the leading causes of pneumonia, meningitis, and otitis media, causing an estimated 7.5 million cases/year in the U.S. and more than 100 million cases/year worldwide. In addition, the currently available polysaccharide vaccines and conjugate vaccines under development are of limited value, particularly in the ability to stimulate isotype switching. The fact that IL-12 induces IgG2a Abs in response to vaccination is particularly exciting, since this is the primary isotype that mediates optimal complement fixation and opsonization in mice. While our results were obtained using CFA as an adjuvant, additional experiments showed that mice immunized with soluble DNP-Ficoll or Menomune and IL-12 also produced heightened IgG2a responses, indicating that IL-12 alone is an effective adjuvant for TI-2 Ab responses (R. M. Buchanan and D. W. Metzger, unpublished observations). We have also found similar effects of IL-12 after immunization with alum, the adjuvant approved for human use. Furthermore, preliminary analyses of Ab specificities to individual serotypes within the vaccine preparations indicate that high levels of IgG2a are induced against serotypes associated with the most problematic organisms. Robbins et al. (32, 33) have provided evidence that protection against encapsulated bacteria is associated with levels of circulating IgG Abs, suggesting that serum IgG2a Abs induced by IL-12 will be effective in mediating bacterial clearance. Thus, our results indicate that IL-12 will be useful as an adjuvant for increasing the protective capacity of current polysaccharide vaccines as well as conjugate vaccines as they become available.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dennis W. Metzger, Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington, Toledo, OH 43614-5806. E-mail address:
3 Abbreviations used in this paper: TI-2, T-independent type 2; TD, T-dependent; TNP, 2,4,6-trinitrophenyl; WT, wild type; KO, knockout; GKO, IFN- KO; HPRT, hypoxanthine phosphoribosyl transferase.
Received for publication February 23, 1998. Accepted for publication July 9, 1998.
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N. Gao, T. Dang, and D. Yuan IFN-{gamma}-Dependent and -Independent Initiation of Switch Recombination by NK Cells J. Immunol., August 15, 2001; 167(4): 2011 - 2018. [Abstract] [Full Text] [PDF]
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 A. Ortqvist Pneumococcal vaccination: current and future issues Eur. Respir. J., July 1, 2001; 18(1): 184 - 195. [Abstract] [Full Text] [PDF]
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S. T. Test, J. Mitsuyoshi, C. C. Connolly, and A. H. Lucas Increased Immunogenicity and Induction of Class Switching by Conjugation of Complement C3d to Pneumococcal Serotype 14 Capsular Polysaccharide Infect. Immun., May 1, 2001; 69(5): 3031 - 3040. [Abstract] [Full Text] [PDF]
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B. P. Arulanandam, R. H. Raeder, J. G. Nedrud, D. J. Bucher, J. Le, and D. W. Metzger IgA Immunodeficiency Leads to Inadequate Th Cell Priming and Increased Susceptibility to Influenza Virus Infection J. Immunol., January 1, 2001; 166(1): 226 - 231. [Abstract] [Full Text] [PDF]
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S. Muthukkumar, J. Goldstein, and K. E. Stein The Ability of B Cells and Dendritic Cells to Present Antigen Increases During Ontogeny J. Immunol., November 1, 2000; 165(9): 4803 - 4813. [Abstract] [Full Text] [PDF]
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T. Kieber-Emmons, B. Monzavi-Karbassi, B. Wang, P. Luo, and D. B. Weiner Cutting Edge: DNA Immunization with Minigenes of Carbohydrate Mimotopes Induce Functional Anti-Carbohydrate Antibody Response J. Immunol., July 15, 2000; 165(2): 623 - 627. [Abstract] [Full Text] [PDF]
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B. P. Arulanandam, J. N. Mittler, W. T. Lee, M. O'Toole, and D. W. Metzger Neonatal Administration of IL-12 Enhances the Protective Efficacy of Antiviral Vaccines J. Immunol., April 1, 2000; 164(7): 3698 - 3704. [Abstract] [Full Text] [PDF]
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A. R. Satoskar, L. M. Stamm, X. Zhang, M. Okano, J. R. David, C. Terhorst, and B. Wang NK Cell-Deficient Mice Develop a Th1-Like Response but Fail to Mount an Efficient Antigen-Specific IgG2a Antibody Response J. Immunol., November 15, 1999; 163(10): 5298 - 5302. [Abstract] [Full Text] [PDF]
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A. R. Satoskar, L. M. Stamm, X. Zhang, A. A. Satoskar, M. Okano, C. Terhorst, J. R. David, and B. Wang Mice Lacking NK Cells Develop an Efficient Th1 Response and Control Cutaneous Leishmania major Infection J. Immunol., June 1, 1999; 162(11): 6747 - 6754. [Abstract] [Full Text] [PDF]
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