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Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;
Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA 70112; and
Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines and mucosal adjuvants added to mucosal vaccines are two factors that can potentially augment the mucosal CTL response elicited by a given mucosal vaccine. The role of cytokines in the regulation of the mucosal CTL response and resistance to mucosal viral transmission is largely unknown, although we have recently shown that the CTL response elicited by an HIV peptide vaccine using cholera toxin (CT)2 as adjuvant was IL-12 dependent and that the addition of rIL-12 to the CT adjuvant greatly enhanced both mucosal Ag-specific CTL and protection against mucosal challenge (1, 18). In addition, in studies of peptide vaccines emulsified in IFA with cytokines given by a s.c. route, we discovered that GM-CSF and IL-12 synergistically enhanced the CTL response (19). Such synergy was also demonstrated by Iwasaki et al. (20) in the context of DNA immunization, but it has not been addressed for a mucosal vaccine. On this basis, we wished to determine whether mucosal vaccination to induce mucosal CTL with a peptide vaccine would also be synergistically enhanced by the addition of GM-CSF with IL-12.
Although CT is a powerful mucosal adjuvant, it causes potentially serious side effects (21). In addition, CT has been shown to down-regulate IL-12 production (22). Therefore, it was of interest to determine the adjuvanticity of a mutant form of Escherichia. coli labile toxin (LT), which should lack these detrimental qualities (23, 24, 25, 26, 27, 28). Accordingly, we examined the mutant LT (designated as LT(R192G); see Refs. 29 and 30) that was constructed using site-directed mutagenesis to create a single amino acid substitution in the proteolytically sensitive region of the biologically active domain (A subunit, residue 192 Arg to Gly; see Refs. 29 and 31). This mutation rendered the toxin insensitive to trypsin activation and consequently greatly diminished its toxicity without altering the intrinsic adjuvanticity characteristic of the native molecule (29, 31). A number of reports published recently have evaluated the efficacy of LT(R192G) as an effective mucosal adjuvant (29, 32, 33, 34, 35). However, the cellular responses measured were systemic, and none of these studies evaluated the induction of CTL in the mucosa itself. In addition, phase I and phase II clinical trials have been conducted with LT(R192G), either alone or in combination with a killed, whole-cell Campylobacter vaccine (36, 37). In the current study, we examined LT(R192G) for its ability to enhance mucosal CTL elicited by a peptide vaccine, both alone and in combination with cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Frederick Cancer Research Center (Frederick, MD). Mice used in this study were 6–12 wk old. Mice were maintained in a specific pathogen-free microisolator environment.
Immunizations
The peptide vaccine used, PCLUS3-18IIIB, (KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK) (38) consists of the multideterminant helper segment PCLUS3 (KQIINMWQEVGKAMYAPPISGQIR) (39) from the CD4 binding domain of HIV-1 IIIB, combined with the immunodominant CTL epitope presented by H-2Dd in BALB/c mice, called P18 IIIB RIQRGPGRAFVTIGK) (40), from the V3 loop of the IIIB strain of HIV-1, as a single continuous peptide. Mice were immunized with two or four doses of PCLUS3-18IIIB (50 µg/mouse for each immunization) on days 0 and 7 or 0, 7, 14, and 21 in combination with CT (7 µg/mouse), LT(R192G) (7 µg/mouse), or wild-type E. coli LT (7 µg/mouse) by intrarectal (IR) administration (18). When cytokines were included, these and the peptide vaccine were mixed together with N-[1-2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), a cationic lipofection agent (Boehringer Mannheim, Indianapolis, IN). Controls received peptide plus DOTAP without cytokines. For s.c. immunization, IFA was used (38, 41, 42).
Cell purification
Two weeks after the last vaccine dose, Ag-specific T cells were isolated from Peyer’s patches (PP) and the spleen (SP). The PPs were carefully excised from the intestinal wall and dissociated into single cells by using collagenase type VIII, 300 U/ml (Sigma, St. Louis, MO) as described (18, 43). Our data showed that most PP CD3+ T cells isolated from normal mice were CD4+, while CD3+CD8+ T cells were less frequent. Further, collagenase did not alter the expression of CD3, CD4, or CD8 on splenic T cells treated with this enzyme. Cells were washed and then layered onto a discontinuous gradient containing 75% and 40% Percoll (Pharmacia Biotech, Uppsala, Sweden). After centrifugation (4°C, 600 x g, 20 min), the interface layer between the 75% and 40% Percoll was carefully removed and washed with incomplete medium. The SP were aseptically removed, and single-cell suspensions were prepared by gently teasing them through sterile screens. The erythrocytes were lysed in Tris-buffered ammonium chloride, and the remaining cells were washed extensively in RPMI 1640 containing 2% FBS.
CTL assay
Immune cells from SP and PP were cultured for 7 days at 5 x 106 per milliliter in 12-well culture plates with 1 µM synthetic CTL epitope peptide P18-I10 (RGPGRAFVTI) (or simply I10), the minimal epitope of P18 IIIB (44, 45, 46), in complete T cell medium (RPMI 1640 containing 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and 5 x 10-5 M 2-ME). On day 3 we added 10% Con A supernatant-containing medium (T-stim; Collaborative Biomedical Products, Bedford, MA) as a source of IL-2 (1). Cytolytic activity of CTL lines was measured by a 4-h assay with 51Cr-labeled targets. Two different cell lines were used as target cells: 1) 15-12 cells (BALB/c 3T3 fibroblasts transfected with HIV-1IIIB gp160 and endogenously expressing HIV gp160), compared with 18 Neo BALB/c 3T3 fibroblasts transfected with NeoR alone as a control, or 2) P815 targets tested in the presence or absence of P18-I10 peptide (1 µM). For testing the peptide specificity of CTL, 51Cr-labeled P815 targets were pulsed for 2 h with peptide at the beginning of the assay. The percent specific 51Cr release was calculated as 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). Maximum release was determined from supernatants of cells that were lysed by addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells (18, 47).
Recombinant vaccinia challenge and determination of viral titer in the ovary
Fourteen days after the last immunization, mice were challenged IR with 2.5 x 107 PFU of vaccinia virus-expressing gp160IIIB (vPE16). Six days after the challenge, the mice were killed and ovaries were removed, homogenized, sonicated, and assayed for vPE16 titer by plating serial 10-fold dilutions on a plate of BSC-2 indicator cells, staining with crystal violet, and counting plaques at each dilution. The minimal detectable level of virus was 100 PFU (1, 48, 49).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously we showed that the ability of a synthetic HIV vaccine
construct given by a systemic route to induce a CTL response is
synergistically enhanced by the coadministration of IL-12 and GM-CSF
(19).3
Therefore, we asked whether the vaccine-induced mucosal CTL response is
similarly enhanced synergistically by IL-12 and GM-CSF when the vaccine
and cytokines are given together by IR administration. Accordingly, we
immunized BALB/c mice IR twice (on days 0 and 7) with the HIV-peptide
construct PCLUS3–18IIIB and CT with or without GM-CSF plus recombinant
murine (rm) IL-12, all together in DOTAP. Two weeks later (day 21) we
examined the CTL response in the PPs (Fig. 1
, left) and SP (Fig. 1, right). Because lamina propria CTL responses have always
mirrored the PP patch responses in this system (1, 18),
they were not measured in these studies. By immunizing mice only twice
instead of four times (as in previous studies), we hoped to be better
able to detect synergy. We found that two IR immunizations with HIV
peptide constructs plus mucosal adjuvant did not induce a significant
HIV-specific CTL response in mucosal sites and systemic lymphoid
tissues under these conditions. Similarly, after just two
immunizations, the HIV peptide construct plus GM-CSF alone was not
effective in the induction of mucosal CTL, and the HIV peptide
construct plus IL-12 alone induced a very modest HIV-specific CTL
response (Fig. 1). In contrast, the peptide vaccine in combination with
GM-CSF and IL-12 together induced a substantial CTL response, markedly
greater than that induced by the peptide alone or in combination with
either of the cytokines singly, in both compartments (Fig. 1). Thus,
GM-CSF synergizes with IL-12 in the induction of mucosal
CD8+ CTL by an HIV peptide vaccine construct
applied to the mucosal surface with CT adjuvant. Use of such synergy
between GM-CSF and IL-12 for induction of mucosal
CD8+ CTL may be a valuable strategy for mucosal
vaccine development.
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, A
and B). To address this question of synergy of cytokines in
the induction of protective mucosal immunity, we immunized BALB/c mice
twice IR on days 0 and 7 with the HIV peptide vaccine and CT together
with GM-CSF and IL-12 mixed with DOTAP. As control groups, we IR
immunized mice twice with the HIV peptide vaccine and CT plus either
GM-CSF or IL-12 singly, or without cytokine, in DOTAP. Two weeks after
the last immunization we challenged the mice with the recombinant
vaccinia virus vPE16, which expresses HIV-1 gp160, and determined virus
titers in the ovaries, where this virus preferentially replicates, 6
days later. The mice immunized only twice with the HIV vaccine and
treated with GM-CSF plus IL-12 showed a reduction of 4 logs in virus
titer compared with unimmunized mice (Fig. 2B). This
reduction was equivalent to that seen after four immunizations without
cytokines (Fig. 2A). In contrast, mice IR immunized with the
HIV peptide construct plus GM-CSF alone or rmIL-12 alone had virus
titers that were not significantly lower than those of unimmunized mice
(Fig. 2). Thus, GM-CSF synergized with IL-12 for enhancing both the
mucosal CTL response and resistance to mucosal transmission of virus.
Given that protection against mucosal viral challenge in this system
has been found to be completely CD8+ T cell
dependent and dependent on CTL in the mucosa (1), we
believe that the enhanced resistance is likely due to enhanced CTL
activity, although we cannot completely rule out a contribution from
other mechanisms.
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In previous studies we found that whereas CT is not essential for IR peptide vaccine induction of mucosal CTL, it can significantly increase the generation of CTL, both in the intestinal lymphoid tissues and in systemic lymphoid organs (18). However, CT causes severe diarrhea in humans and thus cannot be used as an adjuvant for a human vaccine (21). For this reason, we examined the ability of the genetically detoxified E. coli LT designated LT(R192G), which is a less toxic adjuvant than CT, to induce HIV-specific CTL responses in mucosal sites after IR immunization (29, 30).
Accordingly, we immunized BALB/c mice IR with the HIV peptide in the
presence of LT(R192G) on days 0, 7, 14, and 21 and studied the mice
either 2 wk later (day 35) or at 6 mo for memory CTL in PP and SP.
Control groups consisted of mice immunized IR with HIV peptide and CT
or wild-type LT. The level of mucosal CTL responses 2 wk after the last
immunization with LT(R192G) was at least as high as, or even higher
than, the CTL level induced when CT was used as mucosal adjuvant (Fig. 3A). In addition, LT(R192G)
plus HIV peptide vaccine induced mucosal CTL as well as the wild-type
LT (data not shown). Memory CTL were long-lasting, as they were
detected at least 5 mo after the last immunization in both mucosal
(Fig. 3B) and systemic sites (data not shown). Thus, the
synthetic HIV peptide vaccine PCLUS3–18IIIB administered IR with
LT(R192G) can induce strong HIV-specific CTL responses in PPs of the
intestine (Fig. 3) as well as in the SP (data not shown).
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We know that CT can significantly down-regulate the production of
IL-12 and expression of IL-12 receptors in vivo and in vitro, whereas
LT(R192G) was much less potent in decreasing IL-12 in vitro
(22). Therefore, we hypothesized that if LT(R192G) did not
similarly down-regulate IL-12, the greater response induced by peptide
vaccine with LT(R192G) might be due to less limiting IL-12 production.
To test this hypothesis, we asked whether exogenous rmIL-12 can
up-regulate the level of mucosal CTL responses when CT or LT(R192G) are
used as a mucosal adjuvant. We immunized a first group of mice with HIV
peptide vaccine (50 µg/mouse/immunization) with CT (7 µg/mouse)
with or without rmIL12 (1 µg/mouse), all together in DOTAP
(1), and a second group of mice with the peptide vaccine
with LT(R192G) (7 µg/mouse) (29) plus rmIL-12, also all
in DOTAP, using the same protocol as in previous studies. As control
groups, we immunized mice with the peptide vaccine plus either CT or
LT(R192G) without rmIL-12, also in DOTAP. Without rmIL-12 in the
formulation, the vaccine given with LT(R192G) induced higher CTL
responses in the PPs than did the corresponding vaccine given with CT
(Fig. 4). Further, exogenous murine IL-12
significantly up-regulated the level of mucosal CTL induced by HIV
peptide vaccine in the presence of CT (Fig. 4), but only to the level
achieved with LT(R192G) in the absence of exogenous IL-12. Furthermore,
when LT(R192G) was used as the mucosal adjuvant, additional IL-12 did
not induce a further increase in the response, in contrast to CT (Fig. 4). Thus, LT(R192G) alone induced levels of CTL more like those induced
by CT plus rmIL-12.
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). This result
supports the concept that exogenous IL-12 is less essential when
LT(R192G) is used as adjuvant instead of LT and shows the synergy
between adjuvant and cytokine in optimizing the immune response.
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). This result confirms
the hypothesis that endogenous IL-12 is more limiting when CT is used
as adjuvant than when LT(R192G) is used.
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by Ag-specific PP T cells after
mucosal immunization with HIV-peptide construct plus LT(R192G) or CT
One of the mechanisms that can explain the differences in mucosal
adjuvant effect on HIV-specific CTL is the cytokine profile (Th1 vs
Th2) produced by immune T cells after IR immunization with HIV peptide
constructs. If, as shown above, LT(R192G) allows more endogenous IL-12
production than CT, we hypothesized that it should also be associated
with less IL-4 production. To test this hypothesis, we immunized mice
with HIV peptide vaccine in the presence of LT(R192G) or CT on days 0,
7, 14, and 21 and then determined the capacity of T cells obtained from
mice on day 35 to produce IL-4 or IFN-. Immune cells from PPs were
cultured at 5 x 106 per milliliter in
12-well culture plates in complete T cell medium with PCLUS3–18IIIB
(to evaluate the T cell response to both CD4 helper and CD8 CTL
epitopes) or P18-I10 (to evaluate only the latter). One week after such
in vitro stimulation, the culture supernatants were collected and
assayed for IFN- and IL-4. We found that after in vitro stimulation
with PCLUS3–18IIIB, the Ag-specific PP T cells produced less IL-4 when
the mice were immunized with peptide vaccine and wild-type LT or
LT(R192G) than when the mice were immunized using CT as adjuvant (Fig. 7A). Furthermore, although
coadministration of rmIL-12 significantly down-regulated the production
of IL-4 by immune T cells in PPs when either adjuvant was used, the
levels induced when CT was used with IL-12 remained higher than those
induced with LT or LT(R192G) as adjuvant, even without IL-12. Results
similar to those shown in PPs were also found in SP cells after mucosal
immunization with HIV peptide vaccine plus CT or LT(R192G) with or
without IL-12 (data not shown). In contrast, we found that IR
immunization with HIV vaccine plus LT(R192G) induced concentrations of
IFN- produced by HIV peptide-specific CD8+ CTL
in PPs and SP in vitro similar to those induced by immunization with
the peptide vaccine plus CT, and there was little or no effect of
incorporation of IL-12 in the adjuvant (Fig. 7B). Therefore,
the lower levels of IL-4 with LT(R192G) do not reflect simply a weaker
overall cytokine response, but rather a selective reduction in IL-4 in
contrast to IFN-. We conclude that LT(R192G) induces a more
favorable cytokine response than CT in that the former does not inhibit
IL-12 production.
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Given that LT(R192G) was at least as effective as CT as adjuvant
in inducing mucosal CTL, we asked whether vaccination with peptide plus
LT(R192G) would protect against challenge with recombinant vaccinia
virus- expressing HIV-1 gp160. Therefore, we immunized BALB/c mice IR
with the HIV peptide vaccine plus LT(R192G) or CT as mucosal adjuvant
and then challenged the mice IR 2 wk after the last immunization with a
recombinant vaccinia virus-expressing the envelope protein HIV-1 IIIB
gp160. Six days after the challenge, the mice were killed, and the
ovaries were removed and assayed for vaccinia titer (because 6 days
after infection with vaccinia, the ovaries are the site of the highest
titer of virus). We found at least a 4-log reduction of viral titer in
the ovaries of mice that had received an IR immunization with HIV
vaccine plus LT(R192G), comparable to that achieved with CT as adjuvant
(Fig. 8). The reduction in PFU for
LT(R192G) was only marginally greater than that for CT (4.3 vs 5.2
x 104 PFU), consistent with the marginal
difference in IFN- production (Fig. 7
), despite the enhancement in
CTL. This may reflect the importance of IFN- in protection against
this infection, but other protective mechanisms may play a role as
well. Thus, protection from mucosal viral challenge after IR
immunization with HIV peptide plus LT(R192G) was as effective as after
immunization with the HIV peptide vaccine plus CT as mucosal
adjuvant.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of the cytokines to enhance mucosal responses indicates
several features of mucosal immunization that bear on the feasibility
of using this type of immunization in eliciting protection against
mucosal pathogens such as HIV. First, they show that the introduction
of proteins into the rectum of an animal allows the delivery of
the proteins into lymphoid tissues associated with the rectal mucosa
without major loss of biologic activity. This probably is due to the
fact that the microenvironment of the rectum does not contain a large
concentration of proteolytic enzymes that would otherwise destroy the
proteins before they could reach their intended targets. Second, they
suggest that biologically active proteins can readily cross the
epithelium of the rectal mucosa and interact with the rich network of
lymphoid cells that reside in the rectal submucosa. In the latter
regard, while the rectal mucosa does not contain large lymphoid
aggregations such as PPs, it does contain smaller lymphoid cell
aggregations that can give rise to immune responses. Third, and most
germane to the present findings, they show that the two cytokines used
in these studies act by different mechanisms to enhance immune
responses. Based on our prior studies showing that GM-CSF in an
emulsion adjuvant delivered s.c. results in increased APC activity in
the draining lymph nodes,3 it is likely that the
mucosally administered GM-CSF led to enhanced presentation of
administered peptide vaccine in local mucosal lymphoid tissues. In
contrast, IL-12 is known to be a key cytokine for the optimal
differentiation of CTL precursors and IFN--producing cells and thus
is likely to be a promotor of CTL responses in the mucosal tissues,
particularly in situations in which the adjuvant used has the effect of
inhibiting IL-12 production (see further discussion below). On these
bases, it is reasonable to propose that GM-CSF and IL-12 enhance CTL
responses elicited by the mucosal vaccine by addressing different
aspects of CTL development in the mucosal lymphoid tissue.
The separate enhancing activities of GM-CSF and IL-12 were conditioned
by the nature of the mucosal adjuvants used to facilitate the response
elicited by the IR HIV peptide vaccine. The activities of the two
toxin-type mucosal adjuvants employed in these studies are not yet
fully understood. Although both interact with the cell membrane via GM
gangliosides, CT has a high capacity to activate adenylate cyclase and
cause the release of cAMP, whereas the mutant E. coli LT
(LT(R192G)) has a greatly diminished capacity to activate adenylate
cyclase and cause the release of cAMP. Thus, as discussed below,
LT(R192G) is a far less toxic compound than is CT and can be used in
humans. These differences between the two adjuvants may also explain
their somewhat different adjuvant effects, because it has been shown
that CT inhibits IL-12 production and causes skewing of T cell
responses in the direction of Th2 T cell differentiation (Fig. 7). In
addition, CT suppresses production of IL-2 in both resting and
activated Th1 T cells whereas it has little effect on the proliferation
of Th2 T cells or on the latter’s production of IL-4. LT, in contrast,
leads to a more balanced T cell response in that it enhances both Th1
and Th2 T cell differentiation. This is seen in a recent study by
Morris et al. (29), in which it was shown that intranasal
immunization of BALB/c mice with HIV-1 gp160 with LT(R192G) adjuvant
resulted in Th1 and Th2 T cell cytokine production as well as mucosal
IgG and IgA responses (29). In addition, LT(R192G)
enhanced a systemic CTL response to a HIV-1 envelope peptide that
presumably required IL-12 production (29). However, these
studies did not examine mucosal CTL or directly compare CT with
LT(R192G).
The different results obtained with CT and LT(R192G) in these studies must be viewed in the context of an expanding body of work on bacterial enterotoxins as mucosal adjuvants. The most important barrier to the use of these toxins as vaccine components is that they stimulate a massive lumenal secretory response: as little as 5 µg of purified CT administered orally was sufficient to induce significant diarrhea in human volunteers, while ingestion of 25 µg of CT elicited a 20-liter cholera-like purge (53). A number of attempts have been made to alter this toxicity, most of which have focused on eliminating ADP-ribosyltransferase activity of the A subunit and induction of cAMP, which is responsible for the secretory response. The majority of these efforts have involved the use of site-directed mutagenesis to change amino acids associated with the molecular crevice thought to be the site of NAD binding and catalysis. Indeed, a number of studies have shown that replacement of any amino acid involved in NAD-binding and catalysis reduces both ADP-ribosyltransferase activity and toxicity in a variety of biological assay systems (26, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65). The adjuvanticity of some of these mutants has been tested in animal models using a variety of coadministered Ags (26, 59, 65, 66, 67). In addition, Lycke et al. (59) showed that extinction of ADP-ribosyltransferase activity by exchange of K for E at position 112 in LT also resulted in loss of cAMP activation, and of adjuvant activity as well, suggesting that ADP-ribosylation and induction of cAMP are essential for the adjuvant activity of these molecules. These studies appeared to establish a causal linkage between adjuvanticity and enterotoxicity; that is, the accumulation of cAMP responsible for net ion and fluid secretion into the gut lumen appeared difficult to separate from adjuvanticity.
However, a somewhat different picture emerged when LT(R192G) was developed using an alternate approach for dissociation of enterotoxicity from adjuvanticity (31). Like other bacterial toxins that are members of the A-B toxin family, both CT and LT require proteolysis of a trypsin-sensitive bond to become fully active. In these two enterotoxins, that trypsin-sensitive peptide is subtended by a disulfide interchange that joins the A1 and A2 pieces of the A subunit. In theory, if the A1 and A2 pieces cannot separate, A1 may not be able to find its target (adenylate cyclase) on the basolateral surface of the enterocyte or may not assume the conformation necessary to bind or hydrolyze NAD. To exploit these possibilities, Dickinson and Clements (31) constructed by site-directed mutagenesis an LT containing a single amino acid substitution of G for R at position 192 within the disulfide- subtended region of the A subunit separating A1 from A2. This mutation rendered the toxin insensitive to trypsin activation and consequently greatly diminished its toxicity without altering the intrinsic adjuvanticity characteristic of the native molecule. A number of reports published recently have evaluated the efficacy of LT(R192G) as an effective mucosal adjuvant (32, 33, 34, 35, 36), as assessed by Ab responses or systemic cellular responses. In one such study, a randomized, placebo-controlled dose-escalating phase I safety study, LT(R192G) was shown to be safe in volunteers at doses up to 100 µg while retaining immunogenicity comparable to native LT (36). In a follow-up study, LT(R192G) was shown to be a safe and effective adjuvant for a killed Campylobacter jejuni whole-cell vaccine administered orally to volunteers (37). However, these studies did not measure mucosal CTL.
Uncertainty as to the role of cAMP in the adjuvanticity of CT and LT remains in part because of the variability in Ags, routes of administration, and evaluation techniques employed in different laboratories. To address this question directly, Cheng et al. (30) provided a side-by-side comparison of LT, active-site mutants of LT, the protease-site mutant LT(R192G), and recombinant LT B subunit for the ability to induce specific, targeted immunologic outcomes using a single, defined Ag (tetanus toxoid, TT) following two different mucosal routes of immunization (intranasal or oral). They showed that although LT-B given intranasally can induce serum IgG anti-TT, it is not able to induce any significant level of T cell response to TT. Furthermore, while all active-site mutants examined were able to induce Ag-specific Ab responses when administered intranasally with TT, only native LT, LT(A69G), and LT(R192G), which retained the ability to induce production of cAMP, were able to elicit a sufficient T cell response to measure TT-specific production of Th1 and Th2 cytokines. A similar correlation was seen after oral immunization. The ability of different forms of LT to induce Th1 and Th2 responses correlated with their ability to induce cAMP.
In studies consistent with those of Cheng et al. (30), Giuliani et al. (68) compared two active-site mutants, LT(S63K) and LT(A72R), for the ability to function as adjuvants in the intranasal immunization of mice with OVA. In these studies, LT(A72R), which retains some level of enzymatic activity, was a better adjuvant than LT(S63K), which lacks any detectable enzymatic activity. In a follow-up study, Ryan et al. (69) compared LT(S63K) and LT(A72R) as adjuvants for intranasal immunization of mice with an acellular pertussus vaccine. Relevant to the current study, they found that LT(S63K), which lacks enzymatic activity, induced a mixed Th1/Th2 response, whereas LT(A72R), which retains a low level of enzymatic activity, induced a predominantly Th2 response, especially at a low dose.
The data of Cheng et al. (30) and Ryan et al. (69) make it clear that different mutants of LT have different properties that vary depending upon the nature of the mutation and the route of delivery. Whereas many of these mutants suffice for induction of Ag-specific Ab responses after intranasal immunization, to induce Abs by oral immunization, or to induce the best T cell cytokine responses, requires native LT or mutants that retain some cAMP activity. Thus, for example, in contrast to the intranasal immunization results noted above, when administered orally, only those LT mutants that retain some cAMP activity elicit Th1-type responses.
The data presented here on the use of cytokines to enhance mucosal
responses in the presence of HIV peptide vaccine employing CT and
LT(R192G) can be rationalized on the basis of the above considerations.
They show that vaccine adjuvanted by CT results in a response that is
skewed toward Th2 T cell differentiation relative to vaccine adjuvanted
by LT(R192G) in that: 1) the CT-adjuvanted response results in a
greater induction of IL-4 than the LT(R192G)-adjuvanted response (in
the presence or absence of exogenous IL-12); similar results of skewing
of T cell responses to Th2 type were reported by other groups
(70, 71); 2) exogenous IL-12 enhances the CT-adjuvanted
response but not the LT(R192G)-adjuvanted response; and 3) treatment of
mice with anti-IL-12 has a more negative effect on the
CT-adjuvanted response than the LT(R192G)-adjuvanted response,
indicating that endogenous IL-12 production is more limiting in the
former situation than the latter. Taken together, these results
establish that LT(R192G) or similar types of adjuvants that do not have
a down-regulating effect on IL-12 production are inherently more
suitable as adjuvants for vaccines whose main purpose is the induction
of a Th1 T cell response and the elaboration of IL-12-dependent CTLs.
Such adjuvants not only reduce the need to incorporate IL-12 into the
immunization protocol to compensate for untoward adjuvant effects on
IL-12 production, but they also offer the opportunity to further
enhance the intrinsic Th1 effects of the adjuvant and thus further the
induction of CTL responses either in magnitude or in duration. In
support of this conclusion, when we brought the study full circle and
asked whether GM-CSF would synergize with LT(R192G) without exogenous
IL-12 to induce an enhanced CTL response after just two immunizations,
we found that indeed it did (Fig. 5), whereas with CT as adjuvant,
IL-12 was needed in addition to GM-CSF (Fig. 1). This result emphasizes
the fine interplay and even potential synergy between adjuvants and
cytokines in optimizing the immune response. However, it remains
possible that IL-12 will further enhance CTL responses or Th1 cytokine
responses even when LT(R192G) is used, for example, in the case of
fewer immunizations, or would lead to more prolonged CTL memory.
Studies to address these issues are in progress.
In summary, we show here that coadministration of GM-CSF and IL-12 has a synergistic effect on the ability of an HIV peptide vaccine delivered by a mucosal route to increase the level of mucosal CTL induction and to provide protection against mucosal infection by a recombinant vaccinia virus that expresses HIV epitopes. In addition, we show that such synergy is to some extent dependent on the endogenous cytokine milieu created by the vaccine adjuvant, in that an adjuvant such as CT that ordinarily skews responses in the direction of Th2 T cell differention is more greatly helped by the addition of IL-12 than is an adjuvant such as LT(R192G) that does not have this skewing effect. In contrast, we show that GM-CSF still complements LT(R192G) and thus enhances the CTL response even in the absence of exogenous IL-12, in contrast to the case of CT. Thus, it becomes apparent that LT(R192G) is not only a less toxic adjuvant than is CT but is also an adjuvant that has less need for the administration of a compensating cytokine. On this basis, we are currently using LT(R192G) in our studies of peptide HIV vaccines in primates. More generally, we conclude that adjuvant effects on endogenous cytokines alter the need for exogenous cytokines and therefore influence their ability to synergize. Correct pairing of adjuvants and exogenous cytokines can optimize the immune response.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CT, cholera toxin; DOTAP, N-[1-2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; LT, labile toxin; IR, intrarectal; PP, Peyer’s patch; SP, spleen; TT, tenanus toxoid; rm, recombinant murine.
3 J. D. Ahlers, I. M. Belyakov, S. Matsui, and J. A. Berzofsky. Mechanisms of cytokine synergy essential for vaccine protection against viral challenge. Submitted for publication.
Received for publication June 15, 2000. Accepted for publication August 31, 2000.
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synergize with IL-12 to enhance induction of CTL. J. Immunol. 158:3947.[Abstract]
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