We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3–18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.
Recently, we showed that a CTL response could protect against mucosal viral transmission, but only if the CTL were present locally in the mucosa; systemic CTL were not sufficient (1). This observation accords with the facts that protection against rectal SIV challenge strongly correlates with the activity of MHC class I- restricted env-specific CTLs in the gut lamina propria (2) and, in addition, that HIV-1-specific CD8+ lymphocytes were detected in the cervical mucosa of HIV-exposed, seronegative prostitutes in Nairobi (3). These findings suggest that while HIV-specific CTL are important in mediating protective immunity against HIV-1 infection (4, 5, 6, 7, 8), the CTL may need to be present in the genital and gastrointestinal mucosa to prevent natural HIV transmission, which typically occurs through a mucosal barrier (9, 10). Thus, a successful vaccine may need to induce mucosal CTL (1, 2, 3, 11, 12), as well as mucosal Abs including secretory IgA (13, 14), for protection against mucosal exposure to HIV/SIV (15, 16, 17).
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.
Materials and Methods
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.
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).
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 × 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.
Immune cells from SP and PP were cultured for 7 days at 5 × 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 × 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 × (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 × 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).
Synergistic effect of mucosal administration of GM-CSF and IL-12 on the induction of CD8+ CTL responses in PPs and SP, and on resistance to mucosal viral transmission
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.
To determine the relevance to protection, we next asked whether the enhanced mucosal CTL response achieved with the synergistic combination of GM-CSF and IL-12 given with the peptide vaccine was accompanied by an increased level of resistance to viral challenge through a mucosal route. In previous studies we found that induction of mucosal HIV-specific CTL was dependent on endogenous IL-12 (18) and that local resistance to mucosal transmission of a recombinant vaccinia virus expressing HIV gp160 could be enhanced by local mucosal coadministration of IL-12 with the peptide vaccine (1). However, protection was marginal after only two immunizations rather than four (compare Fig. 2, 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. 2,B). This reduction was equivalent to that seen after four immunizations without cytokines (Fig. 2,A). 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.
Induction of mucosal (PP) and systemic (SP) CTL by IR immunization with synthetic HIV peptide construct plus LT(R192G)
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. 3,A). 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. 3,B) 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).
LT(R192G) without IL-12 is as effective as CT plus IL-12 as an adjuvant for HIV-peptide vaccine mucosal induction of CTL
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.
In view of these results and our earlier results showing that GM-CSF synergized with IL-12 in the presence of CT in enhancing the CTL response after just two mucosal immunizations with the peptide vaccine, we hypothesized that perhaps when we used LT, GM-CSF would be sufficient to enhance the response after just two immunizations. To test this hypothesis, we immunized groups of five mice with peptide and either CT or LT(R192G) with or without GM-CSF. The results show that after just two immunizations, when CT is used as adjuvant, even the addition of GM-CSF gives only a marginal CTL response, whereas when LT(R192G) is used, the addition of GM-CSF produces substantial enhancement (Fig. 5). 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.
To further explore the role of endogenous IL-12 in the induction of mucosal CTL when LT(R192G) or CT was used as a mucosal adjuvant, we immunized one group of mice with HIV peptide vaccine plus LT(R192G) and a second group of mice with HIV peptide vaccine plus CT. We treated one subgroup of mice receiving each adjuvant i.p. 1 day before and after each immunization with 0.5 mg monoclonal anti-IL-12 Ab (clone C17.8, a kind gift of G. Trinchieri, The Wistar Institute, Philadelphia, PA) (50). We found that such treatment led to an almost total inhibition of the generation of CTL at both mucosal and systemic sites when CT was used as a mucosal adjuvant, but only modest inhibition of mucosal CTL when LT(R192G) was used as the adjuvant (Fig. 6). This result confirms the hypothesis that endogenous IL-12 is more limiting when CT is used as adjuvant than when LT(R192G) is used.
Production of IL-4 and IFN-γ 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-γ. Immune cells from PPs were cultured at 5 × 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-γ 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. 7,A). 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-γ 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. 7 B). 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-γ. We conclude that LT(R192G) induces a more favorable cytokine response than CT in that the former does not inhibit IL-12 production.
Protection against mucosal challenge with virus after IR immunization with LT(R192G) as mucosal adjuvant
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 × 104 PFU), consistent with the marginal difference in IFN-γ production (Fig. 7), despite the enhancement in CTL. This may reflect the importance of IFN-γ 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.
In previous studies, we showed that CTL responses following systemic (s.c.) immunization with HIV vaccines are enhanced in a synergistic fashion by the inclusion of two different cytokines in the vaccine emulsion (51).3 We now report for the first time that CTL responses elicited by an HIV peptide vaccine (containing adjuvant) delivered via the mucosa (i.e., IR delivery) is also synergistically enhanced by the coadministration of two cytokines, GM-CSF and IL-12. In particular, we show that such coadministration results in the induction of high cytotoxic responses after two immunizations rather that the usual four necessary for responses in the absence of the two cytokines and, in addition, these responses are associated with protection against infection through a mucosal route by a recombinant vaccinia virus encoding HIV epitopes. Protection in this system has been previously shown to be completely dependent on CD8+ T cells (1). Furthermore, the recombinant vaccinia virus used for the challenge studies, unlike HIV-1 itself, does not incorporate gp160 into the virus particle, and therefore is not susceptible to neutralizing Abs specific for gp160 (52). Rather, the gp160 is expressed only in the infected cell. For both these reasons, it is likely that the enhancement of protection by the combination of GM-CSF and IL-12 is due to the corresponding enhancement in induction of CD8+ CTL, but the contribution of other potential mechanisms cannot be excluded.
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-γ-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.
We thank Dr. GiorgioTrinchieri for his kind gift of anti-IL-12 Ab, and Drs. Brian Kelsall and Graham LeGros for critical reading of the manuscript and helpful suggestions.
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.
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.