ABSTRACT
Vaccines are an important public health measure for prevention and treatment of diseases. In addition to the vaccine immunogen, many vaccines incorporate adjuvants to stimulate the recipient's immune system and enhance vaccine-specific responses. While vaccine development has advanced from attenuated organism to recombinant protein or use of plasmid DNA, the development of new adjuvants that safely increase immune responses has not kept pace. Previous studies have shown that the complex mixture of molecules that comprise saline soluble egg antigens (SEA) from Schistosoma mansoni eggs functions to promote CD4+ T helper 2 (Th2) responses. Therefore, we hypothesized that coadministration of SEA with a Listeria vector HIV-1 Gag (Lm-Gag) vaccine would suppress host cytotoxic T lymphocyte (CTL) and T helper 1 (Th1) responses to HIV-1 Gag epitopes. Surprisingly, instead of driving HIV-1 Gag-specific responses toward Th2 type, we found that coadministration of SEA with Lm-Gag vaccine significantly increased the frequency of gamma interferon (IFN-γ)-producing Gag-specific Th1 and CTL responses over that seen in mice administered Lm-Gag only. Analysis of the functionality and durability of vaccine responses suggested that SEA not only enlarged different memory T cell compartments but induced functional and long-lasting vaccine-specific responses as well. These results suggest there are components in SEA that can synergize with potent inducers of strong and durable Th1-type responses such as those to Listeria. We hypothesize that SEA contains moieties that, if defined, can be used to expand type 1 proinflammatory responses for use in vaccines.
INTRODUCTION
Adjuvants are integral and critical aspects of many vaccines. In general, adjuvants activate innate immune cells either as a function of their particulate nature or by ligating pattern recognition receptors on antigen-presenting cells, increasing their overall antigen-processing capabilities and resulting in enhanced vaccine-specific immune responses (1, 2). For immunocompromised or low-responder populations, adjuvants in vaccines increase responder rates. For example, the adjuvant MF59 is incorporated into an influenza vaccine to enhance vaccine efficacy in elderly recipients (3, 4). By enhancing vaccine immunogenicity, adjuvants may allow for dose-sparing in vaccines, driving comparable immune responses with smaller amounts of antigen. Dose sparing is a critical consideration when urgent large-scale vaccination is needed and vaccine production is limited (5–7). Similarly, use of adjuvants may reduce the number of vaccinations required for any given vaccine, easing compliance issues and, in much of the world, logistical challenges. The lack of new, safe adjuvants has limited the potency and efficacy of many existing and new-generation vaccines. Thus, development of functional and safe adjuvants is essential and beneficial in multiple ways.
According to the Centers for Disease Control and Prevention, aluminum gels or aluminum salts are the only adjuvants currently licensed for human use in the United States (8). Alum adjuvants consist of precipitates of aluminum phosphate and/or aluminum hydroxide to which vaccine antigens/organisms are adsorbed (9, 10). Aluminum salts enhance humoral immunity (11) and trigger necrotic cell death and the release of the endogenous danger signal, uric acid (10, 12). Alum has been safely used for decades, though controversy remains concerning the mechanism of action of alum adjuvants and potential toxicity, emphasizing the need for better adjuvants.
Previously, we demonstrated that infection with the helminth parasite Schistosoma mansoni suppressed/eliminated the ability of recipient mice to generate T cell responses to a plasmid DNA HIV-1 vaccine (13, 14). Schistosomes induce CD4+ Th2 biasing and interleukin 10 (IL-10)-mediated immune suppression, primarily by deposition of parasite eggs into host tissues (15–21). Schistosome egg induction of anti-inflammatory responses is essential in reducing hepatic inflammation and is key for host survival (22–27). Similar to schistosome eggs, the saline homogenate of schistosome eggs, soluble egg antigen (SEA), also induces strong CD4+ Th2 responses (15, 22, 23, 28–31). In this regard, coadministration of SEA with a third-party protein antigen led to an increase in vaccine-specific Th2-type responses (32). Therefore, we hypothesized that addition of SEA to a Th1-driving Listeria vector HIV-1 Gag vaccine would suppress the induction of type 1, proinflammatory T cell responses. We tested this hypothesis and found, unexpectedly, that SEA functioned to enhance type 1 proinflammatory T cell responses, enhancing not only Lm-Gag vaccine efficacy, but also Lm-Gag Th1-type responses and expansion of Lm-Gag-specific T cell compartments.
MATERIALS AND METHODS
Biological materials.The vaccine we tested comprised an attenuated strain of Listeria monocytogenes expressing the HIV-1 IIIB Gag protein (Lm-Gag) (33). As a control vaccine, we used the same L. monocytogenes strain expressing the E7 oncoprotein of the human papillomavirus 16 (Lm-E7) (34). All Listeria vector vaccines were grown in BHI supplemented with streptomycin. Five- to 7-week-old female BALB/c mice were purchased from Harlan and Jackson laboratories, housed in specific pathogen-free conditions, and allowed to acclimate for 1 week prior to manipulation. All animal work was performed in accordance with institutional policy and approved by the institutional animal care and use committee.
Preparation of SEA.Approximately 7 weeks postinfection, we removed infected livers from Schistosoma mansoni (PR strain)-infected Swiss Webster mice provided by the National Institute of Allergy and Infectious Diseases (NIAID) schistosomiasis resource center. In addition, we infected female BALB/c mice by intraperitoneal injection of 100 to 150 S. mansoni cercariae. Parasite eggs were isolated from livers of infected mice and combined from both sources. SEA was prepared as previously described (22). The protein concentration of SEA was determined by both the Bradford and bicinchoninic acid (BCA) protein assays (Thermo Scientific).
Vaccination of mice.Six- to 8-week-old BALB/c mice were injected intraperitoneally with 30 μg of SEA or left naive. One week later, mice were primed intravenously with 103 CFU Lm-Gag vaccine, with or without 30 μg of SEA injected intraperitoneally or control Lm-E7 vaccine (matched CFU dose), or left unvaccinated. Mice were boosted in an identical manner 2 weeks after priming. Mice were sacrificed 2 weeks post last vaccination (wplv), unless otherwise indicated.
ELISpot.Splenocytes were harvested, plated at 300 k cells per well in gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay plates (BD) and incubated with complete media (RPMI 1640 supplemented with 10% fetal bovine serum [FBS], 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mM l-glutamine, 5 μM β-mercaptoethanol, and nonessential amino acids) in the presence of 20 μM specific cytotoxic T lymphocyte (CTL) peptide (H2-Kd-restricted AMQMLKETI from HIV-1 IIIB Gag protein), 20 μM irrelevant peptide (H2-Kd-restricted TYQRTRALV from influenza A/PR/8/34 nucleoprotein), 20 μM specific helper peptide (class II-restricted NPPIPVGEIYKRWIILGLNK from HIV-1 IIIB Gag protein), 20 μM listeriolysin O (LLO) peptide (H2-Kd-restricted GYKDGNEYI from L. monocytogenes listeriolysin O), or 1 μg/ml concanavalin A (ConA) (positive control). Peptides were synthesized by Biosynthesis, Inc., at >95% purity and reconstituted in dimethyl sulfoxide (DMSO) prior to dilution in media. After 20 h incubation, ELISpots were performed according to manufacturer's instructions and enumerated using an Immunospot analyzer (CTL).
Cytometric bead array.Splenocytes were harvested, plated at 1.5 million cells per well in 48-well plates, and stimulated with 20 μM peptide, 25 μg/ml SEA, or 1 μg/ml ConA or left unstimulated for 72 h. Cytokine levels in supernatant were measured using a Th1/Th2/Th17 cytometric bead array (CBA) kit according to the manufacturer's instructions (BD).
In vivo cytotoxic T lymphocyte assay.To prepare targets, splenocytes from naive BALB/c mice were fluorescently labeled with 1 μM or 10 μM green (Vybrant CFDA-SE cell tracer kit) fluorescent dye, according to the manufacturer's instructions (Invitrogen). Cells were then pulsed for 2 h with 20 μM irrelevant or CTL peptide, respectively. Targets were mixed and 12 million cells were injected intravenously per mouse. After 18 h, splenocytes were collected and analyzed for target recovery. The level of Gag-specific killing was calculated by the formula: % specific killing = [1 − (rnaive/rimmunized)] ×100, where r = % low carboxyfluorescein succinimidyl ester concentration (CFSElow) cells/% CFSEhigh cells (35).
Flow cytometry.To evaluate different T cell populations, splenocytes were incubated in the presence of phorbol myristate acetate (PMA), ionomycin, and GolgiStop for 6 h. Cells were stained with α-CD3ε, α-CD8a, and α-CD4 antibodies (BD), and then fixed. After membrane permeabilization, intracellular proteins were stained using α-IFN-γ, α-IL-4, α-IL-17A, α-IL-10, or α-FoxP3 antibodies (BD). Live cells (as indicated by LIVE/DEAD fixable dye; Invitrogen) were acquired and analyzed using a BD LSRII flow cytometer running FACSDiva (BD) or FlowJo (TreeStar) software.
Statistical analysis.Statistical analyses were performed using one-way or two-way analysis of variance (ANOVA) with the Newman Keuls or Bonferroni post hoc test, as detailed in the figure legends (Prism GraphPad Software, La Jolla, CA).
RESULTS
SEA coadministration with Listeria vector HIV-1 Gag vaccine enhances proinflammatory Gag-specific vaccine responses.To evaluate the effect of SEA on the immunogenicity of the Lm-Gag vaccine, three animal groups were immunized and analyzed for vaccine-specific responses, including naive unvaccinated, Lm-Gag vaccinated (Lm-Gag), and SEA coadministered Lm-Gag (SEA/Lm-Gag) groups. Two wplv, Gag-specific responses were determined by IFN-γ ELISpot after stimulation with Listeria LLO peptide or HIV Gag-specific CTL and helper peptides. As expected, cells from naive control mice did not respond to any of the peptides. Meanwhile, SEA coadministration significantly enhanced HIV Gag-specific vaccine responses toward immunodominant CTL (Fig. 1A) and helper (Fig. 1B) epitopes compared to the Lm-Gag-only group. There were no significant differences in responses to LLO in the Listeria vector-vaccinated groups (Fig. 1C), most likely due to Listeria's robust induction of LLO-specific cell-mediated responses that were saturated at high levels. An IL-4 ELISpot assay was also performed; however, there were minimal numbers of spots produced for all animal groups (see Fig. S2 in the supplemental material), which indicated that Lm-Gag does not induce Th2-type vaccine responses. In preliminary experiments, we injected mice with 50 μg of SEA (a higher dose) alone, and this did not induce any IFN-γ-producing CTL or T helper 1 responses to HIV-1 Gag (see Fig. S1A). Further, injection of 50 μg SEA alone did not generate CTL effectors capable of Gag-specific killing (see Fig. S1B). As these results showed SEA itself did not induce Gag-specific responses, we did not include an SEA-alone group in the remainder of our studies.
SEA enhances vaccine-specific IFN-γ-producing Th1 and CTLs. Splenocytes from naive and vaccinated mice were collected 2 wplv and immune responses to the immunodominant HIV Gag CD4+ helper (A) and CTL epitopes for HIV Gag (B) and LLO (C) were analyzed by IFN-γ ELISpot. Data were pooled from two similar, independent experiments and results from individual mice (n = 16) were plotted. Statistical analysis was performed using one-way ANOVA with Newman Keuls post hoc test. **, P < 0.01; ***, P < 0.001; ns, not significant.
To validate that the responses measured by ELISpot result from functional CTL effector cells, an in vivo CTL assay was performed. Lm-E7-vaccinated mice were included as the vector control group. As expected, the percent measurable CFSEhigh Gag-specific targets were similar in both the naive and Lm-E7 control groups (Fig. 2A), demonstrating that immunization with the Lm vector does not induce target killing. Mice vaccinated with Lm-Gag alone modestly reduced the level of Gag-specific targets, whereas the SEA/Lm-Gag group had a significant reduction of Gag-specific targets (Fig. 2A). As Gag-specific target recovery was higher than that of irrelevant targets, shown in histograms of naive mice, the actual level of Gag-specific killing by the SEA/Lm-Gag group was higher than the observed reduction in the histogram. The percentages of Gag-specific killing of all individual animals from each group were calculated (Fig. 2B). Killing of Gag-specific target cells in the Lm-Gag-vaccinated group was between 15 and 20%. The SEA/Lm-Gag group significantly increased specific killing to an average of 40%. These results show that addition of SEA to the Lm-Gag vaccine not only significantly enhanced total Gag-specific IFN-γ-producing T cells, but also increased functional Gag-specific CTL effector cells generated by Listeria vector vaccines.
SEA augments functional CTL response of vaccinated mice in vivo. Naive splenocytes were stained with CFSE at low and high concentrations and then pulsed with an irrelevant or immunodominant CTL epitope of HIV Gag, respectively. These targets were injected into naive or vaccinated mice. After 18 h, splenocytes were collected and analyzed for specific killing of targets using flow cytometry. (A) Representative histograms of irrelevant (left peak) and HIV Gag (right peak) targets recovered from each animal group are shown. (B) Representative data from three independent experiments are shown. Levels of specific killing in individual mice (n = 8) were plotted and the groups were compared by one-way ANOVA and Newman-Keuls post hoc test. ***, P < 0.001.
SEA coadministration significantly increases multiple Gag-specific T cell populations and induces vaccine-specific Th1-type cytokine production in vaccinated mice.To investigate total T cell compartment composition in Lm-Gag-vaccinated mice with or without SEA coadministration, splenocytes from naive and vaccinated groups were harvested 2 wplv and analyzed by flow cytometry after polyclonal stimulation with PMA and ionomycin. As shown in Fig. 3, the percentages of all analyzed T cell populations in mice vaccinated with Lm-Gag only were similar to those of naive mice. In contrast, addition of SEA to the Lm-Gag vaccine regimen significantly increased the quantity of Th1, Th2, and two different T regulatory cell (Treg) populations compared to both naive and Lm-Gag-only groups (Fig. 3A, B, D, and E). Th17 cells were unaltered by addition of SEA (Fig. 3C).
SEA increases multiple T cell populations when coadministered with Lm-Gag vaccine. Splenocytes from naive and vaccinated mice were collected 2 wplv and assayed by flow cytometry for Th1 (A), Th2 (B), Th17 (C), and Tregs (D and E) or IFN-γ-secreting CTLs (F). Percentages of each T cell population from individual mice (n = 8) were plotted and statistical differences between groups were analyzed by one-way ANOVA and Newman-Keuls post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
To further evaluate cytokine production, mouse splenocytes were stimulated with HIV Gag-specific peptides or Listeria LLO peptide and different cytokine levels were measured. As shown in Fig. 4, splenocytes from both vaccinated groups (Lm-Gag with or without SEA) produced proinflammatory cytokines, IL-2, IL-6, IFN-γ, and TNF-α in response to Gag helper peptide. Cells from the SEA/Lm-Gag group had significantly higher levels of these cytokines compared to levels from cells of the naive group (Fig. 4). Gag CTL peptide stimulation induced production of IL-6, IFN-γ, and TNF-α but not IL-2 in both the Lm-Gag and SEA/Lm-Gag groups compared to the naive group. There was an enhancement in IFN-γ and TNF-α production in cells from the SEA/Lm-Gag group compared to cells from the Lm-Gag-only group, though this effect was not significant. IL-6, IFN-γ, and TNF-α were also produced in vaccinated groups stimulated with the LLO peptide, with the SEA/Lm-Gag but not Lm-Gag-only group having significantly enhanced production of these cytokines compared to the naive group.
SEA increases vaccine-specific proinflammatory cytokine production in vaccinated mice. Splenocytes from naive and vaccinated mice were collected 2 wplv and stimulated with immunodominant CD4+ helper (square) and CTL epitopes for HIV Gag (circle) and LLO (triangle) for 72 h. Unstimulated controls (open circle) were included for each treatment. Cytokine production levels in supernatants were quantified by CBA. Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
As shown in Fig. S3 in the supplemental material, stimulation with the HIV Gag peptides or LLO peptide did not induce production of IL-17a, IL-4, or IL-10 in splenocytes from any of the groups. This finding is interesting as SEA induces strong CD4+ Th2 responses (15, 22, 23, 28–31). Thus, we examined cytokine production of naive and vaccinated splenocytes restimulated with SEA (see Fig. S4 in the supplemental material). As expected, SEA restimulation induced significant levels of IL-4 and IL-10 cytokines in the SEA/Lm-Gag group, with minimally detectable levels of IL-17A. Interestingly, SEA recall significantly increased levels of Th1-type proinflammatory cytokines, IL-2, IL-6, IFN-γ, and TNF-α in the SEA/Lm-Gag group compared to the naive and Lm-Gag-only groups.
Coadministration of SEA induces durable vaccine-specific responses.To evaluate the duration of vaccine-specific cell-mediated immune responses, splenocytes from vaccinated mice were analyzed by IFN-γ ELISpot at 12 and 20 wplv. As expected, at both 12 and 20 wplv, cells from the Lm-E7 control vaccinated mice did not respond to HIV Gag epitopes and responded only to the LLO peptide, again demonstrating that the Listeria vector does not drive responses to HIV Gag vaccine antigens/peptides (Fig. 5). Similar to our earlier observations, cells from SEA/Lm-Gag-vaccinated mice had significantly enhanced Gag-specific Th1 and CTL responses at both time points compared to cells from Lm-Gag-only mice (Fig. 5A and B). At 20 wplv, the Lm-E7 responses to LLO were significantly higher than the Lm-Gag group; however, at both time points, no significant difference in LLO response was observed between the groups vaccinated with Lm-Gag with or without SEA (Fig. 5C).
SEA improves longevity of vaccine-specific Th1 and CTL responses. Splenocytes from each animal group were collected at 12 (open symbols) and 20 wplv (filled symbols) and immune response to the immunodominant CD4+ helper (A) and CTL epitopes for HIV Gag (B) and LLO (C) were assayed by IFN-γ ELISpot. Data were pooled from two similar, independent experiments and results from individual mice (n = 8) were plotted. Statistical analysis was performed using one-way ANOVA with Newman Keuls post hoc test. *, P < 0.05; ***, P < 0.001.
DISCUSSION
Our starting hypothesis was that incorporation of schistosome SEA to an Lm-Gag vaccine would suppress vaccine-specific CTL and Th1-type responses generated by the Lm-Gag vaccine. However, results clearly demonstrate that coadministration of SEA with Lm-Gag significantly enhanced responses toward immunodominant HIV Gag-specific CTL and T helper 1 epitopes compared to mice vaccinated with Lm-gag alone (Fig. 1). Further analysis of CTL functionality showed that the level of HIV-1 Gag-specific killing was also enhanced in the SEA/Lm-Gag group (Fig. 2). SEA coadministration with Lm-Gag also induced long-lasting vaccine-specific responses (Fig. 5). Collectively, these results suggest that, completely opposite to our expectations, the addition of SEA to the Lm-Gag vaccine had a seemingly synergistic effect with the Lm-Gag vaccine in enhancing type 1, proinflammatory, vaccine-specific T cell responses.
Specifically, we saw a trend of SEA addition in enhancing production of proinflammatory cytokines, producing significantly higher levels of IL-6, IFN-γ, and TNF-α compared to cells from naive mice stimulated with vaccine antigens (Fig. 4). These cytokines were likely secreted by CTL and Th1 cells, which were increased in quantity by the addition of SEA to the Lm-Gag vaccine (Fig. 3A and D). Meanwhile, there was no significant production of vaccine-specific IL-4, IL-17A, and IL-10 cytokines in any of the groups (see Fig. S3 in the supplemental material). These results strengthen our overall observation that SEA significantly enhanced vaccine-specific Th1-type cytokine production when administered in concert with the Lm-Gag vaccine.
SEA is known to drive CD4+ Th2 biasing (15, 22, 23, 28–31), evidenced in our study by the significant increase in Th2 and Treg populations (Fig. 3) associated with significant production of IL-4 and IL-10 in SEA/Lm-Gag mice (see Fig. S4 in the supplemental material). However, levels of IL-6, IFN-γ, and TNF-α were also significantly increased, suggesting an ongoing induction of both Th1- and Th2-type responses when SEA is coadministered. Our observation that SEA functions to enhance Lm-Gag efficacy is exciting and unexpected. How Th2-driving SEA functions to enhance vaccine-specific proinflammatory T cell responses remains to be discovered, as previously reported studies have demonstrated that addition of SEA to protein vaccines enhances Th2-type responses (15, 32, 36).
While immunoparasitologists categorize SEA as a Th2-type and anti-inflammatory-driving mixture of molecules, there is evidence that SEA also induces proinflammatory responses in vivo. Immediately after the onset of egg laying by adult worms, the host immune responses to eggs are predominantly CD4+ Th1-type, coincident with an increase in total numbers of proinflammatory, classically activated macrophages (37). Several reports describe the ability of schistosome antigen(s) to ligate and initiate signaling via host pattern recognition receptors, including the Toll-like receptors (TLRs), driving proinflammatory responses (38–42). Recently, SEA was shown to drive production of proinflammatory mediators from primary human placental trophoblasts (43, 44). Thus, SEA does contain molecules that stimulate proinflammatory responses, and these appear to work in juxtaposition to SEA molecules that drive Th2-type and anti-inflammatory responses. The latter responses occur via SEA molecule ligation of C-type lectins or TLRs (41, 45).
As noted earlier, one possible explanation for our unexpected results is that we are observing a synergy between the proinflammatory components in SEA and the proinflammatory molecules expressed by the Listeria vector used in this study, as Lm-Gag alone did not drive the same levels of vaccine-specific Th1 and CTL responses as those seen in mice immunized with SEA/Lm-Gag. Based on the results of our study, we suggest that vaccination with the Th1 vaccine Lm-Gag and additional SEA injections amplifies proinflammatory vaccine-specific responses, overpowering the Th2-Treg response normally driven by SEA. This allows host vaccine-specific immune responses to polarize toward proinflammatory. The proinflammatory milieu amplifies classical activation of macrophages, promoting recruitment of immune cells and proinflammatory (Th1 and CTL) activation of vaccine-specific CD4+ and CD8+ T cells.
Though SEA has been studied extensively, most of the “immune-activating” components are not yet identified. SEA is a saline homogenate of S. mansoni eggs and therefore contains a myriad of different molecules and classes of compounds, including proteins, lipids, glycolipids, carbohydrates, and nucleic acids. Numerous studies describe the ability of SEA to induce Th2-type and anti-inflammatory responses as being dependent on carbohydrate components (32, 46–50). In contrast, reports on schistosome molecules that drive Th1 proinflammatory responses are scarce. Aksoy et al. has described double-stranded RNAs from schistosome eggs that activate dendritic cells via TLR3 (42), while Duraes et al. found schistosome tegumental molecules that induce IL-12 and TNF-α production in dendritic cells (38). The most recently described proinflammatory molecule from schistosomes is a glycolipid obtained from extracts of adult worms (40). Thus, our next priority is to chemically fractionate SEA and define the actual class(es) of molecules that are responsible for the proinflammatory enhancing activity reported in this study, as well as determining if these observations are limited to the coadministration of SEA with specific types of vector-based vaccines.
ACKNOWLEDGMENTS
Infected animals were provided by BRI via the NIAID schistosomiasis resource center under NIH-NIAID contract no. HHSN272201000005. These materials can be obtained by contacting BEI Resources. We also thank the University of Georgia—College of Veterinary Medicine Cytometry Core Facility.
This work is supported by grants NIH-AI-071883 and NIH-AI-078787 awarded to Donald A. Harn.
The authors C.B., L.S., and D.H. have no conflicts of interest. Y.P. discloses that she has a financial interest in Advaxis, Inc., a vaccine and therapeutic company that has licensed or has an option to license all patents from the University of Pennsylvania that concern the use of Listeria or listerial products as vaccines.
FOOTNOTES
- Received 5 March 2014.
- Returned for modification 22 April 2014.
- Accepted 23 June 2014.
- Accepted manuscript posted online 2 July 2014.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00138-14.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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