www.nature.com/npjvaccines ARTICLE OPEN Adjuvant composition and delivery route shape immune response quality and protective efﬁcacy of a recombinant vaccine for Entamoeba histolytica 1 2,3 2 1 2 2 2 Mayuresh M. Abhyankar , Mark T. Orr , Susan Lin , Mohammed O. Suraju , Adrian Simpson , Molly Blust , Tiep Pham , 2 4 4 5 1 2,3 Jeffrey A. Guderian , Mark A. Tomai , James Elvecrog , Karl Pedersen , William A. Petri Jr. and Christopher B. Fox Amebiasis caused by Entamoeba histolytica is the third leading cause of parasitic mortality globally, with some 100,000 deaths annually, primarily among young children. Protective immunity to amebiasis is associated with fecal IgA and IFN-γ in humans; however, no vaccine exists. We have previously identiﬁed recombinant LecA as a potential protective vaccine antigen. Here we describe the development of a stable, manufacturable PEGylated liposomal adjuvant formulation containing two synthetic Toll-like receptor (TLR) ligands: GLA (TLR4) and 3M-052 (TLR7/8). The liposomes stimulated production of monocyte/macrophage chemoattractants MCP-1 and Mip-1β, and Th1-associated cytokines IL-12p70 and IFN-γ from human whole blood dependent on TLR ligand composition and dose. The liposomes also demonstrated acceptable physicochemical compatibility with the recombinant LecA antigen. Whereas mice immunized with LecA and GLA-liposomes demonstrated enhanced antigen-speciﬁc fecal IgA titers, mice immunized with LecA and 3M-052-liposomes showed a stronger Th1 immune proﬁle. Liposomes containing GLA and 3M-052 together elicited both LecA-speciﬁc fecal IgA and Th1 immune responses. Furthermore, the quality of the immune response could be modulated with modiﬁcations to the liposomal formulation based on PEG length. Compared to subcutaneous administration, the optimized liposome adjuvant composition with LecA antigen administered intranasally resulted in signiﬁcantly enhanced fecal IgA, serum IgG2a, as well as systemic IFN-γ and IL-17A levels in mice. The optimized intranasal regimen provided greater than 80% protection from disease as measured by parasite antigen in the colon. This work demonstrates the physicochemical and immunological characterization of an optimized mucosal adjuvant system containing a combination of TLR ligands with complementary activities and illustrates the importance of adjuvant composition and route of delivery to enhance a multifaceted and protective immune response to amebiasis. npj Vaccines (2018) 3:22 ; doi:10.1038/s41541-018-0060-x INTRODUCTION E. histolytica infection starts with the ingestion of amebic cysts from contaminated food or water, which after excystation form Amebiasis, a diarrheal disease caused by the enteric protozoan trophozoites in the intestinal lumen. These adhere to epithelial parasite Entamoeba histolytica (a biodefense category B patho- cells through interaction of galactose and N-acetyl-D- gen), is responsible for ~50 million infections annually worldwide, galactosamine (Gal/GalNAc)-speciﬁc lectin with host Gal/GalNAc- causes traveler’s diarrhea, and disproportionately affects disad- 1,2 containing glycoconjugates. The trophozoites kill and ingest host vantaged populations. For example, cases of amebiasis disease 7,8 cells in a lectin-dependent manner. Blockade of lectin activity in Mexico from 2000–2010 dramatically outnumbered the with Gal or GalNAc prevents contact-dependent cytolysis. combined number of cases of dengue, tuberculosis, malaria, AIDS, Similarly, monoclonal antibodies against the 170-kDa lectin leishmaniasis, Chagas disease, and multiple other infectious heavy-chain subunit (hgl) completely eliminate the galactose- diseases. Moreover, the risk of death for infected young children speciﬁc adherence of E. histolytica trophozoites to colonic mucins is among the highest of all diarrheal diseases as determined in the in vitro. Vaccination with either parasite-puriﬁed native lectin or Global Enteric Multicenter Study. Although there is currently no recombinant lectin subunits containing parts of cysteine-rich vaccine available or in clinical testing, it is known that human extracellular domain of the heavy chain combined with potent immunity is associated with fecal IgA directed against amebic water-in-oil emulsion or cholera toxin adjuvants provides protec- 10–14 surface lectin and IFN-γ production from peripheral blood tion against amebiasis. Important for vaccine development, mononuclear cells in response to amebic lysate. Preschool the sequence of the hgl genes is nearly completely conserved in children from a Bangladesh cohort showing E. histolytica-speciﬁc isolates of E. histolytica from different continents. gut IgA and systemic IFN-γ response were protected from We have developed and manufactured recombinant subunit 5,6 recurrent infections. vaccine “LecA” (amino acids 578–1154 of hgl). It is a major target 1 2 Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia Health System, Charlottesville, VA, USA; IDRI, 1616 Eastlake Ave E, 3 4 5 Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA; 3M Drug Delivery Systems, 3M Center, 275-3E-10, St. Paul, MN, USA and TECHLAB, Inc., 2001 Kraft Drive, Blacksburg, VA, USA Correspondence: Christopher B. Fox (firstname.lastname@example.org) Received: 19 November 2017 Revised: 26 April 2018 Accepted: 3 May 2018 Published in partnership with the Sealy Center for Vaccine Development Adjuvant composition and delivery route shape MM Abhyankar et al. of cell-mediated and humoral response of immune individuals, its dipalmitoyl phosphatidylethanolamine with PEG MW of 750 sequence is conserved in every E. histolytica isolate ever examined, (DPPE-PEG750), and cholesterol. To determine the roles of GLA and it contains all of the antibody neutralizing epitopes on the and 3M-052, and whether both TLR ligands were necessary for 12,15,17 molecule. However, as a highly pure recombinant antigen, it optimal adjuvant activity, we generated liposomes containing GLA is poorly immunogenic without the aid of adjuvant technology. or 3M-052, or both. To determine the effect of PEG length on Since both IgA and IFN-γ are associated with amebiasis immunity stability and adjuvant activity, we manufactured liposomes in humans, we sought to develop an adjuvant formulation containing the same molar concentration of PEG MW 750 or amenable to parenteral and mucosal delivery with the potential MW 2000. We also evaluated the effects of different TLR ligand to enhance both mucosal antibody as well as Th1 cell responses. concentrations on liposome stability. We note here that, due to Activation of multiple TLRs more closely represents the mechan- reagent availability considerations, it was necessary to switch from isms activated by effective live-attenuated vaccines. TLR4 and the dipalmitoyl PEGylated lipid acyl conﬁguration (DPPE-PEG) TLR7/8 ligand combinations have shown particular promise in used in our previous work to distearoyl (DSPE-PEG) in the present strengthening Th1 immunity and inducing long-lived neutralizing work, unless noted otherwise. 19–21 antibodies. In previous work, we screened various adjuvant formulations for their ability to generate a Th1-indicating IgG2a/ Physicochemical stability IgG1 ratio in mice after subcutaneous administration with LecA, Liposome stability was monitored by dynamic light scattering thus identifying a PEGylated liposomal formulation containing a (particle size and size polydispersity), visual appearance, and combination of synthetic TLR4 (GLA) and TLR7/8 (3M-052) ligands reverse-phase HPLC (GLA and 3M-052 concentrations). The size as a promising candidate. Further evaluation demonstrated that polydisperstity index is a dimensionless number that indicates the this adjuvant formulation upregulated production of IFN-γ and IL- width of the particle size distribution, with values below 0.2 17A, cytokines shown to be protective in a mouse model, after 11,23 indicating a mostly monodisperse distribution. The initial particle subcutaneous administration with adjuvanted LecA. When size of liposomes containing PEG2000 was somewhat smaller than administered via alternating intranasal and subcutaneous routes liposomes containing PEG750, whereas the size polydispersity with LecA, the GLA-3M-052 liposome formulation generated values for PEG2000 liposomes were higher than for PEG750 functional fecal IgA and 34% protective efﬁcacy as assessed by liposomes (Fig. S1). Nevertheless, particle size and size poly- culturing of parasites from cecal contents in a mouse challenge dispersity values for PEG2000 and PEG750 liposomes showed little model of amebic colitis. change over 12 months when stored at 5 °C (Fig. S1). Likewise, the In the present work, we have focused on evaluating the visual appearance of the liposomes was consistently translucent physicochemical stability and immunological effects of liposome and homogeneous and did not change over time when stored at composition and immunization regimen, with the goal of 5 °C. When stored at 37 °C, liposomes containing PEG2000 showed optimizing adjuvant potency. Importantly, these studies demon- greater change in particle size and size polydispersity over time strate that a comprehensive mucosal and systemic immune response necessary for protection against amebic colitis can be compared to liposomes containing PEG750, which remained achieved by a simpliﬁed intranasal-only immunization regimen of remarkably stable (Fig. 1). Increasing the TLR ligand concentra- the adjuvanted vaccine. Furthermore, complementary roles for the tions by 5-fold appeared to result in slightly less stable PEG750 dual TLR ligands and the surprising impact of excipient structure liposomes for some compositions when stored at 37 °C, whereas on adjuvant biological activity and stability are elucidated. no difference was apparent with PEG2000 liposomes (Fig. S2). Together, these ﬁndings have resulted in an optimized vaccine Overall, the presence of one or both TLR ligands did not have composition suitable for advanced development as an amebiasis detrimental effects on the physical stability of the liposomes when vaccine candidate. Moreover, the adjuvant formulation developed stored for 12 months at 5 °C although a slightly higher could be applicable for other enteric diseases requiring mucosal polydispersity value was noted for the PEG750 liposome contain- antibodies and a Th1-type immune response proﬁle. ing high concentrations of both TLR ligands (Fig. S2). Although lower size polydispersity values are generally preferred, the differences in polydispersity values reported here would not be RESULTS expected to signiﬁcantly impact manufacturability of the liposome Liposome compositions compositions described. Formulations were analyzed by HPLC-CAD for GLA content after The liposome formulation developed in our previous work consisted of dipalmitoyl phosphatidylcholine (DPPC), PEGylated manufacture and the GLA concentration was within 20% of the AB 150 0.6 125 0.5 100 0.4 75 0.3 50 0.2 25 0.1 0 0 02468 10 12 0 2 4 6 8 10 12 Months at 37°C Months at 37°C Fig. 1 Effect of PEG length and adjuvant composition on liposome physical stability. a Particle size and b size polydispersity of PEGylated liposomes with indicated compositions and stored at 37 °C. Error bars represent the standard deviation from nine measurements (three measurements from each of three cuvettes) npj Vaccines (2018) 22 Published in partnership with the Sealy Center for Vaccine Development 1234567890():,; Parcle Diameter (Z-ave, nm) Size Polydispersity Index Adjuvant composition and delivery route shape MM Abhyankar et al. target value for all batches. After 12 months of storage at 5 °C, GLA and 0.04 mg/ml 3M-052 to mimic the planned mixing procedure content was generally stable and still within 20% of the target for the in vivo subcutaneous immunization studies described value (Fig. S3). Regarding 3M-052 content, six out of eight batches below (actual measured concentrations ranged from 0.088 to were within the target concentration after manufacture, whereas 0.118 mg/ml for GLA and 0.027 to 0.039 mg/ml for 3M-052). two batches showed low 3M-052 content, indicating that not all of Formulations had a translucent, homogeneous appearance before the 3M-052 was recovered during the manufacturing process in and after mixing with antigen. Overall, there was little (<15%) or these batches. After 12–16 months storage at 5 °C, the 3M-052 no change in particle size over 24 h after mixing with antigen content for all batches was within 20% of the initially measured whether stored at 5 °C or ambient temperature (Fig. S4), although values (Fig. S3), although the data should be interpreted with liposomes containing DSPE-PEG2000 showed a slightly greater caution since different HPLC methods were employed (see tendency to increase in size compared to the liposomes contain- Methods section). ing the shorter PEG length (DSPE-PEG750). Overall, the presence To further investigate the chemical stability of GLA and 3M-052, of GLA, 3M-052, or both agonists together did not appear to affect we subjected the GLA-3M-052-PEG2000 liposome batch contain- the particle size compatibility results. Likewise, size polydispersity ing 0.5 mg/ml GLA and 0.2 mg/ml 3M-052 to storage at elevated values changed little (Fig. S4). SDS-PAGE analysis of the antigen- temperatures (ambient, 37 or 60 °C). This batch had been stored adjuvant mixtures indicated no change in antigen primary for approximately 12 months at 5 °C prior to this study. When structure at any timepoint or storage condition over 24 h, stored at ambient temperature for 8 weeks, no change in GLA or regardless of liposome composition, with a uniform single band 3M-052 content was apparent (Fig. S3). At 37 and 60 °C, the rate of near the expected MW (Fig. S4). When component concentra- GLA loss appeared to be somewhat greater than that of 3M-052, tions were increased to support the intranasal immunization indicating that GLA may be more heat labile than 3M-052 in this composition (resulting in target concentrations of 0.25 mg/ml formulation, although loss of some GLA content would not LecA, 0.25 mg/ml GLA, and 0.1 mg/ml 3M-052 after mixing), necessarily be reﬂected in mouse immunogenicity readouts. liposome particle size increased immediately after mixing with Overall physicochemical stability data indicate that GLA-3M-052 LecA to >30% the size of the adjuvant before mixing (67–87 nm), liposomes could be a candidate for a controlled temperature with only small increase in size thereafter (18 h after mixing the chain scenario (i.e., stable for limited time outside of 2–8°C size was 95 nm); moreover, the SDS-PAGE proﬁle of LecA conditions). remained unchanged in the presence of the adjuvant (data not shown). Overall, these data indicate that the antigen-adjuvant mixture demonstrated acceptable short-term compatibility for up Compatibility with LecA antigen to 24 h at 5 °C or ambient temperature, thus supporting a “bedside Since the adjuvant is designed to be mixed with the antigen mix” approach immediately prior to immunization. immediately prior to immunization, it is important to establish the short-term (e.g., up to 24 h) physicochemical compatibility of the In vitro biological activity antigen-adjuvant mixture. To evaluate the short-term (≤24 h) compatibility of adjuvant and LecA antigen after mixing, stock We evaluated production of the chemokines monocyte chemoat- antigen was diluted in saline and then mixed in 1:1 volume ratio tractant protein 1 (MCP-1) and macrophage inﬂammatory protein 1- with adjuvant containing target concentrations of 0.1 mg/ml GLA beta (Mip-1β), and Th1-associated cytokines IL-12p70 and IFN-γ from A B Diluon factor Diluon factor C D Diluon factor Diluon factor Fig. 2 Cytokine production from human whole blood stimulated by liposomes containing GLA, 3M-052, both TLR ligands, or neither (empty). Stimulated whole blood was analyzed for production of Th1-associated cytokines a IL-12p70 and b IFN-γ, as well as chemokines c Mip-1β and d MCP-1. The x-axes represent serial dilutions with starting concentrations of 4 µg/ml for GLA and 1.5 µg/ml 3M-052. The y-axes represent concentrations of target analytes secreted post whole blood stimulation with error bars representing the standard error of the mean from three blood donors with each individual value averaged from duplicate wells Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 22 Adjuvant composition and delivery route shape MM Abhyankar et al. A B IgG1 IgG2a LecA + + + + + + + LecA + + + + + + + - + - GLA - + - + + - + GLA - + + + 3M-052 - - + - + + 3M-052 - - + + - + + - + + PEG750 - - - - + + + PEG750 - - - + PEG2000 - + + - - - PEG2000 - + + + - - - + LecA + + + + + + + GLA - + - + + - + 3M-052 - - + + - + + PEG750 - - - - + + + + - - PEG2000 - + + - Fig. 3 Biological activity of adjuvant formulation: importance of PEG length and complementary roles for GLA and 3M-052. Five mice per group were immunized three times with a 2-week interval between immunizations using a mixed intranasal/subcutaneous regimen. Mice were euthanized a week after third immunization and samples collected. a Stool supernatants were diluted 250-fold and anti-LecA IgA titer was determined by ELISA. b Plasma samples were diluted 100,000-fold and titers of anti-LecA IgG1 (black circles) and IgG2a (red circles) subtypes were determined by ELISA. c Intracellular IFN-γ levels were measured using ﬂow cytometry as described (negative values obtained after subtracting values from unstimulated cells were considered zero; the outlier test described below was conducted prior to this data transformation). Error bars represent standard error of the mean. For the analysis of the data in plot a, data were analyzed by one-way ANOVA with Sidak’s correction for selected comparisons. For the analysis of the data in plot b, data were log-transformed with a small offset if necessary and Sidak’s correction for selected comparisons was employed. All formulations in plot b elicited statistically increased (p < 0.05) IgG1 and IgG2a responses compared to antigen alone; for clarity in the ﬁgure these statistical bars are not shown. For the analysis of the data in plot c, data were analyzed by Welch’s one-way ANOVA with Games–Howell correction for multiple comparisons; two outliers were identiﬁed by Grubb’s test (α = 0.05) and statistical signiﬁcance was only achieved when they were excluded from analysis: the low value for the 3M-052 PEG2000 group (indicated by the hash mark in the plot), and a high value (11.8%) in the antigen alone control group (data point not shown in plot) human whole blood stimulated with DPPE-PEG750 liposome vehicle T cells (Fig. 3c). Thus, complementary roles for GLA and 3M-052 control, 3M-052 DPPE-PEG750 liposomes, GLA DPPE-PEG750 lipo- were elucidated, motivating inclusion of both TLR ligands in order somes, or GLA-3M-052 DPPE-PEG750 liposomes (Fig. 2). While to elicit a complete proﬁle of mucosal IgA and Th1-type cellular responses varied according to adjuvant dose, overall it was apparent immune responses. The pattern of complementary roles for GLA that 3M-052 liposomes induced higher IL-12p70, MCP-1, Mip-1β,and and 3M-052 was evident regardless of the PEG length that was IFN-γ compared to GLA liposomes. The combination of both ligands employed in the formulation. However, an overall increase in induced equivalent or additive innate chemokine responses, but magnitude of response was apparent due to inclusion of PEG2000 induced a synergistic innate Th1 immune response. compared to PEG750 (Fig. 3a–c). Subsequent studies employed the GLA-3M-052-PEG2000 composition. In vivo biological activity Effect of immunization route on response proﬁle To determine whether GLA and 3M-052 had complementary adjuvant roles and whether PEG length affected vaccine In order to promote generation of both mucosal and systemic immunogenicity, we immunized CBA mice using an alternating immune responses we employed an alternating intranasal and intranasal/subcutaneous/intranasal regimen as described in our subcutaneous immunization regimen in our previous study. To previous publication. Fecal IgA measured 1 week after the 3rd more fully assess the impact of immunization regimen in the immunization indicated that GLA-containing formulations elicited present work, we evaluated fecal IgA, plasma IgG2a:IgG1, and the highest IgA responses with no added beneﬁt due to 3M-052 cytokine (IFN-γ, IL-17A, and IL-2) production from LecA-stimulated inclusion (Fig. 3a). Conversely, plasma IgG2a titers measured splenocytes in mice immunized three times subcutaneously, 1 week after the 3rd immunization demonstrated that the highest intranasally, or alternating combinations of both routes. As responses were elicited by formulations containing 3M-052, expected, the intranasal-only regimen resulted in the highest especially with respect to the IgG2a:IgG1 ratio, with no added fecal IgA levels (Fig. 4a) with little response evident in the mice beneﬁt of GLA inclusion (Fig. 3b). Likewise, T cell responses immunized by the subcutaneous-only regimen. Surprisingly, the analyzed by intracellular cytokine staining indicated that 3M-052- intranasal-only regimen also resulted in the highest IgG2a containing formulations drove higher IFN-γ production from CD4 response (Fig. 4b) coupled with signiﬁcant LecA-speciﬁc cytokine npj Vaccines (2018) 22 Published in partnership with the Sealy Center for Vaccine Development Adjuvant composition and delivery route shape MM Abhyankar et al. A C * * B * IgG1 IgG2a * * Fig. 4 Effect of immunization route on response. Ten mice per group were immunized three times with a 2-week interval between immunizations via intranasal (IN) or subcutaneous (SC) route or a combination of these using GLA-3M-052-PEG2000 liposomes as an adjuvant. Samples were collected 1 week after 3rd immunization. a Stool supernatants were diluted 400-fold to determine LecA-speciﬁc IgA titer by ELISA. b Plasma samples were diluted 100,000-fold to determine titers of IgG1 (black circles) and IgG2a (red circles) subtypes. c Splenocytes were restimulated with LecA for 72 h and production of extracellular cytokines in the culture supernatants was determined by Luminex. Cytokine levels of the unstimulated samples were at the baseline (not shown). Error bars represent standard error of the mean. For clarity, statistical signiﬁcance vs. the adjuvant alone control groups is not represented but is detailed below. For the analysis of the data in plot a, Welch’s one-way ANOVA was employed with Games–Howell correction for multiple comparisons; all of the vaccine groups except for SC + SC + SC were statistically different (p < 0.05) from the adjuvant alone groups. For the analysis of the data in plot b, data were log-transformed and one-way ANOVA with Tukey’s correction for multiple comparisons was employed; all of the vaccine groups were statistically different (p < 0.05) from the adjuvant alone groups for both IgG2a and IgG1, and statistically signiﬁcant differences between vaccine groups represent IgG2a only since no statistical differences were found between vaccine groups for IgG1. For the analysis of the data in plot c, data were log-transformed with a small offset as necessary and Welch’s ANOVA with Games–Howell correction for multiple comparisons was employed (outliers were maintained since they did not affect statistical signiﬁcance); all vaccine groups were statistically different (p < 0.05) from the adjuvant alone groups for IFN-γ production, and the adjuvant alone IN + IN + IN was statistically different from the adjuvant alone SC + SC + SC; for IL-17A production, all vaccine groups except for SC + SC + SC were signiﬁcantly different (p < 0.05) from the adjuvant alone groups; for IL-2 production, all vaccine groups were signiﬁcantly different (p < 0.05) from the adjuvant alone groups production from splenocytes (Fig. 4c). While IFN-γ levels were amebiasis in humans or the mouse model is not known. Overall, highest and comparable between the regimens containing at least LecA + GLA-3M-052-PEG2000 liposomes administered intranasally one dose of intranasal immunization, IL-17A production was resulted in a complete immune response proﬁle considered favored by intranasal priming (Fig. 4c). Interestingly, subcuta- necessary for protective efﬁcacy in humans and/or mice, including neously primed mixed regimen showed signiﬁcantly more IL-2 mucosal IgA and Th1/Th17 cellular immunity. production compared to subcutaneous-only or intranasal-only regimens. Other than IL-2 production, no signiﬁcant beneﬁt was Protection studies apparent in the subcutaneous regimen or the more complicated alternating route regimens compared to the intranasal-only The protective efﬁcacy of LecA + GLA-3M-052-PEG2000 liposomes regimen. Moreover, the signiﬁcance of IL-2 in protection from was evaluated in the mouse model of amebic colitis developed by Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 22 Adjuvant composition and delivery route shape MM Abhyankar et al. e=13% e=38% e=42% * e=59% Fig. 5 Intranasal immunization protected from intestinal amebiasis in a mouse model. Mice were immunized three times using an intranasal or a subcutaneous regimen with a 2-week interval between immunizations. Mice were challenged intracecally with a virulent strain of E. histolytica 4 weeks past 3rd immunization. Mice were euthanized a week after challenge and cecal contents analyzed for a antigen load using ELISA and b live amebae by culture as a measure of sterile protection. Group sample size was 13–15 mice as speciﬁed in the Materials and Methods section. Both LecA-only and adjuvant-only control groups received intranasal immunization. Antigen load in plot a was log- transformed to generate normal distributions, and one data point was identiﬁed as an outlier based on Grubb’s test (the data point is indicated by the hash mark in the plot); the statistical signiﬁcance (p < 0.05) of the intranasal adjuvanted vaccine group vs. the controls was performed using Welch’s ANOVA with Games–Howell’s correction for multiple comparisons. Statistical signiﬁcance was only achieved if the outlier data point was not included in the statistical analysis. Error bars represent standard error of the mean. The efﬁcacies (e) in plot b were calculated with regard to the control groups, although statistical signiﬁcance (p < 0.05) was not achieved for any group after the Bonferroni multiple comparison correction was performed our lab. Mice were immunized three times intranasally or 052-PEG2000 liposomes appears to be improved over 46% seen subcutaneously and subsequently challenged intracecally with E. for LecA + GLA-3M-052-PEG750 liposomes administered using a histolytica 4 weeks following the last immunization. Mice were combination administration regimen reported previously. More- over, an efﬁcacy of 59% based on culture data shown by the euthanized a week after challenge and cecal contents analyzed for antigen load by ELISA as well as for the presence of live ameba by present adjuvanted vaccine appears to be an improvement over culturing in growth medium. Antigen load was reduced by over the 34% reported previously. While caution should be used in 80% (or 90% when outlier data point is excluded) in mice comparing different studies performed at different times, this immunized intranasally compared to control groups as deter- apparent improvement in efﬁcacy is likely due to the optimization mined from the mean cecal LecA values (Fig. 5a). Subcutaneous of liposome composition and immunization route. Recent work has indicated promising potential for TLR4/TLR7-8 immunization, in contrast, was not effective in reducing antigen 19,26–28 load. Furthermore, infection rate in intranasally immunized mice adjuvant formulations. TLR7-8 ligands have particular was reduced by 59% compared to animals receiving adjuvant relevance for vaccines designed for infants. Nevertheless, the increased complexity of such an approach requires demonstration alone, and by 42% compared to animals receiving antigen alone of added beneﬁt of each TLR ligand to justify further develop- (Fig. 5b). Subcutaneous route on the other hand reduced the 30,31 infection rate by 33 and 13% in comparison with adjuvant or ment. It was previously reported that intranasal administration LecA-only control group, respectively. Statistical signiﬁcance (p < of an aqueous nanosuspension of GLA in mice increased mucosal IgA and serum IgG titers, and Th1 and/or Th17 type cellular 0.05) was not achieved in the infection rate group comparisons responses, consistent with our results. Interestingly, in minipigs, (Fig. 5b) after the analysis was corrected for multiple comparisons, intranasal administration of a TLR7/8 ligand (R848) in combination whereas antigen load reduction was statistically signiﬁcant when the outlier data point (indicated by the hash mark in Fig. 5a) was with an aqueous nanosuspension of GLA resulted in reduced serum IgG and IgA responses compared to R848 alone. In removed. contrast, we found that GLA and 3M-052 demonstrated com- plementary roles, with GLA favoring production of mucosal IgA, DISCUSSION whereas 3M-052 drove plasma IgG2a and CD4 T cell IFN-γ The work described here was built on our previously published responses. Such discrepancies could be attributable to signiﬁcant studies to develop a novel protective recombinant adjuvanted differences in formulation compositions, the TLR7/8 ligand vaccine for amebiasis. Important advancements described in the structure (R848 vs. 3M-052), or the animal model. In our hands, present studies include demonstrating unique and complemen- while intranasal immunization with adjuvanted amebiasis vaccine tary roles for the two different TLR ligands employed (GLA and cleared more than 80% cecal antigen upon challenge, subcuta- 3M-052), optimization of the adjuvant formulation composition neous immunization failed to clear antigen. Thus, present data (replacing PEG750 with PEG2000) for enhanced immunogenicity, indicates a superior protection capability of the optimized and simplifying the immunization regimen to three intranasal intranasal formulation. We note here that a potential limitation administrations while maintaining mucosal antibody and systemic in interpretation of the data is the lack of antigen alone or Th1/Th17 immune responses. Together, these improvements adjuvant alone controls for the subcutaneous-only regimen. Also, resulted in enhanced protective efﬁcacy in the amebic colitis E. histolytica cystic form, the infective stage, has not been detected mouse model. Greater than 80% reduction in antigen load in mice. This limits our ability to assess the effect of vaccination on demonstrated here for intranasally administered LecA + GLA-3M- transmission using this model. npj Vaccines (2018) 22 Published in partnership with the Sealy Center for Vaccine Development Adjuvant composition and delivery route shape MM Abhyankar et al. The ability of the GLA-3M-052 liposome adjuvant to elicit model). Increased TLR4 expression in nasal tissue could explain mucosal IgA and systemic Th1/Th17-type immune responses is why intranasally administered TLR4 ligands such as GLA enhance important since these readouts are associated with protection mucosal IgA. Compared to GLA, the TLR7/8 ligand 3M-052 elicited from amebiasis. Moreover, vaccines against other enteric diseases a stronger Th1-type response in the present work, which is such as cryptosporidiosis may require a similar immune response consistent with previous results from our lab where an aluminum proﬁle for protective efﬁcacy. Evaluation of the GLA-3M-052 salt formulation of 3M-052 induced higher levels of antigen- liposome adjuvant with other vaccine antigens and disease speciﬁc IFN-γ, TNF, IL-2, and serum IgG2 compared to the same models will be required to determine suitability for other enteric formulation with GLA. Plasmacytoid dendritic cells produce high diseases. The potential practical beneﬁts of an intranasally levels of type 1 interferons in response to stimulation of TLR7 but 41,43,44 administered vaccine are many, including needle-free delivery, do not express TLR4, which could help explain the potent ability to administer without specialized medical training, and Th1-inducing activity of 3M-052 in the present work. Thus, GLA enabling of both mucosal and systemic immunity. Potential and 3M-052 may be activating distinct subsets of dendritic cells, challenges of intranasal administration in a global health setting resulting in complementary adjuvant activity. Indeed, one report include the difﬁculty in preparing multidose vials without risking found that TLR7 and TLR4 ligands stimulated different levels of IL- contamination between users, and overcoming safety concerns 12 from mouse splenic dendritic cells depending on the cell stemming from an intranasal inﬂuenza vaccine product previously phenotype. The production of cytokines and chemokines from licensed in Europe that was associated with an elevated risk of human whole blood stimulated in vitro by the adjuvant Bell’s palsy (potentially attributable to the E. coli heat-labile toxin formulations is consistent with the Th1-biasing proﬁle of 3M-052 adjuvant component). In this context, it is important to highlight liposomes evident in the in vivo studies. that the TLR ligands described in the present work are synthetic We note that the present work did not evaluate the effect of and highly pure molecules. Mice did not show any visible side liposomes in the absence of TLR ligands in the in vivo studies; effects upon immunization in terms of local reaction or behavior nevertheless, the in vitro assay demonstrated no stimulatory during the course of experiments, although comprehensive activity which is consistent with our previous work on PEGylated toxicity studies have not yet been performed. liposomes with a recombinant inﬂuenza antigen indicating Regarding the effects of PEG length, PEG750 liposomes minimal effects on Th1-type immune responses in mice by the appeared to be more physically stable under accelerated liposomal vehicle in the absence of TLR ligands. Additional conditions than PEG2000 liposomes, and PEG2000 liposomes questions remaining to be investigated include longevity of appeared to be more susceptible to particle size change upon immune response elicited and how it compares to previous mixing with antigen. Although both liposome compositions reports of GLA-based adjuvant formulations. demonstrated acceptable overall stability, the increased physical The present work represents a signiﬁcant step forward from our stability of the PEG750 liposomes compared to PEG2000 previously published results on dual TLR ligand liposome- liposomes is somewhat unexpected since longer PEG length is adjuvanted LecA by identifying complementary roles for GLA expected to provide greater steric stability to liposomes; therefore, and 3M-052, demonstrating enhancement of adjuvant potency by the mechanism for greater stability in the PEG750 liposomes is modulation of liposome PEG length, and maintaining mucosal unclear. Nevertheless, overall increased LecA-speciﬁc immune antibody and systemic Th1/Th17-type immune responses with a responses were evident with PEG2000 liposomes compared to simpliﬁed mucosal immunization regimen. The reﬁned liposome PEG750 liposomes. There is general agreement in the literature composition enhanced LecA-speciﬁc fecal IgA and Th1/Th17 that PEGylation, including PEG length and density, is correlated cellular immunity, resulting in 59% protective efﬁcacy against E. with reduced non-speciﬁc protein adsorption on liposomes and histolytica in mice immunized intranasally. GLA-3M-052-PEG2000 other nanoparticles (the “protein corona” effect), resulting in is a promising clinical adjuvant candidate since it is based on increased circulation times in the case of intravenous drug completely synthetic and biocompatible components that have delivery or faster drainage from the injection site to the lymph been produced under cGMP conditions and are in clinical testing node in the case of intramuscular, subcutaneous, or intranasal with other products. Thus, LecA + GLA-3M-052-PEG2000 repre- immunization. In particular, PEG2000 appears to be more effective sents an exciting vaccine candidate meriting further development 36,37 in this regard than shorter PEG lengths. Interestingly, Pozzi in preparation for potential clinical testing as a ﬁrst-in-human et al. reported that PEG2000 cationic liposomes showed less non- amebiasis vaccine candidate. speciﬁc protein bound to the liposome surface upon exposure to human plasma compared to liposomes decorated with PEG1000; moreover, a higher proportion of the proteins bound to the METHODS PEG2000 liposomes was comprised of opsonization-promoting Preparation of LecA antigen and adjuvant formulation classes of proteins such as IgG and complement, which are 38 LecA antigen was manufactured by TECHLAB, Inc. (Blacksburg, VA) as expected to enhance lymph node retention. Following this line described and stored in phosphate buffered saline at 5 °C. Glucopyr- of reasoning, it is possible that the PEG2000 liposomes in the anosyl lipid adjuvant (GLA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine present work allow more effective lymph node delivery and (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy retention following immunization due to the altered protein (polyethylene glycol)-750] (DSPE-PEG750), and 1,2-distearoyl-sn-glycero- corona composition. Further investigation along these lines is 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE- merited; for instance, delivery and uptake of PEG750 or PEG2000 PEG2000) were obtained from Corden Pharma (Liestal, Switzerland), Avanti liposomes is likely dependent on the density of the PEG layer. Polar Lipids (Alabaster, AL), or Lipoid LLC (Newark, NJ). 3M-052 was provided courtesy of 3M Drug Delivery Systems (St. Paul, MN). Cholesterol Finally, the tissue-speciﬁc expression patterns of TLRs and the and buffer salts were purchased from J.T. Baker (San Francisco, CA) or complex interplay between different innate pathways require Sigma (St. Louis, MO). further study to elucidate the mechanisms underlying the PEGylated liposome formulations were manufactured by combining complementary roles of intranasally administered TLR4 and DPPC, cholesterol, DSPE-PEG750 or DPPE-PEG2000, and 3M-052 and/or TLR7/8 ligands demonstrated here. For instance, ﬁve subsets GLA in chloroform. The lipid molar ratio was 9.8:5.7:0.8 (DPPC:cholesterol: of antigen presenting cells have been recently identiﬁed within PEGylated lipid). The organic solvent from the formulations was then the mouse nasal tissue, the majority of which appear to be of the evaporated for at least 12 h using a rotary evaporator. The lipid ﬁlm was + 40 CD11b phenotype. These cells express TLR4 in some mouse rehydrated in 25 mM ammonium phosphate buffer (pH ~5.7) and strains, whereas there is conﬂicting evidence regarding whether sonicated in a Crest Powersonic CP230D (Trenton, NJ) water bath at this phenotype expresses TLR7 (TLR8 is refractory in the mouse ~60 °C for up to 2 h until the lipid ﬁlm had separated from the sides of the Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 22 Adjuvant composition and delivery route shape MM Abhyankar et al. glass ﬂask. The formulation was then microﬂuidized at 30,000 psi for ~5 were prepared for each donor. Samples were incubated for 24 h at 37 °C/ passes with a recirculating water chiller set at 10 °C. Liposomes were 5% CO in a humidiﬁed incubator and plasma supernatants assayed by ﬁltered through a 0.8/0.2 µm double membrane polyethersulfone ﬁlter and ELISA for the selected cytokines (Mip-1β [R&D Systems, catalog #DY271]; IL- stored at 5 and 37 °C. Smaller batches of liposomes employed in the 12p70 [eBioscience, catalog #88-7126-86]; IFN-γ [eBioscience, catalog #88- in vitro human whole blood assay were made with DPPE-PEG750 instead 7316-86]; MCP-1 [eBioscience, catalog #88-7399-88]). of DSPE-PEG750, hydrated with phosphate-buffered saline instead of ammonium phosphate buffer, and were manufactured by sonication only Immunizations (without microﬂuidization). It should be noted that liposomes intended for Four to 6-week-old male CBA/J mice (Jackson Labs) were used for all the subcutaneous administration were manufactured to contain lower experiments. Typically 5 μg of LecA was mixed with liposome formulations concentrations of GLA and 3M-052 compared to liposomes intended for intranasal administration due to administration volume considerations containing appropriate TLR ligand/s (5 μg GLA; 1–2 μg 3M-052) prior to (100 vs. 20 µL, respectively), whereas phospholipid concentration was immunizations. The volume was brought up with saline to 100 μl for constant regardless of administration route. Thus, liposomes intended for subcutaneous or to 20 μl for intranasal immunization. Subcutaneous subcutaneous administration were manufactured at 7.2 mg/ml DPPC, injections were given in the neck region, whereas intranasal immunizations 1.2 mg/ml DSPE-PEG750 or 2.2 mg/ml DSPE-PEG2000, 2.2 mg/ml choles- were carried out under anesthesia and 10 μl of antigen-adjuvant mix was terol, 0.1 mg/ml GLA, and 0.04 mg/ml 3M-052; liposomes intended for administered per nostril. A 2-week interval was maintained between the intranasal administration were manufactured at these same phospholipid consecutive immunizations for all the regimens, which consisted of three and cholesterol concentrations with 0.5 mg/ml GLA and 0.2 mg/ml 3M-052. immunizations total. For immunogenicity experiments, 5 mice per group All liposomes were mixed 1:1 by volume with LecA/saline prior to were employed for the data shown in Fig. 3, whereas 10 mice per group immunization. were used for the data shown in Fig. 4; for the protective efﬁcacy experiment, 13–15 mice per group were analyzed (13 for the antigen-only control group, 14 for the adjuvant-only control group, and 15 for the Physicochemical characterization of adjuvant formulation adjuvanted vaccine groups). Each mouse experiment was performed once. The concentrations of GLA and 3M-052 were determined by reverse-phase Sample size was determined based on our previous experience evaluating HPLC with charged aerosol detection. The HPLC method was essentially as adjuvant formulations in this model with the aim of using a minimum described previously. For 12–16 month timepoints of 3M-052-containing number of animals possible to generate meaningful results in immuno- formulations, the concentration of 3M-052 was determined by reverse- genicity and efﬁcacy. Animal experiments were not randomized. phase HPLC with UV detection (321 nm); the HPLC method in this case consisted of ﬁrst diluting the formulation 10-fold in isopropanol containing 0.5% triﬂuoroacetic acid and then eluting the sample on a Zorbax Bonus Measurement of immunogenicity RP column (4.6 × 150 mm, 3.5 µm) at 45 °C with a 1 ml/min ﬂow rate while Mice were euthanized a week after ﬁnal immunization for immunogenicity employing a gradient mobile phase consisting of Mobile Phase A (0.1% experiments (stool IgA, plasma IgG, cytokines). Antibody titers were triﬂuoroacetic acid in water), Mobile Phase B (methanol), and Mobile Phase measured by ELISA using 96-well plates coated with 0.5 μg LecA per well. C (isopropanol) as follows: initial (85% A, 15% B), 2.5 min (60% A, 40% B), Horseradish peroxidase-conjugated secondary antibodies speciﬁc for IgA 17.5 min (5% A, 40% B, 55% C), 22.0 min (5% A, 40% B, 55% C), 22.5 min (catalog #1040-05) and IgG (catalog #1071-05 and 1081-05) subtypes were (85% A, 15% B). Liposome particle size and size polydispersity were purchased from Southern Biotechnology and diluted as per the evaluated using a Malvern Instruments (Worcestershire, UK) Zetasizer manufacturer’s instructions. Antibody units were calculated using standard Nano-S or Nano-ZS. The formulation was diluted 10-fold or 100-fold in curves. For the measurement of extracellular cytokines, 250,000 spleno- ultrapure (18.2 MΩ) water in a 1.5 ml polystyrene disposable cuvette. In general, three separate cuvettes were prepared for each sample and size cytes were re-stimulated with 50 μg/ml LecA in 200 μl RPMI1640 complete measurements were then made three times for each cuvette. The data medium (supplemented with 10% heat-activated fetal bovine serum shown represent a single batch of formulation with the indicated (Gemini Labs), 2.05 mM L-Glutamine, 10,000 U/ml Penicillin, and 10 mg/ml composition; multiple batches of each composition were not Streptomycin) for 72 h and undiluted supernatant analyzed by a multiplex manufactured. suspension array system using Luminex beads (Bio-Plex 200, Bio-Rad). For intracellular cytokine staining, 1 × 10 splenocytes were cultured in complete RPMI medium and re-stimulated with 10 μg/ml LecA for 22 h. Physicochemical compatibility of antigen and adjuvant after Anti-CD28 and Golgiplug (BD Biosciences) were added for the last 12 h of mixing stimulation, followed by surface staining with anti CD4 PerCp-Cy5.5 The short-term (≤24 h) physicochemical compatibility of the antigen- (BioLegend, catalog #100433) and anti CD44 APC-Cy7 (BioLegend, catalog adjuvant mixture was evaluated by monitoring particle size, visual #103027). Cells were permeabilized and stained with anti IFN-γ PE-Cy7 appearance, and antigen primary structure immediately after mixing and (BioLegend, catalog #505825) and run on a BD FACSCalibur (BD 4 and 24 h after mixing, with mixtures stored at 5 °C and ambient Biosciences). The data were analyzed with FlowJo software (Fig. S5). All temperature. The antigen was ﬁrst diluted in saline to 0.1 mg/ml, and antibodies were purchased from BD Biosciences. subsequently mixed in 1:1 volume with the liposomal adjuvant formula- tion. Particle size was measured as described above except that one cuvette was prepared instead of three. SDS-PAGE was conducted by Culture conditions and challenge experiments mixing 50 µL sample with 50 µL 4× reducing sample buffer and 100 µL Virulent trophozoites, originally derived from HM1:IMSS (ATCC) and passed 20% SDS. The sample was heated at 90 °C for 5 min and stored at −20 °C, sequentially through mice were maintained in a trypsin-yeast extract-iron after which it was reheated for 1 min at 90 °C and loaded onto a (TYI-S-33) medium supplemented with 2% Diamond vitamins, 13% heat- polyacrylamide gel with Tris-glycine running buffer for 65 m at 180 V, inactivated bovine serum and 100 U/ml penicillin plus 100 μg/ml followed by staining with Coomassie blue. Gels were processed in parallel streptomycin (Invitrogen). Mice from immunized and control groups were and derive from the same experiment, which was performed once challenged intracecally 4 weeks after the ﬁnal boost with 2 million according to a well-established protocol performed regularly in our lab. trophozoites in 150 μl medium following laparotomy. Mice were eutha- nized a week after the challenge. Cecal contents were suspended in 1 ml Human whole blood assay PBS, 300 μl were cultured anaerobically in TYI-S-33 broth at 37 C for 5 days Heparinized human blood samples were collected from three normal, and 200 μl used for antigen load ELISA. Culture results were scored in a healthy male donors using standard phlebotomy techniques. The sample blinded manner. Vaccine efﬁcacy was calculated as 100 × 1−(% of size of three donors was not powered to detect statistical differences but vaccinated mice with infection)/(% of sham mice with infection). rather to represent general trends. This experiment was performed once using a well-established assay regularly employed in our lab. All Fecal antigen detection formulations were serially diluted in two-fold steps in irrigation-grade Fecal antigen in the cecal contents was detected using the E. HISTOLYTICA saline, then 20 μl of each serial dilution was added to 96-well tissue-culture grade U-bottom plates, followed by the addition of 180 μl of whole blood, II ELISA kit (TechLab Inc., catalog #T5017). An optical density at 450 nm of providing a ﬁnal well volume of 200 μl with a top formulation ≥0.05 above the negative control was considered positive. A standard concentration of 4 μg/ml GLA and/or 1.5 µg/ml 3M-052. Duplicate wells curve was generated using puriﬁed LecA. npj Vaccines (2018) 22 Published in partnership with the Sealy Center for Vaccine Development Adjuvant composition and delivery route shape MM Abhyankar et al. Statistical analysis ADDITIONAL INFORMATION Statistical analyses were performed using Graph Pad Prism software and Supplementary information accompanies the paper on the npj Vaccines website Microsoft Excel (www.biostathandbook.com/welchanova.xls). While not (https://doi.org/10.1038/s41541-018-0060-x). pre-established prior to the experiments, the statistical criteria below were followed consistently, and take into account normality, similarity of Competing interests: M.A.T. and J.E. are employees of 3M and 3M-052 is an asset of variance, outliers, and corrections for multiple comparisons. Proportions of 3M’s. W.A.P. is a consultant for TechLab, Inc. and in addition receives royalties for infected and uninfected mice from challenge trials were analyzed using amebiasis diagnostics that are donated in their entirety to the American Society of one-sided Fisher’s exact test with Bonferroni’s correction for multiple Tropical Medicine and Hygiene. K.P. is an employee of TechLab, Inc. and amebiasis comparisons. Antigen load, antibody titers, and cytokine levels were diagnostics are an asset of TechLab’s. C.B.F., M.T.O., S.L., A.S., M.B., T.P., and J.A.G. are analyzed by ﬁrst testing for normality using the D’Agostino & Pearson employees of IDRI and GLA is an asset of IDRI’s. The remaining authors declare no normality test (α = 0.05). If all groups passed the normality test, they were competing interests. then tested for similarity of variance using the Brown–Forsythe test (p < 0.05). If variances between groups was similar, they were analyzed using Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims one-way ANOVA with Tukey’s or Sidak’s correction for multiple compar- in published maps and institutional afﬁliations. isons as indicated. If data did not pass the normality test, the data were log-transformed and re-tested for normality. If the normality test was still not passed, Grubb’s test for outliers (α = 0.05) was employed to remove REFERENCES outliers. If original or log-transformed data did not pass the similarity of 1. 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If material is not included in the improving nanoparticle-based drug and gene delivery. Adv. Drug Deliv. Rev. 99, article’s Creative Commons license and your intended use is not permitted by statutory 28–51 (2016). regulation or exceeds the permitted use, you will need to obtain permission directly 37. Pozzi, D. et al. Effect of polyethyleneglycol (PEG) chain length on the bio-nano- from the copyright holder. To view a copy of this license, visit http://creativecommons. interactions between PEGylated lipid nanoparticles and biological ﬂuids: from org/licenses/by/4.0/. nanostructure to uptake in cancer cells. Nanoscale 6, 2782–2792 (2014). 38. Moghimi, S. M. The effect of methoxy-PEG chain length and molecular archi- © The Author(s) 2018 tecture on lymph node targeting of immuno-PEG liposomes. Biomaterials 27, 136–144 (2006). npj Vaccines (2018) 22 Published in partnership with the Sealy Center for Vaccine Development
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