3M-052 as an adjuvant for a PLGA microparticle-based Leishmania donovani recombinant protein vaccine

Qian Wang,1,2 Meagan A. Barry,1,2 Christopher A. Seid,1,2 Elissa M. Hudspeth,1,2
C. Patrick McAtee,1,2 Michael J. Heffernan1,2
1Department of Pediatrics (Section of Tropical Medicine), National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas
2Texas Children’s Hospital Center for Vaccine Development, Houston, Texas

Received 12 March 2017; revised 7 July 2017; accepted 21 July 2017
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33965

Abstract: It is believed that an effective vaccine against leish- maniasis will require a T helper type 1 (TH1) immune response. In this study, we investigated the adjuvanticity of the Toll-like receptor (TLR) 7/8 agonist 3M-052 in combination with the Leishmania donovani 36-kDa nucleoside hydrolase recombinant protein antigen (NH36). NH36 and 3M-052 were encapsulated in separate batches of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). The loading efficiency for NH36 was 83% and for 3M-052 was above 95%. In vitro stim- ulation of bone marrow-derived dendritic cells, measured by IL-12 secretion, demonstrated that 3M-052 (free or MP-formu- lated) had a concentration-dependent immunostimulatory effect with an optimum concentration of 2 mg/mL. In immuno- genicity studies in BALB/c mice, MP-formulated NH36 and 3M-052 elicited the highest serum titers of TH1-associated IgG2a and IgG2b antibodies and the highest frequency of IFNg-producing splenocytes. No dose dependency was observed among MP/NH36/3M-052 groups over a dose range of 4–60 mg 3M-052 per injection. The ability of MP-formulated NH36 and 3M-052 to elicit a TH1-biased immune response indicates the potential for PLGA MP-formulated 3M-052 to be used as an adjuvant for leishmaniasis vaccines. VC 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000– 000, 2017.

Key Words: immune response, microparticle, poly(lactic-co- glycolic acid), vaccine
How to cite this article: Wang, Q, Barry, MA, Seid, CA, Hudspeth, EM, McAtee, CP, Heffernan, MJ 2017. 3M-052 as an adjuvant for a PLGA microparticle-based Leishmania donovani recombinant protein vaccine. J Biomed Mater Res Part B 2015:00B:000–000.

INTRODUCTION
There is a need for new vaccine strategies against leishman- iasis, a sand fly-transmitted infection with protozoan para- sites of the genus Leishmania.1–3 There are an estimated 12 million cases of leishmaniasis worldwide in three basic clini- cal forms: cutaneous, mucosal, and visceral leishmaniasis. Among all the forms, visceral leishmaniasis is the leading cause of mortality, causing >60,000 deaths annually.4 Current treatments rely on antiparasitic drugs such as sodium stibogluconate, miltefosine, and amphotericin B.1 However, the efficacies of these treatments may vary by species, region of acquisition, and type of leishmaniasis, leading to unsatisfactory clinical results in patients and consequent retreatments.5 In addition, these drugs have significant adverse side effects, and in many areas the drugs have lim- ited availability and are costly.3,5–7 Recently, a computer model was used to simulate the economics of introducing a vaccine for visceral leishmaniasis in Bihar State, India, and it was found that even a modestly protective vaccine was cost-effective.8 Additionally, with the presence of animal reservoirs and the emergence of parasite resistance to che- motherapies, long-term reductions in parasite transmission and disease burden will depend upon widespread availabil- ity of effective human and veterinary vaccines.1

A strong antigen candidate for a Leishmania vaccine is the parasite’s 36-kDa nucleoside hydrolase (NH36, also termed GP36), a highly conserved protein among all Leish- mania species.9 A vaccine consisting of NH36 protein puri- fied from Leishmania donovani was shown to be protective against experimental leishmaniasis in mice.10 Also, studies with a plasmid DNA vaccine encoding L. donovani NH36 have demonstrated cross-protection against various Leish- mania species in mouse and hamster models.11,12 The development of a recombinant NH36 protein vaccine has been pursued within our organization, based on the exten- sive clinical record of protein-based vaccines and the suit- ability of recombinant proteins to scalable production processes. Recombinant proteins, however, generally require adjuvants to enhance their immunogenicity.13 In particular, for a leishmaniasis vaccine, evidence suggests that an Correspondence to: M. J. Heffernan, present address: Fannin Innovation Studio, Houston, Texas 77027; e-mail: [email protected] Contract grant sponsor: Curtis Hankamer Basic Research Fund (Baylor College of Medicine); contract grant number: Junior

Faculty Seed Award effective vaccine would require interleukin 12 (IL-12) pro- duction eliciting a T helper type 1 (TH1)-directed immune response, whereby T cells would produce high levels of IFNg that stimulates macrophages to kill the intracellular Leishmania amastigotes.14 Thus, an adjuvant system is needed in a leishmaniasis vaccine with the ability to induce an antigen-specific immune response with TH1 polarization.

Toll-like receptor (TLR) agonists are an important class of immunopotentiating molecules and have been widely employed in preclinical and clinical studies.15 The imidazo- quinolines are a class of agonists for TLR7 and TLR8 capable of inducing TH1-biased immune responses.16,17 Imi- quimod (a TLR7 agonist) and resiquimod (a TLR7/8 agonist) have been evaluated in numerous clinical trials for treat- ment of various cancers and infectious diseases.18 Also, imi- quimod is commercially available as a topical cream treatment for skin cancer and other skin growths.17 One potential limitation in the use of imiquimod and resiquimod as vaccine adjuvants is that the molecules, when injected in an aqueous buffer, diffuse rapidly away from the site of injection. To address this limitation, the lipophilic derivative 3M-052 was developed as a next-generation TLR7/8 agonist that is retained at the injection site longer.17 Because of the lipophilic nature of 3M-052, liposomes or other lipid-based delivery vehicles have been used for subcutaneous, intra- muscular, or intratumoral injections of 3M-052.19–23

As an alternative to liposomes or lipid-based formula- tions, we propose the use of biodegradable polymer micro- particles (MPs) as a delivery vehicle for 3M-052. Biodegradable MPs have certain properties that are amena- ble to their use as a vaccine delivery platform: (1) they enhance uptake of vaccines by antigen-presenting cells, (2) they create a vaccine depot at the injection site, (3) they have adjustable degradation and release rate by selections of polymer composition, polymer molecular weight, and for- mulation methods,24,25 and (4) they can be freeze-dried for long-term storage.26,27 MPs composed of poly(lactic-co-gly- colic acid) (PLGA) have been used in several FDA-approved drug delivery applications.28 Water-soluble proteins are typ- ically encapsulated in PLGA MPs by a double-emulsion method, whereas lipophilic compounds such as 3M-052 can be encapsulated by a single-emulsion method.

In this study, we evaluated the immunogenicity of a leishmaniasis vaccine consisting of NH36 protein antigen and 3M-052 adjuvant encapsulated in separate PLGA MP batches. The use of separate MP batches facilitates the vac- cine preparation process and allows for independent varia- tion of antigen and adjuvant dosages. The preclinical vaccination study, conducted in BALB/c mice, used a prime- boost regimen with a fixed dosage of NH36 and four differ- ent dosages of 3M-052. The immune response was charac- terized by serum antibody titers and splenocyte IFNg production. We hypothesized that PLGA MP-formulated NH36 protein and 3M-052 would elicit a stronger antigen- specific TH1-biased immune response in comparison to con- trol formulations including soluble or PLGA MP-formulated NH36 protein.

MATERIALS AND METHODS
Materials
Poly(lactic-co-glycolic acid) (PLGA, MW 7000–17,000, RESOMERVR RG 502H), polyvinyl alcohol (PVA, MW 31,000– 50,000, 98–99% hydrolyzed), D-mannitol, dimethyl sulfoxide (DMSO), 2-mercaptoethanol, and concanavalin A (ConA) were purchased from Sigma-Aldrich (Milwaukee, WI). 3M- 052 adjuvant was generously provided by 3M Drug Delivery Systems Division (St. Paul, MN) under a material transfer agreement. Micro BCATM Protein Assay and 3,30,5,50-tetrame- thylbenzidine (TMB) substrate were purchased from Thermo Fisher Scientific Inc. (Rockford, IL). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1, IgG2a, and IgG2b antibodies were purchased from LifeSpan Biosci- ences, Inc. (Seattle, WA). Mouse IFNg ELISPOT kit was pur- chased from Cellular Technology Ltd. (Shaker Heights, OH). The Ready-SET-Go!VR ELISA kit for mouse IL-12 was obtained from eBioscience, Inc. (San Diego, CA). PBS, RPMI- 1640, and sodium dodecyl sulfate (SDS) were purchased from Mediatech Inc. (Manassas, VA). ACK lysis buffer was purchased from Lonza Inc. (Allendale, NJ). Fetal bovine serum (FBS) and penicillin–streptomycin (Pen/Strep) were purchased from HycloneTM GE Healthcare Life Sciences (Logan, UT). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from R&D Systems, Inc. (Minneapolis, MN).
Bone marrow-derived dendritic cell (BMDC) culture medium was prepared with RPMI-1640 medium plus 10% FBS, 100 I.U./mL penicillin, 100 mg/mL streptomycin, 50 mM 2-mercaptoethanol, and 20 ng/mL GM-CSF. Splenocytes were either cultured in the serum-free media supplied with the ELISPOT kit, or in a culture medium prepared with RPMI-1640 medium containing 10% FBS, 100 I.U./mL peni- cillin, and 100 mg/mL streptomycin. PBST was prepared using phosphate-buffered saline (PBS) solution with the addition of 0.05% TweenVR 20.

Animals
BALB/c female mice were obtained from Taconic Farms, Inc. (Hudson, NY). All mice were housed in the Texas Children’s Hospital Feigin Center animal facility, operated by the Cen- ter for Comparative Medicine at Baylor College of Medicine. Experiments were conducted under an animal research pro- tocol approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine.

Expression and purification of NH36
The expression and purification of recombinant protein NH36 was performed as previously reported.29 Briefly, recombinant protein NH36 was derived from the wild-type sequence (Gen- Bank: XP_003860171.1), with four asparagine residues (N39, N77, N89, and N185) mutated to glutamines to reduce hyper- glycosylation.29 The protein was expressed in Pichia pastoris X-33 through an optimized fermentation process in a 14 L scale vessel. NH36 in the supernatant was purified by cation- exchange column packed with CaptoTM SP ImpRes medium (GE Healthcare Life Sciences, Pittsburgh, PA), followed by size-exclusion column packed with Sephacryl S-200 HR resin

ORIGINAL RESEARCH REPORT
(GE Healthcare Life Sciences, Pittsburgh, PA) to remove impu- rities and endotoxin. The protein was LAL tested to ensure that the endotoxin level was below 2 endotoxin units per mil- ligram (2 EU/mg). Encapsulation and characterization of NH36 and 3M-052 in PLGA MPs NH36 protein was encapsulated in PLGA MPs using a water-oil-water double-emulsion method.29,30 Briefly, a working solution with 2 mg of NH36 protein in 100 mL of PBS containing 0.5% PVA and 50 mg/mL D-mannitol was combined with a solution of 100 mg of PLGA in 2 mL of chloroform, and the mixture was emulsified using an IKAVR T25 homogenizer (IKA Works, Inc., Wilmington, NC) at 20,000 rpm for 1 min. The primary emulsion was poured into 13 mL of 5% PVA solution (in 33 PBS) and homoge- nized at 24,000 rpm for another 2 min. The resulting emul- sion was poured into 187 mL of 5% PVA solution (in 33 PBS) and stirred overnight at room temperature to evapo- rate the chloroform. The MPs were collected by centrifuga- tion at 7500g, washed twice using Milli-Q water, re- suspended, and lyophilized. Parallel batches of MPs were pooled during the washing step.

3M-052 adjuvant was encapsulated in PLGA MPs using an oil-in-water single-emulsion method. The adjuvant 3M- 052 was dissolved in chloroform and mixed with 100 mg of PLGA in a total chloroform volume of 1.5 mL and then homogenized with 13 mL of 5% PVA solution (in 33 PBS) at 24,000 rpm for 2 min. The resulting emulsion was poured into 137 mL of 5% PVA solution (in 33 PBS) and stirred for overnight at room temperature to evaporate the chloroform. The MPs were collected, washed, and lyophi- lized using the same method used for the NH36 MPs.

MPs were imaged prior to lyophilization by scanning electron microscopy (SEM, JEOL JSM-6100). The protein encapsulation level was measured by Micro BCATM Protein Assay after MPs were digested in a mixture of DMSO, SDS, and sodium hydroxide solution.30,31 The adjuvant 3M-052 encapsulation level was measured by spectrophotometry at 320 nm after dissolving lyophilized 3M-052 MPs in DMSO at a concentration of 2.5 mg/mL. The concentration of 3M- 052 was determined by a calibration curve established using varying concentrations of 3M-052 in DMSO spiked with 2.5 mg/mL PLGA empty MPs.
The loading level (mg/mg) is defined as the mass of NH36 protein (or 3M-052) divided by the mass of MPs. The loading efficiency (%) is calculated as the actual loading level divided by the nominal loading level, which is defined as the starting NH36 protein (or 3M-052) mass divided by the starting PLGA mass. The MP mass yield is defined as the mass of recovered MPs divided by the starting PLGA mass.

In vitro stimulation of dendritic cells
BMDCs were generated as described through a modified protocol by Inaba et al.32 Briefly, bone marrow cells isolated from tibiae and femurs of BALB/c mice were suspended at a concentration of 2.5 3 106 cells/mL in BMDC culture medium. At day 3, two-thirds of the medium was changed, and at days 5 and 7, fresh medium was added without removing the old medium. At day 8, floating cells were col- lected and were transferred into a 24-well plate at 1 3 106 cells in 1 mL of BMDC culture medium per well. MPs or sol- utions containing 2, 10, or 50 mg of 3M-052 were dispersed in 1 mL of medium and added into cultures. As a control, empty PLGA MPs of an equivalent mass to the highest con- centration of 3M-052 MPs were added to the culture. Treat- ment groups were run in duplicate. After 24 h, supernatants were collected, and IL-12 was quantified by ELISA per the manufacturer’s instructions.

Vaccinations and sample collection
Female BALB/c mice, age 8–10 weeks, were vaccinated with 40 mg of NH36 plus a varying amount of 3M-052 adjuvant (4, 10, 25, or 60 mg) encapsulated in separate PLGA MPs (MP/NH36/3M-052). Control groups included mice immu- nized with PBS, soluble NH36 (NH36), or NH36 MPs (MP/ NH36). The mice were given a prime vaccination at week 0 via subcutaneous injection in the flank area and received a boost vaccination at week 3 on the opposite flank, at the same dosage as the prime. Two weeks after the boost vacci- nation, mice were euthanized. Blood samples were collected via cardiac puncture, and serum samples were separated by centrifugation at 10,000g for 5 min. Serum samples were stored at 2208C before performing serum antibody ELISAs.

Serum antibody ELISA
NH36-specific serum antibodies were measured by ELISA. Briefly, 96-well plates were sensitized overnight at 48C with 100 ng/well of antigen (NH36) in 50 lL of PBS. After wash- ing and blocking, plates were incubated for 2 h with 50 lL of serum samples that were serially diluted from 1:100 to
1:976,562,500 by fivefold dilution in PBST containing 0.1% BSA. After washing with PBST, plates were incubated with
50 lL of a 1:4000 dilution of HRP-conjugated anti-mouse IgG antibodies (IgG1, IgG2a, and IgG2b) for 1 h. Plates were washed, and HRP was quantified by adding 100 lL of TMB substrate. After 8–10 min, the reaction was stopped with 100 lL of 1 M HCl, and the absorbance was measured at 450 nm. Titers were determined by a log-linear interpola- tion of the serum dilution value corresponding to a cut-off absorbance of 0.2.

T-cell IFNg ELISPOT
To perform the T-cell IFNg ELISPOT assay, 2.5 3 105 sple- nocytes were resuspended in 100 mL of serum-free media and placed in 96-well filter plates which were precoated with anti-mouse IFNg antibodies. Splenocytes were restimu- lated for ~24 h with either 100 mL of 100 mg/mL NH36 protein or 100 mL of 4 mg/mL ConA, or left unstimulated with 100 mL of serum-free media. Spots were counted by a CTL-ImmunoSpotVR S6 Micro Analyzer (Cellular Technology Ltd., Shaker Heights, OH).

Statistical analysis
For the animal study, two identical experiments (n 5 5 mice per treatment group) were carried out, and the results were analyzed through a randomized complete block design.33 Results were analyzed using Friedman’s test with Tukey adjustment for post hoc multiple comparisons with a confi- dence interval of 0.95 (p < 0.05) using SAS (SAS Institute Inc., Cary, NC). Data from the PBS (negative control) group
were excluded from the statistical analysis.

RESULTS
Characterization of NH36 and 3M-052 in PLGA MPs Separate batches of NH36- and 3M-052-loaded MPs were made, with the particles having a generally spherical shape as shown in Figure 1(A). NH36-loaded MPs had a size range from 0.4 to 1.5 mm, and 3M-052-loaded MPs had a size range 0.6–2 mm, based on SEM images. NH36-containing MPs were prepared at a target loading level of 20 mg NH36 per mg PLGA, which resulted in MP mass yields of approxi- mately 62.9%, with an average loading level of 16.6 mg of NH36 per mg of particles (83% efficiency). 3M-052- containing MPs were formulated using three target loading levels (8.3, 27.8, and 48.4 mg 3M-052 per mg PLGA), which resulted in MP mass yields ranging from 46 to 67%, with loading efficiencies ranging from 97.5 to 113.3%. It is noted that the loading efficiency was calculated as the actual load- ing level divided by the nominal loading level, which is defined as the starting NH36 protein (or 3M-052) mass divided by the starting PLGA mass. The actual loading level is dependent on the encapsulated ingredient mass and the final product mass of MPs, and therefore, the actual loading level could be higher than the nominal loading level due to
the removal of extraneous PLGA during the preparation step. The loading levels and yields are listed in Table I. Absorbance was used to measure 3M-052 loading, as shown in Figure 1(B). The aromatic rings of 3M-052 show a strong absorbance peak around 320 nm, with minimal interference from the solvent DMSO and empty PLGA particles. The absorbance is linearly correlated with the concentration of 3M-052 in DMSO at a range from 4 to 250 mg/mL, allowing fast and reliable quantification of encapsulated 3M-052. MPs containing 27.1 mg of 3M-052 per mg of particles were used in the two cohorts of animal studies. MPs containing 49.4 mg of 3M-052 per mg of particles were used in the in vitro BMDC stimulation study.

In vitro stimulation of dendritic cells
The ability of soluble or PLGA-encapsulated 3M-052 to stim- ulate IL-12 production, a cytokine which induces IFNg pro- duction and promotes TH1 cell differentiation, was evaluated in BMDC cultures. As shown in Figure 2, both soluble and In vitro activation of dendritic cells by 3M-052 in solution or encapsulated in PLGA MPs. BMDCs were stimulated for 24 h with 3M-052, either soluble or MP-formulated, at a concentration ranging from 0.08 to 50 mg/mL. Treatment groups were run in duplicate. Supernatant IL-12 was quantified by ELISA. PLGA-encapsulated 3M-052 elicited concentration-dependent production of IL-12 with an optimum concentration of 2 mg/ mL. At this concentration, PLGA-encapsulated and soluble 3M-052 elicited IL-12 levels of 629 and 1372 pg/mL, respec- tively. The IL-12 production by BMDCs stimulated with empty PLGA MPs was comparable to that of unstimulated cells. Soluble 3M-052 generally elicited higher IL-12 than cor- responding doses of MP/3M-052.

Mouse immunogenicity studies
Serum antibody ELISA. All mice vaccinated with NH36- containing formulations generated substantial titers of serum antibodies of the subclasses IgG1, IgG2a, and IgG2b (Figure 3). For the IgG1 subclass, all groups vaccinated with MP-formulated NH36 (with or without 3M-052 adjuvant) had a trend of higher geometric mean titers than those vac- cinated with non-MP (soluble) NH36; however, only the MP/NH36 group had statistically higher titers than the solu- ble NH36 group. There were no significant differences in IgG1 mean titers among the MP-formulated NH36 groups, regardless of the presence or dosage of 3M-052. For the IgG2a and IgG2b subclasses, which are TH1-asso- ciated in BALB/c mice,34–36 both subtypes had consistently higher titers with the presence of 3M-052; however, there were no significant differences among the MP/NH36/3M- 052 groups. For IgG2a, all of the MP/NH36/3M-052 vac- cines elicited at least 2500-fold higher mean titers than sol- uble NH36 and 150-fold higher mean titers than MP/NH36 (p < 0.001). For IgG2b, the MP/NH36/3M-052 vaccines had least 125-fold higher titers than soluble NH36 and 12-fold higher mean titers than the MP/NH36 group (p < 0.001). There were no significant differences in IgG2a and IgG2b titers between the MP-formulated NH36 (MP/NH36) group and the non-MP (soluble) NH36 group.

We also examined the ratio of IgG2a titers to IgG1 titers, which indicates the degree to which the immune system is TH1-biased.37 In this comparison, the MP/NH36/3M-052 groups had a significantly higher IgG2a:IgG1 ratio than the groups without 3M-052 [Figure 3(D)], which further con- firmed the TH1 bias. Splenocyte IFNg ELISPOT. The frequencies of antigen- specific IFNg-producing splenocytes were measured by ELI- SPOT as shown in Figure 4. Mice vaccinated with MP/ NH36/3M-052 had at least a 30-fold higher frequency of IFNg-producing splenocytes than the soluble NH36 group and a 5.5-fold higher frequency than the MP/NH36 group. There was no significant difference among the MP/NH36/ 3M-052 groups.

DISCUSSION
The primary objective of this study was to develop a deliv- ery platform for the lipophilic, TH1-inducing adjuvant 3M- 052 using biodegradable PLGA MPs. As 3M-052 is not solu- ble in aqueous buffers, it requires a delivery platform as an injectable vaccine adjuvant. In this study, 3M-052 was for- mulated in PLGA MPs using a single-emulsion, solvent-evap- oration method, with loading levels ranging from 8.3 to 48.4 mg/mg and loading efficiencies ranging from 97.5 to 113.3%. The results indicate that PLGA MPs are a suitable platform for formulating 3M-052 over a broad loading range. In this study, adjuvant 3M-052 and antigen protein NH36 were encapsulated in separate MP batches, which were combined in the desired ratios prior to injection. While our laboratory has developed techniques to encapsu- late hydrophobic and hydrophilic ingredients in the same batch of MPs, we used separate MP batches in this study to facilitate independent dosing of antigen and adjuvant. It would be useful in future studies to evaluate coencapsu- lated antigen/adjuvant in comparison to separate batches of MPs.

The MPs are in the size range 0.6–2.0 mm and thus can be taken up by antigen-presenting cells through phagocytosis and/or macropinocytosis. Although there is some variation in literature reports as to the optimal MP size for antigen- presenting cell uptake and the relationship between particle size and immune response magnitude, researchers have observed that larger particles (0.5–5 mm) require longer trans- port time to the lymph node and elicit weaker TH1-biased responses than smaller particles (<0.2 mm).38–40 Consistent with those reports, our present study found that MPs enhanced TH2-associated IgG1 titers more than TH1-associ- ated IgG2a/2b titers, and that an immunostimulatory adjuvant (3M-052) was required to elicit increased TH1-biased humoral and cellular immune responses. Our group has observed simi- lar results in previous studies with CpG oligodeoxynucleotide adjuvant and protein in MP formulations.29,41

In the immunogenicity study, mice vaccinated with MP/ NH36/3M-052 induced higher levels of TH1-associated IgG2a and IgG2b titers and higher frequencies of IFNg- producing splenocytes, in comparison to the formulations without 3M-052 (NH36 and MP/NH36). In addition, all four dose levels (4–60 mg) of 3M-052 elicited similar humoral and cellular immune responses, suggesting that the optimal Serum antibody responses to vaccination with PLGA MP-formulated NH36 and 3M-052. BALB/c mice were vaccinated by subcutane- ous injection at weeks 0 and 3 with 40 mg of NH36 protein plus varying amounts of 3M-052 adjuvant (4, 10, 25, or 60 mg) encapsulated in sepa- rate PLGA MP (MP/NH36/3M-052). Vaccine controls included PBS, MP-formulated NH36 (MP/NH36), and soluble NH36 (NH36). Mice were vaccinated in two cohorts (n 5 5 for each cohort; cohort 1, open circles; cohort 2, filled circle). Week 5 serum was analyzed by ELISA for NH36- specific antibody subtype titers. (A) IgG1, (B) IgG2a, and (C) IgG2b titers. The titers were set to 1 if they were below the initial dilution. (D) Ratio of IgG2a:IgG1 titers (PBS group not plotted). *p < 0.05, **p < 0.01, and ***p < 0.001. dose of 3M-052 is 4 mg or lower. The TH1 bias of the immune response was further confirmed by the significantly higher ratios of IgG2a:IgG1 titers elicited by the 3M-052- adjuvanted formulations. The enhancement of TH1-type immune responses is attributed to the engagement of 3M- 052 with TLR7 and/or TLR8 in antigen-presenting cells, mainly dendritic cells, leading to secretion of IFNg-regulated cytokines and proinflammatory cytokines, such as IL-12, that induce TH1-type immunity.42 The TH1-enhancing effect of 3M-052 is similar to that seen in a previous study wit. Splenocyte IFNg ELISPOT responses in mice vaccinated with PLGA MP-formulated NH36 and 3M-052. BALB/c mice were vacci- nated by subcutaneous injection at weeks 0 and 3 with 40 mg of NH36 protein plus varying amounts of 3M-052 adjuvant (4, 10, 25, or 60 mg) encapsulated in separate PLGA MP (MP/NH36/3M-052). Vaccine con- trols included PBS, MP-formulated NH36 (MP/NH36), and soluble NH36 (NH36). Mice were vaccinated in two cohorts (n 5 5 for each cohort; cohort 1, open circles; cohort 2, filled circle). Splenocytes were isolated and re-stimulated with 50 mg/mL NH36 protein in vitro for 24 h. The background value of unstimulated splenocytes was sub- tracted. If the number of spots was negative after background subtraction, the number of spots was set to zero. *p < 0.05, **p < 0.01, and ***p < 0.001.

MP formulations containing TLR9 agonist CpG oligodeoxy- nucleotide adjuvant and NH36 protein; in that study, an 80 mg dose of CpG delivered with NH36 in MPs enhanced IgG2a and IgG2b titers in comparison to MP-formulated NH36 without CpG.29 The strong adjuvanticity of 3M-052 exhibited in our study was also observed in other reports17,21–23 in which 3M-052 was formulated into vaccines using lipid-based or combined lipid-AlhydrogelVR delivery platforms. Fox et al. reported that a liposome-AlhydrogelVR formulation with 3M- 052 enhanced the antigen-specific IgG1 and IgG2c titers and frequency of TH1 cytokine-producing CD41 memory cells of a tuberculosis vaccine in mice. Similarly, Vasilakos et al. reported that 3M-052 enhanced the IFNg production and IgG2a titers of an H1N1 influenza vaccine in mice, and Van Hoeven et al. reported that various formulations of 3M-052 provided protection against influenza virus challenge in mice and ferrets. In a study with newborn rhesus macaques, Dowling et al. reported that 3M-052 stimulated generation of TH1-type CD41 cells, activation of antigen-specific B cells, enhancement of antigen-specific antibody titers, and opso- nophagocytic killing, when admixed with pneumococcal con- jugate vaccine 13.

In summary, the results indicate that (1) PLGA MPs are a suitable platform for formulating 3M-052 over a broad loading range and (2) this adjuvant formulation enhancesthe antigen-specific, TH1-biased immune response of an L. donovani NH36 recombinant protein vaccine, which is desir- able for a vaccine against intracellular Leishmania para- sites.43,44 The chosen adjuvant/delivery formulation has a composition that is amenable to clinical translation. The adjuvant 3M-052 is closely related in structure to imiqui- mod, which is used in an FDA-approved topical treatment for skin cancer17 and is undergoing several clinical trials for additional indications.45 Also, PLGA is used in several FDA- approved drug delivery MP formulations.46 By formulating the L. donovani NH36 recombinant protein antigen with 3M-052 and PLGA, we anticipate that this will facilitate the advancement of the proposed leishmaniasis vaccine toward clinical translation.

ACKNOWLEDGMENTS
This work was supported by the Curtis Hankamer Basic Research Fund (Junior Faculty Seed Award at Baylor College of Medicine), as well as by the Texas Children’s Hospital Center for Vaccine Development. We would like to thank 3M Drug Delivery Systems, in particular John Vasilakos, for providing 3M-052 adjuvant for use in these studies.

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