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. Author manuscript; available in PMC: 2016 May 20.
Published in final edited form as: Carbohydr Res. 2014 Dec 3;405:87–92. doi: 10.1016/j.carres.2014.11.007

Sublingual injection of microparticles containing glycolipid ligands for NKT cells and subunit vaccines induces antibody responses in oral cavity

Elizabeth S DeLyria 1,*, Dapeng Zhou 2,*,†, Jun Soo Lee 1, Shailbala Singh 3, Wei Song 1,4, Fenge Li 1, Qing Sun 1, Hongzhou Lu 4, Jinhui Wu 5, Qian Qiao 5, Yiqiao Hu 5, Guodong Zhang 6, Chun Li 7, K Jagannadha Sastry 3, Haifa Shen 6
PMCID: PMC4874192  NIHMSID: NIHMS646748  PMID: 25555750

Abstract

Natural Killer T (NKT) cells are a unique type of innate immune cells which exert paradoxical roles in animal models through producing either Th1 or Th2 cytokines and activating dendritic cells. Alpha-galactosylceramide (αGalCer), a synthetic antigen for NKT cells, was found to be safe and immune stimulatory in cancer and hepatitis patients. We recently developed microparticle-formulated αGalCer, which is selectively presented by dendritic cells and macrophages, but not B cells, and thus can avoid the anergy of NKT cells. In this study, we have examined the immunogenicity of microparticles containing αGalCer and protein vaccine components through sublingual injection in mice. The results showed that sublingual injection of microparticles containing αGalCer and ovalbumin triggered IgG responses in serum (titer >1:100,000), which persisted for more than 3 months. Microparticles containing ovalbumin alone also induced comparable level of IgG responses. However, immunoglobulin subclass analysis showed that sublingually injected microparticles containing αGalCer and ovalbumin induced 20 fold higher Th1 biased antibody (IgG2c) than microparticles containing OVA alone (1:20,000 as compared to 1:1000 titer). Sublingual injection of microparticles containing αGalCer and ovalbumin induced secretion of both IgG (titer >1:1000) and IgA (titer =1:80) in saliva secretion, while microparticles containing ovalbumin alone only induced secretion of IgG in saliva. Our results suggest that sublingual injection of microparticles and their subsequent trafficking to draining lymph nodes may induce adaptive immune responses in mucosal compartments. Ongoing studies are focused on the mechanism of antigen presentation and lymphocyte biology in the oral cavity, as well as the toxicity and efficacy of these candidate microparticles for future applications.

Keywords: Microparticles, alpha-galactosylceramide, Natural Killer T cells, Vaccines, Mucosal Immunity, Antibody Class Switch

Introduction

Mucosal route of vaccination is a current focus of HIV vaccine research. Effective adaptive immune responses at mucosal sites are critical for the success of a prophylactic vaccine. Intranasal, intra-intestinal, intra-vaginal, and intra-rectal routes of immunization have been widely tested in multiple types of vaccines composed of proteins, peptides, DNAs, or adenoviral vector-based components. Evidence of successful induction of protective adaptive immune responses has been reported at both mucosal sites and systemic compartments (1–4).

The oral mucosal associated lymphoid tissues (MALT) are perhaps the least understood area of mucosal immunity. Oral MALT including tonsils and submandibular lymph nodes are known to have anatomical structures and cell types required for effective adaptive immune responses (5–7). Our recent study showed that the oral route of immunization by HIV env peptide vaccines in the presence of an adjuvant which activates Natural Killer T cells (NKT) can induce effective adaptive anti-viral CD8 responses (8).

Adjuvants bridge the innate and adoptive immune responses to prime strong and specific immunity (9–10). NKT cells are important members of the innate immunity that are activated in response to specific glycolipids such as αGalCer presented by dendritic cells (DC) in the context of the CD1d surface molecule (11). The potency of αGalCer delivery (both systemic and mucosal) to enhance immunity against tumors (12) and intracellular infections such as hepatitis (13) and malaria (14) has been well established. We have found orally delivered αGalCer enhanced humoral and cell-mediated immunity to the co-administered antigens (8).

While the mechanisms underlying the potential of αGalCer to serve as a vaccine adjuvant are not fully understood, it is generally believed that the DCs are the critical players in presenting αGalCer and vaccines to NKT cells and adaptive immune cells, respectively (15). It was found that targeting αGalCer and vaccines to the same DC population is critical for the adjuvant effect of αGalCer (16). There is a need for novel formulation of vaccines that can simultaneously deliver αGalCer and protein subunit vaccines to DCs.

Nanoparticle and microparticle formulations of vaccines have been shown to be superior to free form of vaccine (17–19), since these particles are selectively phagocytosed by professional antigen presenting cells, DCs. In this study, we tested the utility of microparticle-formulated αGalCer and subunit vaccines for sublingual injection.

Materials and Methods

Preparation of polylactic acid (PLA)-based microparticles and surface conjugation of streptavidin

The PLA-based microparticles (500 nm in size) were prepared according to our previously published methods (20). Four hundred milligrams of PLA was dissolved in 2 mL of dichloromethane in a glass tube, and 100 μL of Milli-Q water was added to the polymer solution. The polymer solution was then sonicated for 15 s to create the primary emulsion. 4 mL of an aqueous 1% (w/v) solution of PEMA (poly[ethylene-alt-maleic acid]) was added to the tube, and the sonication step was repeated. After the second sonication, the emulsion was poured into 100 mL of 0.3% (w/v) aqueous solution of the same stabilizer used for the second emulsion, under rapid stirring with a magnetic stirrer. The resulting microparticles were stirred in the solution for 3 h to evaporate the organic solvent. The microparticles were then washed three times with Milli-Q water and re-suspended in 4 mL of Milli-Q water for use.

To conjugate streptavidin to microparticles, 12.5 mg microparticles were re-suspended in coupling buffer (50 mM MES, pH 5.2) at 170 μL. 20 μL of EDAC (N-[3-Dimethylaminopropyl]-N′-ethylcarbodiimide hydrochloride) solution (200 g/L) was added to the suspension and incubated at room temperature with continuous mixing for 15 min. Next, 200 μg of streptavidin was added and mixed gently for 1 h at room temperature. The strepatavidin-conjugated microparticles were stored in 10 mM Tris buffer, pH 0.5% with 0.05% bovine serum albumin. 2 different batches of microparticles were synthesized and studied in separate experiments.

Particle size was measured using dynamic light scattering at a 90° scatter angle on a ZetaPLUS particle electrophoresis system (Brookhaven Instruments Corp., Holtsville, NY). Correlation data were analyzed using a BI-9000AT Digital Autocorrelator (Brookhaven), and a nonnegativity-constrained least squares (CONTIN) approximation was used to calculate the particle size distribution (Figure 1). To measure the surface charges of particles, particles were suspended in 50 mM phosphate buffer (pH7.2), and measured in Malvern DIP cell kit (Malvern Instruments, UK) by Malvern Zetasizer Nano ZS system (Table 1).

Figure 1. Size analysis of microparticles.

Figure 1

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Microparticles were resuspended in 10 mM phosphate-buffered saline solution (pH 7.0) and analyzed by dynamic light scattering. Data were representative of 3 different batches of microparticles.

Table 1.

Surface charges of micro-particles after loading of glycolipid adjuvant αGalCer and Ovalbumin

Content of micro-particles Surface charge (mV)
Unloaded particles −21.2+/−0.2
Particles loaded with Ovalbumin −17.3+/−1.9
Particles loaded with αGalCer and Ovalbumin −19.9+/−0.9
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12.5 μg of streptavidin-coated microparticles, 10 μg of biotinylated ovalbumin and/or 2 μg of biotinylated αGalCer were mixed and incubated for overnight at 4 °C under constant shaking. Microparticles were washed by PBS for 3 times by centrifugation at 2000 × g.

Particles were suspended in 50 mM phosphate buffer (pH7.2), and measured in Malvern DIP cell kit (Malvern Instruments, UK) by Malvern Zetasizer Nano ZS system. No significant difference on surface charges were found after the particles were loaded with ovalbumin, or with ovalbumin plus αGalCer. Data were representative of two independent expriments.

Preparation of microparticles containing αGalCer and ovalbumin

The synthesis of biotinylated αGalCer was previously published (21). Ovalbumin was from Sigma (Grade V A5503). Biotinylated ovalbumin was prepared by a EZ-Link Sulfo-NHS-Biotinylation Kit from Pierce Protein Biology products (Rockford, IL). For treatment of each mouse, 12.5 μg of streptavidin-coated microparticles, 10 μg of biotinylated ovalbumin and/or 2 μg of biotinylated αGalCer were mixed and incubated for overnight at 4 °C under constant shaking. Microparticles were washed by PBS for 3 times by centrifugation at 2000 × g.

Examining the rate of binding for biotinylated αGalCer and Ovalbumin

The biotinylated αGalCer, when present in solution, can be measured biologically in an NKT cell stimulation assay. We could not find detectable NKT stimulation activity of the post-binding supernatant after the biotinylated αGalCer was mixed overnight with streptavidin microparticles. Thus the biotinylated αGalCer was considered as 100% bound to microparticles. By ELISA detection with a rabbit polyclonal anti-ovalbumin antibody from Abcam (Cambridge, MA), we could not detect presence of ovalbumin in the post-binding supernatant after biotinylated ovalbumin was mixed overnight with streptavidin microparticles, which suggested 100% of biotinylated ovalbumin were bound to microparticles.

Sublingual and intravenous vaccination

Individual groups of 6 week old C57Bl/6 mice (n=3) were anesthetized by 80mg/kg of ketamine and 10mg/kg of xylazine, before being vaccinated with the soluble form of ovalbumin and αGalCer, the microparticles containing ovalbumin and αGalCer, and control PBS by intravenous or sublingual route of injection. Intravenous injection was via the tail vein in a total volume of 100 μl in PBS. Sublingual injection was underneath the mucous membrane in 20 μl of PBS. All injections were performed with 0.5 ml syringe and 28G1/2 needle (Becton Dickinson and Company, Franklin Lakes, NJ).

Immune monitoring of antibody responses

Saliva flow was induced by an intraperitoneal injection of 20μL of phosphate-buffered saline (PBS) containing isoproterenol (0.02 mg/ml) and pilocarpine (0.05 mg/ml). Saliva was then collected for 10 minutes from the mouse oral cavity using a micropipette. Ovalbumin antigen-specific IgA and IgG levels in serum and saliva were measured using the standard ELISA procedures and isotype-specific secondary antibodies. Goat anti-mouse total IgG, IgG1, IgG2c, IgG2b, IgG3, IgA, and IgE were from Southern Biotech (Birmingham, Alabama).

Statistic analysis of data

The raw ELISA measurements were transformed to the logarithmic scale prior to statistical analysis. Analysis of variance (ANOVA) method was used to estimate and compare the groups for each biomarker. Dunnett’s adjustment was used to control overall type I error rate at 5% for pairwise comparisons against the control group. All tests were two-sided and p-values of 5% or less after adjustment were considered statistically significant. Statistical analysis was carried out using SAS version 9.3 (SAS Institute, Cary, NC).

Results

Sublingual immunization by microparticles triggers systemic antibody responses

Based on our observation that αGalCer conjugated to the surface of nanoparticles and microparticles could avoid the anergy of NKT cells, we have tested its application in delivering protein subunit vaccines by using ovalbumin as a model antigen. As shown in Figure 2, three doses of vaccination by sublingual route using microparticles containing OVA and αGalCer induced OVA-specific IgG titer of 1:200,000 one week after immunization, and this antibody titer lasted for at least 3 months post vaccination. Vaccination by intravenous route using microparticles containing OVA and αGalCer induced 20 to 50 fold weaker serum IgG titer as compared to sublingual route (p<0.05).

Figure 2. Sublingually-delivered microparticles induced sustained antibody responses in mice.

Figure 2

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A. Groups of C57Bl/6 mice (n=3) were vaccinated by sublingual injection of microparticles containing OVA and αGalCer with control groups of different formulations and routes of vaccination; B. Serum IgG titer toward OVA antigen was measured by ELISA at day 21 and day 90 respectively. Data were representative of 3 independent experiments using 3 different batches of microparticles.

We have also compared the sublingual delivery of microparticle-formulated OVA and αGalCer to soluble OVA and αGalCer. The microparticle formulation caused a 10 fold of higher serum IgG titer through sublingual route as compared to mixed soluble OVA and αGalCer (p<0.05).

Interestingly, microparticles containing OVA alone also induced very strong serum IgG titer (1:100,000) when injected sublingually, but only a very weak serum IgG titer (1:4,000) was also observed when microparticles containing OVA were injected intravenously. This finding led us to focus our study on sublingual route of immunization and subsequently analyzed the subclasses of immunoglobulins with or without αGalCer adjuvant.

Subclasses of immunoglobulins induced by sublingually-injected microparticles

αGalCer is a well known NKT agonist which regulates the production of both Th1 and Th2 cytokines, which may influence the outcome of immunoglobulin class switch. In mice, IgG1, IgG2b, and IgE are enhanced by the Th2 cytokines such as IL4, while IgG2c is regulated by Th1 cytokines (e.g. IFNγ). We thus further studied the subclasses of immunoglobulins in mice vaccinated by microparticles.

Serum IgG1

Figure 3 shows that IgG1 was a main type of immunoglobulin induced by sublingual injection of microparticles containing OVA and αGalCer, with a titer of >1:100,000. Sublingual injection of microparticles containing only OVA also induced comparable titer of IgG1 (1:300,000). Sublingual injection of the soluble form of OVA and αGalCer induced much weaker IgG1 (1:5,000).

Figure 3. Sublingually-injected microparticles containing αGalCer and OVA induced 20 fold higher Th1 antibodies (IgG2c) than microparticles containing OVA alone.

Figure 3

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Data were representative of 3 independent experiments using 3 different batches of microparticles.

Serum IgG2c

The highest titer of IgG2c response was induced by sublingual delivery of microparticles containing OVA and αGalCer (1:40,000). In contrast, microparticles containing only OVA induced 20 fold weaker IgG2c response (1:2000, p<0.05).

Serum IgG2b

Sublingual injection of microparticles containing OVA and αGalCer induced IgG2b at a titer of 1:160,000. Sublingual injection of microparticles containing only OVA also induced comparable titer of IgG1 (1:40,000).

Serum IgG3

No IgG3 was detectable in any group of immunized mice.

Serum IgA

Sublingual injection of microparticles containing OVA and αGalCer induced a titer of IgA at 1:1000. IgA could also be detected in groups vaccinated by microparticles containing OVA alone. One way ANOVA analysis by Kruskal-Wallis test could not find significant difference in antibody titers between these 2 groups.

IgE

Sublingual injection of microparticles containing OVA and αGalCer induced a low titer of IgE (1:1000). IgE could also be detected in groups vaccinated by microparticles containing OVA alone by intravenously injected microparticles.

Sublingual immunization by microparticles triggers antibody responses in mucosal compartment

Immune responses in mucosal compartments are critical for protection against microbial infection and antitumor immunity. We have measured the secretion of IgG and IgA in saliva (Figure 4).

Figure 4. Sublingually-injected microparticles containing αGalCer and OVA induced secretion of both IgG and IgA antibodies in mucosal compartments.

Figure 4

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Data were representative of 3 independent experiments using 3 different batches of microparticles.

Mucosal IgG

Sublingually-injected microparticles containing OVA and αGalCer induced secretion of IgG in saliva (1:600). Similar level secretion of IgG in saliva was observed in mice which received sublingual injection of microparticles containing OVA alone. No secreted IgG was detected in saliva for the group sublingually vaccinated by soluble form of αGalCer and Ovalbumin.

Mucosal IgA

Sublingually-injected microparticles containing OVA and αGalCer induced IgA secretion in saliva. The average titer of IgA is 1:80. In contrast, no IgA was detectable in saliva of the group vaccinated by microparticles only containing OVA.

For the group which received sublingual injection of soluble OVA and αGalCer, IgA was detected at lower level in saliva (p<0.05 as compared to microparticles containing αGalCer and ovalbumin).

Discussion

The immune response in mucosal compartments is the first line immune defense against microbes. It is also possibly involved in pathogenesis of mucosal cancer lesions such as oral, throat cancer, and cervical cancer (22). In this study, we examined the role of NKT cell function in jump starting adaptive immunity in the oral cavity. We found that the sublingual injection of vaccine formulation in microparticles induced antibody responses systemically, which suggested that the microparticles have effectively trafficked to draining lymph nodes and primed the naïve T cells and B cells. The antibody responses lasted for more than 3 months. IgG2c was induced, suggesting Th1-cytokine mediated antibody responses. However, weak IgE (titer lower than 1:1000) was also observed, indicating Th2 cytokines were also involved in NKT cell activation. Sublingually-injected microparticles containing OVA and αGalCer induced IgG and IgA secretion in mucosal compartments, which has previously been reported for intranasal immunization by virus antigens with αGalCer as adjuvant (23–25).

The cell types that are involved in inducing adaptive immune responses remain unclear. Microparticles are most likely phagocytosed and presented by macrophages and DCs according to our previous studies (20). It was reported by several groups that cognate help provided by NKT cells could induce short-term antibody responses (23–30) but failed to generate long lived memory pool of B cells (31). For the microparticles which we used as vaccine carrier, Bcl-6-expressing follicular CD4 T helper cells might be the key helper cells which provided help for the class switch and memory B cell differentiation (32–33).

CD8 T cells are essential for antiviral and antitumor immunity. We found sublingual delivery of microparticles triggered effective CD8 responses in the spleen, which could kill tumor cells (data to be published elsewhere). However, the T cells in the mucosal compartment remain to be studied, due to presence of few lymphocytes in the oral cavity. Tools like transgenic T cells (34) and B cells (35) which recognize antigens like ovalbumin and hen egg lysosome are being used to study the biology of these effector cells in oral cavity and other mucosal compartments.

When this work was in progress, Bershteyn et al. reported that strong class-switched, high avidity humoral immune responses can be elicited by subcutaneous vaccination with poly(lactide-co-glycolide) micro- or nano-particles enveloped by PEGylated phospholipid bilayers, with protein antigens covalently anchored to the lipid surface and lipophilic adjuvants inserted in the bilayer coating (36). In our study, we have focused on mucosal route of delivery by sublingual injection, with aim to induce antibody responses in mucosal compartments.

In this study, we have directly injected microparticles sublingually by 28G1/2 needle. This method may be feasible for treating head and neck cancer patients with mucosal lesions. However, it is clearly not applicable for prophylactic vaccines to prevent infectious diseases like HIV and HPV. It remains to be studied whether such microparticles can be delivered through micro injection patches and micro needle delivery systems.

In summary, this study suggests that NKT cells in the oral cavity serve as a critical arm in innate immunity, which exerts unique adjuvant effects in regulating immune memory, class switch of B cells, and mucosal secretion of antibodies.

Highlights.

  • Alpha-galactosylceramide was used as adjuvant in microparticle vaccines

  • Microparticle vaccines containing glycolipid were sublingually injected

  • A 20 fold higher Th1-biased antibody response was induced

  • Induction of IgG and IgA antibody secretion in oral cavity was accomplished

Acknowledgments

We thank Wei (Auston) Wei for statistic analysis of data. ESD is supported by National Cancer Institute training Grant T32CA009598. DZ is supported by The University of Texas MD Anderson Cancer Center and National Institutes of Health grant AI079232. The University of Texas MD Anderson Cancer Center is supported in part by National Institutes of Health grant CA16672.

Footnotes

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